Antibodies or antigen-binding fragments thereof specifically binding to cd20, chimeric antigen receptors and uses thereof

Abstract

The application relates to an antibody or antigen-binding fragment thereof specifically binding to CD20, a chimeric antigen receptor and use thereof. According to ELISA results, 25 positive clones capable of specifically binding to CD20 are obtained through panning of a phage library, and according to sequencing results, two unique scFv sequences are obtained. The two sequences are used to construct CAR-T, wherein the expression level of the scFv in the 2A7 CAR-T is higher than that of other scFv, and compared with other CAR-T cells (including CAR-T20.19 cells), the killing rate of the 2A7 CAR-T is higher under the condition of an effector-to-target ratio of 1:1.

Description

Technical Field

[0001] This article generally covers the fields of biotechnology and medical technology, with a particular focus on an antibody that specifically binds to CD20 or its antigen-binding fragment, chimeric antigen receptors and their uses. Background Technology

[0002] B cells include pre-B cells, early-developing B cells, and mature B cells. Mature B cells differentiate into plasma cells and malignant B cells through terminal differentiation. Most pre-B-cell acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia (CLL), pre-lymphocytic leukemia, hairy cell leukemia, ordinary acute lymphoblastic leukemia, and some non-acute lymphoblastic leukemias highly express CD20. The expression of CD20 on plasma cells further indicates that it can be expressed in various B-cell tumors such as multiple myeloma, plasmacytoma, and Waldenström's tumor. Therefore, CD20 is considered a target for various hematologic malignancies.

[0003] Chimeric antigen receptor T-cell (CAR-T) therapy, as a powerful new adoptive immunotherapy technology, has shown very effective therapeutic effects and has been used to treat a variety of solid and hematological cancers. In particular, CD20-CAR-T therapy for B-cell lymphoblastic leukemia and lymphoma has shown very good clinical therapeutic effects.

[0004] Therefore, antibody sequences targeting CD20 are crucial for the development of antibody drugs and related CAR-T products. Based on this principle, Roche developed rituximab, which was launched in 1997. Over the past few decades, rituximab has been widely used to treat various hematological malignancies and autoimmune diseases. However, rituximab is a human-mouse chimeric antibody, and this incomplete humanization characteristic mainly leads to immunogenic reactions and infusion-related adverse reactions in clinical practice. Subsequently, several companies developed multiple fully human CD20-targeting antibodies, but none have surpassed rituximab in efficacy and safety. Therefore, there is an urgent need in this field to develop fully human antibodies, antigen-binding fragments, and CAR-T cells targeting CD20 that are superior to existing products to improve therapeutic efficacy and reduce side effects. Summary of the Invention

[0005] Based on this, this application provides an antibody or antigen-binding fragment thereof that specifically binds to CD20, comprising: The light chain variable region (VL) includes light chain CDR1 (VL CDR1) shown in SEQ ID NO:1, light chain CDR2 (VL CDR2) shown in SEQ ID NO:2, and light chain CDR3 (VL CDR3) shown in SEQ ID NO:3; and The heavy chain variable region (VH) includes heavy chain CDR1 (VH CDR1) shown in SEQ ID NO:4, heavy chain CDR2 (VH CDR2) shown in SEQ ID NO:5, and heavy chain CDR3 (VH CDR3) shown in SEQ ID NO:6. Among them, any of the above amino acid sequences also includes a derivative sequence that has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining CD20 binding affinity.

[0006] On the other hand, this application also provides a polynucleotide that encodes an antibody or an antigen-binding fragment thereof as described herein.

[0007] On the other hand, this application also provides a vector containing the polynucleotides described herein.

[0008] On the other hand, this application also provides a host cell containing the polynucleotides or vectors described herein.

[0009] On the other hand, this application also provides a chimeric antigen receptor (CAR) that specifically binds to CD20, the chimeric antigen receptor comprising, from the N-terminus to the C-terminus: CD20 binding domain, which contains the antibody or antigen binding fragment described herein; Transmembrane domains; and Cytoplasmic domain.

[0010] On the other hand, this application also provides a polynucleotide that encodes the chimeric antigen receptor described herein.

[0011] On the other hand, this application also provides a vector comprising a polynucleotide encoding the chimeric antigen receptor described herein.

[0012] In another aspect, this application also provides a cell containing a polynucleotide encoding a chimeric antigen receptor described herein or a vector containing a polynucleotide encoding a chimeric antigen receptor described herein.

[0013] On the other hand, this application also provides the use of the antibodies or antigen-binding fragments thereof described herein or the cells described herein in the preparation of a medicament for treating B-cell malignancies.

[0014] On the other hand, this application also provides a method for preparing cells capable of expressing CARs that specifically bind to CD20, comprising transferring the polynucleotides described herein into the cells.

[0015] This application used phage library screening to obtain 25 positive clones that specifically bind to CD20 based on ELISA results. Sequencing revealed two unique scFv sequences. CAR-T cells were constructed using these two sequences. The expression level of scFv in 2A7 CAR-T cells was higher than that of other scFvs, and compared to other CAR-T cells (including CAR-T20.19 cells), 2A7 CAR-T cells exhibited a higher killing rate at an effector-to-target ratio of 1:1.

[0016] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the embodiments described in the description and the accompanying drawings. Attached Figure Description

[0017] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0018] Figure 1 This is an SDS-PAGE electrophoresis result of the CD20 target protein in Example 1 of this application.

[0019] Figure 2 This is a flowchart of affinity screening in Embodiment 1 of this application.

[0020] Figure 3 The CAR in Embodiment 2 of this application is shown. + The binding status of cells to recombinant CD20 protein.

[0021] Figure 4 The CAR in Embodiment 2 of this application is shown. + Cellular expression level (MFI).

[0022] Figure 5 This application illustrates a 1:1 effector-to-target ratio CAR-T cell versus Raji (CD20) CAR-T cell therapy in Example 3. + Results of in vitro cytotoxicity assays of tumor cells.

[0023] Figure 6 This application illustrates a 1:1 effector-to-target ratio CAR-T cell-to-K562 (CD20) ratio in Example 3. - Results of in vitro cytotoxicity assays of tumor cells.

[0024] Figure 7 The amino acid sequence of the 2A7 antibody in this application is shown. Detailed Implementation

[0025] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application pertains. When a quantity, concentration, or other value or parameter is expressed as a range, preferred range, or preferred upper and lower numerical limits, it should be understood that this is equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with any lower or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range.

[0026] When used with a numerical variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within the 95% confidence interval of the mean) or within ±10% of the specified value, or a wider range.

[0027] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0028] The expression "at least one" or "one or more" indicates 1, 2, 3, 4, 5, 6, 7, 8, 9 or more kinds.

[0029] This application provides an antibody or antigen-binding fragment thereof that specifically binds to CD20, comprising: The light chain variable region (VL) includes light chain CDR1 (VL CDR1) shown in SEQ ID NO:1, light chain CDR2 (VL CDR2) shown in SEQ ID NO:2, and light chain CDR3 (VL CDR3) shown in SEQ ID NO:3; and The heavy chain variable region (VH) includes heavy chain CDR1 (VH CDR1) shown in SEQ ID NO:4, heavy chain CDR2 (VH CDR2) shown in SEQ ID NO:5, and heavy chain CDR3 (VH CDR3) shown in SEQ ID NO:6. Among them, any of the above amino acid sequences also includes a derivative sequence that has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining CD20 binding affinity.

[0030] The amino acid sequence is as follows: TSSQGLVDSDGNTYLS (SEQ ID NO:1) KVSYRDY (SEQ ID NO:2) MQSTQWPLT (SEQ ID NO:3) GSSMH (SEQ ID NO:4) RVGLKTETYATTYAASVTG (SEQ ID NO:5) YDYSARRYY (SEQ ID NO:6) When used to refer to antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50-70 kDa, in which the N-terminal portion includes a variable region of approximately 120-130 or more amino acids, and the C-terminal portion includes a constant region. Based on the amino acid sequence of the heavy chain's constant region, the constant region can be one of five different types, called alpha (a), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The different heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When bound to a light chain, these different types of heavy chains produce five well-known classes of antibodies: IgA, IgD, IgE, IgG, and IgM, including four subclasses of IgG called IgG1, IgG2, IgG3, and IgG4. The heavy chain can be a human heavy chain.

[0031] When used to refer to antibodies, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, wherein the amino-terminal portion includes a variable region of approximately 100 to approximately 110 or more amino acids, and the carboxyl-terminal portion includes a constant region. The length of a light chain is approximately 211 to 217 amino acids. Based on the amino acid sequence of the constant region, light chains are classified into two distinct types, called kappa (κ) and lambda (λ). The amino acid sequences of light chains are well known in the art. Light chains can be human light chains.

[0032] The term "variable domain" or "variable region" refers to a portion of the light or heavy chain of an antibody, typically located at the amino terminus of the light or heavy chain, and approximately 120 to 130 amino acids long in the heavy chain and approximately 100 to 110 amino acids long in the light chain. It is responsible for the binding and specificity of each specific antibody to its specific antigen. Variable domains vary significantly in sequence between different antibodies. Sequence variability is concentrated in the CDR (contextually defined domain), while the less variable portion of the variable domain is called the frame region (FR). The CDR of both the light and heavy chains is primarily responsible for the antibody-antigen interaction. Variable regions can be human variable regions.

[0033] A CDR refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the β-sheet frame of an immunoglobulin (Ig or antibody) VH, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the β-sheet frame of an antibody VL. Therefore, a CDR is a variable region sequence scattered within a framework sequence. CDR regions are well known to those skilled in the art and have been defined by various methods / systems. These systems and / or definitions have been developed and refined over many years, including Kabat, Chothia, IMGT, AbM, and Contact.

