KR102829757B1 - Humanized antibody specific for CD22 and chimeric antigen receptor using the same - Google Patents

Abstract

The present invention relates to an antibody specific for humanized CD22 and a chimeric antigen receptor using the same, and more specifically, to an antibody (4F5 antibody) specifically binding to CD22, a chimeric antigen receptor comprising the antibody or CD19xCD22 antibody, a CAR-T cell expressing the chimeric antigen receptor, and a pharmaceutical composition for preventing or treating a disease mediated by B cells comprising the same.
In the present invention, CD22-CAR-T cells and bispecific CD19xCD22-CAR-T cells produced based on a humanized antibody that specifically binds to CD22 were confirmed to effectively recognize the CD22 antigen, thereby activating CAR-T cells, and effectively killing cells expressing CD22.
Furthermore, since it was confirmed that the bispecific CD19xCD22-CAR-T cells exhibited excellent anti-tumor effects in animal models, the humanized antibody-based CD22-CAR-T cells and bispecific CD19xCD22-CAR-T cells that specifically bind to CD22 of the present invention can be usefully utilized as compositions for the prevention or treatment of diseases related to CD22 (or CD19) expression or diseases related to B cells.

Description

Humanized antibody specific for CD22 and chimeric antigen receptor using the same

The present invention relates to an antibody specific for humanized CD22 and a chimeric antigen receptor using the same, and more specifically, to an antibody (4F5 antibody) specifically binding to CD22, a chimeric antigen receptor comprising the antibody or CD19xCD22 antibody, a CAR-T cell expressing the chimeric antigen receptor, and a pharmaceutical composition for preventing or treating a disease mediated by B cells comprising the same.

CD22 is expressed in most B-cell leukemias and lymphomas, including NHL, acute lymphoblastic leukaemia (B-ALL), chronic lymphocytic leukaemia (B-CLL), and especially acute non-lymphocytic leukaemia (ANLL).

For the treatment or diagnosis of diseases related to CD22 expression, antibodies specific for CD22 are being developed. International Patent Publication No. WO1998-041641 discloses a recombinant anti-CD22 antibody having cysteine residues at positions V _H 44 and V _L 100, and International Patent Publication No. WO 1998-042378 discloses an anti-CD22 antibody for the treatment of B-cell malignancy.

As mentioned above, in order to produce antibodies for treatment, mice are mainly used to produce monoclonal antibodies. However, non-human antibodies such as mouse-derived monoclonal antibodies are considered foreign antigens in the human body, so they induce an immune response and have a short half-life, which limits their therapeutic effect.

To solve the above problem, humanized antibodies have been developed in which only the antigen-binding portion of the antibody is replaced with a human antibody. The method of replacing a mouse antibody with a humanized antibody currently in use is to select the most similar human antibody gene for the antibody to be replaced, and replace only the CDR portion of the mouse antibody with the human antibody CDR position using a method called CDR grafting. Such humanized antibodies have the advantage of reducing the immune response in the human body because most of the genes are humanized.

Meanwhile, antibodies specific for various other B cell surface markers (antigens) are being developed for the treatment of B cell disorders or diseases, autoimmune diseases, transplant rejection, etc., and CD19, in addition to the CD22 antigen, is an antigen that is commonly used as a target, and many clinical trials for CAR-T cells targeting it are also in progress. However, the amount of target antigen expression varies depending on cells such as leukemia that do not express CD19, and single CAR or single CAR-T cell treatment targeting only one antigen may cause problems such as loss of the target antigen due to the immune evasion strategy of tumor cells. In fact, CD19-negative relapse, in which CD19 is not expressed, has been observed in patients with B-cell ALL (acute lymphoblastic leukemia) (up to 25% of B-cell ALL patients who initially responded to CD19 CAR-T therapy), and this phenomenon has been identified as a resistance mechanism of tumor cells to CAR-T cell therapy (Maude, SL, et al. , N. Engl. J. Med ., 378:439-448, 2018).

To address this, CAR-T cells targeting dual or multiple antigens are being studied, which may reduce the possibility of antigen-loss variants by targeting two antigens simultaneously.

Accordingly, in the present invention, an antibody (4F5 antibody) that binds to CD22 was selected to reduce an immune response in the human body, and humanized anti-CD22 antibodies (4F5 (V4) and 4F5 (V11)) were produced using the same, and a chimeric antigen receptor and CAR-T cells targeting CD22 were produced using the humanized anti-CD22 antibody of the present invention.

