Anti-DLL3 nanoantibodies, chimeric antigen receptors and their applications

Abstract

The present application relates to an anti-DLL3 nanobody, a chimeric antigen receptor and its application. Specifically, the present application provides a nanobody or an antigen-binding fragment thereof that specifically binds to DLL3, and provides a chimeric antigen receptor, a polynucleotide, a recombinant vector, a host cell, an antibody, an antibody conjugate, a pharmaceutical composition, a kit related to the nanobody or its antigen-binding fragment, and also provides related uses and methods.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an anti-DLL3 nanobody, a chimeric antigen receptor and applications thereof. Background Art

[0002] Small cell lung cancer (SCLC) is a malignant epithelial tumor that originates from the bronchial mucosa or glands and is a type of neuroendocrine carcinoma. It is characterized by small tumor cells, less cytoplasm, finely granular nuclear chromatin, high malignancy, short doubling time, early and extensive metastasis. SCLC accounts for about 15%-20% of all lung cancers and is one of the most aggressive subtypes of lung cancer. The prognosis of small cell lung cancer is usually poor, and the specific survival period is affected by many factors, including cancer stage, treatment method, and the patient's overall health status. The median survival time of patients with limited stage is about 12-18 months, while the median survival time of patients with extensive stage is even shorter.

[0003] DLL3 (Delta-like Ligand 3) is a single-pass transmembrane protein attached to the cell surface and belongs to the Notch ligand family. It consists of 619 amino acids, including a DSL domain, six epidermal growth factor (EGF)-like repeat sequences and a transmembrane domain. The DSL gene sequence is highly conserved in the ligand family and is a necessary structure for binding to the Notch receptor. It is expressed in a variety of neural or neuroendocrine tumors, especially in small cell lung cancer (SCLC), where more than 80% of cases show positive expression of DLL3, while it is almost not expressed in normal adult tissues. This makes DLL3 an ideal target for immunotherapy of SCLC and other DLL3-positive tumors. Due to the high expression of DLL3 on the surface of tumor cells, targeted therapy against DLL3 shows great potential in tumor treatment. SCLC, as a highly lethal and invasive neuroendocrine tumor, is sensitive to chemotherapy and radiotherapy but has a very poor prognosis, with an extremely low five-year survival rate. Therefore, the development of targeted therapy against DLL3 is of great significance for improving the prognosis of tumor patients, especially SCLC patients.

[0004] Heavy chain antibodies (HCAbs) are a special class of antibodies that consist of only two heavy chains and no light chains. This type of antibody was originally discovered in camelids (such as camels, alpacas, etc.) and is a form of antibody naturally produced by camelids during evolution. Heavy chain antibodies are composed of two heavy chains, each of which contains a variable region (VHH) and multiple constant regions (such as CH2, CH3, etc.). However, unlike traditional antibodies, the heavy chain of heavy chain antibodies undergoes specific shearing during gene expression, resulting in the loss of the CH1 region, which makes it impossible to pair with the light chain. VHH (Variable Domain of Heavy Chain of Heavy-Chain Antibody) is the variable region of heavy chain antibodies, that is, the part of the heavy chain responsible for antigen binding. VHH consists of four framework regions (FRs) and three complementary determining regions (CDRs). Its structure is similar to that of the variable region (VH) of human heavy chain antibodies, but it has a longer CDR3 region (3-28 amino acid residues). This structure enables VHH to bind to antigens independently and with high affinity. Since heavy chain antibodies do not have light chains, VHH, as the variable region of heavy chain antibodies, undertakes the task of binding to antigens alone. VHH's high affinity, high stability and easy engineering transformation make it have broad application prospects in the biomedical field. As an emerging drug, VHH has experienced the dual challenges of R&D maturity and market recognition. With its unique small molecule properties, excellent tissue penetration, easy modification and convenient production, VHH shows great potential in targeted therapy, molecular imaging, biosensing and other fields.

[0005] Chimeric antigen receptor (CAR) is a receptor protein constructed by genetic engineering technology, which can give immune cells (such as T cells, NK cells) new antigen recognition and killing capabilities. CAR usually consists of four parts: extracellular specific tumor antigen recognition region, hinge region, transmembrane region and intracellular signal transduction domain. When CAR binds to specific antigens on the surface of tumor cells, it can activate T cells and trigger a series of signal transduction events, ultimately leading to the killing of tumor cells. DLL3-targeted CAR therapy uses DLL3-specific nanoantibodies as the antigen binding domain of CAR to achieve specific recognition and killing of DLL3-positive tumor cells. Currently, a variety of anti-DLL3 CAR therapies have entered the clinical trial stage, initially showing anti-tumor activity and safety. Therefore, the discovery of more highly active DLL3-specific nanoantibodies can provide better and more flexible options for the development of new DLL3 CAR therapies.

[0006] It should be noted that the methods described in this section are not necessarily methods that have been previously conceived or adopted. Unless otherwise indicated, it should not be assumed that any method described in this section is considered to be prior art simply because it is included in this section. Similarly, unless otherwise indicated, the issues mentioned in this section should not be considered to have been recognized in any prior art. Summary of the invention

[0007] In view of this, the present application provides an anti-DLL3 nanobody that can specifically recognize the DLL3 antigen and achieve targeted killing of tumor cells by coupling with drug molecules or directly serving as the antigen binding domain of CAR.

[0008] According to one embodiment of the present application, the present application provides a nanobody or an antigen-binding fragment thereof, wherein the nanobody or the antigen-binding fragment thereof comprises at least the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0009] According to one embodiment of the present application, a chimeric antigen receptor is also provided, wherein the chimeric antigen receptor comprises the nanobody or antigen-binding fragment thereof described in the present application.

[0010] According to one embodiment of the present application, a polynucleotide is also provided, which encodes the nanobody or antigen-binding fragment thereof described in the present application, or the chimeric antigen receptor described in the present application.

[0011] According to one embodiment of the present application, a recombinant vector is also provided, wherein the recombinant vector comprises the polynucleotide described in the present application.

[0012] According to one embodiment of the present application, a host cell is also provided, wherein the host cell comprises the polynucleotide described in the present application and / or the recombinant vector described in the present application.

[0013] According to one embodiment of the present application, an antibody is also provided, which comprises the nanobody or antigen-binding fragment thereof described in the present application.

[0014] According to one embodiment of the present application, an antibody conjugate is also provided, wherein the antibody conjugate comprises the nanobody or antigen-binding fragment thereof described in the present application, and a payload connected to the nanobody or antigen-binding fragment thereof.

[0015] According to one embodiment of the present application, a pharmaceutical composition is also provided, which includes the nanobody or antigen-binding fragment thereof described in the present application, the chimeric antigen receptor described in the present application, the polynucleotide described in the present application, the recombinant vector described in the present application, the host cell described in the present application, the antibody described in the present application, and / or the antibody conjugate described in the present application.

[0016] According to one embodiment of the present application, a kit is also provided, which includes the nanobody or antigen-binding fragment thereof described in the present application, the chimeric antigen receptor described in the present application, the polynucleotide described in the present application, the recombinant vector described in the present application, the host cell described in the present application, the antibody described in the present application, the antibody conjugate described in the present application, and / or the pharmaceutical composition described in the present application.

[0017] According to one embodiment of the present application, the use of the nanoantibody or antigen-binding fragment thereof, the chimeric antigen receptor described in the present application, the polynucleotide described in the present application, the recombinant vector described in the present application, the host cell described in the present application, the antibody described in the present application, the antibody conjugate described in the present application, the pharmaceutical composition described in the present application, and / or the kit described in the present application in the preparation of a drug or reagent for detecting, preventing, alleviating or treating a disease is also provided.

[0018] According to one embodiment of the present application, a detection method for non-diagnostic purposes is also provided, the method comprising using the Nanobody or antigen-binding fragment thereof described in the present application to detect the expression level of DLL3 in a sample.

[0019] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings exemplarily illustrate the embodiments and constitute a part of the specification, and together with the text description of the specification, are used to explain the exemplary implementation of the embodiments. The embodiments shown are for illustrative purposes only and do not limit the scope of the claims. In all drawings, the same reference numerals refer to similar but not necessarily identical elements.

[0021] Figure 1 This is the EC50 flow cytometric graph of different nanobodies targeting DLL3 detected by flow cytometry in Example 2;

[0022] Figure 2 This is a graph showing the EC50 of different nanobodies targeting DLL3 detected by flow cytometry in Example 2;

[0023] Figure 3This is a graph showing the EC50 of different nanobodies targeting DLL3 detected by ELISA in Example 2;

[0024] Figure 4 This is a flow cytometric graph of the specific binding of different Nanobodies targeting DLL3 using flow cytometry in Example 2;

[0025] Figure 5 It is a schematic diagram of the structure of the chimeric antigen receptor carrying the DLL3 antibody in Example 3;

[0026] Figure 6 Construction of lentiviral plasmid map for the full-length DLL3 CAR in Example 3;

[0027] Figure 7 Construction of retroviral plasmid map for the full-length DLL3 CAR in Example 3;

[0028] Figure 8 This is a flow cytometric graph of the CAR positivity of different nano-antibody CAR-T cells targeting DLL3 of donor 105 in Example 5;

[0029] Fig. 9 This is a flow cytometric graph of the CAR positivity of different nano-antibody CAR-T cells targeting DLL3 of donor 108 in Example 5;

[0030] Fig.10 This is a flow cytometric graph of the CAR positivity of different nano-antibody CAR-NK cells targeting DLL3 of donor 105 in Example 5;

[0031] Fig.11 This is a graph showing the killing effect of different nano-antibodies CAR-T cells targeting DLL3 from donor 105 on 293T-DLL3 in Example 6;

[0032] Fig.12 This is a graph showing the killing effect of different nano-antibodies CAR-T cells targeting DLL3 from donor 108 on 293T-DLL3 in Example 6;

[0033] Fig.13 This is a curve diagram of the killing effect of different nano-antibodies CAR-T cells targeting DLL3 of donor 105 on U118-MG-DLL3 in Example 6;

[0034] Fig.14 This is a curve diagram of the second round of killing effect of different nano-antibodies CAR-T cells targeting DLL3 of donor 105 on U118-MG-DLL3 in Example 6;

[0035] Fig.15This is a curve diagram of the killing effect of different nano-antibodies CAR-NK cells targeting DLL3 of donor 105 on U118-MG-DLL3 in Example 6;

[0036] Fig.16 This is a curve diagram of the second round of killing effect of different nano-antibodies CAR-NK cells targeting DLL3 of donor 105 on U118-MG-DLL3 in Example 6;

[0037] Fig.17 This is a three-round killing effect curve of different nano-antibodies CAR-NK cells targeting DLL3 of donor 105 on U118-MG-DLL3 in Example 6;

[0038] Fig.18 This is a bar graph of IFN-γ factor secretion after co-incubation of different nano-antibody CAR-T cells targeting DLL3 from donor 105 with 293T-DLL3 in Example 6;

[0039] Fig.19 Schematic diagram of expressing full-length and truncated human DLL3 proteins on 293T for epitope mapping in Example 7;

[0040] Fig. 20 This is a diagram showing the flow cytometry expression of full-length and truncated human DLL3 on 293T cells detected by Flag tag in Example 7;

[0041] Fig.21 This is a bar graph of cytokine secretion detected by flow cytometry after 24 hours of co-incubation of effector cells DLL3-CAR-T with 293T expressing full-length or truncated DLL3 in Example 7;

[0042] Fig. 22 The EC50 flow cytometric graph of different humanized antibodies targeting DLL3 detected by flow cytometry in Example 8;

[0043] Fig.23 This is a graph showing the EC50 curves of different humanized antibodies targeting DLL3 detected by flow cytometry in Example 8;

[0044] Fig.24 This is a graph showing the EC50 of different humanized antibodies targeting DLL3 detected by ELISA in Example 8;

[0045] Fig.25 This is a flow cytometric graph of specific binding of different humanized antibodies targeting DLL3 detected by flow cytometry in Example 8;

[0046] Fig.26 This is a flow cytometric graph of the CAR positivity of different humanized antibody CAR-T cells targeting DLL3 of donor 105 in Example 9;

[0047] Fig. 27 This is a flow cytometric graph of the CAR positivity rate of different humanized antibody CAR-T cells targeting DLL3 of donor 105 (second batch) in Example 9;

[0048] Fig.28 This is a curve diagram of the killing effect of different humanized antibody CAR-T cells targeting DLL3 of donor 105 on NCI-H446-DLL3 in Example 9;

[0049] Fig.29 This is a curve diagram of the second round of killing effect of different humanized antibody CAR-T cells targeting DLL3 of donor 105 on NCI-H446-DLL3 in Example 9;

[0050] Fig.30 This is a bar graph of GM-CSF (granulocyte-macrophage colony-stimulating factor) secretion of the cytokine after co-incubation of different humanized antibody CAR-T cells targeting DLL3 from donor 105 with NCI-H446-DLL3 in Example 9;

[0051] Fig.31 This is a bar graph of cytokine TNF-α (tumor necrosis factor) secretion after co-incubation of different humanized antibody CAR-T cells targeting DLL3 from donor 105 and NCI-H446-DLL3 in Example 9;

[0052] Fig.32 This is a bar graph of cytokine IFN-γ (interferon-γ) secretion after co-incubation of different humanized antibody CAR-T cells targeting DLL3 from donor 105 with NCI-H446-DLL3 in Example 9;

[0053] Fig.33 This is a graph showing the continuous proliferation curve of DLL3-CAR-T cells of donor 105 targeting DLL3 with different humanized antibodies under multiple rounds of stimulation of NCI-H446-DLL3 tumor cells in Example 9;

[0054] Fig.34 This is a flow cytometric graph of the CAR positivity of different humanized antibody CAR-NK cells targeting DLL3 of donor 102 in Example 9;

[0055] Fig.35 This is a curve diagram of the killing effect of different humanized antibody CAR-NK cells targeting DLL3 of donor 102 on NCI-H446-DLL3 in Example 9;

[0056] Fig.36 This is a curve diagram of the second round of killing effect of different humanized antibody CAR-NK cells targeting DLL3 of donor 102 on NCI-H446-DLL3 in Example 9;

[0057] Fig.37 This is a three-round killing effect curve of different humanized antibody CAR-NK cells targeting DLL3 of donor 102 on NCI-H446-DLL3 in Example 9;

[0058] Fig.38 This is a curve diagram of the four-round killing effect of different humanized antibody CAR-NK cells targeting DLL3 of donor 102 on NCI-H446-DLL3 in Example 9;

[0059] Fig.39 This is a graph showing the five-round killing effect of different humanized antibody CAR-NK cells targeting DLL3 of donor 102 on NCI-H446-DLL3 in Example 9;

[0060] Fig.40 This is a graph showing the six-round killing effect of different humanized antibody CAR-NK cells targeting DLL3 of donor 102 on NCI-H446-DLL3 in Example 9;

[0061] Fig.41 The figure is a flow cytometric graph of iNKT purity detection in donor 702 in Example 9 (A) and a flow cytometric graph of CAR positivity rate of different humanized antibody CAR-iNKT cells targeting DLL3 in donor 702 (B);

[0062] Fig.42 This is a graph showing the continuous proliferation curve of DLL3-CAR-iNKT of donor 702 targeting DLL3 with different humanized antibodies under multiple rounds of stimulation of NCI-H446-DLL3 tumor cells in Example 9;

[0063] Fig.43 This is a curve diagram of the change in tumor size when CAR-T cells with different humanized antibodies targeting DLL3 in Example 10 treat NCI-H446-DLL3 model mice;

[0064] Fig.44 This is a curve diagram of weight change when CAR-T cells with different humanized antibodies targeting DLL3 in Example 10 treat NCI-H446-DLL3 model mice;

[0065] Fig.45 This is a survival curve diagram of CAR-T cells with different humanized antibodies targeting DLL3 in Example 10 when treating NCI-H446-DLL3 model mice;

[0066] Fig.46 This is a flow cytometric graph of the CAR positivity rate of different anti-DLL3 tandem CAR-T cells targeting DLL3 in donor 105 in Example 11;

[0067] Fig.47 This is a graph showing the continuous proliferation curve of donor 105 targeting DLL3 with different anti-DLL3 tandem CAR-T cells under multiple rounds of stimulation of NCI-H446-DLL3 tumor cells in Example 11. DETAILED DESCRIPTION

[0068] Unless otherwise indicated, all numbers used in this specification and claims to indicate content, concentration, ratio, mass, volume, time, temperature, thickness, technical effect, etc. should be understood as modified by the term "about" or "approximately" in any case. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the attached claims are approximate values. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained through the present disclosure, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or in a manner understood by those skilled in the art.