[0034] In some embodiments, the anti-CD20 antibody or its antigen-binding fragment comprises VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 of the antibody or antigen-binding fragment of this application. In some embodiments, the anti-CD20 antibody or its antigen-binding fragment comprises a variant of the antibody or antigen-binding fragment of this application. In some embodiments, the variant of the anti-CD20 antibody or antigen-binding fragment comprises substitutions, additions, and / or deletions of 1 to 30 amino acids in the anti-CD20 antibody or antigen-binding fragment. In some embodiments, the variant of the anti-CD20 antibody or antigen-binding fragment comprises substitutions, additions, and / or deletions of 1 to 25 amino acids in the anti-CD20 antibody or antigen-binding fragment. In some embodiments, the variant of the anti-CD20 antibody or antigen-binding fragment comprises substitutions, additions, and / or deletions of 1 to 20 amino acids in the anti-CD20 antibody or antigen-binding fragment. In some embodiments, variants of the anti-CD20 antibody or antigen-binding fragment include substitutions, additions, and / or deletions of 1 to 15 amino acids in the anti-CD20 antibody or antigen-binding fragment. In some embodiments, variants of the anti-CD20 antibody or antigen-binding fragment include substitutions, additions, and / or deletions of 1 to 10 amino acids in the anti-CD20 antibody or antigen-binding fragment. In some embodiments, variants of the anti-CD20 antibody or antigen-binding fragment include substitutions, additions, and / or deletions of 1 to 5 conserved amino acids in the anti-CD20 antibody or antigen-binding fragment. In some embodiments, variants of the anti-CD20 antibody or antigen-binding fragment include substitutions, additions, and / or deletions of 1 to 3 amino acids in the anti-CD20 antibody or antigen-binding fragment. In some embodiments, the substitutions, additions, and / or deletions of amino acids are conserved amino acid substitutions. In some embodiments, the conserved amino acid substitutions are located in the CDR of the antibody or antigen-binding fragment. In some embodiments, the conserved amino acid substitutions are not located in the CDR of the antibody or antigen-binding fragment. In some embodiments, the conserved amino acid substitutions are located in the frame region of the antibody or antigen-binding fragment.

[0035] In some implementations, it is necessary to modulate the biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector function, glycosylation, immunogenicity, and / or solubility. Those skilled in the art will understand that amino acid changes can alter the post-translational processes of the antibody, such as changing the number or location of glycosylation sites or altering membrane anchoring properties.

[0036] A mutation can be a substitution, deletion, or insertion of one or more nucleotides encoding an antibody or peptide, resulting in a change in the amino acid sequence relative to the native antibody or peptide sequence. In some embodiments, an amino acid substitution is the result of replacing one amino acid with another amino acid having similar structure and / or chemical properties, such as replacing leucine with serine, for example, a conserved amino acid substitution. Insertions or deletions can range from about 1 to 5 amino acids. In some embodiments, substitutions, deletions, or insertions relative to the parent molecule include substitutions of fewer than 25 amino acids, fewer than 20 amino acids, fewer than 15 amino acids, fewer than 10 amino acids, fewer than 5 amino acids, fewer than 4 amino acids, fewer than 3 amino acids, or fewer than 2 amino acids. In some embodiments, variations in the biologically useful and / or relevant amino acid sequences can be determined by systematically inserting, deleting, or substituting into the sequence and detecting the activity of the resulting variant protein compared to the parent protein.

[0037] In some embodiments, variants of the anti-CD20 antibody or antigen-binding fragment disclosed in this application may maintain CD20 binding capacity similar to, the same as, or greater than that of the parent antibody or antigen-binding fragment. In some embodiments, the variant may have at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more identity in the amino acid sequence with the parent antibody or antigen-binding fragment. In some embodiments, variants of the anti-CD20 antibody or antigen-binding fragment include the amino acid sequence of a parent anti-CD20 antibody or antigen-binding fragment having one or more conserved amino acid substitutions. Conserved amino acid substitutions are known in the art, including the substitution of an amino acid having a specific physical and / or chemical property with another amino acid having the same or similar chemical or physical properties.

[0038] In some embodiments, variants of the anti-CD20 antibody or antigen-binding fragment include an amino acid sequence of a parent antibody or antigen-binding fragment having one or more non-conserved amino acid substitutions. In some embodiments, variants of the anti-CD20 antibody or antigen-binding fragment include an amino acid sequence of a parent binding antibody or antigen-binding fragment having one or more non-conserved amino acid substitutions, wherein the one or more non-conserved amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant (e.g., CD20 binding). In some embodiments, one or more conserved amino acid substitutions, and / or one or more non-conserved amino acid substitutions, can enhance the biological activity of the variant, thereby increasing the biological activity of the functional variant compared to the parent binding portion.

[0039] In some implementations, the variants may have 1, 2, 3, 4 or 5 amino acid substitutions in the binding portion of the CDR (e.g., VH CDR1, VH CDR2, VHCDR3, VLCDR1, VL CDR2 and VL CDR3).

[0040] In some embodiments, the anti-CD20 antibody or antigen-binding fragment of this application is chemically modified either naturally or through intervention. In some embodiments, the anti-CD20 antibody or antigen-binding fragment is chemically modified via glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, and / or linking to cellular ligands or other proteins. Any of these chemical modifications can be performed using known techniques. The anti-CD20 antibody or antigen-binding fragment may include one or more amino acid analogs (including, for example, non-natural amino acids), as well as other modifying groups known in the art.

[0041] In some embodiments, the light chain variable region has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:7; and the heavy chain variable region has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0042] DIVLTQTPLSLPVTLGQPASISCTSSQGLVDSDGNTYLSWFHQRPGQSPRRLIYKVSYRDYGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQSTQWPLTFGGGTKVEIK (SEQ ID NO:7) QVQLQESGGGLVQPGGSLTLSCAASGFTFSGSSMHWVRQPSGKGLEWVARVGLKTETYATTYAASVTGRFTISRDDSTNTAFLQMNSLKTEDTAVYYCLAYDYSARRYYWGRGTLVTVSS (SEQ ID NO: 8) In the context of two or more polynucleotides or polypeptides, the terms "identity," "percentage identity," and their grammatical equivalents as used in this application refer to two or more sequences or subsequences that are identical or have a specific percentage of identical nucleotide or amino acid residues, without regard to any conserved amino acid substitutions as part of sequence identity, when compared and aligned (with gaps introduced where necessary) to obtain maximum correspondence. Sequence percentages can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software available for obtaining amino acid or nucleotide sequence alignments are well known in the art. These algorithms or software include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG WisconsinPackage, and variants thereof. In some embodiments, the two polynucleotides or polypeptides provided in this application are substantially identical, meaning that when compared and aligned using sequence comparison algorithms or by visual inspection to obtain maximum correspondence, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide or amino acid residue identity. In some embodiments, identity exists in regions of amino acid sequences of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or any integer value between both. In some embodiments, identity exists in regions longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, these sequences are substantially identical in length across the entire length of the compared sequences, such as the coding region of a target protein or antibody. In some embodiments, identity exists in regions of nucleotide sequences of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases, or any integer value between both. In some embodiments, identity exists in regions longer than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments, these sequences are substantially identical in length across the entire length of the compared sequences (e.g., the nucleotide sequence encoding a target protein).

[0043] In some embodiments, the light chain variable region is the amino acid sequence shown in SEQ ID NO:7, and the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:8.

[0044] In some embodiments, the anti-CD20 antibody or antigen-binding fragment also includes a constant region. It is known in the art that the constant region of an antibody mediates multiple effector functions, and these effector functions can vary depending on the antibody isotype. For example, the C1 region of complement binds to the Fc region of an IgG or IgM antibody (by binding to an antigen) to activate the complement system. Activation of complement plays a crucial role in the opsonization and lysis of cellular pathogens. Activation of complement can also stimulate inflammatory responses and participate in autoimmune hypersensitivity reactions. Furthermore, the Fc region of an antibody can bind to cells expressing Fc receptors (FcRs). Many Fc receptors exist that are specific to different classes of antibodies, including IgG (γ receptor), IgE (ε receptor), IgA (α receptor), and IgM (μ receptor). The binding of antibodies to cell surface Fc receptors triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, killing of cells that lyse antibody-coated target cells (a process known as antibody-dependent cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In some embodiments, the anti-CD20 antibody or antigen-binding fragment of this application includes a constant region of at least one human IgA antibody. In some embodiments, the anti-CD20 antibody or antigen-binding fragment of this application includes a constant region of at least one human IgD antibody. In some embodiments, the anti-CD20 antibody or antigen-binding fragment of this application includes a constant region of at least one human IgE antibody. In some embodiments, the anti-CD20 antibody or antigen-binding fragment of this application includes a constant region of at least one human IgG antibody. In some embodiments, the anti-CD20 antibody or antigen-binding fragment of this application includes a constant region of at least one human IgM antibody. In some embodiments, the anti-CD20 antibody or antigen-binding fragment of this application includes a constant region of at least one human IgG1 antibody. In some embodiments, the anti-CD20 antibody or antigen-binding fragment of this application includes a constant region of at least one human IgG2 antibody. In some embodiments, the anti-CD20 antibody or antigen-binding fragment of this application includes a constant region of at least one human IgG3 antibody. In some embodiments, the anti-CD20 antibody or antigen-binding fragment of this application includes a constant region of at least one human IgG4 antibody.

[0045] In some embodiments, at least one or more constant regions in the anti-CD20 antibody or antigen-binding fragment of this application have been modified or deleted. In some embodiments, the antibody includes modification of one or more of the three heavy chain constant regions (CH1, CH2, or CH3), and / or modification of the light chain constant region (CL). In some embodiments, the heavy chain constant region of the modified antibody includes at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibody includes more than one human constant region. In some embodiments, modification of the constant region includes the addition, deletion, or substitution of one or more amino acids in one or more domains. In some embodiments, one or more domains are partially or completely deleted from the constant region of the modified antibody. In some embodiments, the entire CH2 domain (δCH2 construct) is removed from the antibody. In some embodiments, the deleted constant region is replaced by a short amino acid spacer region to provide some of the molecular flexibility that would normally result from a missing constant region. In some embodiments, the modified antibody includes a CH3 domain directly fused to the antibody hinge region. In some embodiments, the modified antibody includes a peptide spacer region inserted between the hinge region and the modified CH2 and / or CH3 domains.

