Humanized CD20 antibody, related product thereof and use thereof

Abstract

The present application relates to the field of biomedicine, and in particular to a humanized CD20 antibody, a related product thereof, and a use thereof. Specifically, the present application provides a CD20 antibody that specifically binds to CD20. The antibody comprises three complementarity-determining regions CDR1, CDR2, and CDR3; and the complementarity-determining regions CDR1, CDR2, and CDR3 are sequentially as shown in SEQ ID NOs: 1-3 or 11-13. Furthermore, on the basis of the antibody, a CD19 antibody is tandemly linked to construct CAR-T cells and CAR / TRuC-T cells. Also disclosed is a use of the humanized CD20 antibody in cell therapy.

Description

Humanized CD20 antibodies and related products and applications

[0001] Cross-reference to related applications

[0002] This application claims priority to the following patent applications: Patent Application No. 202410679782.X, filed on May 29, 2024, entitled "Humanized Antibody B09 and its Application in the Preparation of CAR-T Cells"; Patent Application No. 202410679752.9, filed on May 29, 2024, entitled "CD20 Single Domain Antibody and its Humanized Antibody F11"; and Patent Application No. 202510395700.3, filed on March 31, 2025, entitled "A CD19CD20 CAR / TRuC-T Cell and Related Products and Applications", the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This application relates to the biomedical field, specifically to humanized CD20 antibodies and related products and applications. Background Technology

[0004] B cells play a crucial role in the body's humoral immune response and antigen presentation. The consequences of B cell disorders are severe, resulting in two main categories of diseases: malignant B-cell hematologic malignancies and autoimmune diseases caused by abnormal B cells recognizing their own antigens. CD20 plays a vital role in the B-cell immune response, appearing from the late pro-B stage, and its expression level gradually increases with B cell maturation. CD19 is a surface protein expressed on B lymphocytes and follicular dendritic cells, belonging to the immunoglobulin (Ig) superfamily. CD19 plays a crucial role in early B-cell development; all B-cell acute leukemias express CD19.

[0005] In CAR-T (chimeric antigen receptor T-cell) therapy, these two antigens have become key targets for treating certain types of B-cell malignancies, such as non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Tandem CAR-T cell therapy, which targets both CD19 and CD20, means simultaneously targeting two antigens within the same CAR structure. The advantages of this design are: 1) Enhanced targeting: By simultaneously targeting two surface antigens, the ability of CAR-T cells to recognize and kill cancer cells can be increased. This is particularly beneficial for tumor cells that may evade attack by a single-target CAR-T cell due to reduced expression of one target. 2) Reduced escape: Tumor cells may evade the immune system by reducing or altering the expression of a particular antigen. By targeting multiple antigens, tandem CAR-T cell therapy can reduce the risk of tumor escape after cell therapy. 3) Improved efficacy: Dual-target CAR-T therapy may provide higher therapeutic efficacy than single-target therapy, especially in cases with limited response to traditional single-target CAR-T therapy. Summary of the Invention

[0006] In view of the fact that the long-term effects of single-target therapy in the current CAR technology for tumor treatment are not ideal, and that the tumor microenvironment affects the therapeutic effect of CAR technology, this application firstly provides a new single-domain antibody that specifically targets CD20 and optimizes it into a humanized antibody. The CD20 single-domain antibody has high affinity and the whole sequence has been humanized, which can realize the production of low immunogenic antibody and CAR-T cells.

[0007] The novel CD20 single-domain antibody provided in this application is tandemly linked with the CD19 antibody obtained through optimized screening to form a bispecific chimeric antigen receptor (CAR). CAR-T cells prepared from this CAR-T cell can specifically recognize CD19 and / or CD20 proteins, killing cells expressing the corresponding protein targets. Specifically, this application provides the following technical solutions:

[0008] product

[0009] In a first aspect, this application provides a CD20 antibody targeting the CD20 protein or an antigen-binding fragment thereof, wherein the amino acid sequences of CDR1, CDR2, and CDR3 in the CD20 antibody or the antigen-binding fragment thereof are as shown in SEQ ID NO.1-3 or SEQ ID NO.11-13, or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same amino acid sequence as SEQ ID NO.1-3 or SEQ ID NO.11-13.

[0010] Optionally, the antibody is humanized.

[0011] The term "humanized" as used in this application refers to a protein molecule that retains only a portion of the complementarity-determining region (CDR) of the variable region of an immunoglobulin molecule derived from a non-human species, while the remaining major variable regions and all constant regions are derived from human sources and are recombinantly designed. Theoretically, humanized antibodies should have less immunogenicity than chimeric antibodies, making them more suitable for large-scale and repeated use.

[0012] Optionally, the CD20 antibody may be a single-domain antibody, or a monoclonal antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, a single-chain Fv (scFV), a Fab fragment, an F (ab') fragment, a disulfide bond Fv (sdFV), an anti-individual genotype antibody, or an epitope binding fragment of such antibodies.

[0013] Optionally, the CD20 antibody is a single-domain antibody.

[0014] The single-domain antibody described in this application may also be called a "nanobody (Nb)," a "variable domain of heavy chain of heavy-chain antibody (VHH antibody)," or a "single-domain antibody (sdAb)," generally referring to a class of antibodies that lacks the antibody light chain and only has the variable domain of the heavy chain. A single-domain antibody may consist of a heavy chain variable domain (VH). The term "heavy chain variable domain" generally refers to the amino-terminal domain of the heavy chain of the antigen-binding fragment. The heavy chain variable domain can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are scattered within more conserved regions called framework regions (FRs). The heavy chain variable domain contains a binding domain that interacts with the antigen, specifically consisting of three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In the CDR region, minor additions, deletions, insertions, substitutions, or modifications of amino acids are also permissible, as long as they retain the ability of the antibody to bind to a specific antigen.

[0015] Optionally, the CD20 antibody has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence shown in any one of SEQ ID NO. 4-9, 14-19.

[0016] Without substantially affecting antibody activity, those skilled in the art can substitute, add, and / or delete (eliminate) any one or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) in the sequence of this application (e.g., SEQ ID NO. 4-9, 14-19) to obtain variants of the functional fragment. These variants are all considered to be included within the scope of protection of this application.

[0017] Furthermore, the "CD20 antibody" described in this application also includes functionally conserved variants or functional variants of the CD20 antibody. Specifically, the functionally conserved variants of the antibody include amino acid substitutions in terms of polarity, hydrogen bond potential, acidity, basicity, hydrophobicity, aromatic groups, etc. The "functionally conserved variant" refers to a variant in which a given amino acid in the antibody according to this application is substituted without impairing the overall conformation and function of the antibody, including the substitution of an amino acid with another amino acid having similar properties (e.g., polarity, hydrogen bond potential, acidity, basicity, hydrophobicity, presence of aromatic groups, etc.). Amino acids with similar properties are well known to those skilled in the art. Specifically, the functional variants include, but are not limited to, derivatives that are substantially similar in primary structural sequence but not present in the parent antibody of this application, such as chemically and / or biochemically modified derivatives in vitro or in vivo. These modifications include, for example, acetylation, acylation, covalent linkage of nucleotides or nucleotide derivatives, covalent linkage of lipids or lipid derivatives, crosslinking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, polyethylene glycolation, proteolytic treatment, phosphorylation, and such functional variants are also included within the scope of protection of this application.

[0018] Optionally, the CD20 antibody comprises or has the amino acid sequence shown in any one of SEQ ID NO. 4-9, 14-19, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence shown in any one of SEQ ID NO. 4-9, 14-19. The sequences shown in SEQ ID NO. 4 and 14 are the amino acid sequences of the original antibody. SEQ ID NO. 5-9 are the amino acid sequences of five humanized CD20 antibodies B09 HM1-HM5 obtained after humanizing the original antibody B09 shown in SEQ ID NO. 4. SEQ ID NO. 15-19 are the amino acid sequences of five humanized CD20 antibodies F11 HM1-HM5 obtained after humanizing the original antibody F11 shown in SEQ ID NO. 14. Among these, B09-HM5, containing the amino acids shown in SEQ ID NO. 9, is the humanized antibody with the best binding affinity obtained after humanizing B09. F11-HM2, containing the amino acids shown in SEQ ID NO. 16, is the humanized antibody with the best binding affinity obtained after humanizing F11.

[0019] Secondly, this application provides a humanized CD19 antibody, the CD19 antibody comprising a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region being shown in SEQ ID NO.34 and the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO.38; or, the amino acid sequence of the light chain variable region being shown in SEQ ID NO.35 and the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO.39.

[0020] Specifically, the light chain variable region having the amino acid sequence shown in SEQ ID NO. 34 is referred to as VL-HM2 in this application, the light chain variable region having the amino acid sequence shown in SEQ ID NO. 35 is referred to as VL-HM3 in this application, the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 38 is referred to as VH-HM1 in this application, the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 39 is referred to as VH-HM2 in this application, and the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 40 is referred to as VH-HM3 in this application; the antibody formed by linking VL-HM2 and VH-HM1 is referred to as VL2+VH1 or abbreviated as CD19-21, 21 in this application; and the antibody formed by linking VL-HM3 and VH-HM2 is referred to as VL3+VH2 or abbreviated as CD19-32, 32 in this application. The nucleic acids encoding VL2+VH1 or VL3+VH2 are shown in SEQ ID NO.43 / 44.

[0021] Optionally, the light chain variable region and the heavy chain variable region are connected by a linker (also known as a fusion protein adapter, adapter, or linker).

[0022] Optionally, the linker may be composed of G (Gly, glycine) and / or S (Ser, serine); the linker may also include other amino acids.

[0023] Optionally, the linker is (GGGGS)n, preferably n=3.

[0024] Specifically, the structure of the CD19 antibody can be: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0025] Thirdly, this application provides a fusion protein containing the CD20 antibody described in the first aspect of this application and the CD19 antibody described in the second aspect of this application.

[0026] Optionally, the CD20 antibody described in the first aspect of this application and the CD19 antibody described in the second aspect of this application are directly or indirectly linked to form a fusion protein via a linker. That is, in the fusion protein, the CD20 antibody described in the first aspect of this application and the CD19 antibody described in the second aspect of this application are separated by one or more amino acids, and the one or more amino acids separating them can also be referred to as a linker.

[0027] More specifically, the fusion protein consists of, from N-terminus to C-terminus, the aforementioned CD20 antibody, a first linker, the light chain variable region of the aforementioned CD19 antibody, a second linker, and the heavy chain variable region of the aforementioned CD19 antibody.

[0028] The first linker and the second linker can be the same or different.

[0029] The linker (also known as a fusion protein linker or linker) described in this application includes (GGGGS)n, (GGGS)n, (SSSSG)n, (GSGSA)n, and (GGSGG)n, etc., and the linker may also include other amino acids.

[0030] Fourthly, this application provides a chimeric antigen receptor, which includes the CD20 antibody described in the first aspect of this application, the CD19 antibody described in the second aspect of this application, or the fusion protein described in the third aspect of this application.