[0069] Although the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximate, the numerical values ​​set forth in the specific embodiments are provided as accurately as possible. However, any numerical value will inherently contain certain errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include each narrower numerical range that falls within the broader numerical range, just as if these narrower numerical ranges were all clearly written herein.

[0070] Unless otherwise specified or contradictory to the context, the terms or expressions used herein should be read in conjunction with the overall content of this article and as understood by those of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0071] As used herein, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B, and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B, and C. The meanings of similar expressions can be deduced analogously.

[0072] When used herein, the expressions “comprises”, “includes” and “comprising” mean that in addition to the listed elements, other elements are not excluded.

[0073] As used herein, "nucleic acid" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof.

[0074] In this application, "polypeptide" and "peptide" are used interchangeably to refer to amino acid polymers of any length. Therefore, polypeptides, oligopeptides, proteins, antibodies and enzymes are all included in the definition of polypeptides.

[0075] "Identity" described in the present application refers to the degree of similarity between a pair of sequences (nucleotides or amino acids).Identity is determined by dividing the number of identical residues by the total number of residues, and multiplying the quotient by 100 to obtain a percentage. Gaps are not counted when evaluating identity. Therefore, two copies of identical sequences have 100% identity, but sequences with deletions, additions or substitutions may have a lower degree of identity. It is known to those skilled in the art that there are some computer programs that can be used to determine the identity of a sequence, such as those using algorithms such as BLAST. BLAST nucleotide searches are performed using the NBLAST program, and BLAST protein searches are performed using the BLASTP program, wherein the default parameters of each program are used.

[0076] The "antibody" described in this application refers to a specific immunoglobulin for an antigenic site. Antibodies can be manufactured according to methods known in the art. Antibodies may be in the form of polyclonal or monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (e.g., bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, fully human antibodies, human antibodies, fusion proteins comprising an antigen binding site of an antibody, and any other modified immunoglobulin molecules comprising an antigen binding site, as long as the antibody exhibits the desired biological binding activity.

[0077] "Nanobody", "VHH", "single domain antibody" and "sdAb" described in this application are used interchangeably to represent the variable domain of a single heavy chain of those types of antibodies found in camelids. In the absence of a light chain, each nanobody has three CDRs, represented by CDR1, CDR2 and CDR3. It is the smallest antigen-binding fragment with complete function. Usually, an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a nanobody (Nanobody, Nb) consisting of only one heavy chain variable region, also known as VHH. Nanobody / single domain antibody, as a new type of small molecule antibody fragment, is cloned from the heavy chain variable region (VHH) of natural heavy chain antibodies of camelids. It has excellent biological properties, a molecular weight of 12-15kDa, which is one-tenth of a complete antibody, and has good tissue penetration, high specificity and good water solubility.

[0078] The "antigen-binding fragment" as used herein refers to one or more parts of an antibody that retain the binding specificity to a target antigen. Antigen-binding fragments include, but are not limited to, VHH, fragments containing CDRs, and the like.

[0079] "Specific binding" as used herein refers to the non-covalent interaction between an antibody or its antigen-binding fragment and an antigen. The strength or affinity of the interaction can be expressed by the equilibrium dissociation constant (KD or Kd) of the antigen and the corresponding antibody: the smaller the KD value, the stronger the binding strength between the epitope and the antibody. The equilibrium dissociation constant (KD) is calculated as the ratio of Koff / kon, where the "association rate constant (Kon)" and "dissociation rate constant (Koff)" can be determined by calculating the concentration and the actual association and dissociation rates (see Nature 361: 186-87 (1993)).

[0080] The precise amino acid sequence boundaries of a given complementarity determining region (CDR) or framework region (FR) can be readily determined using a number of numbering schemes well known in the art, including: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Edition Public Health Service, National Institutes of Health, Bethesda, Maryland ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding sitetopography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGTunique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272 (“AbM” numbering scheme).

[0081] The boundary of given CDR or FR may be different depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, and the Chothia scheme is based on structural information. The numbering of Kabat and Chothia schemes is based on the most common antibody region sequence length, wherein insertion (such as "30a") is provided by inserting letters and deletion occurs in some antibodies. These two schemes place some insertions and deletions (indel) in different positions, thereby producing different numbering. The Contact scheme is based on the analysis of complex crystal structures, and is similar to the Chothia numbering scheme in many respects. The AbM scheme is a compromise between Kabat and Chothia definitions, and it is based on the scheme used by the AbM antibody modeling software of Oxford Molecular.

[0082] Therefore, unless otherwise specified, it should be understood that the "CDR" of a given antibody or a region thereof (such as its variable region) encompasses CDRs defined by any of the above schemes or other known schemes. For example, where a specific CDR (e.g., CDR3) is specified to contain a given amino acid sequence, it should be understood that such a CDR may also have the sequence of a corresponding CDR (e.g., CDR3) defined by any of the above schemes or other known schemes. Similarly, unless otherwise specified, it should be understood that the FR of a given antibody or a region thereof (such as its variable region) encompasses FRs defined by any of the above schemes or other known schemes. Unless otherwise specified, the numbering scheme used to define the boundaries of CDRs and FRs herein adopts the IMGT scheme.

[0083] The "chimeric antigen receptor" or "CAR" described in this application refers to a group of polypeptides that, when in an immune effector cell, provide the cell with specificity for a target cell (e.g., a cancer cell) and provide intracellular signal generation. In some embodiments, CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen binding domain comprises a nanobody provided in this application.

[0084] The "vector" described in this application refers to a self-replicating DNA molecule that transfers an exogenous target gene into a host organism, and is often in the form of a circular double-stranded DNA molecule. Typical vectors include plasmids, viruses, bacteriophages, cosmids, and minichromosomes. Among them, plasmids are the most common form of vectors, which refer to circular double-stranded DNA that can accept exogenous nucleic acid fragments and replicate in prokaryotic or eukaryotic cells.

[0085] The term "expression vector" and "recombinant vector" used herein are interchangeable and refer to a vector containing an exogenous gene and a regulatory element for expression in a designated host organism. Introducing an expression vector into a suitable host organism can enable it to express the inserted target gene.

[0086] "Foreign" or "heterologous" as used herein are used interchangeably and refer to a source different from the native (original) organism, such as an organism from another species. "Heterologous gene" or "foreign gene" as used herein refers to a gene that does not naturally occur in the host organism and is introduced into the host organism by gene transfer.

[0087] "Transformation" as described in this application refers to the transfer of foreign genes into host organisms such as host cells, resulting in genetically stable inheritance. The transformed gene can be in the form of a plasmid retained in the host organism, or it can be integrated into the host organism genome. Host organisms containing transformation genes are referred to as "transgenic" or "recombinant" or "transformed" organisms or "engineered" organisms. Expression vectors can be used to transform host organisms using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, and the steps used are well known in the art. If necessary, methods such as microinjection, electroporation or liposome packaging can also be used. This is a well-known technology in the art and will not be described in detail here.

[0088] As used herein, "relieve" and "treat" and their synonyms refer to the improvement of a disease, disorder, and / or condition. "Relieve" and "treat" can be an improvement in at least one measurable physical parameter, which is not necessarily recognizable to the patient. "Relieve" and "treat" can also be physically (e.g., stabilizing recognizable symptoms), physiologically (e.g., stabilizing physical parameters), or both, inhibiting the development of a disease, disorder, and / or condition. "Relieve" and "treat" can also be slowing down the development or reversal of a disease, disorder, and / or condition.

[0089] As used herein, "prevent," "prevent," and its synonyms refer to delaying the onset of a particular disease, disorder, and / or condition, or symptoms associated with such disease, disorder, and / or condition, or reducing the risk of acquiring such disease, disorder, and / or condition.

[0090] The "sample" described in this application can refer to a biological sample, including, for example, blood and other body fluids, including but not limited to peripheral blood, serum, plasma, urine and saliva; and also includes solid tissue samples, such as biopsy specimens, especially those containing cancer cells. In certain embodiments, samples containing cancer cells such as tumor tissue or cancer cell lines are the most preferred sample types to be used in the present method.

[0091] In order to make the above-mentioned objects, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below.

[0092] Nanobodies or antigen-binding fragments thereof

[0093] According to one embodiment of the present application, the present application provides a nanobody or an antigen-binding fragment thereof, wherein the nanobody or the antigen-binding fragment thereof comprises at least the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0094] In some embodiments, the nanobody or its antigen-binding fragment specifically binds to human DLL3. In some embodiments, the human DLL3 comprises the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are sites where the nanobody specifically binds to human DLL3. Therefore, those skilled in the art can know from the disclosure of this application that antibodies or their antigen-binding fragments comprising the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 can effectively bind to human DLL3.

[0095] In some embodiments, the Nanobody or antigen-binding fragment thereof comprises a heavy chain variable region. In some embodiments, the heavy chain variable region comprises a complementary determining region (CDR). In some embodiments, the complementary determining region comprises an amino acid sequence as shown below: the amino acid sequence of CDR1 is shown in SEQ ID NO: 1; the amino acid sequence of CDR2 is shown in SEQ ID NO: 2; and the amino acid sequence of CDR3 is shown in SEQ ID NO: 3.

[0096] In some embodiments, the Nanobody or antigen-binding fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 4, 14-17, 24. In some embodiments, the Nanobody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 4, 14-17, 24.

[0097] In some embodiments, the nano antibody or its antigen binding fragment can be a humanized antibody or a chimeric antibody. In some embodiments, the nano antibody or its antigen binding fragment can be a monoclonal antibody. In some embodiments, the nano antibody or its antigen binding fragment can be a monovalent antibody, a bivalent antibody or a multivalent antibody. In some embodiments, the nano antibody or its antigen binding fragment can be a part of a bispecific antibody or a multispecific antibody. In some embodiments, the nano antibody or its antigen binding fragment can be fused with other fragments or molecules. In some embodiments, the antigen binding fragment can be a Fab.

[0098] The Nanobodies or antigen-binding fragments thereof disclosed herein may contain one or more glycosylation sites. As is well known to those skilled in the art, the presence of one or more glycosylation sites in the variable region may enhance antibody immunogenicity or alter the pharmacokinetics of the antibody due to altered antigen binding.

[0099] Chimeric Antigen Receptor

[0100] According to one embodiment of the present application, a chimeric antigen receptor is also provided, wherein the chimeric antigen receptor comprises the nanobody or antigen-binding fragment thereof described in the present application.

[0101] In some embodiments, the chimeric antigen receptor further comprises a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain.

[0102] In some embodiments, the chimeric antigen receptor comprises a signal peptide. In some embodiments, the signal peptide comprises the signal peptides of the following molecules: α chain and β chain of T cell receptor, CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG6. In some embodiments, the signal peptide is a CD8α signal peptide. In some embodiments, the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 42.

[0103] In some embodiments, the extracellular antigen binding domain comprises the nanobody provided in the present application. The nanobody provided in the present application can be coupled to other parts by methods well known in the art to construct a chimeric antigen receptor.

[0104] In some embodiments, the chimeric antigen receptor comprises a hinge region. In some embodiments, the hinge region comprises the hinge region of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, IL-11 receptor. In some embodiments, the hinge region is a CD8α hinge region. In some embodiments, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO: 43.

[0105] In some embodiments, the chimeric antigen receptor comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises the transmembrane domain of the following molecules: CD8, CD28, CD27, CD137, IgG1, IgG4, 4-1BB, PD-1, CD34, CD3ε, CD8α, IL-2 receptor, IL-7 receptor, IL-11 receptor. In some embodiments, the transmembrane domain is a CD8α transmembrane domain. In some embodiments, the amino acid sequence of the CD8α transmembrane domain is as shown in SEQ ID NO: 44.

[0106] In some embodiments, the chimeric antigen receptor comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain includes the costimulatory signaling domains of the following molecules: 4-1BB, CD28, ICOS, CD27, CD19, LIGHT, CD40, OX40, CD2, CD226, CD278. In some embodiments, the costimulatory signaling domain is a 4-1BB costimulatory signaling domain. In some embodiments, the amino acid sequence of the 4-1BB costimulatory signaling domain is as shown in SEQ ID NO: 45.

[0107] In some embodiments, the chimeric antigen receptor comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of the following molecules: CD3ζ, FcεRIγ, CD27, CD28, CD134, CD137, MyD88, CD40, OX40, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d, FYN. In some embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain. In some embodiments, the amino acid sequence of the CD3ζ intracellular signaling domain is shown in SEQ ID NO: 46.

[0108] In some embodiments, the chimeric antigen receptor further comprises a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain in series. In some embodiments, the extracellular antigen binding domain comprises two nanobodies or antigen-binding fragments thereof described herein.

[0109] In some embodiments, the chimeric antigen receptor comprises a CD8α signal peptide, a nanobody or an antigen-binding fragment thereof described in the present application, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB co-stimulatory signal domain, and a CD3ζ intracellular signaling domain, which are sequentially connected in series.

[0110] Polynucleotide, recombinant vector, host cell

[0111] According to one embodiment of the present application, a polynucleotide is also provided, which encodes the nanobody or antigen-binding fragment thereof described in the present application, or the chimeric antigen receptor described in the present application.

[0112] Due to the degeneracy of codons, those skilled in the art can know that there are many nucleic acid sequences that can encode the nanobodies or chimeric antigen receptors provided in the present application, which are not limited here. Those skilled in the art can perform appropriate codon optimization and select appropriate nucleic acid sequences for expressing the nanobodies or chimeric antigen receptors provided in the present application according to the different expression purposes or hosts.

[0113] In some preferred embodiments, the polynucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 9, 19-22, 25. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 9, 19-22, 25.