[0046] In some embodiments, the anti-CD20 antibody or antigen-binding fragment includes an Fc region. In some embodiments, the Fc region is fused via a hinge. The hinge may be an IgG1 hinge, an IgG2 hinge, or an IgG3 hinge. The amino acid sequences of the Fc regions of human IgG1, IgG2, IgG3, and IgG4 are known to those skilled in the art. In some cases, Fc regions with amino acid variations have been found in natural antibodies. In some embodiments, the modified antibody (e.g., the modified Fc region) provides altered effector functions that, in turn, affect the biological properties of the antibody. For example, in some embodiments, the deletion or inactivation of the constant region (through point mutation or other means) reduces the binding of the modified antibody to the Fc receptor during circulation. In some embodiments, constant region modification reduces the immunogenicity of the antibody. In some embodiments, constant region modification increases the serum half-life of the antibody. In some embodiments, constant region modification reduces the serum half-life of the antibody. In some embodiments, constant region modification reduces or eliminates ADCC and / or complement-dependent cytotoxicity (CDC) of the antibody. In some embodiments, specific amino acids in the human IgG1 Fc region are replaced with corresponding IgG2 or IgG4 residues, reducing effector function (e.g., ADCC and CDC) in the modified antibody. In some embodiments, the antibody does not have one or more effector functions (e.g., an effectorless antibody). In some embodiments, the antibody has no ADCC activity and / or no CDC activity. In some embodiments, the antibody does not bind to Fc receptors and / or complement factors. In some embodiments, the antibody has no effector function. In some embodiments, constant region modification increases or enhances the antibody's ADCC and / or CDC. In some embodiments, the constant region is modified to eliminate disulfide bonds or oligosaccharide moieties. In some embodiments, the constant region is modified to add / replace one or more amino acids, thereby providing one or more cytotoxic, oligosaccharide, or carbohydrate linkage sites. In some embodiments, the anti-CD20 antibody or antigen-binding fragment includes a variant Fc region that is genetically engineered by substitution at specific amino acid positions compared to the native Fc region.

[0047] In some embodiments, the antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, IgG, bispecific antibodies, multispecific antibodies, and bispecific T-cell conjugates (TCEs); preferably, the antibody or its antigen-binding fragment is scFv.

[0048] In some embodiments, this application provides an antibody or antigen-binding fragment thereof that specifically binds to CD20 (e.g., human CD20). In some embodiments, this application provides an anti-CD20 antibody. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody provided by this application may be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0049] In some embodiments, this application provides an antigen-binding fragment of an anti-CD20 antibody. In some embodiments, the antigen-binding fragment provided in this application can be a single-domain antibody (sdAb), a heavy-chain antibody (HCAb), Fab, Fab', F(ab')2, Fv, a single-chain variable fragment (scFv), or (scFv)2. In some embodiments, the antigen-binding fragment of the anti-CD20 antibody is a single-domain antibody (sdAb). In some embodiments, the antigen-binding fragment of the anti-CD20 antibody is a heavy-chain antibody (HCAb). In some embodiments, the antigen-binding fragment of the anti-CD20 antibody is Fab. In some embodiments, the antigen-binding fragment of the anti-CD20 antibody is Fab'. In some embodiments, the antigen-binding fragment of the anti-CD20 antibody is F(ab')2. In some embodiments, the antigen-binding fragment of the anti-CD20 antibody is Fv. In some embodiments, the antigen-binding fragment of the anti-CD20 antibody is scFv. In some embodiments, the antigen-binding fragment of the anti-CD20 antibody is a disulfide-linked scFv[(scFv)2]. In some implementations, the antigen-binding fragment of the anti-CD20 antibody is a bispecific antibody (dAb).

[0050] In some embodiments, the anti-CD20 antibody or antigen-binding fragment provided in this application comprises a recombinant antibody or antigen-binding fragment. In some embodiments, the anti-CD20 antibody or antigen-binding fragment provided in this application comprises a monoclonal antibody or antigen-binding fragment. In some embodiments, the anti-CD20 antibody or antigen-binding fragment provided in this application comprises a polyclonal antibody or antigen-binding fragment. In some embodiments, the anti-CD20 antibody or antigen-binding fragment provided in this application comprises a camelid antibody or antigen-binding fragment (e.g., camel, dromedary camel, and llama). In some embodiments, the anti-CD20 antibody or antigen-binding fragment provided in this application comprises a chimeric antibody or antigen-binding fragment. In some embodiments, the anti-CD20 antibody or antigen-binding fragment provided in this application comprises a humanized antibody or antigen-binding fragment. In some embodiments, this application provides a human scFv for anti-CD20.

[0051] In some embodiments, the anti-CD20 antibody or antigen-binding fragment provided in this application is an isolated antibody or antigen-binding fragment. In some embodiments, the anti-CD20 antibody or antigen-binding fragment provided in this application is substantially purified.

[0052] In some embodiments, the anti-CD20 antibody or antigen-binding fragment provided in this application comprises a multispecific antibody or antigen-binding fragment. In some embodiments, the anti-CD20 antibody or antigen-binding fragment provided in this application comprises a bispecific antibody or antigen-binding fragment. In some embodiments, this application provides a bispecific T-cell conjugate (TCE). A TCE is a bispecific antibody that binds to both a T-cell antigen (such as CD3) and a tumor antigen. TCEs have been shown to induce targeted lysis of tumor cells and B cells, thus providing a huge potential therapeutic approach for cancer, autoimmune diseases, and other diseases. In some embodiments, this application provides a TCE that specifically binds to both CD3 and CD20. In some embodiments, the TCE comprises the anti-CD20 antibody or antigen-binding fragment provided in this application. In some embodiments, the TCE comprises the anti-CD20 scFv provided in this application.

[0053] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment; preferably, the antibody or its antigen-binding fragment is a human antibody or antigen-binding fragment.

[0054] On the other hand, this application also provides a polynucleotide that encodes an antibody or an antigen-binding fragment thereof as described herein.

[0055] As used herein, the term "encoding" and its grammatical equivalents refer to the inherent property of a specific nucleotide sequence in a polynucleotide or nucleic acid, such as a gene, cDNA, or mRNA, which serves as a template for the synthesis of other polymers and macromolecules having specific nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or specific amino acid sequences in biological processes, and the resulting biological characteristics. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein, then the gene encodes that protein. Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are mutually degenerate or encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0056] The terms “polynucleotide,” “nucleic acid,” and their interchangeable grammatical equivalents in this application refer to nucleotide polymers of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be bound to the polymer by DNA or RNA polymerases.

[0057] On the other hand, this application also provides a vector containing the polynucleotides described herein.

[0058] As used herein, the term "vector" and its grammatical equivalents refer to a carrier for carrying genetic material (e.g., a polynucleotide sequence) that can be introduced into a host cell, where it can be replicated and / or expressed. Applicable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers operable for stable integration into the host cell chromosome. Furthermore, a vector may include one or more selectable marker genes and appropriate expression control sequences. The selectable marker genes may be capable of, for example, providing resistance to antibiotics or toxins, supplementing nutritional deficiencies, or providing critical nutrients absent in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., known in the art. When two or more polynucleotides are to be co-expressed, both polynucleotides may be inserted, for example, in a single expression vector or in separate expression vectors. For single-vector expression, the encoded polynucleotide may be operatively linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of polynucleotides into host cells can be confirmed using methods known in the art. Those skilled in the art will understand that sufficient amounts of polynucleotides are expressed to produce the desired product, and will further understand that expression levels can be optimized using methods known in the art to achieve adequate expression.

[0059] A variety of expression host / vector combinations can be used. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as those from *E. coli*, including pCR1, pBR322, pMB9, and their derivatives, as well as plasmids with a broader host range, such as M13 and other filamentous single-stranded DNA bacteriophages.

[0060] On the other hand, this application also provides a host cell containing the polynucleotides described herein.

[0061] Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblasts), C127 (derived from mouse mammary tumors), 3T3 (derived from mouse fibroblasts), CHO (derived from Chinese hamster ovaries), HeLa (derived from human cervical cancer), BHK (derived from hamster kidney fibroblasts), HEK-293 (derived from human embryonic kidney) cell lines and their variants. Mammalian expression vectors may include non-transcriptional elements (e.g., origin of replication), suitable promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanking non-transcriptional and 5' or 3' untranslated sequences (e.g., necessary ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, and transcription termination sequences). Expression of recombinant proteins in insect cell culture systems (such as baculoviruses) also provides a powerful method for producing proteins that are correctly folded and have biological functions. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art.

[0062] On the other hand, this application also provides a chimeric antigen receptor (CAR) that specifically binds to CD20, the chimeric antigen receptor comprising, from the N-terminus to the C-terminus: CD20 binding domain, which contains the antibody or antigen binding fragment described herein; Transmembrane domains; and Cytoplasmic domain.

[0063] In some implementations, the CD20 binding domain is an scFv containing the following structures: The light chain variable region (VL) includes light chain CDR1 (VL CDR1) shown in SEQ ID NO:1, light chain CDR2 (VL CDR2) shown in SEQ ID NO:2, and light chain CDR3 (VL CDR3) shown in SEQ ID NO:3; and The heavy chain variable region (VH) includes heavy chain CDR1 (VH CDR1) shown in SEQ ID NO:4, heavy chain CDR2 (VH CDR2) shown in SEQ ID NO:5, and heavy chain CDR3 (VH CDR3) shown in SEQ ID NO:6. Among them, any of the above amino acid sequences also includes a derivative sequence that has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining CD20 binding affinity.

[0064] In some implementations, the CD20 binding domain is an amino acid sequence as shown in SEQ ID NO:9.