[0031] Optionally, the chimeric antigen receptor further comprises a hinge region. Optionally, the hinge region comprises the hinge regions of the following molecules: CD8α, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, and IL-11 receptor. Optionally, the hinge region is the CD8α hinge region, with specific amino acids as shown in SEQ ID NO.22, obtained by transcription and translation of the DNA shown in SEQ ID NO.27.

[0032] Optionally, the chimeric antigen receptor further comprises a transmembrane domain. Optionally, the transmembrane domain includes transmembrane domains of the following molecules: CD8α, CD28, 4-1BB, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-7 receptor, and IL-11 receptor. Optionally, the transmembrane domain is the CD8α transmembrane domain, with specific amino acids as shown in SEQ ID NO. 23, obtained by transcription and translation of the DNA shown in SEQ ID NO. 28.

[0033] Optionally, the chimeric antigen receptor further comprises a co-stimulatory signaling domain. Optionally, the co-stimulatory signaling domain comprises the co-stimulatory signaling domains of the following molecules: 4-1BB (CD137), CD19, CD4, CD27, CD28, ICOS (CD278), CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226. Optionally, the co-stimulatory signaling domain is a 41BB co-stimulatory signaling domain, with specific amino acids as shown in SEQ ID NO.24, obtained by transcription and translation of the DNA shown in SEQ ID NO.29.

[0034] Optionally, the chimeric antigen receptor further comprises an intracellular signal transduction domain. Optionally, the intracellular signal transduction domain includes intracellular signal transduction domains of the following molecules: CD3ζ, CD3γ, ZAP70, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, and CD66d. Optionally, the intracellular signal transduction domain is a CD3ζ (CD3z) intracellular signal transduction domain, with specific amino acids as shown in SEQ ID NO. 25, obtained by transcription and translation of the DNA shown in SEQ ID NO. 30.

[0035] Optionally, the chimeric antigen receptor is composed of the CD20 antibody described in the first aspect, the CD19 antibody described in the second aspect of this application, or the fusion protein described in the third aspect of this application, along with a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signal transduction domain.

[0036] Optionally, the chimeric antigen receptor, from N-terminus to C-terminus, comprises: the CD20 antibody of the first aspect / the CD19 antibody of the second aspect of this application / the fusion protein of the third aspect of this application - CD8 hinge region - CD8 transmembrane domain - 41BB co-stimulatory domain - CD3ζ intracellular signal transduction domain.

[0037] When the chimeric antigen receptor of this application contains both CD19 and CD20 antibodies, it can be called a bispecific chimeric antigen receptor.

[0038] Fifthly, this application provides a fusion protein targeting CD19 and CD20, the fusion protein comprising a chimeric antigen receptor targeting CD19 and a TCR fusion construct targeting CD20;

[0039] The chimeric antigen receptor targeting CD19 contains an antibody targeting CD19;

[0040] The CD20-targeting TCR fusion construct contains a single-domain antibody targeting CD20;

[0041] The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody targeting CD19 are shown in SEQ ID NO.45-47, respectively.

[0042] The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody targeting CD19 are shown in SEQ ID NO.49, HTS, and SEQ ID NO.50, respectively.

[0043] The single-domain antibody targeting CD20 is the CD20 antibody or its antigen-binding fragment described in the first aspect of this application;

[0044] Optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the single-domain antibody targeting CD20 are shown in SEQ ID NO.1-3, respectively.

[0045] Furthermore, the chimeric antigen receptor targeting CD19 is placed first, followed by the TCR fusion construct targeting CD20;

[0046] Optionally, the fusion protein is obtained by sequentially tandemly connecting a chimeric antigen receptor targeting CD19, T2A, and a TCR fusion construct targeting CD20; optionally, the chimeric antigen receptor targeting CD19 further comprises a hinge region, a transmembrane region, a co-stimulatory signaling domain, and an intracellular signal transduction domain; optionally, the hinge region is selected from the hinge regions of the following molecules: CD8, CD28, IgG1, IgG4, 41BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, or IL-11 receptor; optionally, the transmembrane region is selected from the transmembrane regions of the following molecules: CD8, CD28, IgG1, IgG4, 41BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, or IL-11 receptor. 1. Receptor; Optionally, the co-stimulatory signaling domain is selected from the co-stimulatory signaling domains of the following molecules: 41BB, CD27, CD19, CD4, CD28, CD278, CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, or B7H3; Optionally, the intracellular signal transduction domain is selected from the intracellular signal transduction domains of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, or CD66d;

[0047] Optionally, the hinge region is a CD8 hinge region; optionally, the transmembrane region is a CD8 transmembrane region; optionally, the co-stimulatory signaling domain is a 41BB co-stimulatory signaling domain; optionally, the intracellular signal transduction domain is a CD3ζ intracellular signal transduction domain; optionally, the amino acid sequence of the heavy chain variable region of the antibody targeting CD19 is shown in SEQ ID NO. 39; optionally, the amino acid sequence of the light chain variable region of the antibody targeting CD19 is shown in SEQ ID NO. 35; optionally, the amino acid sequence of the single-domain antibody targeting CD20 is shown in SEQ ID NO. 9; optionally, the chimeric antigen receptor targeting CD19 is formed by sequentially connecting the antibody targeting CD19, the CD8 hinge region, the CD8 transmembrane region, the 41BB co-stimulatory signaling domain, and the CD3ζ intracellular signal transduction domain; optionally, the amino acid sequences of the CD8 hinge region, the CD8 transmembrane region, the 41BB co-stimulatory signaling domain, and the CD3ζ intracellular signal transduction domain are respectively shown in SEQ ID NO. 39. Shown in NO.53-56;

[0048] Optionally, the CD20-targeting TCR fusion construct further comprises a linker and a TCR complex subunit fusion portion; optionally, the linker is selected from G4S, (G4S)2, (G4S)3, or EAAAK; optionally, the TCR complex subunit fusion portion is selected from CD3ε, CD3γ, or CD3δ; optionally, the linker is G4S; optionally, the TCR complex subunit fusion portion is CD3ε; optionally, the CD20-targeting TCR fusion construct is obtained by sequentially tandemly connecting a CD20-targeting single-domain antibody, G4S, and CD3ε; optionally, the amino acid sequence of CD3ε is as shown in SEQ ID NO. 57; optionally, the fusion protein is obtained by sequentially tandemly connecting an antibody targeting CD19, a CD8 hinge region, a CD8 transmembrane region, a 41BB co-stimulatory signaling domain, a CD3ζ intracellular signal transduction domain, T2A, a CD20-targeting single-domain antibody, G4S, and CD3ε;

[0049] Optionally, the N-terminus of the fusion protein further includes an effervescent signal peptide; optionally, the effervescent signal peptide is selected from effervescent signal peptide CD8a-SP or effervescent signal peptide GMCSF-SP; optionally, the amino acid sequences of the effervescent signal peptide CD8a-SP and the effervescent signal peptide GMCSF-SP are as shown in SEQ ID NO. 60-61, respectively.

[0050] In this application, CD3z is the same as CD3ζ, and CD3e is the same as CD3ε.

[0051] In some implementations, the Linker is a connector commonly used in the art. The Linker is not limited to the G4S specifically used in the embodiments of this application, and can be any one of (GGGGS)n, (GGGS)n, (SSSSG)n, (GSGSA)n, (GGSGG)n or other connectors, where n can be any integer between 1 and 10.

[0052] In the specific implementation of this application, this application has experimentally verified for the first time that the connection order of the chimeric antigen receptor targeting CD19 first and the TCR fusion construct targeting CD20 last in the fusion protein has a significant impact on the effect of the T cells modified by the fusion protein. The T cells (CD19 CAR & CD20 TRuC-T) prepared based on the connection order of the chimeric antigen receptor targeting CD19 first and the TCR fusion construct targeting CD20 last have significantly better therapeutic effects. That is, the fusion protein provided in the fifth aspect of this application has achieved unexpected technical effects.

[0053] On the other hand, this application provides a nucleic acid molecule that encodes one or more of the following: the CD20 antibody of the first aspect, the CD19 antibody of the second aspect, the fusion protein of the third aspect, the chimeric antigen receptor of the fourth aspect, or the fusion protein of the fifth aspect.

[0054] Optionally, the nucleotide sequence of the antibody targeting CD19 in the fusion protein of the fifth aspect is shown in SEQ ID NO. 62; optionally, the nucleotide sequence of the single-domain antibody targeting CD20 in the fusion protein of the fifth aspect is shown in SEQ ID NO. 10; optionally, the nucleotide sequences of the CD8 hinge region, CD8 transmembrane region, 41BB co-stimulatory signaling domain, CD3ζ intracellular signal transduction domain, and CD3ε in the fusion protein of the fifth aspect are shown in SEQ ID NO. 63-67, respectively.

[0055] In some embodiments, the nucleic acid molecule may comprise natural, non-natural, or modified nucleotides; and it may comprise natural, non-natural, or modified internucleotide linkages, such as aminophosphate linkages or thiophosphate linkages, instead of phosphodiester linkages present between unmodified oligonucleotide nucleotides. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, it may be suitable for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions; therefore, nucleic acids formed by using insertions, deletions, inversions, and / or substitutions based on the nucleic acid molecules provided in this application are also included within the scope of protection of this application.

[0056] On the other hand, this application provides a recombinant vector containing the aforementioned nucleic acid molecules.

[0057] Furthermore, the recombinant vector includes a cloning vector and an expression vector.

[0058] Optionally, the recombinant vector includes a DNA vector, an RNA vector, a plasmid, or a virus-derived vector; optionally, the virus-derived vector includes a lentiviral vector, a retroviral vector, adenovirus vector, adeno-associated virus vector, poxvirus vector, or herpesvirus vector.

[0059] In some embodiments, this application does not impose particular limitations on the vector, and its selection depends on the desired function. Non-limiting examples of vectors include plasmid vectors, virus-derived vectors, bacteriophage vectors, and other vectors conventionally used for, for example, genetic engineering. Various plasmids and vectors can be constructed based on methods well known to those skilled in the art.

[0060] On the other hand, this application provides a genetically modified cell, wherein the genetically modified cell contains or expresses one or more of the following: the CD20 antibody described in the first aspect of this application, the CD19 antibody described in the second aspect of this application, the fusion protein described in the third aspect of this application, the chimeric antigen receptor described in the fourth aspect of this application, the fusion protein described in the fifth aspect of this application, the aforementioned nucleic acid molecules, or the aforementioned recombinant vectors;

[0061] Alternatively, the cells may be prepared from the aforementioned recombinant vector. For example, the vector described in this application may be introduced into the cells, and the vector described in this application may be introduced into the cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0062] The "genetically modified cell" described in this application may include progeny cells from a single cell. Due to natural, accidental, or intentional mutations, progeny cells may not be morphologically or genomically identical to the original parent cell, but they may express the CD20 antibody described in the first aspect of this application, the CD19 antibody described in the second aspect of this application, the fusion protein described in the third aspect of this application, the chimeric antigen receptor described in the fourth aspect of this application, the fusion protein described in the fifth aspect of this application, the aforementioned nucleic acid molecules, or the aforementioned recombinant vectors.