[0114] According to one embodiment of the present application, a recombinant vector is also provided, the recombinant vector comprising the polynucleotide described in the present application. Recombinant vectors suitable for expressing the nanobodies described in the present application are well known in the art and are not limited here.

[0115] In some embodiments, the recombinant vector includes a cloning vector, an expression vector. Both viral and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (typically with expression cassettes for expressing proteins or RNA) and human artificial chromosomes (see, for example, Harrington et al., Nat Genet. [Natural Genetics] 15: 345, 1997, the contents of which are hereby incorporated by reference herein). Virus-derived vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxvirus vectors, and herpesvirus vectors.

[0116] In some embodiments, the recombinant vector further comprises a promoter. The promoter may be any suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the antibody. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular proteins or polypeptides that are homologous or heterologous to the host cell.

[0117] The recombinant vector of the present application can be constructed by methods known in the art. For example, appropriate restriction sites can be added to both ends of the polynucleotide of the present application according to the restriction sites contained in the backbone vector used, and then loaded into the backbone vector.

[0118] According to one embodiment of the present application, a host cell is also provided, the host cell comprising the polynucleotide described in the present application and / or the recombinant vector described in the present application. The host cell can be selected according to the type of the expression vector. The polynucleotide and / or the expression vector can be delivered into the host cell using any suitable method known in the art, which is not limited here.

[0119] In some embodiments, mammalian host cells are used to express and produce the nano antibodies or their antigen-binding fragments, chimeric antigen receptors of the present application. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines containing exogenous expression vectors. These include any normal non-immortal or normal or abnormal immortal animals or human cells. For example, many suitable host cell lines capable of secreting complete immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells and hybridomas. Exemplary host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells (e.g., HEK293, HEK293T, HEK293F), monkey kidney (COS) cells (e.g., COS-1, COS-7), baby hamster kidney (BHK) cells (e.g., BHK-21), African green monkey kidney cells (e.g., BSC-1), HeLa cells, human hepatocellular carcinoma cells (e.g., Hep G2), myeloma cells (e.g., NS0, 653, SP2 / 0), lymphoma cells, oocytes, and cells from transgenic animals (e.g., mammary epithelial cells), or any derivative, immortalized, or transformed cells thereof.

[0120] In some embodiments, the host cell is an immune cell, including but not limited to T cells, B cells, NK cells, NKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof. In some preferred embodiments, the immune cell is a T cell or a NK cell.

[0121] In some embodiments, the host cell also carries an exogenous cytokine coding sequence. In some embodiments, the host cell also expresses another chimeric antigen receptor that recognizes a different tumor antigen; or it also expresses a chemokine receptor; or it also expresses a protein that can block TGF beta signaling; or it also expresses a safety switch. In some embodiments, the chemokine receptors include: CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CCR4, CCR5, CCR6.

[0122] Antibodies, Antibody Conjugates

[0123] According to one embodiment of the present application, an antibody is also provided, which comprises the nanobody or antigen-binding fragment thereof described in the present application.

[0124] In some embodiments, the antibody is a full-length antibody, which includes the nano antibody provided herein, and further includes a light chain portion. Those skilled in the art can select a suitable light chain portion as needed, and use methods known in the art to couple the nano antibody provided herein with the light chain portion to form a full-length antibody.

[0125] In some embodiments, the antibody is a multispecific antibody, which comprises a nanobody provided herein, and further comprises one or more second antibodies or antigen-binding fragments thereof that specifically bind to other antigens. In some embodiments, the second antibody or antigen-binding fragment thereof is selected from a full-length antibody, Fab, Fab'', (Fab'')2, Fv, scFv, scFv-scFv, miniantibody, double antibody or sdAb. Those skilled in the art can select a suitable second antibody or antigen-binding fragment thereof as needed, and couple the nanobody provided herein to the second antibody or antigen-binding fragment thereof using methods known in the art to form a multispecific antibody.

[0126] According to one embodiment of the present application, an antibody conjugate is also provided, wherein the antibody conjugate comprises the nanobody or antigen-binding fragment thereof described in the present application, and a payload connected to the nanobody or antigen-binding fragment thereof.

[0127] In some embodiments, the nanobody or its antigen binding fragment can be directly connected to the payload or indirectly connected. In some embodiments, the nanobody or its antigen binding fragment is indirectly connected to the payload through a linker. In some embodiments, the antibody conjugate further comprises a linker for connecting the nanobody or its antigen binding fragment to the payload.

[0128] In some embodiments, the linker can be a cleavable linker or a non-cleavable linker. For example, an acid-labile linker (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020) can be used.

[0129] In some embodiments, the cleavable linker may include a peptide unit comprising 2-20 amino acids, preferably a peptide unit selected from -valine-citrulline-(-Val-Cit-), -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -valine-alanine-(-Val-Ala-), -valine-lysine-(-Val-Lys-), -valine-arginine-(-Val-Arg-), -phenylalanine-citrulline-(-Phe-Cit-), -phenylalanine-lysine-(-Phe-Lys-), -phenylalanine-arginine-(-Phe-Arg-), and combinations thereof.

[0130] In some embodiments, the payload comprises a cytotoxin, a PROTAC, a molecular glue, a polymer, a protein (including but not limited to an antibody or an antigen-binding fragment thereof), a drug, a radioisotope, a nucleic acid compound, a glucocorticoid, or a detectable marker.

[0131] In some embodiments, the effective load includes a cytotoxin or a radioactive isotope. Suitable radioactive isotopes and cytotoxins are known in the art, and those skilled in the art can select suitable types as needed, and use methods known in the art to couple the nanobodies provided herein with cytotoxins or radioactive isotopes.

[0132] In some non-limiting embodiments, the antibody conjugate comprises a nanobody provided herein and a cytotoxin to form an antibody-drug conjugate (ADC). In some embodiments, examples of cytotoxins include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine, lomustine, 1-methylnitrosourea, cyclophosphamide, mechlorethamine, busulfan, dibromomannitol, streptozocin, mitomycin, cis-dichlorodiamine platinum, cisplatin, carboplatin, daunorubicin, doxorubicin, detoxorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone , actinomycin D, bleomycin, calicheamicin, mithramycin, anthramycin, vincristine, vinblastine, paclitaxel, ricin, Pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, teniposide, colchicine, dihydroxyanthracenedione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane, interferon, and combinations thereof.

[0133] In some non-limiting embodiments, the antibody conjugate comprises a nanobody and a radioactive isotope provided in the present application to form a radionuclide drug conjugate (RDC). Examples of radioactive isotopes that can be used in the present application include, but are not limited to, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, 99mTc, 123I, 18F, and 68Ga.

[0134] In addition to the antibodies and antibody conjugates disclosed above, the nanobodies or antigen-binding fragments thereof disclosed herein can also be conjugated with other factors by chemical methods or by genetic engineering. These factors provide the effect of targeting the antibody to the desired functional site or improve or provide other properties for the antibody.

[0135] Pharmaceutical compositions, kits

[0136] According to one embodiment of the present application, a pharmaceutical composition is also provided, which includes the nanobody or antigen-binding fragment thereof described in the present application, the chimeric antigen receptor described in the present application, the polynucleotide described in the present application, the recombinant vector described in the present application, the host cell described in the present application, the antibody described in the present application, and / or the antibody conjugate described in the present application.

[0137] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically or physiologically acceptable carrier. The carrier can be any compatible physiologically acceptable non-toxic substance suitable for delivering the polypeptide, polynucleotide or recombinant vector provided by the present application into a mammal (eg, a human).

[0138] The term "pharmaceutically acceptable carrier" refers to a carrier, diluent or adjuvant used for the preparation or administration of the polypeptide, polynucleotide or recombinant vector provided in the present application, which is not an essential active ingredient and has no excessive toxicity after administration. Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art.

[0139] Among them, "physiologically acceptable carrier" refers to a carrier or diluent or adjuvant that does not cause significant stimulation to the organism and does not eliminate the pharmaceutical activity and properties of the polypeptide, polynucleotide or recombinant vector provided by the application. Suitable physiologically acceptable carriers are also well known to those of ordinary skill in the art.

[0140] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the excipient comprises at least one of a solubilizer, a disintegrant, a wetting agent, a stabilizer, a thickener, a diluent, a buffer, and a flavoring agent.

[0141] In some non-limiting embodiments, the carriers and / or excipients used in the pharmaceutical compositions of the present application may include, for example, liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, suspending agents, dispersants, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0142] According to one embodiment of the present application, a kit is also provided, which includes the nanobody or antigen-binding fragment thereof described in the present application, the chimeric antigen receptor described in the present application, the polynucleotide described in the present application, the recombinant vector described in the present application, the host cell described in the present application, the antibody described in the present application, the antibody conjugate described in the present application, and / or the pharmaceutical composition described in the present application.

[0143] use

[0144] According to one embodiment of the present application, the use of the nanoantibody or antigen-binding fragment thereof, the chimeric antigen receptor described in the present application, the polynucleotide described in the present application, the recombinant vector described in the present application, the host cell described in the present application, the antibody described in the present application, the antibody conjugate described in the present application, the pharmaceutical composition described in the present application, and / or the kit described in the present application in the preparation of a drug or reagent for detecting, preventing, alleviating or treating a disease is also provided.

[0145] In some embodiments, the disease includes a disease associated with abnormal expression of DLL3. The nanobodies or antigen-binding fragments thereof that specifically bind to DLL3 provided in the present application have good binding ability to DLL3, high specificity and affinity, and can effectively treat diseases associated with abnormal expression of DLL3.

[0146] As known in the art, there are a variety of diseases accompanied by abnormal DLL3 expression (e.g., increased expression levels), including but not limited to: small cell lung cancer (DOI: 10.1056 / NEJMoa2307980), IDH-mutated glioma (DOI: 10.1158 / 1078-0432.CCR-18-2312), small cell bladder cancer (DOI: 10.1158 / 1078-0432.CCR-18-1278), neuroendocrine prostate cancer (DOI: 10.1038 / s41585-019-0190-6), large cell neuroendocrine carcinoma (DOI: 10.1126 / scitranslmed.aac9459), the disclosures of which are incorporated herein by reference in their entirety.

[0147] In some preferred embodiments, the disease comprises a tumor or cancer. In some preferred embodiments, the disease comprises lung cancer, brain glioma, bladder cancer, prostate cancer, neuroendocrine cancer.

[0148] In the uses described in the present application, the dosage of the nano antibodies or chimeric antigen receptors provided herein can depend on several factors, including the severity and responsiveness of the symptoms, the route of administration, the treatment time (several days to several months to several years), and the time to improve the symptoms. Those skilled in the art can adjust the dosage regimen to provide a therapeutic response according to the specific circumstances of the patient. For example, a single administration can be performed, several separate doses can be administered within a predetermined time period, or the dosage can be reduced or increased as indicated by the treatment situation. The specifications of the dosage are determined by the specific therapeutic effect to be achieved. The dosage value can also vary with the type and severity of the condition to be alleviated. For any particular subject, the specific dosage regimen can be adjusted over time according to individual needs and the professional judgment of the treating clinician.

[0149] Non-diagnostic testing methods

[0150] According to one embodiment of the present application, a detection method for non-diagnostic purposes is also provided, the method comprising using the Nanobody or antigen-binding fragment thereof described in the present application to detect the expression level of DLL3 in a sample.

[0151] In some embodiments, the detection method comprises the following steps: mixing the nanobody with the sample; and detecting the expression level of DLL3 in the sample.

[0152] Nanobodies or antigen-binding fragments thereof disclosed herein can be chemically or genetically labeled to provide detectable nanobodies, detectable antibodies comprising detectable moieties. Detectable moieties include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions.

[0153] In some embodiments, the method for detecting the expression level of DLL3 in the sample includes enzyme-linked immunosorbent assay, immunoblotting, flow cytometry and / or immunostaining. Those skilled in the art can select a suitable method as needed, and the implementation steps of these detection methods are all known in the art and are not limited here.

[0154] In some embodiments, the sample may refer to a biological sample, including blood and other body fluids (e.g., peripheral blood, serum, plasma, urine, and saliva); and also includes solid tissue samples (e.g., biopsy specimens). In some preferred embodiments, a sample containing blood (e.g., serum or plasma) is the most preferred sample type in this application.

[0155] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory to each other), and the various embodiments formed by such combination are all considered as part of the disclosure of this application.

[0156] The exemplary embodiments of the present application will be described below in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered to be merely exemplary and are by no means intended to limit the scope of protection of the present application. The scope of protection of the present application is limited only by the claims. Therefore, it should be recognized by those of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope of the present application. Similarly, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0157] Example

[0158] If no specific techniques or conditions are specified in this embodiment, the techniques or conditions described in the literature in this field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained through commercial purchase.

[0159] Example 1: Preparation of Nanobodies Targeting DLL3

[0160] In this example, a yeast-displayed nanoantibody library was constructed and panned, and ELISA was used for preliminary screening. The specific steps are as follows:

[0161] 1.1 Construction of yeast-displayed nanoantibody library

[0162] Alpacas were immunized with recombinant protein of the extracellular segment of DLL3 to stimulate B cells to express antigen-specific nanoantibodies. After the serum titer was detected by ELISA, peripheral blood was drawn; lymphocytes were separated, total RNA was extracted, and then reverse transcribed into cDNA. The VHH sequence was amplified from the cDNA sample using a single-domain antibody cloning primer combination and subcloned into the yeast display vector pYDisplay. EBY100 competent cells were electroporated to construct a single-domain antibody yeast display library; the concentration was adjusted and the cells were aliquoted and frozen in a -80℃ refrigerator for later use.

[0163] 1.2 Screening of yeast-displayed nanoantibody library

[0164] First, the antigen was modified with Biotin, and the single-domain antibody yeast cells constructed in 1.1 were incubated with the labeled target under certain conditions to fully bind the single-domain antibody to the target. The incubated yeast cells were prepared into a single cell suspension, and after one round of magnetic bead sorting and enrichment, multiple rounds of FACS sorting were performed to select the cell population with the strongest fluorescence signal for enrichment. After each round of screening, the enriched yeast cells were amplified and used for the next round of screening. Single clones were isolated from the enriched yeast cells, and the expression of the single clones was verified. The DLL3 antigen was used for detection to confirm that the single domain antibody expressed by it could bind to the target. Using the obtained yeast as a template, the gene sequence of the nano antibody VHH was amplified, and the second-generation sequencing was performed to obtain 5 anti-DLL3 nano antibodies, which were named DLL3-1-A7, DLL3-1-B12, DLL3-1-C11, DLL3-1-D9 and DLL3-2-D3. Among them, the sequence of the obtained nano antibody DLL3-1-B12 is shown in Table 1 below.

[0165] Table 1 Antibody DLL3-1-B12 related sequences

[0166]

[0167] Example 2: Expression, purification, EC50 and specificity identification of nanobodies targeting DLL3

[0168] In this example, the five candidate antibodies obtained in Example 1 were expressed, purified, and the EC50 of the antibodies was detected by ELISA, and the specificity and affinity of the antibodies binding to cells were detected by flow cytometry. The steps included:

[0169] 2.1 VHH Nanobody Expression and Purification

[0170] The five candidate antibodies obtained in Example 1 were cloned into the eukaryotic expression plasmid pcDNA3.4, expressed in CHO cells, and purified by Protein A / G affinity chromatography.