[0065] DIVLTQTPLSLPVTLGQPASISCTSSQGLVDSDGNTYLSWFHQRPGQSPRRLIYKVSYRDYGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQSTQWPLTFGGGTKVEIKGTGGGGSGGGGSGG GGSQVQLQESGGGLVQPGGSLTLSCAASGFTFSGSSMHWVRQPSGKGLEWVARVGLKTETYATTYAASVTGRFTISRDDSTNTAFLQMNSLKTEDTAVYYCLAYDYSARRYYWGRGTLVTVSS (SEQ ID NO:9) As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid protein or polypeptide containing a binding moiety (e.g., an antibody) linked to a signaling or activation domain of an immune cell (e.g., T cell). In some embodiments, a CAR is a synthetic receptor capable of retargeting T cells to tumor surface antigens. CARs can provide antigen binding and immune cell activation functions for immune cells (e.g., T cells). CARs can utilize the antigen-binding properties of monoclonal antibodies to specifically and reactively redirect T cells to selected targets in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition enables CAR-expressing T cells to have antigen recognition capabilities independent of antigen processing, thereby circumventing tumor escape mechanisms.

[0066] The anti-CD20 antibody or antigen-binding fragment of this application can be used as part of a chimeric antigen receptor (CAR) or T-cell receptor (TCR), which can be expressed in immune effector cells used to treat cancer. Thus, this application also provides CARs and TCRs that specifically bind to CD20 (e.g., human CD20), immune effector cells expressing such CARs or TCRs, and the uses of such cells.

[0067] In some implementations, the transmembrane domains are derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, or CD154.

[0068] In some embodiments, the transmembrane domain of the CAR provided in this application may be derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, T cell receptor (TCR) α chain, TCRβ chain, TCRζ chain, CD3ε, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, or other polypeptides expressed in immune effector cells. In some embodiments, the transmembrane domain of the CAR provided in this application includes a transmembrane region, which is the transmembrane region of CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, T cell receptor (TCR) α chain, TCRβ chain, TCRζ chain, CD3ε, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, or other transmembrane regions of polypeptides expressed in immune effector cells.

[0069] In some embodiments, the transmembrane domain of the CAR provided in this application originates from CD8. In some embodiments, the transmembrane domain includes the transmembrane region of CD8. In some embodiments, the transmembrane domain originates from CD28. In some embodiments, the transmembrane domain includes the transmembrane region of CD28. In some embodiments, the transmembrane domain originates from CD3ζ. In some embodiments, the transmembrane domain includes the transmembrane region of CD3ζ. In some embodiments, the transmembrane domain originates from CD4. In some embodiments, the transmembrane domain includes the transmembrane region of CD4. In some embodiments, the transmembrane domain originates from 4-1BB. In some embodiments, the transmembrane domain includes the transmembrane region of 4-1BB. In some embodiments, the transmembrane domain originates from OX40. In some embodiments, the transmembrane domain includes the transmembrane region of OX40. In some embodiments, the transmembrane domain originates from ICOS. In some embodiments, the transmembrane domain includes the transmembrane region of ICOS. In some embodiments, the transmembrane domain originates from CTLA-4. In some embodiments, the transmembrane domain includes the transmembrane region of CTLA-4. In some embodiments, the transmembrane domain is derived from PD-1. In some embodiments, the transmembrane domain includes the transmembrane region of PD-1. In some embodiments, the transmembrane domain is derived from LAG-3. In some embodiments, the transmembrane domain includes the transmembrane region of LAG-3. In some embodiments, the transmembrane domain is derived from 2B4. In some embodiments, the transmembrane domain includes the transmembrane region of 2B4. In some embodiments, the transmembrane domain is derived from BTLA. In some embodiments, the transmembrane domain includes the transmembrane region of BTLA. In some embodiments, the transmembrane domain is derived from the TCRα chain. In some embodiments, the transmembrane domain includes the transmembrane region of the TCRα chain. In some embodiments, the transmembrane domain is derived from the TCRβ chain. In some embodiments, the transmembrane domain includes the transmembrane region of the TCRβ chain. In some embodiments, the transmembrane domain is derived from the TCRζ chain. In some embodiments, the transmembrane domain includes the transmembrane region of the TCRζ chain. In some embodiments, the transmembrane domain is derived from CD3ε. In some embodiments, the transmembrane domain includes the transmembrane region of CD3ε. In some embodiments, the transmembrane domain is derived from CD45. In some embodiments, the transmembrane domain includes the transmembrane region of CD45. In some embodiments, the transmembrane domain is derived from CD5. In some embodiments, the transmembrane domain includes the transmembrane region of CD5. In some embodiments, the transmembrane domain is derived from CD8. In some embodiments, the transmembrane domain includes the transmembrane region of CD8. In some embodiments, the transmembrane domain is derived from CD9. In some embodiments, the transmembrane domain includes the transmembrane region of CD9. In some embodiments, the transmembrane domain is derived from CD16. In some embodiments, the transmembrane domain includes the transmembrane region of CD16.In some embodiments, the transmembrane domain is derived from CD22. In some embodiments, the transmembrane domain includes the transmembrane region of CD22. In some embodiments, the transmembrane domain is derived from CD33. In some embodiments, the transmembrane domain includes the transmembrane region of CD33. In some embodiments, the transmembrane domain is derived from CD37. In some embodiments, the transmembrane domain includes the transmembrane region of CD37. In some embodiments, the transmembrane domain is derived from CD64. In some embodiments, the transmembrane domain includes the transmembrane region of CD64. In some embodiments, the transmembrane domain is derived from CD80. In some embodiments, the transmembrane domain includes the transmembrane region of CD80. In some embodiments, the transmembrane domain is derived from CD86. In some embodiments, the transmembrane domain includes the transmembrane region of CD86. In some embodiments, the transmembrane domain is derived from CD134. In some embodiments, the transmembrane domain includes the transmembrane region of CD134. In some embodiments, the transmembrane domain is derived from CD154. In some embodiments, the transmembrane domain includes the transmembrane region of CD154.

[0070] In some embodiments, the transmembrane domain may be synthetic, in which case it primarily comprises hydrophobic residues such as leucine and valine. Optionally, the transmembrane domain may be derived from peptides not naturally expressed in immune effector cells, provided that the transmembrane domain is capable of transducing signals from the antigen bound to the CAR to intracellular signaling domains and / or co-stimulatory domains. In some embodiments, the transmembrane domain may include a triplet of phenylalanine, tryptophan, and valine at each end. Optionally, short oligonucleotides or peptide linkers, preferably between 2 and 10 amino acids in length, may form a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine conjugates provide a highly suitable linker.

[0071] In some implementations, the cytoplasmic domain includes a signal transduction domain derived from CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.

[0072] The cytoplasmic domain of the CAR provided in this application may contain a signal transduction domain that functions in immune effector cells expressing the CAR. Such a signal transduction domain may, for example, be derived from CD3ζ, Fc receptor γ, FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, or DAP12. The signal transduction domain may also be a combination of signal transduction domains derived from CD3ζ, Fc receptor γ, FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, or DAP12. A signal transduction domain derived from a protein or polypeptide refers to a domain of a protein or polypeptide responsible for activating immune effector cells (e.g., T cells), or a fragment thereof that retains its activating function. Generally, signal transduction domains induce persistent, transport, and / or effector functions in transduced immune effector cells (e.g., T cells). The signal transduction domain of a protein or polypeptide can be an intracellular domain of that protein or polypeptide. In some embodiments, the signal transduction domain includes intracellular domains of CD3ζ, FcRγ, FcγRIIA, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.

[0073] In some embodiments, the cytoplasmic domain of the CAR provided in this application includes a signal transduction domain derived from CD3ζ. In some embodiments, the signal transduction domain includes an intracellular domain of CD3ζ. In some embodiments, the cytoplasmic domain includes a signal transduction domain derived from FcRγ. In some embodiments, the signal transduction domain includes an intracellular domain of FcRγ. In some embodiments, the cytoplasmic domain includes a signal transduction domain derived from FcγRIIa. In some embodiments, the signal transduction domain includes an intracellular domain of FcγRIIa. In some embodiments, the cytoplasmic domain includes a signal transduction domain derived from FCRβ. In some embodiments, the signal transduction domain includes an intracellular domain of FCRβ. In some embodiments, the cytoplasmic domain includes a signal transduction domain derived from CD3γ. In some embodiments, the signal transduction domain includes an intracellular domain of CD3γ. In some embodiments, the cytoplasmic domain includes a signal transduction domain derived from CD3δ. In some embodiments, the signal transduction domain includes an intracellular domain of CD3δ. In some embodiments, the cytoplasmic domain includes a signal transduction domain derived from CD3ε. In some embodiments, the signal transduction domain includes an intracellular domain of CD3ε. In some embodiments, the cytoplasmic domain includes a signal transduction domain derived from CD5. In some embodiments, the signal transduction domain includes an intracellular domain of CD5. In some embodiments, the cytoplasmic domain includes a signal transduction domain derived from CD22. In some embodiments, the signal transduction domain includes an intracellular domain of CD22. In some embodiments, the cytoplasmic domain includes a signal transduction domain derived from CD79a. In some embodiments, the signal transduction domain includes an intracellular domain of CD79a. In some embodiments, the cytoplasmic domain includes a signal transduction domain derived from CD79b. In some embodiments, the signal transduction domain includes an intracellular domain of CD79b. In some embodiments, the cytoplasmic domain includes a signal transduction domain derived from DAP10. In some embodiments, the signal transduction domain includes an intracellular domain of DAP10. In some embodiments, the cytoplasmic domain includes a signal transduction domain derived from DAP12. In some embodiments, the signal transduction domain includes an intracellular domain of DAP12.

[0074] In some implementations, the cytoplasmic domain further includes a co-stimulatory domain derived from CD28, 4-1BB (CD137), OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, CD43, or any combination thereof.