[0063] Optionally, the cells are human cells; optionally, the cells are immune cells; optionally, the cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and / or peripheral blood mononuclear cells; optionally, the cells are T cells. Specifically, when the T cells contain or express the chimeric antigen receptor described in the fourth aspect of this application, they can be called CAR-T cells; when the T cells contain or express the fusion protein described in the fifth aspect of this application, they can be called CAR / TRuC-T cells (i.e., T cells modified with fusion protein).

[0064] On the other hand, this application provides an antibody-drug conjugate comprising a cytotoxic agent or tag, and containing the CD20 antibody and / or CD19 antibody described in this application.

[0065] Optionally, the cytotoxic agent may be MMAF or MMAE; alternatively, the tag may be a fluorescent agent.

[0066] On the other hand, this application provides a pharmaceutical composition comprising one or more of the following: the CD20 antibody described in the first aspect of this application, the CD19 antibody described in the second aspect of this application, the fusion protein described in the third aspect of this application, the chimeric antigen receptor described in the fourth aspect of this application, the fusion protein described in the fifth aspect of this application, the aforementioned nucleic acid molecules, the aforementioned recombinant vector, the aforementioned cells, or the aforementioned antibody-drug conjugates.

[0067] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient. The pharmaceutically acceptable carrier and / or excipient are manufactured in Remington's Pharmaceutical Sciences (19...). th As detailed in ed., 1995, these substances are used as needed to aid in the stability of the drug or to help enhance the activity of the active ingredient. The formulation that can be used in such a pharmaceutical composition can be in the form of the original compound itself, or optionally in the form of a pharmaceutically acceptable salt thereof. The pharmaceutical composition thus formulated can be administered in any suitable manner known to those skilled in the art, as needed. The pharmaceutical composition may also comprise one or more of the group consisting of hormones, targeted small molecule formulations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0068] In some embodiments, the pharmaceutically or physiologically acceptable carrier and / or excipients may comprise a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). The dosage of the active ingredient is a therapeutically effective amount. Furthermore, one or more of the aforementioned CD20 antibody, CD19 antibody, fusion protein, chimeric antigen receptor, fusion protein, nucleic acid molecule, recombinant carrier, cell, and antibody-drug conjugate may also be used with other therapeutic agents.

[0069] Optionally, the pharmaceutical composition contains T cells expressing the aforementioned chimeric antigen receptor.

[0070] In some exemplary embodiments, such sterile injectable fluids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0071] The pharmaceutical compositions provided in this application can be formulated into various dosage forms as needed, and can be administered by clinicians based on factors such as the type, age, weight, general disease condition, and route of administration of the tested patients to determine the beneficial dosage for the patient. The route of administration can be, for example, injection or any other suitable route of administration known to those skilled in the art.

[0072] Optionally, the pharmaceutical composition contains immune cells expressing the chimeric antigen receptor described in this application, particularly CAR-T cells, which can be transported, used, stored, and prepared in a suitable solution or cell culture medium.

[0073] On the other hand, this application provides a biological agent comprising the aforementioned pharmaceutical composition;

[0074] Optionally, the dosage form of the biological agent is selected from lyophilized powder for injection, liquid suspension, liposome formulation, microsphere formulation, or gel formulation.

[0075] In some embodiments, suitable pharmaceutical compositions or biological agents are administered in forms suitable for parenteral administration, such as by injection or infusion, for example by rapid injection or continuous infusion, intravenous, inhalable, or subcutaneous forms. When the product is intended for injection or infusion, it may be in the form of a suspension, solution, or emulsion in an oily or aqueous medium and may contain formulation agents such as suspending agents, preservatives, stabilizers, and / or dispersants.

[0076] In some embodiments, the pharmaceutical composition or biological agent may be formulated into various dosage forms as needed, and the dosage that is beneficial to the patient may be determined by a physician based on factors such as patient type, age, weight and general disease condition, and method of administration. Skilled physicians are usually able to easily determine the prescription and the dosage and method of administration that are effective for the desired treatment and / or prevention.

[0077] On the other hand, this application provides a detection kit containing labeled antibodies, which are the aforementioned CD19 antibody and / or CD20 antibody.

[0078] Optionally, the label may include radionuclides, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, and photosensitizing diagnostic agents.

[0079] In some embodiments, labeled antibodies can be assembled into a kit for the detection of CD19 and / or CD20. The kit also includes containers, instructions for use, buffers, etc. In other embodiments, the kit further includes a lysis medium for dissolving the sample to be tested, universal reagents and buffers required for the detection, such as various buffer solutions, detection labels, detection substrates, etc. This detection kit can be an in vitro diagnostic device.

[0080] The term "labeled antibody" in this application refers to an antibody containing one or more labels at its C-terminus, N-terminus, or any position within the molecule. These labels can be any suitable labels known to those skilled in the art, such as radioactive, fluorescent, or chemiluminescent labels.

[0081] application

[0082] On the other hand, this application provides the use of any one or more of the following products in the treatment of CD20-related cancers, and in the preparation of drugs for CD20-related cancers:

[0083] The CD20 antibody described in the first aspect of this application, the fusion protein described in the third aspect of this application, the chimeric antigen receptor described in the fourth aspect of this application, the fusion protein described in the fifth aspect of this application, the aforementioned nucleic acid molecules, the aforementioned recombinant vectors, the aforementioned cells, the aforementioned antibody-drug conjugates, the aforementioned pharmaceutical compositions, and the aforementioned biological agents.

[0084] On the other hand, this application provides the use of any one or more of the following products in the treatment of CD19-related cancers, and in the preparation of drugs for CD19-related cancers:

[0085] The CD19 antibody described in the second aspect of this application, the fusion protein described in the third aspect of this application, the chimeric antigen receptor described in the fourth aspect of this application, the fusion protein described in the fifth aspect of this application, the aforementioned nucleic acid molecules, the aforementioned recombinant vectors, the aforementioned cells, the aforementioned antibody-drug conjugates, the aforementioned pharmaceutical compositions, and the aforementioned biological agents.

[0086] On the other hand, this application provides the use of the CD20 antibody described in the first aspect of this application, the fusion protein described in the third aspect of this application, or the aforementioned kit in the detection of CD20 and in the preparation of CD20-related cancer diagnostic products.

[0087] On the other hand, this application provides the use of the CD19 antibody described in the second aspect of this application, the fusion protein described in the third aspect of this application, or the aforementioned kit in the detection of CD19 and in the preparation of CD19-related cancer diagnostic products.

[0088] method

[0089] On the other hand, this application provides a method for preventing and / or treating CD20-related cancers, comprising administering to a subject in need an effective amount of the CD20 antibody described in the first aspect of this application, the fusion protein described in the third aspect of this application, the chimeric antigen receptor described in the fourth aspect of this application, the fusion protein described in the fifth aspect of this application, the aforementioned nucleic acid molecules, the aforementioned recombinant vector, the aforementioned cells, the aforementioned antibody-drug conjugate, and the aforementioned pharmaceutical composition.

[0090] Optionally, the method involves administering an effective amount of CAR-T cells and / or CAR / TRuC-T cells to the subject, wherein the CAR contains the CD20 antibody or its antigen-binding fragment as described in the first aspect of this application and / or the CD19 antibody as described in the second aspect of this application, and the CAR / TRuC is the fusion protein as described in the fifth aspect of this application.

[0091] On the other hand, this application provides a method for preventing and / or treating CD19-related cancers, comprising administering an effective amount of the aforementioned CD19 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, antibody-drug conjugate, or pharmaceutical composition to a subject in need.

[0092] Optionally, the method involves administering an effective amount of CAR-T cells and / or CAR / TRuC-T cells to the subject, wherein the CAR contains the CD20 antibody or its antigen-binding fragment as described in the first aspect of this application and / or the CD19 antibody as described in the second aspect of this application, and the CAR / TRuC is the fusion protein as described in the fifth aspect of this application.

[0093] In a specific embodiment of this application, the chimeric antigen receptor-modified T cells (CAR-T cells) or fusion protein-modified T cells (CAR / TRuC-T cells) are administered via infusion. It should be noted that the scope of protection of this application is not limited by the specific method of administration or dosage. As long as the antibody, fusion protein, CAR-T cells, CAR / TRuC-T cells, pharmaceutical composition, or biological agent described in this application produces the expected therapeutic and / or preventative effect in a subject, it falls within the scope of protection of this application.

[0094] The term "effective amount" in this application refers to an amount of an active compound or a material, composition or dosage form containing the active compound that, when administered according to the desired treatment regimen, is effective in producing certain desired therapeutic effects commensurate with a reasonable benefit / risk ratio.

[0095] Specifically, the method involves administering an effective amount of CAR-T cells and / or CAR / TRuC-T cells to a subject, wherein the CAR contains the CD20 antibody or its antigen-binding fragment as described in the first aspect of this application and / or the CD19 antibody as described in the second aspect of this application, and the CAR / TRuC is the fusion protein as described in the fifth aspect of this application.

[0096] In this application, the term "application" generally refers to any route known in the art, specifically including intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral, or mucosal administration. Pharmaceutical carriers and formulations or compositions are also well known in the art.

[0097] In this application, the terms "effective amount" or "effective dose" generally refer to an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutic effective amount" or "therapeutic effective dose" of a drug or therapeutic agent is generally any amount of drug that, when used alone or in combination with another therapeutic agent, promotes disease remission (proven by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of damage or disability resulting from the disease).

[0098] On the other hand, this application provides a method for detecting CD20 protein or diagnosing CD20-related cancers, the method comprising contacting a sample with the CD20 antibody described in the first aspect of this application or the fusion protein described in the third aspect of this application and detecting the complex, wherein detecting the complex indicates CD20 protein expression in the sample.

[0099] On the other hand, this application provides a method for detecting CD19 protein or diagnosing CD19-related cancers, the method comprising contacting a sample with the CD19 antibody described in the second aspect of this application and the fusion protein described in the third aspect of this application and detecting the complex, wherein detecting the complex indicates CD19 protein expression in the sample.

[0100] Optionally, the sample may include any type of sample from the subject, such as bodily fluids, tissues, or cells.

[0101] Optionally, the detection is performed on an ex vivo sample in vitro.

[0102] Optionally, the detection is not for diagnostic purposes.

[0103] Optionally, the detection can be qualitative, quantitative, or semi-quantitative.

[0104] General concept

[0105] The term "T cell" or "T lymphocyte" as used in this application can refer to any T cell, such as cultured T cells, primary T cells, or T cells derived from mammals (preferably primates, species including monkeys, dogs, or humans). T cells can be any type of T cell, including NKT cells, and can be at any developmental stage. If derived from mammals, T cells can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids.

[0106] The terms “CD20-related cancer” or “CD20-positive cancer” used in this application include lymphoma, leukemia, or autoimmune diseases, and more specifically include non-Hodgkin's lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, B-cell lymphoma, multiple sclerosis, rheumatoid arthritis, marginal zone lymphoma, small lymphocytic lymphoma, lupus nephritis, mantle cell lymphoma, systemic lupus erythematosus, pemphigus, immune thrombocytopenic purpura, limb nephropathy, B-cell leukemia, acute lymphoblastic leukemia, microscopic polyangiitis, kidney transplant rejection, and nephrotic syndrome.