[0171] Five purified anti-DLL3 VHH antibodies were obtained, including anti-DLL3-1-A7 VHH antibody, anti-DLL3-1-B12 VHH antibody, anti-DLL3-1-C11 VHH antibody, anti-DLL3-1-D9 VHH antibody, and anti-DLL3-2-D3 VHH antibody. The absorbance value of OD280 was detected by a microplate reader and the concentration was calculated. The purity and molecular weight were detected by SDS-PAGE gel.

[0172] 2.2 EC50 detection by flow cytometry or ELISA

[0173] 2.2.1 EC50 by flow cytometry

[0174] The median effective concentration (EC50) of the five anti-DLL3 VHH antibodies obtained in step 2.1 of this example was determined by flow cytometry (FACS, Fluorescence Activated Cell Sorting) to reflect the affinity of the antibodies for the DLL3 antigen.

[0175] Using 293F cells (Chinese Academy of Sciences Cell Bank, SCSP-688S) and 293F cells stably transfected with human DLL3 (293F / Human DLL3), DLL3 target antibody was gradiently diluted (starting concentration 30ug / ml, 3-fold gradient dilution 11 points) and co-incubated with 293F / Human DLL3, and Goat anti-Human IgG (H+L)-Alexa Flour 647 (1:1000 dilution) was used as the secondary antibody. Flow cytometry was used to detect and record the changes in the binding intensity at each concentration. As the antibody concentration increases, the cell effect usually presents a dose-effect curve, where EC50 is the drug concentration that causes 50% of the maximum effect on the curve. The measurement results are shown in Figure 2. Figure 1 Flowchart and Figure 2 EC50 curve is shown. Figure 2 It can be seen that the anti-DLL3-1-B12 antibody exhibits the highest binding ability, with the smallest EC50 value of 0.03063ug / mL, followed by the positive control (BI-905711) antibody and the anti-DLL3-1-C11 antibody, with EC50 values ​​of 0.06872ug / mL and 0.08562ug / mL, respectively.

[0176] 2.2.2 ELISA EC50

[0177] The five anti-DLL3 VHH antibodies obtained in step 2.1 of this example were measured by enzyme-linked immunosorbent assay (ELISA) for half effective concentration (EC50) to evaluate the affinity of these antibodies for DLL3 antigen.

[0178] The experimental principle is based on the efficient catalytic effect of enzymes. The results of the immune reaction are amplified by enzyme labeling technology, making the determination method extremely sensitive. ELISA uses enzyme-labeled anti-antibodies (anti-human immunoglobulin antibodies) to detect the test antibody bound to the solid phase antigen. On the solid phase carrier, the antigen-antibody complex is separated from other substances in the liquid through a washing step. After that, the enzyme-labeled antigen or antibody is added to bind to the complex on the solid phase carrier. After the addition of the substrate for the enzyme reaction, the substrate is catalyzed by the enzyme to generate a colored product, and the amount of the product is directly related to the amount of the test antibody in the specimen. According to the depth of the color reaction, the antibody can be qualitatively or quantitatively analyzed to obtain the EC50 value of each antibody, reflecting its affinity for the DLL3 antigen.

[0179] The results are shown in Table 2 and Figure 3 As shown. Figure 3 It can be seen that the anti-DLL3-1-B12 antibody exhibits the highest binding ability, with the smallest EC50 value of 0.03631ug / mL, followed by the anti-DLL3-2-D3 antibody and the positive control antibody BI-905711 (sequence as shown in SEQ ID NO: 5), with EC50 values ​​of 0.04482ug / mL and 0.5789ug / mL, respectively.

[0180] Table 2 EC50 values ​​of ELISA test

[0181]

[0182] 2.3 Flow cytometry specificity

[0183] CHO-S cells (Jin Shaoyuan Biotechnology, JSY-CC2001), CHO-S cells stably transfected with human DLL3 (CHO-S / Human DLL3) and CHO-S cells stably transfected with mouse DLL3 (CHO-S / Mouse DLL3) were used to evaluate the antibody binding cell specificity by flow cytometry. 3×10^5 / well of CHO-S, CHO-S / Human DLL3 and CHO-S / Mouse DLL3 were mixed with the purified anti-DLL3 VHH antibody obtained in step 2.1 of this example, incubated in an ice bath for 30 minutes, and then incubated with Goat anti-human lgG Fc PE (1:500 dilution) antibody for 30 minutes. The results were detected by flow cytometry. Figure 4Among them, 1-A7, 1-B12, 1-C11, 1-D9 and 2-D3 antibodies specifically bind to CHO-S / Human DLL3, and have different degrees of weak binding to CHO-S / Mouse DLL3. 1-A7, 1-B12, 1-C11 and 2-D3 do not bind to CHO-S, but 1-D9 has weak binding to CHO-S; the negative control does not bind to CHO-S, CHO-S / Human DLL3, and CHO-S / Mouse DLL3. This shows that the anti-DLL3 nanoantibody can specifically recognize the DLL3 antigen on the cell surface.

[0184] Example 3: Construction of chimeric antigen receptor plasmid (CAR plasmid) targeting DLL3 and viral packaging and concentration

[0185] In this Example 3, lentiviral and retroviral vectors were constructed and packaged to obtain lentivirus and retrovirus.

[0186] 3.1 Construction of lentiviral and retroviral vectors

[0187] First, according to step 2.1 in Example 2, the anti-DLL3 VHH antibody sequence is further constructed to form a chimeric antigen receptor (i.e., a chimeric antigen receptor carrying DLL3 VHH). The schematic diagram of the structure of the chimeric antigen receptor is shown in FIG. Figure 5 As shown, it mainly includes the following parts: CD8a signal peptide, nanoantibody specific to DLL3 (anti-DLL3 VHH), CD8a hinge region, transmembrane region, co-stimulatory molecule and immune receptor tyrosine activation motif (CD3ζ), and its amino acid sequence is shown in Table 3 below.

[0188] Table 3 CAR-related sequences

[0189]

[0190] Secondly, the nucleic acid encoding the CAR fragment was cloned into a lentiviral and retroviral vector to establish a full-length CAR construct in a single coding frame, wherein the lentiviral vector uses the human EF1a promoter for expression. The resulting CAR backbone vector was named "pLV-DLL3-CAR-XX" or "pRV-DLL3-CAR-XX". The vector map is shown in Figure 6 and 7 As shown. Lentiviral plasmids expressing chimeric antigen receptors of 1-A7, 1-B12, 1-C11, 1-D9 and 2-D3, VH-VL (positive control BI-905711), VL-VH (positive control), AMG757 (positive control) and Flag (negative control) were constructed, and the sequences involved are shown in Table 4 below.

[0191] Table 4 Viral vector related sequences

[0192]

[0193] 3.2 Lentivirus packaging and concentration

[0194] 1) 293T cells in logarithmic growth phase were inoculated with 1×10 7 Incubate overnight in a T75 flask at 37°C, 5% CO2 to prepare for virus packaging in DMEM containing 10% fetal bovine serum;

[0195] 2) Dissolve 8 μg of the lentiviral vector pLV-DLL3-CAR obtained in step 3.1 of this example, 4 μg of the auxiliary plasmid pSPAX2, and 8 μg of the envelope plasmid VSVG in 0.5 mL of serum-free opti-MEM culture medium, mix well, and obtain a DNA mixture;

[0196] 3) Dissolve 40ug PEI pro (1 ug / uL) in 0.5mL serum-free opti-MEM culture medium, vortex at 1000rpm for 5 seconds, and incubate at 25℃ for 5min to obtain a PEI mixed solution;

[0197] 4) Add the PEI mixture obtained in step 3) to the DNA mixture in step 2), vortex mix or gently mix immediately after addition, and incubate at 25°C for 25 minutes to obtain a transfection complex;

[0198] 5) Add 1 mL of the transfection complex obtained in step 4) dropwise to the 293T cell culture in step 1). After 6 hours, replace with fresh culture medium. After 48 hours, collect the supernatant of the virus solution, centrifuge at 1000g / 5min to remove cell debris, concentrate with an Amicon® Ultra filter (Milipore), and aliquot and store at -80°C.

[0199] 6) Determine the efficiency of transduction by flow cytometry.

[0200] 3.3 Retroviral packaging and concentration

[0201] 1) 293T cells in logarithmic growth phase were inoculated with 1×10 7 Incubate overnight in a T75 flask at 37°C, 5% CO2 to prepare for virus packaging in DMEM containing 10% fetal bovine serum;

[0202] 2) Dissolve 10 μg of the retroviral vector pRV-DLL3-CAR obtained in step 3.1 of this example and 10 μg of the packaging plasmid pCL-10A1 (Novus Biologicals, NBP2-29542) in 0.5 mL of serum-free opti-MEM culture medium, mix well, and obtain a DNA mixture;

[0203] 3) Dissolve 40ug PEI pro (1 ug / uL) in 0.5mL serum-free opti-MEM culture medium, vortex at 1000rpm for 5 seconds, and incubate at 25℃ for 5min to obtain a PEI mixed solution;

[0204] 4) Add the PEI mixture obtained in step 3) to the DNA mixture in step 2), vortex mix or gently mix immediately after addition, and incubate at 25°C for 25 minutes to obtain a transfection complex;

[0205] 5) Add 1 mL of the transfection complex obtained in step 4) dropwise to the 293T cell culture in step 1). After 6 hours, replace with fresh culture medium. After 48 hours, collect the supernatant of the virus solution, centrifuge at 1000g / 5min to remove cell debris, concentrate with an Amicon® Ultra filter (Milipore), and aliquot and store at -80°C.

[0206] 6) Determine the efficiency of transduction by flow cytometry.

[0207] Example 4: Construction of overexpression cell lines

[0208] In this embodiment, 293F cells (293F / Human DLL3), CHO cells (CHO-S / Human DLL3, CHO-S / Mouse DLL3), 293T cells (293T / Human DLL3), NCI-H446 (NCI-H446 / Human DLL3), and U118-MG (U118-MG / Human DLL3) cells overexpressing DLL3 are constructed, including the following:

[0209] 8ug of DLL3-PpyRE9-clover plasmid (synthesized by Nanjing GenScript), 4μg of auxiliary plasmid pSPAX2 and 8ug of envelope plasmid VSVG were co-transfected to obtain DLL3 lentivirus in the same manner as in Example 3.3. 293F cells were cultured at 1×10 6The cells were inoculated into 6-well plates and 1 mL of DLL3-GFP lentivirus was added to obtain 293F cells overexpressing DLL3 (abbreviated as 293F-DLL3). A similar protocol was used to obtain CHO-S-DLL3, CHO-S-mDLL3, 293T-DLL3, NCI-H446-DLL3 and U118-MG-DLL3 cells.

[0210] Example 5: Construction of CAR cells targeting DLL3 and determination of CAR positivity

[0211] 5.1 Construction of Nanoantibody CAR-T Cells Targeting DLL3

[0212] In this example, the lentivirus prepared in step 3.2 of Example 3 is used to transfect T lymphocytes, comprising the following steps:

[0213] 1) Adjust human PBMC to a density of 1.5 × 10 using T cell culture medium (X-VIVO 15 + 10% FBS + 200U / mL IL-2). 6 cells / mL, and Dynabeads magnetic beads (gibco) were added at a cell number and beads ratio of 1:3 for 48 h to obtain activated T cells.

[0214] 2) Collect activated T cells and adjust the cell density to 3×10 5 cells / mL, add the lentivirus obtained in step 3.2 of Example 3 at a multiplicity of infection (MOI) of 1, and add Protamine (fish protein) to a final concentration of 10ug / mL; 32°C, 2500rpm, centrifuge for 90min, culture overnight at 37°C, 5% CO2 environment, replace with fresh culture medium, and subculture every 3 days.

[0215] 3) 2 days after T cell infection, 3×10 5T cells were centrifuged at 4°C and 500g for 5 min, the supernatant was discarded, and the cells were washed once with flow cytometry buffer; 50 μL of buffer was added to resuspend the cells, 0.5 uL of MonoRab™ Rabbit Anti-HumanizedVHH Antibody [PE], mAb (Nanjing GenScript, A02171) was added to the experimental group, 0.5 uL of G4S Linker (E7O2V) Rabbit mAb (Alexa Fluor 647 Conjugate)® (Cell Signaling Technology, 69782) was added to the positive control group, and 0.5 uL of anti-DYKDDDDK (Biolegend, 637322) was added to the negative control group. The cells were incubated on ice for 30 min; after washing once with buffer, 300 μL of buffer was added to resuspend the cells.

[0216] The expression rate of chimeric antigen receptors on T lymphocytes was detected by flow cytometry. Figure 8 (Donor 105) and Fig. 9 (Donor 108). T cells not infected with the virus were used as a control and were treated in the same manner as above.

[0217] from Figure 8 It can be seen that the infection efficiency of CAR-T cells in each group of donor 105 was 29.52%, 51.59%, 60.11%, 1.35%, 46.34%, 36.83% and 35.12%, respectively, indicating that CAR-T cells were successfully constructed.

[0218] from Fig. 9 It can be seen that the infection efficiency of CAR-T cells in each group of donor 108 was 30.52%, 69.92%, 66.25%, 1.16%, 54.79%, 34.79% and 42.23%, respectively, indicating that CAR-T cells were successfully constructed.

[0219] 5.2 Construction of Nanoantibody CAR-NK Cells Targeting DLL3

[0220] In this embodiment, the retrovirus prepared in step 3.3 of embodiment 3 is used to transfect NK cells, comprising the following steps:

[0221] 1) NK cells were isolated from PBMC (peripheral blood mononuclear cells were purchased from Zhejiang Free Trade Zone Maishun Biotechnology Co., Ltd., catalog number PB003F-W, and NK cells were cultured from peripheral blood mononuclear cells according to the method published in https: / / doi.org / 10.1084 / jem.20201529) and activated and cultured.

[0222] 2) Adjust the NK cells on D6 to 3×10 with NK cell culture medium (CTS+5%PLTGold+1000IU / ml IL-2+10 ng / mL IL-15). 5 cells / mL, add the retrovirus obtained in step 3.3 of Example 3 at a multiplicity of infection (MOI) of 1, add Protamine (fish protein) to a final concentration of 10ug / mL; 32°C, 2500rpm, centrifuge for 90min, culture overnight at 37°C, 5% CO2 environment, replace with fresh culture medium, and subculture every 3 days.

[0223] 3) 2 days after NK cell infection, 3×10 5 NK cells were centrifuged at 4°C and 500g for 5 min, the supernatant was discarded, and the cells were washed once with flow cytometry buffer; 50 μL of buffer was added to resuspend the cells, 0.5 uL of MonoRab™ Rabbit Anti-Humanized VHH Antibody [PE], mAb (Nanjing GenScript, A02171) was added to the experimental group, 0.5 uL of G4S Linker (E7O2V) Rabbit mAb (Alexa Fluor 647 Conjugate)® (Cell Signaling Technology, 69782) was added to the positive control group, and 0.5 uL of anti-DYKDDDDK (Biolegend, 637322) was added to the negative control group. The cells were incubated on ice for 30 min; after washing once with buffer, 300 μL of buffer was added to resuspend the cells.