[0075] In some embodiments, the cytoplasmic domain of the CAR provided in this application further includes two co-stimulatory domains. Such co-stimulatory domains can provide enhanced activation of immune effector cells (e.g., T cells). The co-stimulatory signaling domains can be derived from, for example, CD28, 4-1BB (CD137), OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, TIGIT, GITR, TLR, DR3, or CD43. Co-stimulatory domains derived from proteins or peptides refer to the domains of proteins or peptides responsible for providing enhanced activation of immune effector cells (e.g., T cells), or fragments that retain their activation function. In some embodiments, the co-stimulatory domains of the CAR provided in this application include intracellular domains of CD28, 4-1BB (CD137), OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, TIGIT, GITR, TLR, DR3, or CD43. In some embodiments, the CAR cytoplasmic domain provided in this application includes a co-stimulatory domain derived from CD28. In some embodiments, the co-stimulatory domain includes an intracellular domain of CD28. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from 4-1BB. In some embodiments, the co-stimulatory domain includes an intracellular domain of 4-1BB. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from OX40. In some embodiments, the co-stimulatory domain includes an intracellular domain of OX40. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from ICOS. In some embodiments, the co-stimulatory domain includes an intracellular domain of ICOS. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from DAP10. In some embodiments, the co-stimulatory domain includes an intracellular domain of DAP10. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from 2B4. In some embodiments, the co-stimulatory domain includes an intracellular domain of 2B4. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from CD27. In some embodiments, the co-stimulatory domain includes an intracellular domain of CD27. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from CD30. In some embodiments, the co-stimulatory domain includes an intracellular domain of CD30. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from CD40. In some embodiments, the co-stimulatory domain includes an intracellular domain of CD40. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from CD2. In some embodiments, the co-stimulatory domain includes an intracellular domain of CD2. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from CD7.In some embodiments, the co-stimulatory domain includes an intracellular domain of CD7. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from LIGHT. In some embodiments, the co-stimulatory domain includes an intracellular domain of LIGHT. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from TIGIT. In some embodiments, the co-stimulatory domain includes an intracellular domain of TIGIT. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from GITR. In some embodiments, the co-stimulatory domain includes an intracellular domain of GITR. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from TLR. In some embodiments, the co-stimulatory domain includes an intracellular domain of TLR. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from DR3. In some embodiments, the co-stimulatory domain includes an intracellular domain of DR3. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from CD43. In some embodiments, the co-stimulatory domain includes an intracellular domain of CD43.

[0076] In some embodiments, the cytoplasmic domain of the CAR may include two costimulatory domains derived from two costimulatory receptors, such as CD28 and 4-1BB, or CD28 and OX40, or other combinations of costimulatory ligands disclosed in this application.

[0077] In some implementations, the chimeric antigen receptor also includes a CD8 hinge located between the antibody or antigen-binding fragment and the transmembrane domain.

[0078] In some embodiments, the CAR may also include spacers or sequences that interconnect the domains of the CAR. For example, spacers may be included between a signal peptide and an antigen-binding domain, between an antigen-binding domain and a transmembrane domain, between a transmembrane domain and an intracellular domain, and / or between domains within an intracellular domain, such as between a stimulatory domain and a co-stimulatory domain. The spacers may be flexible enough to allow various domains to interact with other peptides, for example, allowing the antigen-binding domain to have directional flexibility to facilitate antigen recognition. The spacers may be, for example, a hinge region from IgG, a CH2CH3 (constant) region of an immunoglobulin, and / or a portion of CD3 (differentiation cluster 3), or certain other sequences suitable as spacers. In some embodiments, the CAR disclosed in this application includes a hinge domain connecting the CD20-binding domain and the transmembrane domain. In some embodiments, the hinge domain includes a CD8 hinge structure. In some embodiments, the hinge domain includes a CD28 hinge structure.

[0079] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence as shown in SEQ ID NO:10.

[0080] MALPVTALLLPLALLLHAARPDIVLTQTPLSLPVTLGQPASISCTSSQGLVDSDGNTYLSWFHQRPGQSPRRLIYKVSYRDYGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQSTQWPLTF GGGTKVEIKGTGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLTLSCAASGFTFSGSSMHWVRQPSGKGLEWVARVGLKTETYATTYAASVTGRFTISRDDSTNTAFLQMNSLKTEDTAVYYCL AYDYSARRYYWGRGTLVTVSSSEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:10) In some embodiments, the chimeric antigen receptor has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO:10.

[0081] On the other hand, this application also provides a polynucleotide that encodes the chimeric antigen receptor described herein.

[0082] On the other hand, this application also provides a vector comprising a polynucleotide encoding the chimeric antigen receptor described herein.

[0083] In another aspect, this application also provides a cell containing a polynucleotide encoding a chimeric antigen receptor described herein or a vector containing a polynucleotide encoding a chimeric antigen receptor described herein.

[0084] In some implementations, the cells are immune effector cells.

[0085] In some implementations, the cells are T cells, NK cells, NKT cells, macrophages, or granulocytes.

[0086] As used in this application and as understood in the art, "immune effector cells" refers to cells of hematopoietic origin that play a direct role in an immune response against a target, such as a pathogen, cancer cell, or foreign substance. Immune effector cells include T cells, B cells, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, and basophils.

[0087] In some embodiments, the immune effector cells are selected from the group consisting of T cells, B cells, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, and basophils. In some embodiments, the immune effector cells provided in this application are selected from the group consisting of T cells, NK cells, NKT cells, macrophages, neutrophils, and granulocytes. In some embodiments, the immune effector cells provided in this application are T cells. In some embodiments, the immune effector cells provided in this application are NK cells. In some embodiments, the immune effector cells provided in this application are NKT cells. In some embodiments, the immune effector cells provided in this application are macrophages. In some embodiments, the immune effector cells provided in this application are neutrophils. In some embodiments, the immune effector cells provided in this application are granulocytes.

[0088] In some embodiments, the immune effector cells provided in this application can be genetically engineered. In some embodiments, the genetically engineered immune effector cells provided in this application are isolated. In some embodiments, the genetically engineered immune effector cells provided in this application are substantially purified.

[0089] In some embodiments, this application provides immune effector cells capable of recombinantly expressing the polypeptides (e.g., antibodies or CARs) disclosed herein. This application also provides immune effector cells (e.g., T cells) comprising polynucleotides or vectors, the polynucleotides encoding the polypeptides (e.g., antibodies or CARs) disclosed herein, and the vectors comprising the polynucleotides disclosed herein. In some embodiments, this application provides immune effector cells (e.g., T cells) comprising polynucleotides encoding the anti-CD20 antibody or antigen-binding fragment disclosed herein. In some embodiments, this application provides immune effector cells (e.g., T cells) capable of recombinantly expressing the anti-CD20 antibody or antigen-binding fragment disclosed herein. In some embodiments, this application provides immune effector cells comprising polynucleotides encoding the anti-CD20 CAR disclosed herein. In some embodiments, this application provides immune effector cells capable of recombinantly expressing the anti-CD20 CAR disclosed herein (immune effector cells such as T cells; immune effector cells capable of recombinantly expressing anti-CD20 CAR such as anti-CD20 CAR-T cells). In some embodiments, this application provides immune effector cells comprising polynucleotides encoding the anti-CD20 TCR disclosed herein. In some embodiments, this application provides immune effector cells capable of recombinantly expressing the anti-CD20 TCR disclosed herein (immune effector cells such as T cells; immune effector cells capable of recombinantly expressing anti-CD20 TCR such as anti-CD20 TCR T cells).

[0090] In some embodiments, the immune effector cells provided in this application are T cells. T cells can be cytotoxic T cells, helper T cells, or γδT cells, CD4+ / CD8+ double-positive T cells, CD4+ T cells, CD8+ T cells, CD4 / CD8 double-negative T cells, CD3+ T cells, naive T cells, effector T cells, cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, Th0 cells, Th1 cells, Th2 cells, Th3 (Treg) cells, Th9 cells, Th17 cells, Thαβ helper cells, Tfh cells, stem cell-like central memory TSCM cells, central memory TCM cells, effector memory TEM cells, effector memory TEMRA cells, or γδT cells. In some embodiments, the T cells are cytotoxic T cells. In some embodiments, the T cells are genetically engineered. In some embodiments, the T cells provided in this application are isolated. In some embodiments, the T cells provided in this application are substantially purified.

[0091] In some embodiments, the genetically engineered cells provided in this application are derived from cells isolated from a subject. As used herein, genetically engineered cells "derived from" source cells refer to genetically engineered cells obtained by acquiring source cells and genetically manipulating them. Source cells can be from natural sources. For example, source cells can be primary cells isolated from a subject. The subject can be an animal or a human. Source cells can also be cells that have been passaged or genetically manipulated in vitro.

[0092] In some embodiments, the genetically engineered cells provided in this application are derived from cells isolated from the human body. Immune effector cells (e.g., T cells) can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cell lines available in the art can be used. In some embodiments, the genetically engineered cells provided in this application are derived from cells isolated from peripheral blood. In some embodiments, the genetically engineered cells provided in this application are derived from cells isolated from bone marrow. In some embodiments, the genetically engineered cells provided in this application are derived from cells isolated from peripheral blood mononuclear cells (PBMCs).

[0093] In some embodiments, the genetically engineered cells provided in this application are derived from cells differentiated in vitro from stem cells or progenitor cells. In some embodiments, the stem cells or progenitor cells are selected from the group consisting of T cell progenitor cells, hematopoietic stem / progenitor cells, hematopoietic pluripotent progenitor cells, embryonic stem cells, and induced pluripotent cells. In some embodiments, the genetically engineered cells provided in this application are derived from cells differentiated in vitro from T cell progenitor cells. In some embodiments, the genetically engineered cells provided in this application are derived from cells differentiated in vitro from hematopoietic stem / progenitor cells. In some embodiments, the genetically engineered cells provided in this application are derived from cells differentiated in vitro from hematopoietic pluripotent progenitor cells. In some embodiments, the genetically engineered cells provided in this application are derived from cells differentiated in vitro from embryonic stem cells. In some embodiments, the genetically engineered cells provided in this application are derived from cells differentiated in vitro from induced pluripotent cells.