[0107] The term "CD19-related cancer" used in this application includes lymphoma and leukemia; exemplary, it includes, but is not limited to: non-Hodgkin's lymphoma, follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, marginal zone lymphoma, Burkitt's lymphoma, B-cell acute lymphoblastic leukemia, acute mixed phenotype leukemia, Waldenström macroglobulinemia, hairy cell leukemia, plasmablastic lymphoma, primary central nervous system lymphoma, and mediastinal large B-cell lymphoma. Optionally, the CD19-related cancer is leukemia; optionally, the CD19-related cancer is chronic myeloid leukemia. Specifically, in specific embodiments of this application, chronic myeloid leukemia cell lines are used as target cells for experimental verification.

[0108] The term "chimeric antigen receptor" or "CAR" as used in this application generally comprises three main parts: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain is responsible for recognizing antigens and, in this application, may be a CD20 antibody and / or a CD10 antibody. The extracellular domain may also typically include a hinge region. The main function of the transmembrane domain is to anchor the CAR molecule to the cell membrane, connecting the extracellular and intracellular domains of the CAR molecule. The intracellular domain may include a co-stimulatory domain and / or an intracellular signal transduction domain. The CAR of this application may include linkers added between the various domains for appropriate spacing and conformation of the molecule. CAR is an engineered receptor that can be implanted into immune effector cells with arbitrary specificity; in this application, it specifically refers to T cells, i.e., CAR-T cells.

[0109] The "hinge region" of this application includes a hinge region derived from one or more proteins selected from the group consisting of: CD28, CD8, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8A, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof. In a specific embodiment of this application, the CD8 hinge region is used as an example for construction.

[0110] The "transmembrane domain" described in this application includes transmembrane domains derived from one or more proteins selected from the group consisting of: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM, and variants thereof. In a specific embodiment of this application, the CD8 transmembrane domain is used as an example for construction.

[0111] The "co-stimulatory domain cell" described in this application includes intracellular co-stimulatory signal transduction domains derived from one or more proteins selected from the group consisting of: CD28, 4-1BB (CD137), CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, MyD88, and variants thereof. In a specific embodiment of this application, the 41BB co-stimulatory domain is used as an example for construction.

[0112] The "intracellular signaling domain" described in this application includes intracellular signal transduction domains derived from one or more proteins selected from the group consisting of: CD3ζ, ZAP70, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12, and a domain containing at least one ITAM. In a specific embodiment of this application, the CD3ζ intracellular signal transduction domain is used as an example for construction.

[0113] The "TCR fusion construct (TRuC)" described in this application is a novel antigen receptor design aimed at reprogramming T cells to specifically recognize and kill tumor cells. TRuC consists of a specific ligand antibody fused to a T cell receptor (TCR) subunit. It typically contains a single-domain antibody or other antibody fragment that specifically recognizes tumor surface antigens, fused to the CD3ε subunit of the TCR via a flexible linker peptide.

[0114] The TRuC construct is cloned into vectors such as lentiviruses and transduced into T cells. The expressed fusion protein integrates into the endogenous TCR complex, replacing the natural CD3ε subunit. When TRuC-T cells recognize specific antigens on the surface of tumor cells, they can activate T cells in a human leukocyte antigen (HLA)-independent manner, initiating the T cell's killing function and specifically killing tumor cells. By utilizing the entire TCR's signal transduction capabilities, TRuC-T cells can trigger more effective signal transduction during tumor cell recognition and killing, promoting T cell transport, long-term persistent function, and anti-tumor activity.

[0115] The term "nucleic acid molecule" as used in this application refers to DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.

[0116] The term "expression vector" as used in this application refers to a vector containing recombinant polynucleotides and an expression control sequence that effectively links the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or in an in vitro expression system. Expression vectors include all those known in the art, including clomids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0117] In some embodiments, the expression vector according to this application can direct the replication and expression of the nucleic acid molecule of this application in a host. The expression vector can be, for example, a cloning vector, a binary vector, or an integrative vector. Expression includes the transcription of the nucleic acid molecule, such as transcription into translatable mRNA.

[0118] In some implementations, non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vectors include pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (EdgeBiosystems), pTriEx-Hygro (Novagen), and pCINeo (Promega). Non-restrictive examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all from Invitrogen). Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos, and various mammalian and avian cells is the multipurpose expression vector pCS2+.

[0119] In some embodiments, the vector may contain one or more origins of replication (ori) and genetic systems for cloning or expression, one or more markers for selection in the host (e.g., antibiotic resistance), and one or more expression cassettes. Additionally, established methods can be used to link the coding sequences contained in the vector with transcriptional regulatory elements and / or other amino acid coding sequences. Such regulatory sequences are well known to those skilled in the art and include, but are not limited to, regulatory sequences ensuring transcription initiation, internal ribosome entry sites (IRES), and optional regulatory elements ensuring transcription termination and transcript stability. Non-limiting examples of such regulatory elements ensuring transcription initiation include promoters, translation initiation codons, enhancers, insulators, and / or regulatory elements ensuring transcription termination, which are included downstream of the nucleic acid molecule of this application. Further examples include Kozak sequences and intercalation sequences flanking donor and acceptor sites for RNA splicing, nucleotide sequences encoding secretion signals, or signal sequences depending on the expression system used, capable of guiding the expressed protein into a cell compartment or culture medium. The vector may also contain additional expressible polynucleotides encoding one or more protein chaperones to facilitate proper protein folding.

[0120] The term "host cell" as used in this application refers to cells that can be used to introduce expression vectors, including but not limited to: prokaryotic cells such as *Escherichia coli* or *Bacillus subtilis*, fungal cells such as yeast cells or *Aspergillus*, insect cells such as S2 *Drosophila* cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells. In a specific embodiment of this application, the host cell is preferably an immune cell.

[0121] In some embodiments, the immune cells include, but are not limited to: T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, or any combination thereof, preferably T cells. In addition, the “host cell” mentioned in this application may include a single cell or a population of cells, that is, the “genetically modified host cell” mentioned above in this application includes a single genetically modified host cell and a population of genetically modified host cells.

[0122] The term "treatment" as used in this application refers to the complete or partial improvement or reduction of a disease or condition, symptom, adverse effect or consequence, or associated phenotype. Desired therapeutic effects include, but are not limited to: prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of cancer metastasis, reduction of the rate of disease progression, improvement or mitigation of the disease condition, and relief or improvement of prognosis. This term does not imply a complete cure of the disease or complete elimination of any symptoms, or an effect on all symptoms or outcomes.

[0123] The “prevention” described in this application includes providing preventive effects against the onset or recurrence of the disease in a subject who may be susceptible to the disease but has not yet been diagnosed with it.

[0124] The term "effective amount" as used in this application includes "therapeutic effective amount" and "preventive effective amount." "Therapeutic effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Therapeutic effective amount can vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's immune system, the patient's general characteristics such as age, weight, and sex, the method of drug administration, and other concurrent treatments. "Preventive effective amount" refers to an amount sufficient to prevent, stop, or delay the onset of the disease.

[0125] In some embodiments, the dosage and frequency (single or multiple doses) of the pharmaceutical composition or biologic administered to the subject may vary depending on a variety of factors, such as whether the mammal has another disease and the route of administration; the subject's age, sex, health status, weight, body mass index, and diet; the nature and severity of the symptoms of the disease being treated (e.g., cancer symptoms and the severity of such symptoms), the type of concurrent treatment, complications arising from the treated disease, or other health-related problems. Other treatment regimens or agents may be used in combination with the pharmaceutical composition or biologic and treatment methods described in this application. Adjustments and manipulations of the established dosage (e.g., frequency and duration) are entirely within the capabilities of those skilled in the art.

[0126] In some embodiments, the pharmaceutical composition or biological agent may be in the form of any of the following: tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Furthermore, it can be administered once or multiple times. In this case, the pharmaceutical composition or biological agent is administered in the form of a liquid formulation, powder, aerosol, capsule, vaginal tablet, capsule, or suppository. Routes of administration may include, but are not limited to: intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, local, intranasal, intrapulmonary, rectal, etc. When administered orally, it may be formulated with a coating to protect the active ingredient in the pharmaceutical composition or biological agent from degradation in the stomach. Furthermore, the active ingredient can be administered via any device capable of transfer to target cells. In specific embodiments, the pharmaceutical composition or biological agent provided in this application can be formulated into various dosage forms as needed, and the clinician can determine the beneficial dose for the patient based on factors such as the type, age, weight, general disease condition, and route of administration. The administration method may be injection or any other suitable administration method known to those skilled in the art.

[0127] The term "subject" as used in this application includes both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles. In some embodiments, the "subject" is preferably a human. Attached Figure Description

[0128] Figure 1 shows the results of the binding affinity detection between the original B09 antibody and the humanized antibody HM1-5 and CD20.

[0129] Figure 2 shows the binding affinity test results of CD20 antibodies at different concentrations.

[0130] Figure 3 shows the EC50 detection results of the original B09 antibody and the humanized antibody HM1-5.

[0131] Figure 4 shows the results of the binding affinity detection between the original F11 antibody and the humanized antibody HM1-5 and CD20.

[0132] Figure 5 shows the binding affinity test results of CD20 antibodies at different concentrations.

[0133] Figure 6 shows the EC50 detection results of the original F11 antibody and the humanized antibody HM1-5.

[0134] Figure 7 is a schematic diagram of the CAR structure built based on B09-HM5.

[0135] Figure 8 is a schematic diagram of the CAR structure built based on F11-HM2.

[0136] Figure 9 shows the cytotoxicity of CAR-T cells prepared from CAR cells as shown in Figures 7-8 against K562-CD20 cells.

[0137] Figure 10 is a schematic diagram of the structure of Fmc63-Leu16 CAR.

[0138] Figure 11 shows the results of the comparative detection of the killing power of CAR-T cells prepared from CAR and Fmc63-Leu16 CAR as shown in Figure 7-8 against K562-CD20 cells.

[0139] Figure 12 shows the results of CD19 humanized antibody detection of loss of negative control CHO-K1 cells.

[0140] Figure 13 shows the results of detecting the loss of CHO-K1-CD19 cells by CD19 humanized antibody.

[0141] Figure 14 shows the binding affinity of CD19 humanized antibodies at different concentrations.

[0142] Figure 15 shows the EC50 detection results of CD19 humanized antibody.

[0143] Figure 16 shows the results of affinity testing for VL2+VH1 and VL3+VH2.

[0144] Figure 17 is a schematic diagram of the structure of CAR constructed based on CD19 humanized antibody.

[0145] Figure 18 shows the results of detecting the killing power of CAR-T cells constructed and prepared using VL2+VH1 and VL3+VH2 antibodies against K562-CD19 cells.

[0146] Figure 19 is a schematic diagram of the structure of CAR constructed based on bispecific chimeric antigen receptor.