[0224] Flow cytometry was used to detect the expression rate of chimeric antigen receptors in NK cells. The results are shown in Fig.10 (Donor 105) NK cells not infected with the virus were used as a control and were treated in the same manner as above.

[0225] from Fig.10 It can be seen that the infection efficiency of CAR-NK cells in each group was 36.16%, 41.68%, 80.96%, 1.20%, 67.81%, 77.88% and 24.55%, respectively, indicating that CAR-NK cells were successfully constructed.

[0226] Example 6: Evaluation and screening of CAR cell cytotoxicity and cytokine secretion assays targeting DLL3

[0227] 6.1 In vitro toxicity experiment of CAR-T cells targeting DLL3

[0228] In this example, the CAR-T cells prepared in Example 5 were used to perform an in vitro toxicity experiment, which included the following steps:

[0229] 6.1.1 In vitro toxicity experiment of DLL3-targeted nanoantibody CAR-T cells on 293T-DLL3

[0230] 1) Plating: 1×10 293T-DLL3 cells prepared in Example 4 were inoculated 4 The cells were plated in a 96-well plate and the culture medium was X-VIVO 15+10% FBS, 50ul / well.

[0231] 2) Co-incubation: The CAR-T cells prepared in Example 5 were added to a 96-well plate at effector-target ratios of 8:1, 4:1, 2:1, 1:1 and 1:2, and the culture medium was X-VIVO 15 + 10% FBS, 50ul / well, and 3 replicate wells were set, and the average value of the 3 replicate wells was taken. The detection time was after 24h of co-incubation.

[0232] 3) The experimental groups and control groups are as follows:

[0233] Experimental groups: target cells + effector cells (1-A7 / 1-B12 / 1-C11 / 1-D9 / 2-D3);

[0234] Positive group: target cells + effector cells (VH-VL);

[0235] Negative group: target cells + effector cells (Flag / untransduced T cells, i.e., Control);

[0236] Target cell control group: target cells were cultured alone;

[0237] Blank group: culture medium.

[0238] 4) Detection: Use Bright-LiteTM Luciferase Assay System kit (Nanjing Novezan Biotech Co., Ltd., DD1204-02) for detection. For details, refer to the Bright-LiteTM Luciferase Assay System kit instructions. The cytotoxicity calculation formula is:

[0239] Killing rate (%) = (1-(fluorescence value of target cells in the experimental group-fluorescence value of the blank group)) / (fluorescence value of target cells in the control group-fluorescence value of the blank group)) × 100%

[0240] In this experiment, the fluorescence value is inversely proportional to the amount of cell death. The more cell death, the lower the fluorescence value. When the number of cell deaths in the well reaches a certain number, that is, the CAR-T killing reaches a plateau, the fluorescence value will no longer decrease. The results are shown in Fig.11 and Fig.12 .

[0241] from Fig.11(Donor 105) and Fig.12 (Donor 108) It can be seen that the CAR-T cells constructed in this application have killing activity against 293T-DLL3, indicating that the CAR-T cells constructed in this application have good tumor killing ability. The results showed that 1-B12 in DLL3-CAR-T showed the best killing effect on 293T cells with positive DLL3 expression, which was much higher than others.

[0242] 6.1.2 In vitro toxicity experiment of DLL3-targeted nanoantibody CAR-T cells against U118-MG-DLL3

[0243] 1) Plating: 1×10 U118-MG-DLL3 cells prepared in Example 4 were inoculated 4 The cells were plated in 96-well plates, the culture medium was X-VIVO 15+10% FBS, 50ul / well, 2 plates in total.

[0244] 2) Co-incubation: The above-screened and new control CAR-T cells were added into a 96-well plate at effector-target ratios of 8:1, 4:1, 2:1, 1:1, 1:2 and 1:4. The culture medium was X-VIVO 15+10% FBS, 50ul / well, and 3 replicate wells were set.

[0245] 3) The experimental groups and control groups are as follows:

[0246] Experimental group: target cells + effector cells (1-B12);

[0247] Positive group 1: target cells + effector cells (AMG757);

[0248] Positive group 2: target cells + effector cells (VH-VL);

[0249] Positive group 3: target cells + effector cells (VL-VH);

[0250] Negative group: target cells + effector cells (Flag / untransduced T cells, i.e., Control);

[0251] Target cell control group: target cells were cultured alone;

[0252] Blank group: culture medium.

[0253] 4) Detection: After 24 hours of co-incubation, one of the plates was tested using the Bright-LiteTM Luciferase Assay System kit (Nanjing Novezan Biotech Co., Ltd., DD1204-02). For details, refer to the Bright-LiteTM Luciferase Assay System kit instructions. The cytotoxicity calculation formula is:

[0254] Killing rate (%) = (1-(fluorescence value of target cells in the experimental group-fluorescence value of the blank group)) / (fluorescence value of target cells in the control group-fluorescence value of the blank group)) × 100%

[0255] In this experiment, the fluorescence value is inversely proportional to the amount of cell death. The more cell death, the lower the fluorescence value. When the number of cell deaths in the well reaches a certain number, that is, the CAR-T killing reaches a plateau, the fluorescence value will no longer decrease.

[0256] 5) Second round of killing: After 24 h of co-incubation, another plate was inoculated with U118-MG-DLL3 cells (1×10 4 / well) culture medium was X-VIVO 15+10% FBS, 50ul / well, and after 24h, the test was performed again using the method in 4), i.e., the second round of killing (48h).

[0257] For specific results, see Fig.13 (First round) and Fig.14 (Round 2). Fig.13 (Donor 105) It can be seen that 1-B12 in DLL3-CAR-T showed the best killing effect on U118-MG cells with positive DLL3 expression, and was higher than other positive controls. Fig.14 This advantage reappeared in the second round of killing.

[0258] 6.2 In vitro toxicity experiment of CAR-NK cells targeting DLL3

[0259] In this example, multiple rounds of in vitro toxicity experiments on U118-MG-DLL3 were carried out using the DLL3-targeting Nanobody CAR-NK cells prepared in Example 5, including the following steps:

[0260] 1) Plating: 1×10 U118-MG-DLL3 cells prepared in Example 4 were inoculated 4 The cells were plated in 96-well plates with the culture medium of CTS+5%PLTGold, 50ul / well, for a total of 3 plates.

[0261] 2) Co-incubation: The CAR-NK cells prepared in Example 5 were added to a 96-well plate at effector-target ratios of 16:1, 8:1, 4:1, 2:1, 1:1 and 1:2, and the culture medium was CTS+5% PLTGold, 50ul / well, and 3 replicate wells were set, and the average value of the 3 replicate wells was taken. One plate was taken for detection every 24h.

[0262] 3) The experimental groups and control groups are as follows:

[0263] Experimental groups: target cells + effector cells (1-A7 / 1-B12 / 1-C11 / 1-D9 / 2-D3);

[0264] Positive group: target cells + effector cells (VH-VL);

[0265] Negative group: target cells + effector cells (Flag / untransduced NK, i.e., Control);

[0266] Target cell control group: target cells were cultured alone;

[0267] Blank group: culture medium.

[0268] 4) Detection: Use Bright-LiteTM Luciferase Assay System kit (Nanjing Novezan Biotech Co., Ltd., DD1204-02) for detection. For details, refer to the Bright-LiteTM Luciferase Assay System kit instructions. The cytotoxicity calculation formula is:

[0269] Killing rate (%) = (1-(fluorescence value of target cells in the experimental group-fluorescence value of the blank group)) / (fluorescence value of target cells in the control group-fluorescence value of the blank group)) × 100%

[0270] The fluorescence value is inversely proportional to the amount of cell death in this experiment. The more cell death, the lower the fluorescence value. When the number of cell deaths in the well reaches a certain number, that is, CAR-NK killing reaches a plateau, the fluorescence value will no longer decrease.

[0271] 5) Second round of killing: After 24 h of co-incubation, two plates were inoculated with U118-MG-DLL3 cells (1×10 4 / well) The culture medium was CTS+5%PLTGold, 50ul / well. After 24h, one of the plates was tested again using the method in 4), which was the second round of killing.

[0272] 6) Three rounds of killing: After 24 h of co-incubation, the last plate was inoculated with U118-MG-DLL3 cells (1×10 4 / well) The culture medium was CTS+5%PLTGold, 50ul / well. After 24h, the test was performed again using the method in 4), i.e., three rounds of killing.

[0273] Results Fig.15 (One round), Fig.16 (Second round) and Fig.17 (three-wheeled). Figure 15-17 (Donor 105) It can be seen that among DLL3-CAR-NK, 1-B12 showed the best killing effect on U118-MG cells with positive DLL3 expression. And in each round of killing, DLL3-CAR-NK-1-B12 continued to outperform other sequences.

[0274] 6.3 Detection of IFN-γ release by CAR-T cells with nanoantibodies targeting DLL3

[0275] The CBA cytokine detection kit (BD Bioscience) was used to detect the secretion of cytokines (such as IFN-γ) during the killing process of the nanoantibody DLL3-CAR-T cells targeting DLL3 prepared in Example 5. The specific operation steps are as follows:

[0276] 1) Preparation of cell suspension: Collect the cell suspension of target cell 293T-DLL3 and effector cell (DLL3-CAR-T targeting DLL3) separately. Centrifuge at 1500rpm for 5min and discard the supernatant. Add 3mL PBS to resuspend and centrifuge at 1200rpm for 5min. Resuspend the effector cells and target cells with 1mL X-VIVO 15+10% FBS medium, mix well, and count the cells using Countstar.

[0277] 2) Cell mixing and incubation: Set the effector cell: target cell (E:T) ratio to 10:1. Add 1×10 target cells to each well of a 96-well plate. 5 , effector cells 1×10 6 , with a total volume of 50 μL. Seal the 96-well plate with sealing film, centrifuge at 250 g (up 3 times and down 1 times) for 5 min. Incubate in a 37°C constant temperature incubator for 24 h.

[0278] 3) Supernatant collection and cytokine detection: Collect the supernatant into a 1.5mL EP tube and centrifuge at 1500rpm for 3min. Prepare the standard: dissolve the freeze-dried powder of the standard with 2mL of standard diluent, let stand at room temperature for 15min, and then dilute it by 2 times to a total of 10 gradients. Add 50μL of standard or sample to a new EP tube, add 50μL of magnetic beads and 50μL of detection antibody, vortex to mix evenly, and incubate at room temperature for 3h. Wash twice with Wash Buffer, discard the supernatant, add 200μL Wash Buffer to resuspend, and detect the cytokine IFN-γ on the machine. Repeat the experiment 3 times to detect the content of cytokines in the supernatant.

[0279] 4) The experimental groups and control groups are as follows:

[0280] Experimental groups: target cells + effector cells (1-A7 / 1-B12 / 1-C11 / 1-D9 / 2-D3);

[0281] Positive group: target cells + effector cells (AMG757);

[0282] Negative group: target cells + effector cells (Flag / untransduced T, i.e., T);

[0283] Results: The cytokine secretion levels of the effector cell-targeted DLL3 nanoantibody DLL3-CAR-T and the target cell 293T-DLL3 were detected by flow cytometry after 24 hours of co-incubation. Fig.18 As shown, 1-B12 secreted the highest IFN-γ. The CBA test results showed that after the nanoantibody DLL3-CAR-T came into contact with DLL3-positive cells, it was stimulated by the antigen to secrete a large amount of anti-tumor factor IFN-γ.

[0284] The above in vitro results prove that DLL3-CAR-T cells can be stimulated and activated by DLL3-positive target cells in vitro and produce a series of cytokines related to immune activation, while achieving a good killing effect on target cells.

[0285] The above in vitro results prove that DLL3-CAR-T and DLL3-CAR-NK constructed based on the DLL3-targeting nanoantibody 1-B12 have significant killing advantages against DLL3-positive cells and can efficiently secrete IFN-γ.

[0286] Example 7: Epitope identification of DLL3 antibodies

[0287] 7.1 Generation of 293T cells expressing truncated DLL3

[0288] A panel of 293T cells expressing full-length (DLL3) and various truncated human DLL3 was used to determine the domains recognized by each DLL3-targeting antibody. The extracellular domain of human DLL3 can be subdivided into distinct subdomains defined by the following amino acid positions:

[0289] Signal peptide: 1-26

[0290] N-terminal: 27-175

[0291] DSL: 176-215

[0292] EGF-1: 216-249

[0293] EGF-2: 274-310

[0294] EGF-3: 312-351

[0295] EGF-4: 353-389

[0296] EGF-5: 391-427

[0297] EGF-6: 429-465

[0298] To generate truncated DLL3 proteins for epitope mapping, the sequences of the corresponding eight extracellular domains of human DLL3 (including the signal peptide plus N-terminus, DSL, EGF1, EGF2, EGF3, EGF4, EGF5 and EGF6) were deleted one by one, starting from the N-terminus of the antigen.

[0299] like Fig.19 As shown, 293T cells expressing truncated human DLL3 with an N-terminal Flag tag were established, and the corresponding coding sequences of the full-length human DLL3-ECD (Extracellular Domain) and seven truncated human N-terminal DLL3-ECD forms (sequences see Table 5 below) were cloned into the plasmid pRV-P2A-puro-kan.

[0300] Table 5 Full-length and truncated DLL3 amino acid sequences

[0301]

[0302] For cell surface expression of human DLL3, the original signal peptide was used, while for cell surface expression of truncated human N-terminal DLL3, the mouse IgG heavy chain signal peptide (MGWSCIILFLVATATGVHS) was used, followed by a Flag tag. All cloning procedures were performed according to standard protocols (Sambrook, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)). Retrovirus encoding full-length or truncated human DLL3 was generated by co-transfecting 293T cells with pRV-XX-P2A-puro-kan vector and pCL-10A1 (Novus Biologicals, NBP2-29542) vector for each construct. Two days after transfection, the supernatant containing viral particles was collected and used to transduce 293T cells with 5 μg / ml protamine. 24 hours after transduction, the constructed 293T cells were drug screened with 1 μg / ml Puromycin. After the drug screening, the expression of truncated DLL3 was verified by FACS using anti-DYKDDDDK (Biolegend, 637322) antibody. Full-length 293T-DLL3 cells were incubated with anti-DYKDDDDK (Biolegend, 637322) antibody as a negative control. Samples were measured by flow cytometry. Fig. 20As shown, the expression of Flag was detected in 293T cells expressing truncated human DLL3, indicating that the construction was successful.

[0303] 7.2 Epitope mapping of DLL3-targeting antibodies

[0304] The method of 6.3 for the secretion of cytokines (such as IFN-γ) by the nanoantibody DLL3-CAR-T cells targeting DLL3 during the killing process is used for positioning, and the binding domain of each clone is determined using the 293T cells expressing full-length or truncated DLL3 in 7.1, and IFN-γ is secreted when the site is recognized. For example, if the nanoantibody DLL3-CAR-T cells recognize (i.e., have IFN-γ secretion) all truncated cells including EGF3, but do not recognize (i.e., do not secrete IFN-γ) any truncated cells without EGF3, then such clones recognize EGF3.