[0094] In some embodiments, this application provides a cell population comprising the cells disclosed in this application. The cells disclosed in this application may include polynucleotides encoding the polypeptides disclosed in this application, or recombinantly express the polypeptides disclosed in this application. The polypeptide may be an anti-CD20 antibody or antigen-binding fragment, an anti-CD20 CAR, or an anti-CD20 TCR. The cell population may be a homologous cell population. The cell population may be a heterogeneous cell population. In some embodiments, the cell population may be a heterogeneous cell population comprising any combination of the cells disclosed in this application. In some embodiments, the cell population is derived from peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocytes (PBLs), tumor-infiltrating lymphocytes (TILs), cytokine-induced killer cells (CIKs), lymphokine-activated killer cells (LAKs), or bone marrow-infiltrating lymphocytes (MILs). In some embodiments, the cell population provided in this application is derived from PBMCs. In some embodiments, the cell population provided in this application is derived from PBLs. In some embodiments, the cell population provided in this application is derived from TILs. In some embodiments, the cell population provided in this application is derived from CIKs. In some embodiments, the cell population provided in this application is derived from LAK cells. In some embodiments, the cell population provided in this application is derived from MILs. The cell population may be genetically engineered to recombinantly express the polypeptide (e.g., antibody or CAR) disclosed in this application. In some embodiments, this application provides a cell population comprising a polynucleotide or a vector, the polynucleotide encoding the polypeptide (e.g., antibody or CAR) disclosed in this application, and the vector having the polynucleotide disclosed in this application. In some embodiments, this application provides a cell population comprising a polynucleotide encoding the anti-CD20 antibody or antigen-binding fragment disclosed in this application. In some embodiments, this application provides a cell population recombinantly expressing the anti-CD20 antibody or antigen-binding fragment disclosed in this application. In some embodiments, this application provides a cell population comprising a polynucleotide encoding the anti-CD20 CAR disclosed in this application. In some embodiments, this application provides a cell population capable of recombinantly expressing the anti-CD20 CAR disclosed in this application (e.g., anti-CD20 CAR-T cells). In some embodiments, this application provides a cell population comprising a polynucleotide encoding the anti-CD20 TCR disclosed herein. In some embodiments, this application provides a cell population capable of recombinantly expressing the anti-CD20 TCR disclosed herein (e.g., anti-CD20TCR T cells).

[0095] On the other hand, this application also provides the use of the antibodies or antigen-binding fragments thereof described herein or the cells described herein in the preparation of a medicament for treating B-cell malignancies.

[0096] In some implementations, antibodies or their antigen-binding fragments or cells are used in combination with additional therapies.

[0097] Combination therapy using agents with different mechanisms of action can produce additive or synergistic effects. Combination therapy allows for lower doses of each agent than those used in single therapy, thereby reducing toxic side effects and / or increasing the therapeutic index of the agents disclosed in this application. Combination therapy can reduce the likelihood of the development of drug-resistant cancer cells. In some embodiments, the additional treatment results in an increase in the therapeutic index of the cell or pharmaceutical composition of this application. In some embodiments, the additional treatment results in a reduction in the toxicity and / or side effects of the cell or pharmaceutical composition of this application. In some embodiments, the anti-CD20 antibody or its antigen-binding fragment, cell, or pharmaceutical composition of this application may be administered in combination with the additional treatment. In some embodiments, the additional treatment may be surgical resection, radiotherapy, or chemotherapy.

[0098] Additional treatment may be administered before, simultaneously with, or after the administration of the anti-CD20 antibody or its antigen-binding fragment, cells, or pharmaceutical composition of this application. Combination administration may include co-administration, administration in a single pharmaceutical formulation or using separate formulations, or administration sequentially in any order, but generally over a period of time so that all active agents can exert their biological activity simultaneously. Those skilled in the art can readily determine an appropriate regimen for the administration of the pharmaceutical composition of this application and the combined additional treatment based on the needs of the treated subject, including the timing and dosage of the additional pharmaceutical agents used in the combination treatment.

[0099] In some implementations, the B-cell malignancy is selected from multiple myeloma, Waldenström macroglobulinemia, Hodgkin lymphoma, or non-Hodgkin lymphoma.

[0100] Diseases that can be treated with the anti-CD20 antibody or antigen-binding fragment, immune effector cells, or pharmaceutical composition provided in this application include any disease or condition related to CD20, as well as any disease or condition that specifically expresses CD20 and / or uses CD20 as a therapeutic target (collectively, "CD20-related diseases or conditions"). Cancers associated with CD20 expression include hematologic malignancies such as multiple myeloma, Waldenström macroglobulinemia, and Hodgkin's lymphoma and non-Hodgkin's lymphoma.

[0101] In some implementations, CD20-related diseases or conditions are B-cell-related disorders. In other implementations, CD20-related diseases or conditions include glioblastoma, lymphomatoid granuloma, post-transplant lymphoproliferative disorders, immunomodulatory disorders, heavy-chain disease, primary or immune-cell-related amyloidosis, or monoclonal gammaglobulinosis of undetermined significance.

[0102] In certain diseases and conditions, CD20 is expressed on malignant cells and in cancers. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is a B-cell malignancy. In some embodiments, the cancer is lymphoma, leukemia, or plasma cell malignancy. The lymphomas covered in this application include, but are not limited to, Burkitt lymphoma (e.g., endemic or sporadic Burkitt lymphoma), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, Waldenström macroglobulinemia, follicular lymphoma, small non-cleaved cell lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), marginal zone lymphoma, splenic lymphoma, nodal monocytic B-cell lymphoma, immunoblastic lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, pulmonary B-cell angiocentric lymphoma, small lymphocytic lymphoma, primary mediastinal B-cell lymphoma, lymphoplasmacytic lymphoma (LPL), or mantle cell lymphoma (MCL). The leukemias covered in this application include, but are not limited to, chronic lymphocytic leukemia (CLL), plasma cell leukemia, or acute lymphoblastic leukemia (ALL).

[0103] The plasma cell malignancies covered in this application include, but are not limited to, multiple myeloma (MM) and plasmacytoma.

[0104] In some embodiments, the anti-CD20 antibody or antigen-binding fragment, immune effector cells, or pharmaceutical composition provided in this application can be used to treat MM. In some embodiments, the MM to be treated is non-secretory MM. In some embodiments, the MM is smoldering MM. In some embodiments, the disease or condition is relapsed and / or refractory multiple myeloma (R / R MM).

[0105] Among these diseases, treatable CD20-related disorders or conditions (e.g., cancers that express CD20) include, but are not limited to, neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma (e.g., multiple myeloma), stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal cancer, and head and neck cancer.

[0106] The anti-CD20 antibody or its antigen-binding fragment, immune effector cells, and pharmaceutical composition provided in this application may be administered to a subject by any method known in the art, including but not limited to intrapleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal administration, or other parenteral administration routes, such as by injection or infusion, or direct administration to the thymus. As used in this application, the phrase "parenteral administration" refers to a route of administration other than enteral and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intraperitoneal injections and infusions. In some embodiments, subcutaneous administration is used. In some embodiments, intravenous administration is used. In some embodiments, oral administration is used. In one embodiment, the cells provided in this application can be locally delivered to a tumor using well-known methods, including but not limited to hepatic or aortic pumps; perfusion of the limbs, lungs, or liver; in the portal vein; via venous shunt; or in cavities or blood vessels near the tumor. In another embodiment, the cells provided in this application can be administered systemically. In a preferred embodiment, the cells are administered locally at the tumor site. The cells can also be administered intratumorally, for example, by direct injection at the tumor site and / or into the tumor vascular system. For example, in the case of malignant pleural diseases, mesothelioma, or lung cancer, intrapleural administration is preferred. Those skilled in the art can select an appropriate administration method based on the type and / or location of the tumor to be treated. The cells can be introduced by injection or catheter. In one embodiment, intrapleural administration is performed on a subject in need, for example, using an intrapleural catheter. Optionally, an expansion / or differentiation agent can be administered to the subject before, during, or after cell administration to increase the in vivo cell generation provided in this application.

[0107] This application also provides pharmaceutical compositions comprising the anti-CD20 antibody or antigen-binding fragment disclosed herein. This application further provides pharmaceutical compositions comprising genetically engineered immune effector cells disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the anti-CD20 antibody or antigen-binding fragment disclosed herein, and further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a pharmaceutically effective amount of the genetically engineered cells disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is useful in immunotherapy.

[0108] On the other hand, this application also provides a method for preparing cells capable of expressing CARs that specifically bind to CD20, comprising transferring the polynucleotides described herein into the cells.

[0109] This application describes several embodiments, but these descriptions are exemplary and not limiting, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature or element of any embodiment may be used in combination with, or may replace, any feature or element of any other embodiment.

[0110] This application includes and contemplates combinations of features and elements known to those skilled in the art. The embodiments, features, and elements disclosed in this application can also be combined with any conventional features or elements to form unique inventive solutions. Any feature or element of any embodiment can also be combined with features or elements from other inventive solutions to form another unique inventive solution. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented individually or in any suitable combination. Therefore, the embodiments are not limited except by the limitations imposed by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0111] Furthermore, in describing representative embodiments, the specification may have presented methods and / or processes as a specific sequence of steps. However, the method or process should not be limited to the specific order of steps described herein, to the extent that it does not depend on such a specific order. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation of the claims. Moreover, the claims concerning the method and / or process should not be limited to the steps performed in the written order, and those skilled in the art will readily understand that these orders can be varied and still remain within the spirit and scope of the embodiments of this application.

[0112] Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, then generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer are followed. Unless otherwise defined or stated, all technical and scientific terms used in this application have the same meaning as those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this application.