Furthermore, a bispecific chimeric antigen receptor (Bivalent CAR or Bispecific CAR) targeting CD19 as well as CD22 and a bispecific CAR-T cell using the same were produced, and it was confirmed that the chimeric antigen receptor was normally expressed in the CD22-CAR-T cells and bispecific CD19xCD22-CAR-T cells produced in the present invention, thereby completing the present invention.

Accordingly, the purpose of the present invention is to provide a humanized antibody specific for CD22, a polynucleotide encoding the antibody, a vector expressing the antibody, and a recombinant cell transformed with the vector.

Another object of the present invention is to provide a chimeric antigen receptor comprising a humanized antibody specific for CD22, a polynucleotide encoding the chimeric antigen receptor, a vector comprising the polynucleotide, and an immune effector cell expressing the chimeric antigen receptor comprising the polynucleotide or the vector.

Another object of the present invention is to provide a bispecific chimeric antigen receptor comprising antibodies specific for CD19 and CD22, a polynucleotide encoding the bispecific chimeric antigen receptor, a vector comprising the polynucleotide, and an immune effector cell expressing the bispecific chimeric antigen receptor comprising the polynucleotide or the vector.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease mediated by B cells, comprising the immune effector cells.

To achieve the above-mentioned purpose,

The present invention comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12; or

Provided is a humanized antibody or fragment thereof that specifically binds to CD22, comprising a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.

In addition, the present invention provides a polynucleotide encoding a humanized antibody or fragment thereof that specifically binds to CD22.

In addition, the present invention provides a vector comprising a polynucleotide encoding a humanized antibody or a fragment thereof that specifically binds to CD22.

Additionally, the present invention provides a recombinant cell producing a humanized antibody or fragment thereof that specifically binds to CD22 transformed with the vector.

To achieve other purposes,

The present invention relates to a chimeric antigen receptor (CAR) comprising a CD22-binding domain; a transmembrane domain; a costimulatory domain; and an intracellular signal transduction domain.

The CD22-binding domain comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12; or

A chimeric antigen receptor targeting CD22 is provided, characterized in that it is a humanized antibody or fragment thereof that specifically binds to CD22, comprising a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.

In a preferred embodiment of the present invention, the transmembrane domain is a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1, the costimulatory domain is a protein selected from the group consisting of CD28, 4-1BB, OX-40 and ICOS, and the signaling domain may be derived from CD3ζ.

In another preferred embodiment of the present invention, a hinge region may be additionally included, located between the C-terminus of the CD22-binding domain and the N-terminus of the transmembrane domain, wherein the hinge region may be derived from CD8α.

Additionally, the present invention provides a polynucleotide encoding the chimeric antigen receptor.

Additionally, the present invention provides a vector comprising a polynucleotide encoding the chimeric antigen receptor.

In addition, the present invention provides an immune effector cell expressing the chimeric antigen receptor, comprising a polynucleotide encoding the chimeric antigen receptor or a vector comprising the polynucleotide.

To achieve another goal,

The present invention relates to a CD19-binding domain and a CD22-binding domain;

transmembrane domain;

costimulatory domain; and

A bispecific chimeric antigen receptor (CAR) containing an intracellular signal transduction domain,

The above CD22-binding domain is characterized in that it is an antibody or a fragment thereof that specifically binds to CD22, consisting of a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 6.

In a preferred embodiment of the present invention, the CD19-binding domain and the CD22-binding domain may be linked in the following order: light chain variable region of an antibody that specifically binds to CD19 - heavy chain variable region of an antibody that specifically binds to CD22 - light chain variable region of an antibody that specifically binds to CD22 - heavy chain variable region of an antibody that specifically binds to CD19.

In another preferred embodiment of the present invention, the light chain variable region of the antibody that specifically binds to CD19 may include a CDR1 region represented by an amino acid sequence of SEQ ID NO: 44, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 45, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 46, and preferably may be represented by an amino acid sequence of SEQ ID NO: 48.

In another preferred embodiment of the present invention, the heavy chain variable region of the antibody that specifically binds to CD19 may include a CDR1 region represented by an amino acid sequence of SEQ ID NO: 41, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 42, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 43, and preferably may be represented by an amino acid sequence of SEQ ID NO: 47.

In another preferred embodiment of the present invention, the transmembrane domain is a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1, the costimulatory domain is a protein selected from the group consisting of CD28, 4-1BB, OX-40 and ICOS, and the signaling domain may be derived from CD3ζ.