[0305] The cytokine secretion levels of the effector cell-targeted DLL3 nanoantibody DLL3-CAR-T and 293T expressing full-length or truncated DLL3 were detected by flow cytometry after 24 hours of co-incubation. The results are as follows Fig.21 As shown, the test results show that the five candidate nanoantibody sequences can only secrete IFN-γ when they bind to the full-length DLL3, while the positive control (AMG757) anti-DLL3 antibody recognizes the EGF3 domain, which is consistent with the literature report. The above in vitro results prove that the recognition site of the five candidate nanoantibody sequences is located at the N-terminus of DLL3.

[0306] Example 8: Nanobody humanization and related validation

[0307] 8.1 Humanization of Nanobodies

[0308] Based on the screening experiment results of Example 6, Nanobody 1-B12 was determined, and the selected Nanobody 1-B12 was humanized using CDR transplantation technology (see, for example, U.S. Patent No. 5,225,539). Briefly, the camel VHH sequence was compared with the sequence in the protein database of the Research Cooperative for Structural Bioinformatics (RCSB). For each camel VHH, a homology model was generated based on the closest VH structure. Residues located near the CDR or buried inside the molecule (i.e., the side chain solvent accessible surface area is less than 15%) were identified from the model structure. Next, each camel VHH sequence was BLAST-aligned in the NCBI human germline V gene database to identify the human VH germline sequence (i.e., human acceptor) with the highest identity to the VHH (see, for example, Foote and Winter, J. Mol. Biol. 224:487-499 (1992); Morea V et al., Methods 20:267-279 (2000); Chothia C et al., J. Mol. Biol. 186:651-663 (1985)). In the CDR transplantation method, the CDRs of the human acceptor are replaced by the CDRs of the camel VHH to produce a direct transplantation sequence. Typically, direct transplantation of antibodies will lose binding activity, which requires restoration by replacing framework residues critical to antibody activity with non-human residues. Amino acid residues located near the CDRs or buried in the molecule usually play an important role in the activity and structure of the antibody and should therefore be used as potential back mutation sites. Using this method, a series of humanized variants (HM1 / HM2 / HM3 / HM4) were designed and synthesized. After synthesizing the humanized antibody sequence, a humanized antibody expression vector was constructed. The expression vector was subjected to a large plasmid extraction to prepare a transfection-grade plasmid. In the design stage, the model was used to predict the possible glycosylation sites, antibody aggregation, immunogenic sites, post-translational modifications, etc. of humanized antibodies. The glycosylation sites, immunogenic sites, deamidation, isomerization oxidation and glycosylation were modified through structural models to avoid physical and chemical properties that are not conducive to antibody development, such as antibody aggregation, isoelectric point and other problems, thereby improving the binding activity and developability of the modified antibodies.

[0309] The humanized variants (HM1 / HM2 / HM3 / HM4) obtained above were expressed, purified, and the EC50 of the antibodies was detected by ELISA, and the specificity and affinity of the antibodies were detected by flow cytometry. The steps include:

[0310] 8.2 Humanized Antibody Expression and Purification

[0311] The humanized variant antibodies (HM1 / HM2 / HM3 / HM4) were cloned into the eukaryotic expression plasmid pcDNA3.4, expressed in CHO cells, and purified by Protein A / G affinity chromatography.

[0312] Four purified anti-DLL3 humanized antibodies were obtained, including anti-DLL3-1-B12-HM1 antibody, anti-DLL3-1-B12-HM2 antibody, anti-DLL3-1-B12-HM3 antibody, and anti-DLL3-1-B12-HM4 antibody. The absorbance value of OD280 was detected by an ELISA instrument and the concentration was calculated. The purity and molecular weight were detected by SDS-PAGE gel. The sequences of the obtained humanized antibodies are shown in Table 6 below.

[0313] Table 6 Humanized antibody related sequences

[0314]

[0315] 8.2.1 EC50 by flow cytometry

[0316] The four humanized antibodies and nanobody DLL3-1-B12 obtained in step 8.1 of this example were subjected to median effective concentration (EC50) determination by flow cytometry (FACS, Fluorescence Activated Cell Sorting) in the above example 2.2.1. The determination results are as follows: Fig. 22 Flowchart and Fig.23 EC50 curve is shown. Fig.23 It can be seen that the EC50 values ​​of anti-DLL3-1-B12-HM1 antibody, anti-DLL3-1-B12-HM2 antibody, anti-DLL3-1-B12-HM3 antibody, anti-DLL3-1-B12-HM4 and anti-DLL3-1-B12 VHH are 0.16 ug / mL, 0.2875 ug / mL, 0.1458 ug / mL, 0.2973 ug / mL and 0.1534 ug / mL, respectively.

[0317] 8.2.2 ELISA EC50

[0318] The 4 humanized antibodies and nanobody DLL3-1-B12 obtained in step 8.1 of this example were measured for half effective concentration (EC50) by enzyme-linked immunosorbent assay (ELISA) method in the above example 2.2.2. The results are shown in Table 7 and Fig.24 As shown. Fig.24It can be seen that the EC50 values ​​of anti-DLL3-1-B12-HM1 antibody, anti-DLL3-1-B12-HM2 antibody, anti-DLL3-1-B12-HM3 antibody, anti-DLL3-1-B12-HM4 and anti-DLL3-1-B12 VHH are 0.2944ug / mL, 0.7601ug / mL, 0.3192ug / mL, 0.5082ug / mL and 0.09415ug / mL, respectively.

[0319] Table 7 ELISA detection EC50 values

[0320]

[0321] 8.3 Flow cytometry specificity

[0322] CHO-S cells (Jin Shaoyuan Biotechnology, JSY-CC2001) were used to evaluate the antibody binding cell specificity by flow cytometry. 3×10^5 / well of CHO-S cells were mixed with the purified anti-DLL3 humanized antibody obtained in step 8.1 of this example and the screened nanoantibody 1-B12 at 10ug / ml or 30ug / ml, incubated in an ice bath for 30min, and then incubated with Goat anti-human lgG Fc PE (1:500 dilution) antibody for 30min. Detection by flow cytometry, the results are shown in Fig.25 All antibodies did not bind to CHO-S, and combined with the EC50 result of 8.2, it can be shown that the anti-DLL3 nanobody in the present application can specifically recognize the DLL3 antigen on the cell surface.

[0323] Example 9: Generation of humanized CAR, cellular cytotoxicity and cytokine secretion assays to evaluate screening

[0324] 9.1.1 Generation of humanized anti-DLL3 CAR and construction of humanized anti-DLL3-CAR-T cells

[0325] Humanized VHH is used to construct a full-length CAR construct. From the N-terminus to the C-terminus, the full-length CAR contains a CD8a signal peptide, a humanized VHH region that binds to DLL3, a CD8a hinge domain, a CD8a transmembrane domain, a 41BB intracellular domain, and a CD3 intracellular domain. Subsequently, the nucleic acid encoding the CAR fragment is cloned into a lentiviral vector to construct a full-length CAR construct in a single coding frame and expressed using the human EF1α promoter. The optimized CAR backbone vector is named "pLV-DLL3-CAR-XX or pRV-DLL3-CAR-XX".

[0326] In this example, the humanized sequence obtained in Example 8 and the 1-B12 sequence screened in Example 6 were used to prepare a lentivirus or retrovirus according to the process in Example 3 to transfect T lymphocytes, comprising the following steps:

[0327] 1) Adjust human PBMC to a density of 1.5 × 10 using T cell culture medium (X-VIVO 15 + 10% FBS + 200U / mL IL-2). 6 cells / mL, and Dynabeads magnetic beads (gibco) were added at a cell number and beads ratio of 1:3 for 48 h to obtain activated T cells.

[0328] 2) Collect activated T cells and adjust the cell density to 3×10 5 cells / mL, add the above-prepared lentivirus according to the multiplicity of infection (MOI) of 1, add Protamine (fish protein) to a final concentration of 10ug / mL; 32℃, 2500rpm, centrifuge for 90min, culture overnight at 37℃, 5% CO2 environment, replace with fresh culture medium, and subculture every 3 days.

[0329] 3) 2 days after T cell infection, 3×10 5 T cells were centrifuged at 4°C and 500g for 5 min, the supernatant was discarded, and the cells were washed once with flow cytometry buffer; 50 μL of buffer was added to resuspend the cells, and 0.5 uL of MonoRab™ Rabbit Anti-Humanized VHH Antibody [PE], mAb (Nanjing GenScript, A02171) or MonoRab™ Rabbit Anti-Camelid VHHCocktail [iFluor 647], mAb (Nanjing GenScript, A01994) was added to the experimental group, 0.5 uL of G4SLinker (E7O2V) Rabbit mAb (Alexa Fluor 647 Conjugate)® (Cell Signaling Technology, 69782) was added to the positive control group, and 0.5 uL of anti-DYKDDDDK (Biolegend, 637322) was added to the negative control group. The cells were incubated on ice for 30 min; after washing once with buffer, 300 μL of buffer was added to resuspend the cells.

[0330] The expression rate of chimeric antigen receptors on T lymphocytes was detected by flow cytometry. Fig.26 and Fig. 27 (Donor 105). T cells not infected with the virus were used as controls and were treated in the same manner as above.

[0331] from Fig.26 It can be seen that the infection efficiency of CAR-T cells of each group (1-B12-HM1 / 1-B12-HM2 / 1-B12-HM3 / 1-B12-HM4 / T3 / Flag) of donor 105 prepared by retrovirus was 38.33%, 46.31%, 39.61%, 23.66%, 32.84% and 13.35%, respectively, indicating that CAR-T cells were successfully constructed.

[0332] from Fig. 27 It can be seen that the infection efficiency of CAR-T cells in each group (1-B12 / 1-B12-HM1 / 1-B12-HM2 / 1-B12-HM3 / 1-B12-HM4 / AMG757 / Flag) of donor 105 prepared by lentivirus was 26.78%, 26.80%, 33.38%, 21.40%, 28.03%, 14.67% and 36.36%, respectively, indicating that CAR-T cells were successfully constructed.

[0333] 9.1.2 Evaluation of in vitro activity of humanized DLL3-CAR-T cells

[0334] The efficacy of humanized CAR was evaluated via CAR-T cell-mediated tumor cell killing, cytokine release, and long-term stimulation assays.

[0335] 9.1.2.1 In vitro cytotoxicity assay

[0336] In this example, multiple rounds of in vitro toxicity experiments on NCI-H446-DLL3 were carried out using the humanized antibody CAR-T cells targeting DLL3 prepared in Example 9.1.1, including the following steps:

[0337] 1) Plating: 1×10 NCI-H446-DLL3 cells prepared in Example 4 were inoculated 4 The cells were plated in 96-well plates with the culture medium of X-VIVO 15+10% FBS, 50ul / well, for a total of 2 plates.

[0338] 2) Co-incubation: The CAR-T cells prepared in Example 9.1.1 were added to a 96-well plate at effector-target ratios of 8:1, 4:1, 2:1, 1:1, 1:2 and 1:4. The culture medium was X-VIVO 15 + 10% FBS, 50ul / well. Three replicate wells were set up and the average of the three replicate wells was taken. One plate was taken for detection every 24h.

[0339] 3) The experimental groups and control groups are as follows:

[0340] Experimental groups: target cells + effector cells (1-B12-HM1 / 1-B12-HM2 / 1-B12-HM3 / 1-B12-HM4);

[0341] Positive group: target cells + effector cells (AMG757);

[0342] Negative group: target cells + effector cells (Flag / untransduced T cells, i.e., Control);

[0343] Target cell control group: target cells were cultured alone;

[0344] Blank group: culture medium.

[0345] 4) Detection: Use Bright-LiteTM Luciferase Assay System kit (Nanjing Novezan Biotech Co., Ltd., DD1204-02) for detection. For details, refer to the Bright-LiteTM Luciferase Assay System kit instructions. The cytotoxicity calculation formula is:

[0346] Killing rate (%) = (1-(fluorescence value of target cells in the experimental group-fluorescence value of the blank group)) / (fluorescence value of target cells in the control group-fluorescence value of the blank group)) × 100%

[0347] In this experiment, the fluorescence value is inversely proportional to the amount of cell death. The more cell death, the lower the fluorescence value. When the number of cell deaths in the well reaches a certain number, that is, the CAR-T killing reaches a plateau, the fluorescence value will no longer decrease.

[0348] 5) Second round of killing: After 24 h of co-incubation, another plate was inoculated with NCI-H446-DLL3 cells (1×10 4 / well) culture medium is X-VIVO 15+10% FBS, 50ul / well. After 24h, one of the plates is tested again using the method in 4), i.e., the second round of killing.

[0349] Results Fig.28 (One round) and Fig.29 (Round 2). Figure 28-29 (Donor 105) It can be seen that in the in vitro anti-tumor activity evaluation, the four humanized antibody CAR-T cells targeting DLL3 all showed good efficacy. After two rounds of killing experiments, there was no clear difference between these CAR-T cells.

[0350] 9.1.2.2 Cytokine secretion assay

[0351] The secretion of cytokines (such as GM-CSF, TNF-α, IFN-γ) by the humanized antibody CAR-T cells targeting DLL3 prepared in Example 9.1 during the killing process was detected using the CBA cytokine detection kit (BD Bioscience). The specific operation steps are as follows:

[0352] 1) Preparation of cell suspension: Collect the cell suspension of target cells NCI-H446-DLL3 and effector cells (humanized antibody DLL3-CAR-T cells targeting DLL3) separately. Centrifuge at 1500rpm for 5min and discard the supernatant. Add 3mL PBS to resuspend and centrifuge at 1200rpm for 5min. Resuspend the effector cells and target cells in 1mL X-VIVO 15+10% FBS medium, mix well, and count the cells using Countstar.

[0353] 2) Cell mixing and incubation: Set the effector cell: target cell (E:T) ratio to 10:1. Add 1×10 target cells to each well of a 96-well plate. 5 , effector cells 1×10 6 , with a total volume of 50 μL. Seal the 96-well plate with sealing film, centrifuge at 250 g (up 3 times and down 1 times) for 5 min. Incubate in a 37°C constant temperature incubator for 36 h.

[0354] 3) Supernatant collection and cytokine detection: Collect the supernatant into a 1.5mL EP tube and centrifuge at 1500rpm for 3min. Prepare the standard: dissolve the freeze-dried powder of the standard with 2mL of standard diluent, let stand at room temperature for 15min, and then dilute it by 2 times to a total of 10 gradients. Add 50μL of standard or sample to a new EP tube, add 50μL of magnetic beads and 50μL of detection antibody, vortex to mix evenly, and incubate at room temperature for 3h. Wash twice with Wash Buffer, discard the supernatant, add 200μL Wash Buffer to resuspend, and detect the cytokines GM-CSF, TNF-α, and IFN-γ on the machine. Repeat the experiment 3 times to detect the content of cytokines in the supernatant.