[0113] Example The materials used in the embodiments are shown below: 1. Materials Primary human umbilical cord blood T lymphocytes: Primary human T cells were isolated from umbilical cord blood. T cells were stimulated with anti-CD3 / CD28 Dynabeads magnetic beads (Life Technologies, Grand Island, NY) and cultured in R10 medium supplemented with 100 IU / mL IL-2 (RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% HEPES, 1% GutaMAX, 1% penicillin and streptomycin, 1% MEM NEAA and 1% sodium pyruvate).

[0114] Cell lines: The following cell lines were cultured in 1640 medium and used in related experiments: Raji cells (human B lymphoma cells) and K562 (human leukemia cells).

[0115] 2. Methods mRNA production: 1) Oligonucleotide synthesis and purification: 50-100 μM oligonucleotides were synthesized using the solid-phase phosphoramidite method. 1) Synthesizing short oligonucleotide fragments of bp, followed by desalting and purification; 2) PCR amplification: Amplifying double-stranded nucleotide fragments using bridge PCR, and confirming the size of the amplified product by gel electrophoresis; 3) Enzymatic assembly and plasmid construction: Introducing specific circularization recognition sequences on both sides of the target sequence, and seamlessly splicing the fragments with the plasmid backbone (such as pLevo-CR2) based on enzymatic reactions of DNA polymerase, restriction exonuclease, and ligase, and verifying the fragment insertion by enzyme digestion; 4) Plasmid transformation and production: Transforming recombinant plasmids into competent E. coli cells, plating on LB plates, screening for positive clones, expanding single colonies of selected positive clones, and verifying the sequence accuracy by Sanger sequencing after plasmid extraction; 5) Plasmid template preparation: Digesting the correct circular plasmids to generate a linear DNA template containing complete circularization elements, and purifying the fragments using magnetic bead recovery; 6) In vitro transcription and splicing / circularization: Using linear DNA as a template, in T7... In vitro transcription was performed using an RNA polymerase system with a mixture of nucleoside triphosphates (NTPs). After preliminary purification, the product was digested with RNase R to remove uncircularized linear RNA and residual template. 7) The product was purified by magnetic beads or HPLC, and then capillary electrophoresis was performed to confirm the size and purity of the RNA. QC testing was passed.

[0116] T cells were stimulated with CD3 / CD28 Dynabeads on day 0. Cells were then cultured in R10 medium containing 100 IU / mL IL-2, with the medium replaced with fresh medium every two days. On day 12, CD20 CAR mRNA was electroporated. T cells were collected, washed twice with PBS, resuspended in OPTI-MEM medium, and the cell density adjusted to 1.0 × 10⁶ cells / day. 8 50 μg of CAR mRNA was mixed with 0.1 mL of cell suspension at a concentration of 1 cell / mL (0.1 mL). The mixture was transferred to a 0.2 cm electroporation cuvette with the following parameters: voltage 500 V, single pulse duration 0.7 ms. After electroporation, the cells were immediately transferred to pre-warmed complete culture medium for resuscitation and cultured at 37°C and 5% CO2. Twenty-four hours after mRNA electroporation of T cells, cells were collected and incubated with fluorescently labeled CD20 antigen, followed by flow cytometry analysis. The percentage of CAR-positive cells in the total T cell population was determined by flow cytometry, and the relative expression level of CAR on the T cell surface was characterized by the average fluorescence intensity of this positive population.

[0117] Twenty-four hours after T cells were electroporated with mRNA, an in vitro tumor cell killing assay was performed to assess specific cytotoxicity. Transfected CAR-T cells were collected and co-incubated with target cells at a 1:1 ratio for 20 hours. Target cells included the human Burkitt lymphoma cell line Raji (specific target cells) expressing CD20 and the human chronic myeloid leukemia cell line K562 (non-specific control target cells) not expressing CD20. The lactate dehydrogenase (LDH) release assay was used to detect the killing effect of CAR-T cells on tumor cells, verifying the in vitro targeted killing activity and antigen-dependent nature of CD20 CAR-T cells. The LDH assay used the Cytotoxicity LDH Assay Kit (Cat. No.: HY-K1090) from MCE, and the sample wells, high control wells, high control blank wells, low control wells, and background blank wells were set according to the kit instructions. Four replicates were set for each group. In 96-well plates, effector cells and target cell suspensions were seeded at an effector-to-target ratio of 1, with a final volume of 100 μL per well. The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 22 hours. After 22 hours of incubation, 10 μL of Lysis Solution was added to each of the high control and high control blank wells, and 10 μL of culture medium was added to each of the low control wells. After gentle mixing, the plates were incubated at 37°C in a 5% CO2 incubator for another 30 minutes. After incubation, 50 μL of culture medium supernatant was carefully aspirated from each well, retaining the remaining 50 μL of culture medium and cells as the assay material. 50 μL of Working Solution was added to each well, and the mixture was vortexed to mix. The plates were incubated at room temperature in the dark for 30 minutes. After stopping the reaction by adding 50 μL of Stop Solution to each well, the absorbance (OD value) at 490 nm was immediately measured using a microplate reader. Specific cytotoxicity % was calculated using the following formula: [(XZ) / (YZ)] × 100%. Where X: OD value of sample well - OD value of background blank well; Y: OD value of high control well - OD value of high control blank well; Z: OD value of low control well - OD value of background blank well.

[0118] Example 1. Preparation of anti-CD20 antibody Anti-CD20 antibodies were prepared and screened using a fully human antibody phage display library (scFv format) by Beijing Zhihe Xinchuang Biotechnology Co., Ltd. 1) Target molecule identification: Take an SDS-PAGE pre-cast gel, dilute 2 μg of target protein with an appropriate amount of PBS, add SDS-PAGE loading buffer to the sample, boil and lyse for 5 min, then add to the loading wells for SDS-PAGE. After electrophoresis, add staining solution and place on a horizontal shaker at room temperature for 2-4 h. Pour out the staining solution, add destaining solution, and place on a horizontal shaker at room temperature for 1-2 h. Observe the results and take pictures, such as... Figure 1 Verify the molecular weight and concentration of the target molecule.

[0119] 2) Detection of target molecule biotinylation effect: Take 3 wells of the ELISA plate, dilute the target protein with coating buffer, 0.1 μg / well, add 50 μL / well; add 50 μL of coating buffer to the second well; dilute other validated biotinylated proteins in the third well under the same conditions as a positive control. Incubate overnight at 4 ℃ or 1 h at 37 ℃. Wash the plate 3 times with PBS. Add blocking buffer, 300 μL / well, incubate at 37 ℃ for 1 h. Wash the plate 3 times with PBS. Add HRP-streptavidin diluted 1:800 in 1% M PBS, incubate at 37 ℃ for 1 h. Wash the plate 4 times with 0.1% PBST. Develop with TMB chromogenic solution, 50 μL / well, stop with 2M H2SO4, and detect at 450 nm. The results are shown in Table 1.

[0120] Table 1. Detection of target protein biotinylation Primary antibody: HRP-streptavidin 3) Friendly Selection ① Select a single clone of Escherichia coli strain TG1, incubate it overnight in an incubator, and obtain a single-clone plate for later use.

[0121] ② Select a single TG1 clone and incubate it in 10 mL of 2YT liquid medium at 37 ℃ and 250 rpm until OD≈0.5, and use it to measure the titer.

[0122] ③ Add streptavidin magnetic beads to EP tube ①, and rotate to block for 1 h with 1 mL of 2% M-PBS; remove the supernatant, add the target protein biotinylated human CD20 (20-291) Protein (ACRO) diluted with PBS at a concentration of 30 μg / mL, and block again with 1 mL of 2% M PBS for 1 h; ④ Add 100 μL of streptavidin magnetic beads to EP tube ②, block with 1 mL of 2% M PBS for 1 h; aspirate the supernatant, add the antibody library HNScL1.2 phage lib (Zhihe Xinchuang) diluted with 1% M PBS, with an addition volume of 1.00 × 10⁻⁶. 13 PFU (Particle Fusion) is used to remove the background of magnetic beads. ⑤ Remove the supernatant from EP tube ① and the contents of EP tube ②, and add them to EP tube ① to bind at room temperature for 1 h; ⑥ Wash 9 times with 0.1% PBST, then wash once with PBS, with gentle shaking for 1 min each time. Add 100 μL of elution buffer per well, shake gently at room temperature for 8 min, aspirate, and add stop solution to obtain the elution product. Measure the titer of the elution product.

[0123] ⑦ Inoculate TG1 monoclonal antibodies into 20 mL of 2YT medium and incubate at 37 ℃ and 250 rpm until OD≈0.5. Add the elution product and mix well, then incubate at 7 ℃ for 30 min. Add 4 μL of Amp (final concentration 20 μg / mL) and incubate at 37 ℃ and 180 rpm for 60 min. Add the appropriate amount of M13KO7 helper phage (helper phage number: bacterial number ≥ 10:1) and incubate at 37 ℃ for 20 min. Add 30 mL of 2YT medium and 6 μL of Amp (final concentration 20 μg / mL) and incubate at 180 rpm for 1 h. Centrifuge at 5000 rpm for 10 min, resuspend the bacterial cells in 50 mL of 2YT + Amp (final concentration 50 μg / mL) + Kan (final concentration 25 μg / mL), and incubate overnight at 30 ℃ and 220 rpm.

[0124] ⑧ Transfer the culture to a centrifuge tube and centrifuge at 12000 rpm for 10 min at 4 ℃. Transfer the supernatant to another centrifuge tube and centrifuge again under the same conditions. Transfer the supernatant to a new centrifuge tube, add 1 / 4 volume of precipitation buffer, and incubate at 4 ℃ for 4 h. Centrifuge at 12000 rpm for 20 min at 4 ℃ and discard the supernatant. Resuspend the precipitate in 0.5 mL PBS, centrifuge at 12000 rpm for 10 min at 4 ℃, and transfer the supernatant to another new centrifuge tube; this is the amplification product. Measure the titer of the amplification product. This completes the first round of selection.

[0125] ⑨ The second to fourth rounds of selection will follow.