[0147] Figure 20 shows the cytotoxicity of CAR-T cells prepared as shown in Figure 19 against K562-CD19 and K562-CD20 cells.

[0148] Figure 21 shows the statistical results of the fold expansion of cultured cells.

[0149] Figures 22-25 show the results of CAR-T cell apoptosis detection on days 5, 8, 10, and 12.

[0150] Figure 26 shows the results of CAR-T cell secretion of IL2 and IFN-γ on days 5, 7, and 12 of culture.

[0151] Figure 27 is a schematic diagram of the structural design corresponding to CD19 CAR / CD20 TRuC-T provided in this application.

[0152] Figure 28 shows the structural diagrams of CD19 CAR-T, CD20 CAR-T, CD19 CAR & CD20 CAR-T, CD19-CD20 CAR-T, CD19 CAR & CD20 TRuC-T, and CD20 CAR & CD19 TRuC-T.

[0153] Figure 29 compares the killing effects of CD19 CAR-T, CD20 CAR-T, CD19 CAR & CD20 CAR-T, CD19-CD20 CAR-T, CD19 CAR & CD20 TRuC-T, CD20 CAR & CD19 TRuC-T, and T cells on the cell lines Raji and Daudi, which express both CD19 and CD20.

[0154] Figure 30 compares the killing effects of CD19 CAR-T, CD20 CAR-T, CD19 CAR & CD20 CAR-T, CD19-CD20 CAR-T, CD19 CAR & CD20 TRuC-T, CD20 CAR & CD19 TRuC-T, and T cells on K562 cell lines that overexpress only CD19 or CD20, and Raji cell lines that have CD19 or CD20 knocked out.

[0155] Figure 31 compares the response rates of CD20 or CD19 in each group of CD19 CAR-T, CD20 CAR-T, CD19 CAR & CD20 CAR-T, CD19-CD20 CAR-T, CD19 CAR & CD20 TRuC-T, CD20 CAR & CD19 TRuC-T, and T cell activation assays.

[0156] Figure 32 shows the results of evaluating the inhibitory effects of CAR-T cells with CD19 CAR-T (CAR19), CD19 CAR&CD20 CAR-T (DUAL), CD19-CD20 CAR-T (Tandem CAR 20&19), and CD19 CAR&CD20 TRuC-T (CAR19&TRuC20) structures on mouse lymphoma cells. Detailed Implementation

[0157] The present application will be further described below with reference to specific embodiments. However, the scope of protection of the present application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present application, based on the technical solution and inventive concept of the present application, should be covered within the scope of protection of the present application. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can be obtained commercially.

[0158] Example 1: Recognition and binding of B09 antibody and 5 humanized antibodies to the CD20 target.

[0159] Step 1: Screening antibodies and their humanization

[0160] An antibody B09 (a single-domain antibody with the CDRs shown in SEQ ID NO. 1-3 and the full-length amino acid sequence shown in SEQ ID NO. 4) specifically targeting CD20 was obtained through screening. The humanization sequence of the B09 antibody was further optimized. The humanization degree scoring system was T20score: http: / / abAnalyzer.lakepharma.com. Five antibodies HM1-HM5 with scores higher than 85 were selected.

[0161] The amino acid sequences of HM1-HM5 are shown in SEQ ID NO.5-9, where the CDR (Complementarity-determining region) sequence is identical to that of the original antibody B09, as shown in SEQ ID NO.1-3, but the framework region (FR) is different from that of the original antibody B09. The T20 scores are shown in the table below.

[0162] The designed humanized antibodies were synthesized and subcloned into the pcDNA3.4-IgG1Fc expression vector. After the vector was verified by sequencing, endotoxin-free plasmids were prepared and the antibodies were expressed.

[0163] The steps of vector expression are as follows:

[0164] 1) Remove the LVTransm transfection reagent and antibody expression vector from the freezer, thaw at room temperature, and mix thoroughly by pipetting. Remove the PBS and warm it to room temperature. Transfer 4 mL of PBS to one well of a 6-well plate, add 20 μg of pcDNA3.4-IgG1Fc, mix thoroughly by pipetting, then add 60 μL of LVTransm, immediately mix by pipetting, and let stand at room temperature for 10 minutes.

[0165] 2) Add the above DNA / LVTransm complex to 20 mL of 293F cells and gently shake to mix thoroughly. Incubate the cells at 37°C in a 5% CO2 incubator.

[0166] 3) After continuous culture for 5 days, the supernatant of the culture medium was collected by centrifugation, filtered through a 0.45 μm filter membrane, and the supernatant was collected to purify the antibody using a Protein A affinity column. The concentration of the purified antibody was determined and SDS-PAGE denaturing electrophoresis was performed simultaneously.

[0167] Step 2, Bonding Force Test

[0168] Flow cytometry detection method:

[0169] 1) Resuscitate CHO and CHO-CD20 cells in liquid nitrogen and adjust the cell state to the logarithmic growth phase;

[0170] 2) Divide the cells into several portions, with each portion containing 3 × 10 cells. 5 Add 100 μL of antibody expression supernatant or humanized antibody (30 μg / ml, 3-fold dilution for 10 spots) to each cell, mix thoroughly, and incubate at room temperature for 1 hour;

[0171] 3) Centrifuge at 800xg at room temperature for 5 minutes, remove the supernatant containing antibodies, and wash the cells 3 times with PBS;

[0172] 4) Add 100 μL of PE-Anti-human IgG (1:500 dilution), mix thoroughly, and incubate at room temperature in the dark for 45 min;

[0173] 5) Centrifuge at 800xg at room temperature for 5 minutes, remove the supernatant containing the secondary antibody, and wash the cells 3 times with PBS;

[0174] 6) Resuspend cells in 200 μL PBS for flow cytometry analysis. Prepare CHO and CHO-CD20 cells, incubate each with 100 μL of antibody expression supernatant, then incubate with PE anti-human IgG fluorescent secondary antibody for flow cytometry detection.

[0175] Further flow cytometry analysis using the above methods showed that both B09 and its humanized antibodies HM1-HM5 specifically bind to CD20, and the binding affinity of humanized antibodies HM1-HM5 to CD20 was superior to that of the original antibody B09 (Figure 1).

[0176] Based on the binding strength and degree of humanization, humanized antibodies HM3 and HM5 were selected, purified, and then reproduced according to the above protocol at different concentrations (Figure 2). FACS EC50 was detected (Figure 3). The results showed that the binding activity of the 8-B09 humanized antibody was consistent with that of the original antibody, while HM5 had stronger flow cytometry binding.

[0177] Example 2: Recognition and binding of F11 antibody and 5 humanized antibodies to the CD20 target.

[0178] Step 1: Screening CD20 antibodies and their humanization

[0179] An antibody specifically targeting CD20, F11 (a single-domain antibody with the CDRs shown in SEQ ID NO.11-13 and the full-length amino acid sequence shown in SEQ ID NO.14), was obtained through screening. The humanization sequence of the F11 antibody was further optimized. The humanization degree scoring system used the T20 score (http: / / abAnalyzer.lakepharma.com) to score the antibody. Five antibodies with scores higher than 85 were selected. The T20 scores of F11-HM1-HM5 are shown in Table 1.

[0180] Table 1 T20 Scores

[0181] The amino acid sequences of F11-HM1-HM5 are shown in SEQ ID NO.15-19, wherein the CDR (Complementarity-determining region) sequence is identical to that of the original antibody F11, as shown in SEQ ID NO.11-13, but the framework region (FR) is different from that of the original antibody F11.

[0182] The obtained antibody F11 and its humanized antibody were synthesized and subcloned into the pcDNA3.4-IgG1Fc expression vector. After the vector was verified by sequencing, an endotoxin-free plasmid was prepared and the antibody was expressed. The vector expression steps were the same as in Example 1.

[0183] Further flow cytometry analysis using the method described in Example 1 showed that both F11 and its humanized antibodies HM1-HM5 specifically bound to CD20, and the binding affinity of humanized antibodies HM1-HM5 to CD20 was superior to that of the original antibody F11 (Figure 4).

[0184] Based on the binding strength and degree of humanization, humanized antibodies HM1, 2, and 5 were selected, purified, and then reproduced according to the above protocol at different concentrations (Figure 5). FACS EC50 was then detected (Figure 6). The results showed that the binding activity of humanized antibody F11 was consistent with that of the original antibody, while HM2 had stronger flow cytometry binding. F11-HM2 was selected for further application and verification.

[0185] Example 3: CAR vectors were constructed using B09-HM5 and F11-HM2 to verify the targeted killing effect of CD20.

[0186] Step 1: Constructing the carrier

[0187] The pCDH-EF1a lentiviral expression plasmid containing CAR was constructed using B09-HM5 (nucleic acid sequence as shown in SEQ ID NO.10, amino acid sequence as shown in SEQ ID NO.9) and F11-HM2 (nucleic acid sequence as shown in SEQ ID NO.20, amino acid sequence as shown in SEQ ID NO.16). The structure of the CAR is shown in Figures 7-8. Specifically, the CD8 signal peptide-HM5 antibody-CD8 hinge region-CD8 transmembrane domain (TM)-41BB co-stimulatory domain-CD3ζ intracellular signal transduction domain (marked as CD3z in the figure) are linked sequentially. That is, the amino acid sequence of the CAR expressed on the vector is SEQ ID NO.21, 9 / 16, 22-25 linked sequentially, and its encoding nucleic acid sequence is SEQ ID NO.26, 10 / 20, 27-30 linked sequentially. EGFRt is also linked to the CAR structure via T2A for subsequent fluorescence assay to determine the positivity rate.

[0188] Step 2: Preparation of lentivirus

[0189] The lentiviral system plasmids (the above pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) were transfected into adherent 293T cells in logarithmic growth phase. The cell culture supernatant was harvested 48-72 hours after transfection, concentrated and filtered to obtain CAR lentivirus, which was stored at -80℃ for later use.

[0190] Step 3: Isolate peripheral blood mononuclear cells

[0191] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood. T cells were isolated using human CD3 / 28 magnetic beads and transduced with the virus within 72 hours of activation. After 24 hours of transduction, the medium was changed and the cells were cultured until day 8. Cells were collected by centrifugation and resuspended in physiological saline. Flow cytometry was used to identify EGFRt molecules on the surface of CAR-T cells, ensuring a positive rate of greater than 30%. If the positive rate was lower, EGFRt-PE magnetic beads were used for enrichment and sorting.

[0192] Step 4, Lethality Detection

[0193] The killing effect was performed using a 5:1 effector-to-target ratio. K562 cells and K562 cells overexpressing CD20 (K562-CD20) were used as target cells. CAR-T killing was performed for 16 hours and LDH was detected.

[0194] The test results are shown in Figure 9. The results demonstrate that CAR-T cells constructed based on the B09-HM5 and F11-HM2 sequences have the ability to specifically kill K562 cells overexpressing CD20. In the figure, NCT cells are negative control T cells.

[0195] Example 4: The killing effect of B09-HM5 / F11-HM2 CAR-T is higher than that of Fmc63-Leu16 CAR-T.