[0355] 4) The experimental groups and control groups are as follows:

[0356] Experimental groups: target cells + effector cells (1-B12 / 1-B12-HM1 / 1-B12-HM2 / 1-B12-HM3 / 1-B12-HM4);

[0357] Positive group: target cells + effector cells (T3, the sequence of which is shown in Table 8 below);

[0358] Negative group: target cells + effector cells (Flag / untransduced T, i.e., 105T);

[0359] The cytokine secretion level of the humanized antibody CAR-T targeting DLL3 in effector cells was detected by flow cytometry after incubation with the target cell NCI-H446-DLL3 for 36 hours. The results are as follows Figure 30-32 As shown, 1-B12-HM2 showed the highest secretion levels of GM-CSF, TNF-α, and IFN-γ, followed closely by 1-B12-HM1. Therefore, when the nanoantibody DLL3-CAR-T encounters DLL3-positive cells and is stimulated by antigens, they will release a large amount of anti-tumor factors, including GM-CSF (granulocyte-macrophage colony stimulating factor), TNF-α (tumor necrosis factor) and IFN-γ (interferon-γ).

[0360] Table 8 Positive control T3 related sequences

[0361]

[0362] 9.1.2.3 Determination of CAR-T Expansion by Long-term Stimulation Assay

[0363] To further distinguish the strength of humanized antibodies, multiple rounds of killing of NCI-H446-DLL3 were carried out using humanized antibody CAR-T cells targeting DLL3 prepared in Example 9.1.1. The continuous killing assay involves repeatedly exposing CAR-T cells to their target cells, causing the CAR-T cells to undergo proliferation, and in some cases, differentiation and exhaustion. This assay is used to select the best clones with high target cell lysis and proliferation capabilities after several rounds of exposure to target cells.

[0364] On day 0, exactly 3 × 10 5 NCI-H446-DLL3 cells were seeded into 24-well plates to ensure stable establishment of monolayer cells. At the same time, CAR-T cells were carefully counted and the same number of 3 × 10 cells was seeded into fresh medium without cytokines. 5 Then, on day 3 or 4, the viable CAR-T cells in each well were accurately counted and a new layer of 3×10 5 A monolayer of NCI-H446-DLL3 cells was plated on an existing 3×10 5 The operation was repeated on 10 CAR-T cells to achieve multiple rounds of stimulation. The expansion multiple after each stimulation was calculated using a scientific method, i.e. [number of surviving CAR-T cells on day 3 or day 4] / 3×10 5(That is, the amount of CAR-T cells inoculated on day 0 for each stimulation). In order to normalize the cells discarded at each new stimulation, the cumulative expansion factor is determined by a more rigorous formula [(N round expansion factor) × ((N+1) round expansion factor) ...]. After multiple rounds of stimulation, the expansion factor of different CAR-T constructs is accurately calculated to better compare and analyze the performance of different constructs.

[0365] The experimental groups and control groups are as follows:

[0366] Experimental groups: target cells + effector cells (1-B12 / 1-B12-HM1 / 1-B12-HM2 / 1-B12-HM3 / 1-B12-HM4);

[0367] Positive group: target cells + effector cells (T3, the sequence of which is shown in Table 8 above);

[0368] Negative group: target cells + effector cells (Flag / untransduced T, i.e., 105T);

[0369] like Fig.33 As shown, DLL3-CAR-T cells continued to proliferate after multiple rounds of stimulation of NCI-H446-DLL3 tumor cells, but after three rounds of Flag and 105T, and after four rounds of 1-B12 and 1-B12-HM3, cells showed negative growth and the experiment was not continued; 1-B12-HM1 and 1-B12-HM2 still had cell killing ability in the seventh round, but had no proliferation ability and showed negative growth. 1-B12-HM2 had the best continuous proliferation and the highest proliferation multiple, followed by 1-B12-HM1.

[0370] In summary, 1-B12-HM2 and 1-B12-HM1 showed superior performance in in vitro antitumor activity, cytokine secretion, and sustained proliferation ability.

[0371] 9.2.1 Construction of humanized anti-DLL3 CAR-NK cells

[0372] In this example, the humanized sequence obtained in Example 8 and the 1-B12 sequence screened in Example 6 were used to prepare a retrovirus according to the process in Example 3 to transfect NK cells, including the following steps:

[0373] 1) NK cells were isolated from PBMC (peripheral blood mononuclear cells were purchased from Zhejiang Free Trade Zone Maishun Biotechnology Co., Ltd., catalog number PB003F-W, and NK cells were cultured from peripheral blood mononuclear cells according to the method published in https: / / doi.org / 10.1084 / jem.20201529) and activated and cultured.

[0374] 2) Adjust the NK cells on D6 to 3×10 with NK cell culture medium (CTS+5%PLTGold+1000IU / ml IL-2+10 ng / mL IL-15). 5 cells / mL, add the above-prepared retrovirus at a multiplicity of infection (MOI) of 1, add Protamine (fish protein) to a final concentration of 10ug / mL; 32℃, 2500rpm, centrifuge for 90min, culture overnight at 37℃, 5% CO2 environment, replace with fresh culture medium, and subculture every 3 days.

[0375] 3) 2 days after NK cell infection, 3×10 5 NK cells were centrifuged at 4°C and 500g for 5 min, the supernatant was discarded, and the cells were washed once with flow cytometry buffer; 50 μL of buffer was added to resuspend the cells, 0.5 uL of MonoRab™ Rabbit Anti-CamelidVHH Cocktail [iFluor 647], mAb (Nanjing GenScript, A01994) antibody was added to the experimental group, 0.5 uL of G4SLinker (E7O2V) Rabbit mAb (Alexa Fluor 647 Conjugate)® (Cell Signaling Technology, 69782) antibody was added to the positive control group, and 0.5 uL of anti-DYKDDDDK (Biolegend, 637322) antibody was added to the negative control group. The cells were incubated on ice for 30 min; after washing once with buffer, 300 μL of buffer was added to resuspend the cells.

[0376] Flow cytometry was used to detect the expression rate of chimeric antigen receptors in NK cells. The results are shown in Fig.34 NK cells not infected with the virus were used as controls and were treated in the same manner as above.

[0377] from Fig.34 It can be seen that the infection efficiency of CAR-NK cells in each group was 6.59%, 14.57%, 13.35%, 13.53%, 34.19% and 12.37%, respectively, indicating that CAR-NK cells were successfully constructed.

[0378] 9.2.2 Evaluation of in vitro activity of humanized anti-DLL3 CAR-NK cells

[0379] In this example, multiple rounds of in vitro toxicity experiments on NCI-H446-DLL3 were carried out using the humanized antibody CAR-NK cells targeting DLL3 prepared in Example 9.2.1, including the following steps:

[0380] 1) Plating: 1×10 NCI-H446-DLL3 cells prepared in Example 4 were inoculated 4 The cells were plated in 96-well plates with the culture medium of CTS+5%PLTGold, 50ul / well, for a total of 6 plates.

[0381] 2) Co-incubation: The CAR-NK cells prepared in Example 9.2.1 were added to a 96-well plate at effector-target ratios of 8:1, 4:1, 2:1, 1:1, 1:2 and 1:4. The culture medium was CTS+5%PLTGold, 50ul / well. Three replicate wells were set up and the average of the three replicate wells was taken. One plate was taken for detection every 24h.

[0382] 3) The experimental groups and control groups are as follows:

[0383] Experimental groups: target cells + effector cells (1-B12-HM1 / 1-B12-HM2 / 1-B12-HM3 / 1-B12-HM4);

[0384] Positive group: target cells + effector cells (AMG757);

[0385] Negative group: target cells + effector cells (Flag / untransduced NK, i.e., Control);

[0386] Target cell control group: target cells were cultured alone;

[0387] Blank group: culture medium.

[0388] 4) Detection: Use Bright-LiteTM Luciferase Assay System kit (Nanjing Novezan Biotech Co., Ltd., DD1204-02) for detection. For details, refer to the Bright-LiteTM Luciferase Assay System kit instructions. The cytotoxicity calculation formula is:

[0389] Killing rate (%) = (1-(fluorescence value of target cells in the experimental group-fluorescence value of the blank group)) / (fluorescence value of target cells in the control group-fluorescence value of the blank group)) × 100%

[0390] The fluorescence value is inversely proportional to the amount of cell death in this experiment. The more cell death, the lower the fluorescence value. When the number of cell deaths in the well reaches a certain number, that is, CAR-NK killing reaches a plateau, the fluorescence value will no longer decrease.

[0391] 5) Second round of killing: After 24 h of co-incubation, another five plates were inoculated with NCI-H446-DLL3 cells (1×10 4 / well) The culture medium was CTS+5%PLTGold, 50ul / well. After 24h, one of the plates was tested again using the method in 4), which was the second round of killing.

[0392] 6) Three rounds of killing: After 24 h of co-incubation, four plates were inoculated with NCI-H446-DLL3 cells (1×10 4 / well) culture medium is CTS+5%PLTGold, 50ul / well. After 24h, one of the plates is tested again using the method in 4), i.e. three rounds of killing.

[0393] 7) Four rounds of killing: After 24 h of co-incubation, three plates were inoculated with NCI-H446-DLL3 cells (1×10 4 / well) culture medium is CTS+5%PLTGold, 50ul / well. After 24h, one of the plates is tested again using the method in 4), i.e. four rounds of killing.

[0394] 8) Five rounds of killing: After 24 h of co-incubation, two plates were inoculated with NCI-H446-DLL3 cells (1×10 4 / well) culture medium was CTS+5%PLTGold, 50ul / well. After 24h, one of the plates was tested again using the method in 4), i.e., five rounds of killing.

[0395] 9) Sixth round of killing: After 24 h of co-incubation, the last plate was inoculated with NCI-H446-DLL3 cells (1×10 4 / well) The culture medium was CTS+5%PLTGold, 50ul / well. After 24h, the test was performed again using the method in 4), i.e., six rounds of killing.

[0396] Results Fig.35 (One round), Fig.36 (Second round), Fig.37 (three-wheel), Fig.38 (four-wheel), Fig.39 (Five rounds) and Fig.40 (Six rounds). Figure 35-40 It can be seen that in the CAR-NK cell study targeting DLL3, we evaluated the anti-tumor activity of four humanized antibody CAR-NK cells in vitro. Since naked NK cells themselves have strong anti-tumor activity, multiple rounds of killing experiments were performed to better evaluate our sequence. The results showed that these four CAR-NK cells had better anti-tumor efficacy and sustained killing ability than the Flag-negative group, and could continue to kill for six rounds. In the multiple rounds of killing experiments, 1-B12-HM2 had the highest killing efficiency in each round at a low-efficiency target ratio of 1:4, which can provide a reference for the sequence screening of 1-B12-HM2.

[0397] 9.3.1 Construction of humanized anti-DLL3-CAR-iNKT cells

[0398] In this example, iNKT lymphocytes were transfected with a lentivirus prepared according to the process in Example 3 using the humanized sequence obtained in Example 8 and the 1-B12 sequence screened in Example 6, comprising the following steps:

[0399] 1) Isolation of iNKT cells: iNKT cells were isolated from PBMC (peripheral blood mononuclear cells were purchased from Zhejiang Free Trade Zone Maishun Biotechnology Co., Ltd., catalog number PB003F-W, refer to DOI: 10.1038 / s41467-023-44310-y for obtaining iNKT cells from peripheral blood mononuclear cells) and activated in culture. The culture medium was X-VIVO 15+10% FBS+200U / mL IL-2+10ng / ml IL15.

[0400] 2) According to experience, the purity of iNKT cells reaches a high level when cultured to D13. 0.5uL of APC anti-human TCR Vα24-Jα18 (iNKT cell) Antibody (Biolegend, 342908) and 0.5uL of Brilliant Violet 421™ anti-human CD3 Antibody (Biolegend, 317344) were used to test the purity of iNKT cells at D13. The results are as follows: Fig.41 As shown in A, the proportion of CD3+iNKT+ in donor 702 has reached 96.69%.

[0401] 3) Collect the above high-purity iNKT cells and adjust the cell density to 3×10 5 cells / mL, add the above-prepared lentivirus according to the multiplicity of infection (MOI) of 1, add Protamine (fish protein) to a final concentration of 10ug / mL; 32℃, 2500rpm, centrifuge for 90min, culture overnight at 37℃, 5% CO2 environment, replace with fresh culture medium, and subculture every 3 days.

[0402] 4) 2 days after iNKT cell infection, 3×10 5iNKT cells were centrifuged at 4°C and 500 g for 5 min, the supernatant was discarded, and the cells were washed once with flow cytometry buffer; 50 μL of buffer was added to resuspend the cells, and 0.5 uL of APC anti-human TCR Vα24-Jα18 (iNKT cell) Antibody (Biolegend, 342908) and 0.5 uL of Brilliant Violet 421™anti-human CD3 Antibody (Biolegend, 317344) were added to all groups. 0.5 uL of MonoRab™Rabbit Anti-Humanized VHH Antibody [PE], mAb (Nanjing GenScript, A02171) or MonoRab™Rabbit Anti-Camelid VHH Cocktail [iFluor 647], mAb (Nanjing GenScript, A01994) was added to the experimental group, and 0.5 uL of G4S Linker (E7O2V) Rabbit mAb (Alexa Fluor 647 Conjugate)® (Cell Signaling Technology, 69782), and 0.5uL of anti-DYKDDDDK (Biolegend, 637322) antibody was added to the negative control group and incubated on ice for 30min; after washing once with buffer, 300μL of buffer was added to resuspend the cells.

[0403] Flow cytometry was used to detect the expression rate of chimeric antigen receptors on iNKT lymphocytes. Fig.41 As shown in B. iNKT cells not infected with the virus were used as a control and were treated in the same manner as above.

[0404] from Fig.41 As shown in Figure B, the infection efficiency of CAR-iNKT cells in each group (1-B12-HM1 / 1-B12-HM2 / T3 / Flag) of donor 702 prepared by lentivirus was 8.82%, 8.30%, 6.89% and 8.54%, respectively, indicating that CAR-iNKT cells were successfully constructed.

[0405] 9.3.2 Testing the expansion function of CAR-iNKT cells through long-term stimulation

[0406] In order to further distinguish the strength of humanized antibodies, multiple rounds of killing of NCI-H446-DLL3 were carried out using humanized antibody CAR-iNKT cells targeting DLL3 prepared in Example 9.3.1. The continuous killing assay involves repeatedly exposing CAR-iNKT cells to their target cells, causing the CAR-iNKT cells to undergo proliferation, and in some cases, differentiation and exhaustion. This protocol is used to select the best clones with high target cell lysis and proliferation capabilities after several rounds of exposure to target cells.

[0407] On day 0, exactly 3 × 10 5 NCI-H446-DLL3 cells were seeded into 24-well plates to ensure stable establishment of monolayer cells. At the same time, CAR-iNKT cells were carefully counted and the same number of 3 × 10 cells was seeded into fresh medium without cytokines. 5 10 active CAR-iNKT cells were seeded on top of the NCI-H446-DLL3 cell layer. Then, on day 3 or 4, the viable CAR-iNKT cells in each well were accurately counted and a new layer of 3×10 5 A monolayer of NCI-H446-DLL3 cells was plated on an existing 3×10 5 The operation was repeated to achieve multiple rounds of stimulation. The expansion multiple after each stimulation was calculated using a scientific method, i.e., [number of surviving CAR-iNKT cells on day 3 or day 4] / 3×10 5 (That is, the amount of CAR-iNKT cells inoculated on day 0 for each stimulation). In order to normalize the cells discarded at each new stimulation, the cumulative expansion fold is determined by a more rigorous formula [(N round expansion fold) × ((N+1) round expansion fold) ...]. After multiple rounds of stimulation, the expansion fold of different CAR-iNKT constructs is accurately calculated to better compare and analyze the performance of different constructs.