[0126] ⑩ The flowchart for the four-round affinity selection is as follows: Figure 2 As shown.

[0127] 4) Preparation of monoclonal phages ① Add 100 μL of 2YT+A+G medium (final concentration: Amp: 100 μg / mL, Glu: 0.1%) to each well of a 96-well culture plate (plate ①). Pick single clones from the successfully eluted petri dishes, and inoculate a total of 372 single clones into each well. Cover the culture plate ① after inoculation, seal it with medical breathable tape, and incubate overnight at 37 ℃ and 220 rpm.

[0128] ② Prepare a new 96-well culture plate (plate ②), and add 100 μL of 2YT+A medium (final concentration Amp: 100 μg / mL) to each well. Transfer 20 μL of bacteria from culture plate ① to the corresponding well in culture plate ②. Cover culture plate ② and seal it with medical breathable tape. Incubate at 37 ℃ and 220 rpm until OD≈0.5.

[0129] ③ Dilute M13KO7 to the required concentration with 2YT, 20 μL / well, incubate at 37 ℃ for 30 min, then gently shake at 180 rpm for 30 min. Add 2YT+A+K (final concentration Amp: 50 μg / mL, Kan: 25 μg / mL), 100 μL / well. Cover the culture plate ②, seal with medical breathable tape, and incubate overnight at 30 ℃ and 220 rpm.

[0130] ④ Take the plate ② that has been incubated overnight, add 4%M PBS, 80 μL / well, cover it, seal it with medical breathable tape, and centrifuge horizontally at 4000 rpm for 10 min.

[0131] 5) Phage ELISA: Using monoclonal phages as samples and rabbit anti-M13 phage-HRP as the secondary antibody, phage ELISA detection is performed. Tables 2-3 below provide representative readings for a 96-well plate of anti-human CD20 monoclonal phage ELISA. Clones in bold and underlined text are positive clones. Plate ① is coated with the CD20 target protein, while plate ② is uncoated.

[0132] Table 2: Culture Plates ① Among them, F12 was the negative control (coated with the target site, with the helper phage M13KO7 as a control), G12 was the blank control (coated with the target site, with 1% M-PBS added instead of the phage), H12 was the positive control (directly coated with M13KO7), and the secondary antibody was HRP-rabbit anti-M13KO7.

[0133] Table 3: Culture Plate ② Among them, F12 was the negative control (coated with the target site, with the helper phage M13KO7 as a control), G12 was the blank control (coated with the target site, with 1% M-PBS added instead of the phage), H12 was the positive control (directly coated with M13KO7), and the secondary antibody was HRP-rabbit anti-M13KO7.

[0134] 6) Sequencing and Sequence Analysis: Based on the ELISA results, 25 positive clones were selected and sent to a sequencing company for sequencing. Sequence analysis was performed based on the sequencing results. Sequences containing different amino acids were identified as unique sequences, resulting in two unique sequences. HCDR3 was then divided into two groups, as shown in Table 4.

[0135] Table 4. Grouping of Unique Sequences Example 2. Preparation and characterization of CD20-CAR-T We constructed two different anti-CD20 CAR-Ts (2A7 CAR-T and 1A9 CAR-T) using the aforementioned anti-CD20 scFv. The CAR-T20.19 product (Sibiman Bio-derived scFv, disclosed in patent CN108395482A) (SEQ ID NO:15) was used as a control. All tested CARs possessed a 4-1BB co-activation domain.

[0136] The 2A7 CAR-T sequence is as follows: MALPVTALLLPLALLLHAARPDIVLTQTPLSLPVTLGQPASISCTSSQGLVDSDGNTYLSWFHQRPGQSPRRLIYKVSYRDYGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQSTQWPLTF GGGTKVEIKGTGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLTLSCAASGFTFSGSSMHWVRQPSGKGLEWVARVGLKTETYATTYAASVTGRFTISRDDSTNTAFLQMNSLKTEDTAVYYCL AYDYSARRYYWGRGTLVTVSSSEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:10) It contains the following structural fields: CD8 α-chain N-terminal signal peptide: MALPVTALLLPLALLLHAARP (SEQ ID NO:11) Anti-CD20 scFv: DIVLTQTPLSLPVTLGQPASISCTSSQGLVDSDGNTYLSWFHQRPGQSPRRLIYKVSYRDYGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQSTQWPLTFGGGTKVEIKGTGGGGSGGGGS GGGGSQVQLQESGGGLVQPGGSLTLSCAASGFTFSGSSMHWVRQPSGKGLEWVARVGLKTETYATTYAASVTGRFTISRDDSTNTAFLQMNSLKTEDTAVYYCLAYDYSARRYYWGRGTLVTVSS (SEQ ID NO:9) CD8 Hinge & TM (Hinge Zone and Transmembrane Zone): EFTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:12) 4-1BB co-stimulatory domain: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:13) CD3zeta signal transduction domain: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:14) The CAR-T 20.19 sequence is as follows (only the scFv differs from 2A7; the other parts of the sequence are identical): MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGT RLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGN YYYGMDVWGQGTTVTVSSSEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:15) Table 5: Anti-CD20 CAR-T, CAR% and expression level T cells were transduced via mRNA to express different anti-CD20 CARs. Table 5 above shows the CAR-T cells used in the studies disclosed in this application, the percentage of CAR-expressing cells, and their respective expression levels. Figure 3 and Figure 4 The proportion of CAR+ cells and their expression levels are shown ("MFI" represents mean fluorescence intensity). Among the two scFvs generated in this application, the expression level of 2A7 is higher than that of the other scFvs.

[0137] Example 3. Specific killing of CD20+ tumor cells by CD20 CAR-T cells The CAR-T cells were co-cultured with Raji cells (specific target cells) and K562 cells (non-specific control target cells) at an effector-to-target ratio of 1:1. The cytotoxic activity against tumor cells was detected using the lactate dehydrogenase (LDH) release assay. The results showed that CAR-T cells exhibited significant specific killing activity against Raji cells. Figure 5 ), while no significant non-specific killing effect was detected in K562 cells ( Figure 6In the two types of CAR-T cells constructed, the 2A7 CAR-T cell showed a significantly higher specific killing rate against Raji cells at an effector-to-target ratio of 1:1 than other CAR-T cells (including CAR-T20.19 cells). Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the application. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of this application.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CD20, characterized in that, Include: A light chain variable region (VL), the light chain variable region comprising light chain CDR1 (VL CDR1) shown in SEQ ID NO:1, light chain CDR2 (VL CDR2) shown in SEQ ID NO:2, and light chain CDR3 (VL CDR3) shown in SEQ ID NO:3; and The heavy chain variable region (VH) includes heavy chain CDR1 (VH CDR1) shown in SEQ ID NO:4, heavy chain CDR2 (VH CDR2) shown in SEQ ID NO:5, and heavy chain CDR3 (VH CDR3) shown in SEQ ID NO:

6. Among them, any of the above amino acid sequences also includes a derivative sequence that has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining CD20 binding affinity.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:7; the heavy chain variable region has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

8.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The light chain variable region is the amino acid sequence shown in SEQ ID NO:7, and the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:

8.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, IgG, bispecific antibody, multispecific antibody and bispecific T cell conjugate (TCE); preferably, the antibody or its antigen-binding fragment is scFv.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The antibody or its antigen-binding fragment is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment; preferably, the antibody or its antigen-binding fragment is a human antibody or antigen-binding fragment.

6. A polynucleotide, characterized in that, The polynucleotide encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-5.

7. A carrier, characterized in that, The vector comprises the polynucleotide of claim 6.

8. A host cell, characterized in that, The host cell comprises the polynucleotide of claim 6 or the vector of claim 7.

9. A chimeric antigen receptor (CAR) that specifically binds to CD20, characterized in that, The chimeric antigen receptor comprises, from the N-terminus to the C-terminus: CD20 binding domain, wherein the CD20 binding domain comprises the antibody or antigen binding fragment according to any one of claims 1-5; Transmembrane domains; and Cytoplasmic domain.

10. The chimeric antigen receptor according to claim 9, characterized in that, The transmembrane domains are derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, or CD154.

11. The chimeric antigen receptor according to claim 9 or 10, characterized in that, The cytoplasmic domain includes a signal transduction domain derived from CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.

12. The chimeric antigen receptor according to any one of claims 9-11, characterized in that, The cytoplasmic domain further includes a co-stimulatory domain derived from CD28, 4-1BB (CD137), OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, CD43, or any combination thereof.

13. The chimeric antigen receptor according to any one of claims 9-12, characterized in that, The chimeric antigen receptor further includes a CD8 hinge located between the antibody or antigen-binding fragment and the transmembrane domain.

14. The chimeric antigen receptor according to claim 9, characterized in that, The chimeric antigen receptor contains an amino acid sequence as shown in SEQ ID NO:

10.

15. A polynucleotide, characterized in that, The polynucleotide encodes the chimeric antigen receptor according to any one of claims 9-14.

16. A carrier, characterized in that, The vector comprises the polynucleotide of claim 15.

17. A cell characterized in that, The cell comprises the polynucleotide of claim 15 or the vector of claim 16.

18. The cell according to claim 17, characterized in that, The cells in question are immune effector cells.

19. The cell according to claim 17 or 18, characterized in that, The cells are T cells, NK cells, NKT cells, macrophages, or granulocytes.

20. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5 or the cell of any one of claims 17-19 in the preparation of a medicament for treating B-cell malignancies.

21. The use according to claim 20, characterized in that, The antibody or its antigen-binding fragment, or the cell, is used in combination with an adjunct therapy.

22. The use according to claim 20 or 21, characterized in that, The B-cell malignant tumors are selected from multiple myeloma, Waldenström macroglobulinemia, Hodgkin lymphoma, or non-Hodgkin lymphoma.

23. A method for preparing cells capable of expressing CARs that specifically bind to CD20, characterized in that, This includes introducing the polynucleotide of claim 15 into cells.

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