[0196] Step 1: Preparation of CD19-CD20 tandem CAR-T cells (Fmc63-Leu16 CAR-T)

[0197] The connection method of the Bispecific anti-CD20, anti-CD19 CAR T cells disclosed in the article "Bispecific anti-CD20, anti-CD19 CAR T cells for relapsed B cell malignancies: a phase 1dose escalation and expansion trial" is shown in Figure 10.

[0198] Step 2: Comparison of lethal effects

[0199] Compare the CAR-T cells prepared based on B09-HM5 / F11-HM2 in Example 2 with the Fmc63-Leu16 CAR-T cells prepared above.

[0200] The killing assay used a 1:1 effector-target ratio. K562 cells overexpressing CD20 (K562-CD20) were used as target cells. After 24 hours of CAR-T killing, the results showed that the CAR-T cells constructed with the B09-HM5 / F11-HM2 sequence had the ability to specifically kill CD20-overexpressing K562 cells, and the ability was higher than that of Fmc63-Leu16CAR-T (Figure 11).

[0201] Example 5: CD19 antibody fmc63 and its humanized antibody

[0202] Step 1: Humanization of the variable regions of heavy and light chains

[0203] The CD19 antibody fmc63 (the amino acid sequence of the light chain variable region is shown in SEQ ID NO.31, the heavy chain variable region is shown in SEQ ID NO.32, where the CDR sequence is marked in Table 2, its all-antibody form is marked as Chimeric in the subsequent results figure, and its single-chain antibody form is marked as Fmc63scfv in the subsequent results figure) was humanized. The frame sequences with a score higher than 80 were screened according to the T20 scoring system described in Example 1. The T20 scores are shown in Table 3.

[0204] Table 2. Amino acid sequence and CDR marker of fmc63

[0205] Table 3 T20 Scores

[0206] The heavy and light chains of the designed humanized antibody were codon-optimized and then synthesized separately. The heavy chain was subcloned into the pcDNA3.4-IgG1 expression vector, and the light chain was subcloned into the pcDNA3.4-IgKc expression vector. After the vectors were verified by sequencing, endotoxin-free plasmids were prepared.

[0207] Step 2, Antibody Screening

[0208] The humanized VH and VL antibody expression vectors were combined in pairs to obtain a total of 15 antibodies, which were named VL1+VH1, VL1+VH2, VL1+VH3, VL1+VH4, VL1+VH5, VL2+VH1, VL2+VH2, VL2+VH3, VL2+VH4, VL2+VH5, VL3+VH1, VL3+VH2, VL3+VH3, VL3+VH4, VL3+VH5, VL4+VH1, VL4+VH2, VL4+VH3, VL4+VH4, VL4+VH5, VL5+VH1, VL5+VH2, VL5+VH3, VL5+VH4, and VL5+VH5. For example, VL1+VH1 represents a complete antibody consisting of VL-HM1 as the light chain variable region shown in SEQ ID NO.33 and VH-HM1 as the heavy chain variable region shown in SEQ ID NO.38. The same applies to other names.

[0209] The above 15 antibodies, along with Chimeric and FMC63scfV, were transiently transfected into 293F cells for expression. Flow cytometry fluorescence sorting (FACS) was then used to detect the binding of the prepared humanized antibodies to the target proteins. The cells used for detection were CHO-K1 (Figure 12) and CHO-K1-CD19 (Figure 13). In the figures, the primary antibody consisted of 100 μL of supernatant and one positive antibody (10 μg / ml FMC63 scfv), and the secondary antibody was PE anti-human IgG.

[0210] Based on the results in Figures 12-13, VL2+VH1, VL2+VH2, VL2+VH3, VL3+VH1, VL3+VH2, and VL3+VH3 were selected for further FACS EC50 detection (Figure 14). According to the FACS EC50 detection, the binding activity of the candidate antibodies was consistent with that of the chimeric antibodies. The average MFI value was calculated based on the results of flow cytometry. The higher the MFI value, the higher the affinity of the corresponding humanized antibody for the CD19 antigen (Figure 15).

[0211] Step 3: Further Screening

[0212] Further affinity assays were performed on VL2+VH1 and VL3+VH2, and compared with the original antibody fmc63. The specific steps were as follows: the HIS1K sensor was immobilized with CD19-His (Kaika Biotechnology, CD1-HM119) at a concentration of 5 μg / ml for 350 s. The buffer was PBST (PBS + 0.02% Tween 20), and Chmeric / VL2+VH1 / VL3+VH2 were diluted to 12.5, 6.25, 3.13, 1.56, 0.7813, and 0 nM. Affinity assay: equilibration for 60 s, binding for 180 s, dissociation for 180 s, and detection temperature at 25℃. The results are shown in Figure 16.

[0213] Example 6: Constructing a CAR based on VL2+VH1 and VL3+VH2 to verify targeted killing.

[0214] Step 1: Constructing the carrier

[0215] The pCDH-EF1a lentiviral expression plasmids for expressing CARs from VL2+VH1 and VL3+VH2 obtained in Example 4 were constructed according to the structure shown in Figure 17. Specifically, the CAR structure was constructed according to step 1 of Example 2, replacing the antibody in step 1 of Example 2 with the VL2+VH1 (encoding nucleic acid sequence as shown in SEQ ID NO. 43, where positions 322-366 encode GGGGSGGGGSGGGGS, and GGGGSx3 acts as a linker connecting the heavy chain variable region and the light chain variable region) and VL3+VH2 (encoding nucleic acid sequence as shown in SEQ ID NO. 44, where positions 322-366 encode GGGGSGGGGSGGGS, and GGGGSx3 acts as a linker connecting the heavy chain variable region and the light chain variable region) and VL3+VH2 (encoding nucleic acid sequence as shown in SEQ ID NO. 44, where positions 322-366 encode GGGGSGGGGSGGGS, and GGGGSx3 acts as a linker connecting the heavy chain variable region and the light chain variable region) obtained in Example 4.

[0216] Step 2: Preparation of lentivirus

[0217] The lentiviral system plasmid (a mixture of pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G plasmids at a mass ratio of 3:2:1) was transfected into adherent 293T cells in logarithmic growth phase. The cell culture supernatant was harvested 48-72 hours after transfection, concentrated, filtered, and stored at -80°C for later use. It was then transfected into peripheral blood mononuclear cells according to the method described in Example 2.

[0218] Step 3, Lethality Detection

[0219] The killing assay used a 5:1 effector-to-target ratio, with K562 cells and K562 cells overexpressing CD19 (K562-CD19) as target cells. CAR-T cell killing was performed for 16 hours, and LDH assays were performed. The results, shown in Figure 18, demonstrate that CAR-T cells constructed using the VL2+VH1 and VL3+VH2 sequences have the ability to specifically kill CD19-overexpressing K562 cells. In the figure, NCT represents negative control T cells, and FMC63-LEU16 represents CD19-CD20 tandem CAR-T cells (i.e., the CD19-CD20 tandem CAR-T cells mentioned in Example 3).

[0220] Example 7: Fmc63-VL3VH2 tandem with B09-HM5 / F11-HM2 CAR-T targeted killing of K562-CD19, K562-CD20, and K562-CD19 / CD20 cells:

[0221] Step 1: Construct vectors based on Fmc63-VL3+VH2 and B09-HM5 / F11-HM2 and prepare CAR-T cells.

[0222] The B09-HM5 / F11-HM2 antibody (amino acid and nucleic acid sequences as shown in SEQ ID NO. 9-10 and 16-20, respectively) screened in Examples 1-3 was tandemly linked with the VL3+VH2 antibody (amino acid sequences shown in SEQ ID NO. 35 and 39 were used as the light chain and heavy chain variable regions, respectively, and the nucleic acid sequence is shown in SEQ ID NO. 44) screened in Examples 4-5. The specific linking order of the components in the tandem structure is B09HM5-linker-VL3-GGGGSx3-VH2. This tandem structure was used to replace the antibody in step 1 of Example 2 to construct a CAR. The constructed CAR structure is shown in Figure 19 (i.e., the bispecific chimeric antigen receptor described in this application).

[0223] Step 2, Lethality Detection

[0224] The plasmid containing the CAR gene Fmc63-VL3VH2 and B09-HM5 / F11-HM2 tandemly constructed in the above steps was expressed, lentivirus was prepared, and CAR-T cells were obtained after cell infection. The killing efficacy against K562 cells (24h) overexpressing CD19, CD20, or CD19+CD20 was tested using a 5:1 effector-to-target ratio.

[0225] Figure 20 shows that the CAR-T cells constructed using the Fmc63-VL3VH2 tandem B09-HM5 / F11-HM2 sequence have the ability to specifically kill K562 cells expressing CD19 or CD20, as well as those co-expressing CD19 and CD20. In the legend, NCT represents the control T cells, and 32-B09HM5 is the CAR-T cell constructed using the Fmc63-VL3VH2 tandem B09-HM5 / F11-HM2 sequence.

[0226] Example 8: Tonic signaling detection

[0227] Two humanized CD20 single-domain antibodies and VL3VH2 humanized antibodies were selected and used to construct second-generation bispecific antibody CAR lentiviral expression vectors. The second-generation CAR is the CAR referred to in Example 2 above, which is CD8 signal peptide-antibody-CD8 hinge region-CD8 transmembrane domain-41BB co-stimulatory domain-CD3ζ intracellular signal transduction domain. The antibody and its figure in the results are shown in Table 4.

[0228] Table 4. Antibody names, related SEQ ID NOs, and their markings in the results figures for CAR.

[0229] After constructing CAR expression plasmids from the above antibodies, expressing them, preparing lentiviruses, infecting cells to obtain CAR-T cells, performing the following tests, and marking the results according to the legend in Table 4.

[0230] 1. Cell expansion fold detection

[0231] The CAR-T cells prepared above were cultured and further tested. The specific steps of cell culture were as follows: an appropriate amount of X-Vivo 15 medium (containing 200 IU / mL IL2, 10 ng / mL IL7, and 5 ng / mL IL15) was added, the cells were resuspended with a pipette, and the cell density was adjusted to 0.5-0.7 × 10^6 cells / mL.

[0232] From day 5 to day 13, cell counts were performed daily to determine the cell number. The initial cell count on day D0 was used as the baseline to calculate the CAR cell expansion fold. The results are shown in Figure 21.

[0233] 2. Apoptosis detection

[0234] A subset of CAR-T cells cultured on days 5, 8, 10, and 12 were collected, centrifuged, washed with pre-cooled PBS, resuspended in 300 μL of 1×Binding Buffer, and then stained with 5 μL of Annexin V-PE and 5 μL of 7AAD. After mixing, the cells were incubated in the dark for 15 minutes. Blank T cells were used as the blank group without any dye. 400 μL of 1×Binding Buffer was added, and the cells were analyzed by flow cytometry. The results on days 5, 8, 10, and 12 are shown in Figures 22-25.