[0408] The experimental groups and control groups are as follows:

[0409] Experimental groups: target cells + effector cells (1-B12-HM1 / 1-B12-HM2);

[0410] Positive group: target cells + effector cells (T3, the sequence of which is shown in Table 8 above);

[0411] Negative group: target cells + effector cells (Flag / untransduced iNKT, i.e., 702iNKT);

[0412] like Fig.42As shown, DLL3-CAR-iNKT continued to proliferate under multiple rounds of stimulation of NCI-H446-DLL3 tumor cells, but Flag and 702iNKT cells showed negative growth after two rounds and the experiment was not continued; 1-B12-HM1 and 1-B12-HM2 still had cell killing ability in the seventh round, but had no proliferation ability, 1-B12-HM2 showed negative growth, 1-B12-HM1 and T3 almost did not proliferate, and the overall relative proliferation multiple of 1-B12-HM1 was the best.

[0413] Example 10: In vivo activity evaluation of humanized DLL3 CAR-T cells in NCI-H446-DLL3 model mice

[0414] To test the anti-tumor activity of humanized DLL3-CAR-T cells, an evaluation was performed in the NCI-H446-DLL3 tumor model. NCI-H446-DLL3 cells were mixed with BD Matrigel Matrix (BD, 354234) at a ratio of 1:1 and 200 μL of 5×10 6 Cell / matrigel suspension of NCI-H446-DLL3 cells. Starting from day 3 after implantation, tumor growth was monitored by measurement using a digital caliper. Tumor size was calculated using the formula tumor volume = (width^2 × length / 2). When the tumor was palpable (100 mm 3 ) About 6 days after implantation, mice were randomly divided into 4 groups based on tumor volume. One day after the mice were randomly divided into groups (day 0), Flag (negative control), T3 (positive control), 1-B12-HM1 / 1-B12-HM2 (experimental group) DLL3-CAR-T cells were counted according to standard procedures. The cells were resuspended in PBS + 1% FBS and injected through the tail vein at a volume of 200 μL per mouse. 7 Total cells (5×10 6 CAR-positive T cells). Tumors were monitored every 3-4 days after tumor cell implantation and when tumor volume reached approximately 3000 mm 3 Or the mice were euthanized when the body weight decreased by more than 30% until the end of the study.

[0415] exist Figure 43-45In the NCI-H446-DLL3 animal model experiment shown, we found that both 1-B12-HM1 and 1-B12-HM2 showed significant tumor inhibition effects, which were not significantly different from the control group T3. In terms of weight changes, the weight of animals treated with 1-B12-HM2 remained relatively stable without significant fluctuations; while the weight of animals in the 1-B12-HM1 and T3 groups showed a downward trend. Regarding the observation of the survival cycle, the experimental results showed that no animals in the 1-B12-HM2 group died during the entire observation period, showing an extremely high survival rate; in contrast, one animal died in the 1-B12-HM1 group, and three animals died in the T3 group. This data further highlights the advantages of 1-B12-HM2 in improving animal survival rates. In summary, this experiment shows that 1-B12-HM1 and 1-B12-HM2 both have good tumor inhibitory effects in the NCI-H446-DLL3 animal model, and 1-B12-HM2 is more outstanding in maintaining animal weight stability and improving survival rate.

[0416] Example 11: Evaluation of in vitro activity of anti-DLL3 tandem CAR-T cells

[0417] 11.1 Anti-DLL3 tandem CAR generation and construction of anti-DLL3 tandem CAR-T cells

[0418] To improve the anti-tumor efficacy of CAR-T, three tandem CARs (HM1-HM2, HM2-1-C11, and HM2-2-D3) were constructed. The full-length CAR contains from the N-terminus to the C-terminus: CD8a signal peptide, humanized VHH region binding to DLL3, G4S Linker (GGGGSGGGGSGGGGS), nanoantibodies specific for DLL3 (anti-DLL3 VHH), CD8a hinge domain, CD8a transmembrane domain, 41BB intracellular domain, and CD3 intracellular domain. Subsequently, the nucleic acid encoding the CAR fragment was cloned into a lentiviral vector, thereby constructing a full-length CAR construct in a single coding frame and expressing it with the help of the human EF1α promoter. The optimized CAR backbone vector was named "pLV-DLL3-CAR-XX".

[0419] In this example, the lentivirus prepared by the process in Example 3 using the tandem sequence constructed above is used to transfect T lymphocytes, comprising the following steps:

[0420] 1) Adjust human PBMC to a density of 1.5 × 10 using T cell culture medium (X-VIVO 15 + 10% FBS + 200U / mL IL-2). 6cells / mL, and Dynabeads magnetic beads (gibco) were added at a cell number and beads ratio of 1:3 for 48 h to obtain activated T cells.

[0421] 2) Collect activated T cells and adjust the cell density to 3×10 5 cells / mL, add the above-prepared lentivirus according to the multiplicity of infection (MOI) of 1, add Protamine (fish protein) to a final concentration of 10ug / mL; 32℃, 2500rpm, centrifuge for 90min, culture overnight at 37℃, 5% CO2 environment, replace with fresh culture medium, and subculture every 3 days.

[0422] 3) 2 days after T cell infection, 3×10 5 T cells were centrifuged at 4°C and 500g for 5 min, the supernatant was discarded, and the cells were washed once with flow cytometry buffer; 50 μL of buffer was added to resuspend the cells, 0.5 uL of G4S Linker (E7O2V) Rabbit mAb (Alexa Fluor 647 Conjugate)® (Cell Signaling Technology, 69782) was added to the experimental group, and 0.5 uL of anti-DYKDDDDK (Biolegend, 637322) antibody was added to the negative control group, and incubated on ice for 30 min; after washing once with buffer, 300 μL of buffer was added to resuspend the cells.

[0423] The expression rate of chimeric antigen receptors on T lymphocytes was detected by flow cytometry. Fig.46 T cells not infected with the virus were used as controls and were treated in the same manner as above.

[0424] from Fig.46 It can be seen that the infection efficiency of CAR-T cells in each group (HM1-HM2 / HM2-1-C11 / HM2-2-D3 / Flag) of donor 105 was 17.76%, 24.21%, 42.01% and 23.95%, respectively, indicating that CAR-T cells were successfully constructed.

[0425] 11.2 Determination of anti-DLL3 tandem CAR-T expansion by long-term stimulation assay

[0426] In order to further distinguish the strength of anti-DLL3 tandem antibodies, the targeted DLL3 tandem CAR-T cells prepared in Example 11.1 were used to carry out multiple rounds of killing of NCI-H446-DLL3.

[0427] On day 0, exactly 3 × 10 5NCI-H446-DLL3 cells were seeded into 24-well plates to ensure stable establishment of monolayer cells. At the same time, CAR-T cells were carefully counted and the same number of 3 × 10 cells was seeded into fresh medium without cytokines. 5 Then, on day 3 or 4, the viable CAR-T cells in each well were accurately counted and a new layer of 3×10 5 A monolayer of NCI-H446-DLL3 cells was plated on an existing 3×10 5 The operation was repeated on 10 CAR-T cells to achieve multiple rounds of stimulation. The expansion multiple after each stimulation was calculated using a scientific method, i.e. [number of surviving CAR-T cells on day 3 or day 4] / 3×10 5 (That is, the amount of CAR-T cells inoculated on day 0 for each stimulation). In order to normalize the cells discarded at each new stimulation, the cumulative expansion factor is determined by a more rigorous formula [(N round expansion factor) × ((N+1) round expansion factor) ...]. After multiple rounds of stimulation, the expansion factor of different CAR-T constructs is accurately calculated to better compare and analyze the performance of different constructs.

[0428] The experimental groups and control groups are as follows:

[0429] Experimental groups: target cells + effector cells (1-B12-HM1-HM2, 1-B12-HM2-1-C11, and 1-B12-HM2-2-D3);

[0430] Negative group: target cells + effector cells (Flag / untransduced T, i.e., 105T);

[0431] like Fig.47 As shown in the figure, in multiple rounds of stimulation experiments of NCI-H446-DLL3 tumor cells, the performance of each group of cells showed significant differences. Specifically, the 1-B12-HM1-HM2 combination was quickly eliminated under early stimulation, and its proliferation ability was limited, failing to achieve a 100-fold increase. In contrast, the 1-B12-HM2-2-D3 combination showed excellent proliferation ability. Not only was it the best in all groups for continuous proliferation, but after 12 rounds of expansion, its highest proliferation multiple far exceeded 7×10 6 The next best combination was 1-B12-HM2-1-C11, which also showed strong proliferation potential and successfully expanded for 10 rounds, with the highest proliferation multiple exceeding 1×10 6 The sequence of the obtained tandem antibody 1-B12-HM1-HM2 is shown in Table 9 below.

[0432] Table 9 Anti-DLL3 tandem antibody amino acid sequences

[0433]

[0434] In summary, this application elaborates on the complete process from the construction of a yeast display nanoantibody library to the preparation of DLL3-targeted nanoantibody CAR-T and CAR-NK cells, as well as a comprehensive evaluation of their in vitro and in vivo activities. Specifically, the yeast display nanoantibody library was successfully constructed by first immunizing alpacas with the recombinant protein of the DLL3 extracellular segment. Subsequently, five anti-DLL3 nanoantibodies were obtained by screening with ELISA and flow cytometry (FACS). These candidate antibodies were cloned into eukaryotic expression plasmids, expressed in CHO cells, and purified by Protein A / G affinity chromatography. The affinity and specificity of the antibodies were detected by ELISA and flow cytometry, and it was determined that the DLL3-1-B12 antibody had the highest binding ability. Next, the DLL3 nanoantibody was fused with the CAR domain to construct a lentiviral vector. The lentivirus was packaged by transfecting 293T cells and concentrated for subsequent experiments. A variety of cell lines were infected with lentivirus to construct a cell line that stably expressed DLL3. CAR cells targeting DLL3 were successfully constructed by infecting T cells with lentivirus and NK cells with retrovirus. The anti-tumor activity of CAR cells was evaluated by cytotoxicity experiments and cytokine secretion assays. The experimental results showed that DLL3-1-B12 showed excellent anti-tumor effects in both CAR-T and CAR-NK cells. The DLL3-1-B12 antibody was humanized by CDR transplantation technology to obtain multiple variants (HM1 / HM2 / HM3 / HM4). The EC50 and specificity of the humanized antibody were verified, and the results showed that the humanized antibody maintained good binding ability. Humanized CAR-T, CAR-NK and CAR-iNKT cells all showed excellent anti-tumor activity in in vitro experiments, among which DLL3-1-B12-HM2 performed best when testing CAR-T and CAR-NK, and DLL3-1-B12-HM1 when testing CAR-iNKT. In the NPSG mouse NCI-H446-DLL3 tumor model, DLL3-1-B12-HM2 CAR-T cells significantly inhibited tumor growth, effectively maintained body weight stability, and improved animal survival. To further enhance the anti-tumor effect, HM1-HM2, HM2-1-C11, and HM2-2-D3 tandem CARs were constructed. Multiple rounds of killing experiments showed that HM2-2-D3 tandem CAR performed best in terms of proliferation ability and anti-tumor activity.

[0435] In summary, DLL3-1-B12 and its humanized variants DLL3-1-B12-HM1 or DLL3-1-B12-HM2 showed excellent anti-tumor effects in CAR-T, CAR-NK and CAR-iNKT cells, especially in in vivo experiments, significantly inhibiting tumor growth and improving animal survival. Preliminary exploration of tandem CARs showed that HM2-2-D3 has strong proliferation ability and anti-tumor activity in vitro, providing a new and effective strategy for further improving the efficacy of CAR therapy.

[0436] It should be noted that the above are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Although specific implementations have been described, for the applicant or other persons skilled in the art, there may be or are currently no foreseeable substitutions, modifications, changes, improvements, and substantial equivalents of the above-mentioned implementations. Therefore, the attached claims submitted and the claims that may be amended are intended to cover all such substitutions, modifications, changes, improvements, and substantial equivalents. It is important that with the evolution of technology, many of the elements described herein may be replaced by equivalent elements that appear after the present application.

Claims

1. A nanobody or an antigen-binding fragment thereof, characterized in that: The Nanobody or its antigen-binding fragment specifically binds to human DLL3, and the Nanobody or its antigen-binding fragment comprises a complementarity determining region (CDR), the amino acid sequence of which is as follows: The amino acid sequence of CDR1 is shown in SEQ ID NO: 1; The amino acid sequence of CDR2 is shown in SEQ ID NO: 2; and The amino acid sequence of CDR3 is shown in SEQ ID NO:

3.

2. The Nanobody or antigen-binding fragment thereof according to claim 1, characterized in that The Nanobody or antigen-binding fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 4, 14-17.

3. The Nanobody or antigen-binding fragment thereof according to claim 1, characterized in that The human DLL3 comprises the amino acid sequence shown in SEQ ID NO:

26.

4. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 3, and the chimeric antigen receptor further comprises: A signal peptide, wherein the signal peptide is a CD8α signal peptide, and the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 42; A hinge region, wherein the hinge region is a CD8α hinge region, and the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO: 43; A transmembrane domain, wherein the transmembrane domain is a CD8α transmembrane domain, and the amino acid sequence of the CD8α transmembrane domain is shown in SEQ ID NO: 44; A costimulatory signaling domain, wherein the costimulatory signaling domain is a 4-1BB costimulatory signaling domain, and the amino acid sequence of the 4-1BB costimulatory signaling domain is shown in SEQ ID NO: 45; and The intracellular signaling domain is a CD3ζ intracellular signaling domain, and the amino acid sequence of the CD3ζ intracellular signaling domain is shown in SEQ ID NO:

46.

5. A polynucleotide, characterized in that The polynucleotide encodes the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the chimeric antigen receptor according to claim 4.

6. The polynucleotide according to claim 5, characterized in that The polynucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 9, 19-22.

7. A recombinant vector, characterized in that: The recombinant vector comprises the polynucleotide according to claim 5 or 6.

8. A host cell, characterized in that The host cell comprises the polynucleotide according to claim 5 or 6, and / or the recombinant vector according to claim 7.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 3, the chimeric antigen receptor according to claim 4, the polynucleotide according to claim 5 or 6, the recombinant vector according to claim 7, and / or the host cell according to claim 8.

10. A kit, characterized in that: The kit comprises the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 3, the chimeric antigen receptor according to claim 4, the polynucleotide according to claim 5 or 6, the recombinant vector according to claim 7, the host cell according to claim 8, and / or the pharmaceutical composition according to claim 9.

11. Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 3 and / or the chimeric antigen receptor according to claim 4 in the preparation of a medicament for alleviating or treating a disease, wherein the disease is small cell lung cancer in which DLL3 is overexpressed.

12. A detection method for non-diagnostic purposes, characterized in that: The method comprises detecting the expression level of DLL3 in a sample using the Nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 3.

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