In another preferred embodiment of the present invention, a hinge region may be additionally included, located between the C-terminus of the CD22-binding domain and the N-terminus of the transmembrane domain, wherein the hinge region may be derived from CD8α.

The present invention also provides a polynucleotide encoding the bispecific chimeric antigen receptor.

Additionally, the present invention provides a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor.

In addition, the present invention provides an immune effector cell expressing the bispecific chimeric antigen receptor, comprising a polynucleotide encoding the bispecific chimeric antigen receptor or a vector comprising the polynucleotide.

To achieve another goal,

The present invention provides a pharmaceutical composition for preventing or treating a disease mediated by B cells, comprising a humanized antibody or fragment thereof that specifically binds to CD22; or the immune effector cell.

In a preferred embodiment of the present invention, the disease mediated by B cells can be selected from the group consisting of tumor, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt's lymphoma, and mantle cell lymphoma.

In the present invention, a humanized antibody that specifically binds to CD22 was produced, and using the same, a single CAR-T cell targeting CD22 and a dual-specific CAR-T cell targeting CD19 and CD22 were produced.

It was confirmed that the CD22-CAR-T cells and bispecific CD19xCD22-CAR-T cells manufactured in the present invention effectively recognized the CD22 antigen, thereby activating CAR-T cells, and effectively killed cells expressing CD22.

Furthermore, since it was confirmed that the bispecific CD19xCD22-CAR-T cells exhibited excellent anti-tumor effects in animal models, the humanized antibody-based CD22-CAR-T cells and bispecific CD19xCD22-CAR-T cells that specifically bind to CD22 of the present invention can be usefully utilized as compositions for the prevention or treatment of diseases related to CD22 (or CD19) expression or diseases related to B cells.

Figure 1 is data confirming the binding affinity of 4F5 antibody (mouse) and humanized 4F5 antibody (4F5 (V4) and 4F5 (V11)) selected in the present invention to CD22 by FACS.
Figure 2 is a schematic diagram showing a chimeric antigen receptor (single CAR) targeting CD22.
Figure 3 is a schematic diagram showing a method for producing CD22-CAR-T cells using a lentivirus expressing CD22-CAR.
Figure 4 is a schematic diagram showing (A) a method for producing CD22-CAR cells using peripheral blood mononuclear cells (PBMCs) and (B) a method for confirming the binding ability of the produced CD22-CAR-T cells to CD22 peptide.
Figure 5 shows data confirming the CD22 binding ability of CD22-CAR-T cells based on humanized anti-CD22 antibodies, 4F5 (V4) and 4F5 (V11).
Figure 6 shows data confirming the killing effect of U2932 cells (CD22 expressing cells) and K562 cells (CD22 non-expressing cells) by CD22-CAR-T cells based on humanized anti-CD22 antibodies, 4F5 (V4) and 4F5 (V11).
Figure 7 is a schematic diagram showing a bispecific chimeric antigen receptor (Bispecific CAR) targeting CD19 and CD22.
Figure 8 is a schematic diagram showing a method for producing CD19xCD22-CAR expressing cells using a lentivirus expressing a bispecific CD19xCD22-CAR targeting CD19 and CD22.
Figure 9 is a schematic diagram showing (A) a method for transfecting a HEK293 cell line with a CD19xCD22-CAR expressing lentivirus and (B) a method for confirming the binding ability of the transfected HEK293 cells to CD19 peptide and CD22 peptide and the level of CD19xCD22-CAR expression.
Figure 10 shows data confirming the expression levels of CD19x4F5-CAR, CD19x4F5(V4)-CAR, and CD19x4F5(V11)-CAR in HEK293FT cells transduced with lentivirus expressing CD19xCD22-CAR.
Figure 11 is a schematic diagram showing a method for producing CD19xCD22-CAR-T cells using a lentivirus expressing CD19xCD22-CAR.
Figure 12 is a schematic diagram showing (A) a method for producing CD19xCD22-CAR-T cells using peripheral blood mononuclear cells (PBMCs) and (B) a method for confirming the binding ability of the produced CD19xCD22-CAR-T cells to CD19 peptide and CD22 peptide.
Figure 13 shows data confirming the activation of CD19xCD22-CAR-T cells. It was confirmed that CD19xCD22-CAR-T cells simultaneously bind to CD22 peptide and CD19 peptide in CD3+ CD19xCD22-CAR-T cells.
Figure 14 shows data confirming the killing effect of CD19xCD22-CAR-T cells on K562 cells (CD19 and CD22 non-expressing cells), K562-CD19 cells (CD19 expressing cells), K562-CD22 cells (CD22 expressing cells), and K562-CD19/CD22 cells (CD19 and CD22 expressing cells).