[0235] 3. Cytokine secretion level detection

[0236] On days 5, 7, and 12, 5 × 10⁵ cells were harvested, washed twice with PBS, resuspended in 1 ml of blank X-Vivo15 medium, and incubated at 37°C in a 5% CO₂ incubator for 24 h. The supernatant was then collected by centrifugation and stored at -80°C. After all supernatant was collected, the secretion of IL-2 and IFN-γ was detected according to the instructions of the IL-2 and IFN-γ ELISA kit.

[0237] Results: Cells were cultured in blank X-vivo15 for 24 h at D5, D7 and D12, and the secretion of IL2 and IFN-γ was detected by taking the supernatant. According to the detection results, CAR-T cells secreted almost no IL-2 but secreted IFN-γ, and the secretion increased with the increase of culture time (Figure 26).

[0238] The results in the above three aspects show that the CAR-T cells prepared by tandemly combining the CD20 antibody and CD19 antibody screened in this application have weak tonic signaling, which can ensure normal expansion of CAR-T cells and a normal apoptosis ratio, while not releasing IL-2 in the absence of activation, and the IFN-γ release level is controllable.

[0239] Example 9: Construction of a novel type of CD19CD20 CAR / TRuC-T cells and verification of their killing effect

[0240] 1. Experimental Methods

[0241] According to the structural design diagram in Figure 27, different types of CAR-T structures were designed and synthesized into pCDH-EF1a lentiviral expression plasmids (see the plasmid structure diagram in Figure 28). The plasmid designs are as follows: (1) CD19 CAR-T; (2) CD20 CAR-T; (3) CD19 CAR & CD20 CAR-T; (4) CD19-CD20 CAR-T; (5) CD19 CAR & CD20 TRuC-T; (6) CD20 CAR & CD19 TRuC-T.

[0242] Among them, (1) the CD19 CAR-T is obtained by connecting CD19 scfv, CD8 hinge, CD8 Tm, 41BB, and CD3z in series; (2) the CD20 CAR-T is obtained by connecting CD20 vhh, CD8 hinge, CD8 Tm, 41BB, and CD3z in series; (3) the CD19 CAR & CD20 CAR-T is obtained by connecting CD19 scfv, CD8 hinge, CD8 Tm, 41BB, CD3z, T2A, CD20 vhh, CD8 hinge, CD8 Tm, 41BB, and CD3z in series; (4) the CD19-CD20 CAR-T is obtained by connecting CD20 vhh, G4S, CD19 scfv, CD8 hinge, CD8 Tm, 41BB, and CD3z in series; (5) the CD19 CAR & CD20 TRuC-T is obtained by connecting CD19 scfv, CD8 hinge, CD8 Tm, 41BB, and CD3z in series; Tm, 41BB, CD3z, T2A, CD20 vhh, G4S, CD3e are connected in series; (6) The CD20 CAR & CD19 TRuC-T are connected in series by CD20 vhh, CD8 hinge, CD8 Tm, 41BB, CD3z, T2A, CD19 scfv, G4S, CD3e.

[0243] The exosome signal peptide that can be selected for the protein is either the exosome signal peptide CD8a-SP or the exosome signal peptide GMCSF-SP. The amino acid sequence information of CD19 scfv and CD20 vhh is shown in Table 5 below. The nucleotide sequences of CD19 scfv and CD20 vhh are shown in SEQ ID NO. 62 and 10, respectively. The amino acid sequences of CD8 Hinge, CD8 Tm, 41BB, CD3z, CD3e, G4S linker, T2A, exosome signal peptide CD8a-SP, and exosome signal peptide GMCSF-SP are shown in SEQ ID NO. 53-61, respectively. The nucleotide sequences of CD8 Hinge, CD8 Tm, 41BB, CD3z, CD3e, G4S linker, T2A, exosome signal peptide CD8a-SP, and exosome signal peptide GMCSF-SP are shown in SEQ ID NO. 63-71, respectively.

[0244] Table 5. Amino acid sequences of CD19 scfv and CD20 vhh

[0245] The lentiviral system plasmid (pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) was transfected into adherent 293T cells in logarithmic growth phase. The cell culture supernatant was harvested 48-72 hours after transfection, concentrated and filtered to obtain CAR lentivirus, which was stored at -80℃ for later use.

[0246] Peripheral blood was collected from patients or healthy volunteers via leukoablation, followed by separation of PBMCs using Ficoll density gradient centrifugation. EasySep was then used to analyze the results. TM Human T Cell Isolation Kit (STEMCELL, #17951) for T cell isolation. It is formulated with 10 ng / mL IL-7 (nearshore protein, GMP-C086), 5 ng / mL IL-15 (nearshore protein, GMP-C016), and ImmunoCult. TM After normal activation of T cells using X-vivo (Lonza) medium containing the Human CD3 / CD28 / CD2 T Cell Activator (STEMCELL, #10970) antibody for 2-3 days, lentivirus transduction was performed. The medium was then replaced with one lacking the activating antibody, and amplification was continued for 9-11 days. CAR-T cell positivity was assessed, and the positivity rate of each group was adjusted to be consistent by incorporating T cells from the same batch.

[0247] After co-incubating the above CAR-T cells with Raji and Daudi cells expressing GFP at a 1:1 effector-target ratio for 24 hours, cell viability was detected, with the cells in the control group (T cells) used as a reference.

[0248] 2. Experimental Results

[0249] The results are shown in Figure 29. The results show that the CD19 CAR & CD20 TRuC-T structure 5 significantly killed the cell lines Raji and Daudi, which express both CD19 and CD20, and significantly reduced cell viability. This effect was significantly better than that of other cell groups.

[0250] Validation of the sensitivity of a class of CD19CD20 CAR / TRuC-T cells constructed in Examples 10 and 9 to CD19 or CD20.

[0251] 1. Experimental Methods

[0252] Plasmids overexpressing human CD19 and CD20 were constructed using pLV3-CMV-target-3×FLAG-CopGFP-Puro. Lentiviral cells were then packaged and infected with K562 cells. Puromycin sorting was used to construct K562 cell lines that individually overexpressed CD19 or CD20. Using the CRISPR-Cas9 system, corresponding sgRNAs for CD19 / CD20 were selected from the database to construct CD19 or CD20 knockout cell lines in Raji. The above four cell lines were incubated overnight with CAR-T cells of different structures constructed in Example 9 at a 1:1 effector-target ratio to verify the cell killing effect.

[0253] 2. Experimental Results

[0254] The results are shown in Figure 30. The results show that the CD19 CAR & CD20 TRuC-T structure is most sensitive to CD20 and can effectively recognize cells expressing either CD19 or CD20 alone.

[0255] Validation of the response rate of a class of CD19CD20 CAR / TRuC-T cells constructed in Examples 11 and 9 to CD19 or CD20

[0256] 1. Experimental Methods

[0257] Using Biotinylated Human CD19(20-291)Protein,Fc,Avitag TM(Arco,CD9-H82F6),Biotinylated Human CD20 / MS4A1 Full Length Protein,His,Avitag TM (Acro,CD0-H82E5), the activating medium was activated by adjusting the antigen concentration with PBS, and the activation of several groups of CAR-T cells constructed in Example 9 above was detected to detect the T cell activation marker CD69.

[0258] 2. Experimental Results

[0259] The results are shown in Figure 31. The results show that the CD19 CAR & CD20 TRuC-T structure 5 has the highest response rate to CD20, while the response rate to CD19 is similar to that of the conventional CD19 CAR.

[0260] Verification of the tumor-killing effect of a class of CD19CD20 CAR / TRuC-T cells constructed in Examples 12 and 9 on tumors in vivo.

[0261] 1. Experimental Methods

[0262] The inhibitory effects of CAR-T cells with the structures CD19 CAR-T (CAR19), CD19 CAR&CD20 CAR-T (DUAL), CD19-CD20 CAR-T (Tandem CAR 20&19), and CD19 CAR&CD20 TRuC-T (CAR19&TRuC20) constructed in Example 1 on mouse lymphoma cells were evaluated. Female NCG mice, 6-8 weeks old, were intravenously infused with 5E5 luciferase-Raji per mouse. Five days later, the corresponding CAR-T cells (1E6 per mouse) were infused. In vivo imaging was performed every seven days to observe tumor growth.

[0263] 2. Experimental Results

[0264] The results are shown in Figure 32. The results show that the CD19 CAR & CD20 TRuC-T structure 5 has the most significant inhibitory effect on lymphoma in mice.

Claims

1. A CD20 antibody or an antigen-binding fragment thereof targeting CD20 protein, wherein the amino acid sequences of CDR1, CDR2 and CDR3 are as set forth in any one of SEQ ID NOs. 1-3 or SEQ ID NOs. 11-13, respectively, or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NOs. 1-3 or SEQ ID NOs. 11-13, respectively. Optionally, the antibody is humanized. Optionally, the CD20 antibody is a single-domain antibody, a monoclonal antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, a single-chain Fv, a Fab fragment, a F(ab') fragment or a disulfide-bond Fv. Optionally, the CD20 antibody is a single-domain antibody. Optionally, the CD20 antibody has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs. 4-9 or SEQ ID NOs. 14-19.

2. The CD20 antibody or the antigen-binding fragment thereof of claim 1, comprising or having an amino acid sequence set forth in any one of SEQ ID NOs. 4-9 or SEQ ID NOs. 14-19, or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs. 4-9 or SEQ ID NOs. 14-19.

3. A humanized CD19 antibody, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO. 34, and the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO. 38; or the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO. 35, and the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO.

39. Optionally, the light chain variable region and the heavy chain variable region are connected by a linker. Optionally, the structure of the CD19 antibody is: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

4. A fusion protein comprising the CD20 antibody or the antigen-binding fragment thereof of claim 1 or 2 and the CD19 antibody of claim 3. ​ Optionally, the fusion protein comprises, from N-terminus to C-terminus, the CD20 antibody or antigen binding fragment thereof of claim 1 or 2, a first linker, the light chain variable region of the CD19 antibody of claim 3, a second linker, and the heavy chain variable region of the CD19 antibody of claim 3. Optionally, the first linker and the second linker are the same or different.

5. A chimeric antigen receptor comprising the CD20 antibody or antigen binding fragment thereof of claim 1 or 2, the CD19 antibody of claim 3, or the fusion protein of claim 4. Optionally, the chimeric antigen receptor further comprises a hinge region. Optionally, the hinge region comprises a hinge region of CD8a, CD28, CD34, 4-1BB, OX40, CD3e, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor. Optionally, the hinge region is a CD8a hinge region. Optionally, the CD8a hinge region has the amino acid sequence of SEQ ID NO.

22. Optionally, the chimeric antigen receptor further comprises a transmembrane domain. Optionally, the transmembrane domain comprises a transmembrane domain of CD8a, CD28, 4-1BB, CD34, CD3e, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-7 receptor, IL-11 receptor. Optionally, the transmembrane domain is a CD8a transmembrane domain. Optionally, the CD8a transmembrane domain has the amino acid sequence of SEQ ID NO.

23. Optionally, the chimeric antigen receptor further comprises a costimulatory signaling domain. Optionally, the costimulatory signaling domain comprises a costimulatory signaling domain of 4-1BB, CD19, CD4, CD27, CD28, ICOS, CD8a, CD8b, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226. Optionally, the costimulatory signaling domain is a 41BB costimulatory signaling domain. Optionally, the 41BB costimulatory signaling domain has the amino acid sequence of SEQ ID NO.

24. Optionally, the chimeric antigen receptor further comprises an intracellular signaling domain. Optionally, the intracellular signaling domain comprises an intracellular signaling domain of CD3zeta, CD3gamma, ZAP70, CD3delta, CD3epsilon, FcRgamma, FcRbeta, TCRzeta, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, Fc epsilon RI, DAP10, DAP12, CD66d. Optionally, the intracellular signaling domain is a CD3zeta intracellular signaling domain. Optionally, the CD3ζ intracellular signaling domain has the amino acid sequence shown in SEQ ID NO.

25.

6. The chimeric antigen receptor of claim 5, which is composed of the CD20 antibody or antigen binding fragment thereof of claim 1 or 2, the CD19 antibody of claim 3, or the fusion protein of claim 4, and a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain; Optionally, the chimeric antigen receptor is sequentially composed of the CD20 antibody or antigen binding fragment thereof of claim 1 or 2, the CD19 antibody of claim 3, or the fusion protein of claim 4, a CD8 hinge region, a CD8 transmembrane domain, a 41BB costimulatory domain, and a CD3ζ intracellular signaling domain, from N-terminus to C-terminus.

7. A fusion protein targeting CD19 and CD20, which comprises a chimeric antigen receptor targeting CD19, and a TCR fusion construct targeting CD20; the chimeric antigen receptor targeting CD19 comprises an antibody targeting CD19; the TCR fusion construct targeting CD20 comprises a single-domain antibody targeting CD20; the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody targeting CD19 are shown in SEQ ID NO. 45-47, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody targeting CD19 are shown in SEQ ID NO. 49, HTS, and SEQ ID NO. 50, respectively; the single-domain antibody targeting CD20 is the CD20 antibody or antigen binding fragment thereof of claim 1 or 2; Optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the single-domain antibody targeting CD20 are shown in SEQ ID NO. 1-3, respectively.

8. The fusion protein of claim 7, wherein the chimeric antigen receptor targeting CD19 is in front, and the TCR fusion construct targeting CD20 is behind. Optionally, the fusion protein is sequentially obtained by concatenation of the chimeric antigen receptor targeting CD19, T2A, and the TCR fusion construct targeting CD20. Optionally, the chimeric antigen receptor targeting CD19 further comprises a hinge region, a transmembrane region, a costimulatory signaling domain, and an intracellular signaling domain; Optionally, the hinge region is selected from the hinge region of CD8, CD28, IgG1, IgG4, 41BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, or IL-11 receptor; Optionally, the transmembrane region is selected from the transmembrane region of CD8, CD28, IgG1, IgG4, 41BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, or IL-11 receptor; Optionally, the co-stimulatory signaling domain is selected from the co-stimulatory signaling domain of 41BB, CD27, CD19, CD4, CD28, CD278, CD8a, CD8b, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, or B7H3; Optionally, the intracellular signaling domain is selected from the intracellular signaling domain of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, TCR zeta, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, Fc epsilon RI, DAP10, DAP12, or CD66d; Optionally, the hinge region is a CD8 hinge region; Optionally, the transmembrane region is a CD8 transmembrane region; Optionally, the co-stimulatory signaling domain is a 41BB co-stimulatory signaling domain; Optionally, the intracellular signaling domain is a CD3 zeta intracellular signaling domain; Optionally, the amino acid sequence of the heavy chain variable region of the antibody targeting CD19 is set forth in SEQ ID NO. 39; Optionally, the amino acid sequence of the light chain variable region of the antibody targeting CD19 is set forth in SEQ ID NO. 35; Optionally, the amino acid sequence of the single-domain antibody targeting CD20 is set forth in SEQ ID NO. 9; Optionally, the chimeric antigen receptor targeting CD19 is obtained by concatenation of the antibody targeting CD19, the CD8 hinge region, the CD8 transmembrane region, the 41BB co-stimulatory signaling domain, and the CD3 zeta intracellular signaling domain in sequence; Optionally, the amino acid sequences of the CD8 hinge region, the CD8 transmembrane region, the 41BB co-stimulatory signaling domain, and the CD3 zeta intracellular signaling domain are set forth in SEQ ID NOs. 53-56, respectively; Optionally, the TCR fusion construct targeting CD20 further comprises a Linker and a TCR complex subunit fusion moiety; Optionally, the Linker is selected from G4S, (G4S)2, (G4S)3, or EAAAK; Optionally, the TCR complex subunit fusion moiety is selected from CD3 epsilon, CD3 gamma, or CD3 delta; Optionally, the Linker is G4S; Optionally, the TCR complex subunit fusion moiety is CD3 epsilon; Optionally, the TCR fusion construct targeting CD20 is obtained by concatenation of the single-domain antibody targeting CD20, G4S, and CD3 epsilon in sequence; Optionally, the amino acid sequence of the CD3 epsilon is set forth in SEQ ID NO. 57; Optionally, the fusion protein is obtained by concatenation of the antibody targeting CD19, the CD8 hinge region, the CD8 transmembrane region, the 41BB co-stimulatory signaling domain, the CD3 zeta intracellular signaling domain, T2A, the single-domain antibody targeting CD20, G4S, and CD3 epsilon in sequence; Optionally, the N-terminus of the fusion protein further comprises a membrane-out signal peptide; Optionally, the membrane export signal peptide is selected from the group consisting of membrane export signal peptide CD8a-SP and membrane export signal peptide GMCSF-SP. Optionally, the amino acid sequences of the membrane export signal peptide CD8a-SP and the membrane export signal peptide GMCSF-SP are shown in SEQ ID NO. 60 and 61, respectively.

9. A nucleic acid molecule encoding one or more of the CD20 antibody or antigen binding fragment thereof of claim 1 or 2, the CD19 antibody of claim 3, the fusion protein of claim 4, the chimeric antigen receptor of claim 5 or 6, or the fusion protein of claim 7 or 8.

10. A recombinant vector comprising the nucleic acid molecule of claim 8. Optionally, the recombinant vector comprises a cloning vector, an expression vector. Optionally, the recombinant vector comprises a DNA vector, an RNA vector, a plasmid, a viral-derived vector. Optionally, the viral-derived vector comprises a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxviral vector, a herpesviral vector.

11. A genetically modified cell comprising or expressing one or more of the CD20 antibody or antigen binding fragment thereof of claim 1 or 2, the CD19 antibody of claim 3, the fusion protein of claim 4, the chimeric antigen receptor of claim 5 or 6, the fusion protein of claim 7 or 8, the nucleic acid molecule of claim 9, or the recombinant vector of claim 10. Alternatively, the cell is prepared from the recombinant vector of claim 10. Optionally, the cell is a human cell. Optionally, the cell is an immune cell. Optionally, the cell comprises a T cell, a B cell, a natural killer cell, a macrophage, an NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte, and / or a peripheral blood mononuclear cell.

12. The cell of claim 11, wherein the cell is a T cell.

13. An antibody drug conjugate comprising a cytotoxic agent or a label, and the CD20 antibody or antigen binding fragment thereof of claim 1 or 2 and / or the CD19 antibody of claim 3. Optionally, the cytotoxic agent is MMAF or MMAE. Optionally, the label is a fluorescent agent.

14. A pharmaceutical composition comprising one or more of the CD20 antibody or antigen binding fragment thereof of claim 1 or 2, the CD19 antibody of claim 3, the fusion protein of claim 4, the chimeric antigen receptor of claim 5 or 6, the fusion protein of claim 7 or 8, the nucleic acid molecule of claim 9, the recombinant vector of claim 10, the cell of claim 11 or 12, or the antibody drug conjugate of claim 13. Optionally, the pharmaceutical composition further comprises a pharmaceutically or physiologically acceptable carrier and / or excipient. Optionally, the pharmaceutically or physiologically acceptable carrier and / or excipient is a sterile injectable liquid. Optionally, the sterile injectable liquid is selected from the group consisting of water for injection, bacteriostatic water for injection, sodium chloride solution, dextrose solution, a solution containing a surfactant, a pH buffered solution, Ringer's solution, and any combination thereof. Optionally, the pharmaceutical composition contains T cells expressing the chimeric antigen receptor of claim 5 or 6 or expressing the fusion protein of claim 7 or 8.

15. A biological preparation comprising the pharmaceutical composition of claim 14.

16. A detection kit comprising a labeled antibody, the antibody being the CD20 antibody or antigen-binding fragment thereof of claim 1 or 2 and / or the CD19 antibody of claim 3. Optionally, the label comprises a radionuclide, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, a photosensitizing diagnostic agent.

17. A method of preventing and / or treating a CD20-associated cancer, comprising administering to a subject in need thereof an effective amount of one or more of the CD20 antibody or antigen-binding fragment thereof of claim 1 or 2, the fusion protein of claim 4, the chimeric antigen receptor of claim 5 or 6, the fusion protein of claim 7 or 8, the nucleic acid molecule of claim 9, the recombinant vector of claim 10, the cell of claim 11 or 12, the antibody drug conjugate of claim 13, the pharmaceutical composition of claim 14, or the biological preparation of claim 15. Optionally, the method is administering to the subject an effective amount of CAR-T cells and / or CAR / TRuC-T cells, the CAR having the CD20 antibody or antigen-binding fragment thereof of claim 1 or 2 and / or the CD19 antibody of claim 3, and the CAR / TRuC being the fusion protein of claim 7 or 8.

18. A method of preventing and / or treating a CD19-associated cancer, comprising administering to a subject in need thereof an effective amount of one or more of the CD19 antibody of claim 3, the fusion protein of claim 4, the chimeric antigen receptor of claim 5 or 6, the fusion protein of claim 7 or 8, the nucleic acid molecule of claim 9, the recombinant vector of claim 10, the cell of claim 11 or 12, the antibody drug conjugate of claim 13, the pharmaceutical composition of claim 14, or the biological preparation of claim 15. Optionally, the method is administering to the subject an effective amount of CAR-T cells and / or CAR / TRuC-T cells, the CAR having the CD20 antibody or antigen-binding fragment thereof of claim 1 or 2 and / or the CD19 antibody of claim 3, and the CAR / TRuC being the fusion protein of claim 7 or 8.

19. A method for detecting a CD20 protein or diagnosing a CD20-associated cancer, the method comprising contacting a sample with the CD20 antibody or antigen-binding fragment thereof of claim 1 or 2 or the fusion protein of claim 4 and detecting a complex, wherein detection of the complex indicates CD20 protein expression in the sample.

20. A method for detecting CD 19 protein or diagnosing CD 19 related cancer, the method comprising contacting a sample with the CD 19 antibody or antigen-binding fragment thereof of claim 3 or the fusion protein of claim 4 and detecting a complex, wherein detection of the complex indicates CD 19 protein expression in the sample.

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