Hereinafter, the present invention will be described in detail.

Antibody that binds specifically to CD22

The present invention relates to a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12; or

It relates to a humanized antibody or fragment thereof that specifically binds to CD22, comprising a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.

In the present invention, the term "humanized antibody" refers to an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human and/or is made using one of the techniques for making human antibodies.

In the present invention, the antibody may be a monoclonal antibody. In the present invention, the term "monoclonal antibody", also called a monoclonal antibody or monoclonal antibody, is an antibody produced by a single antibody-forming cell and has a characteristic of having a uniform primary structure (amino acid sequence). It recognizes only one antigenic determinant and is generally produced by culturing a hybridoma cell that fuses a cancer cell and an antibody-producing cell, but it can also be produced using another recombinant protein expression host cell using the secured antibody gene sequence. In addition, the antibody can be used by humanizing the remaining portion except for the CDR portion, if necessary.

In the present invention, the term "CDR", or "complementarity determining region", refers to a non-contiguous antigen binding site found within the variable regions of both the heavy and light chain regions.

In the present invention, the term "antibody" may be used not only for a complete form having two full-length light chains and two full-length heavy chains, but also for a fragment of an antibody molecule. A fragment of an antibody molecule means a fragment having at least a peptide tag (epitope) binding function, and includes scFv, Fab, F(ab'), F(ab') ₂ , a single domain, etc.

Among antibody fragments, Fab has a structure having variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine residues of the hinge region of Fab' form a disulfide bond. Fv is the minimum antibody fragment having only the heavy chain variable region and the light chain variable region. In the double-chain Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by a disulfide bond, and in the single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are covalently linked, generally via a peptide linker. These antibody fragments can be obtained using a proteolytic enzyme, or preferably, can be produced by genetic recombination technology.

The monoclonal antibody specifically binding to CD22 of the present invention can be produced using the entire CD22 protein or a partial peptide as an immunogen (or antigen). More specifically, first, a CD22 protein, a fusion protein comprising a CD22 protein, or a carrier comprising a CD22 protein as an immunogen is injected once or more into the subcutaneous, muscular, intravenous, calcaneal or peritoneal cavity of a mammal other than a human, together with an adjuvant (e.g., Freund's adjuvant), as an immunostimulant, if necessary, thereby causing immunization. The mammal other than a human is preferably a mouse, a rat, a hamster, a guinea pig, a chicken, a rabbit, a cat, a dog, a pig, a goat, a sheep, a donkey, a horse or a cow (including a transgenic animal engineered to produce antibodies derived from other animals, such as a transgenic mouse that produces human antibodies), and more preferably a mouse, a rat, a hamster, a guinea pig, a chicken or a rabbit. Immunization is performed 1 to 4 times about every 1 to 21 days from the first immunization, and antibody-producing cells can be obtained from the immune-primed mammal about 1 to 10 days after the final immunization. The number of times and the time interval for immunization can be appropriately changed depending on the characteristics of the immunogen used.

The production of a hybridoma secreting a monoclonal antibody can be performed according to the method of Keira and Mirstein et al. (Nature, 1975, Vol. 256, p. 495-497) or a method similar thereto. The hybridoma can be produced by cell fusion of any one selected from the group consisting of the spleen, lymph node, bone marrow or tonsil collected from an animal other than an immunosensitized human, preferably the spleen, with myeloma cells derived from a mammal having no autoantibody production ability. The mammal may be a mouse, a rat, a guinea pig, a hamster, a chicken, a rabbit or a human, and preferably may be a mouse, a rat, a chicken or a human.

Cell fusion is performed using, for example, a fusion promoter such as polyethylene glycol or Sendai virus, or a method using an electric pulse. For example, antibody-producing cells and infinitely proliferating mammalian cells are suspended in a fusion medium containing a fusion promoter at a ratio of about 1:1 to 1:10, and incubated in this state at about 30 to 40°C for about 1 to 5 minutes. As the fusion medium, a typical general one such as MEM medium, RPMI1640 medium, and Iscove's Modified Dulbecco's Medium may be used, and it is preferable to exclude serum such as bovine serum.

The method for screening the hybridoma clones producing the above monoclonal antibodies includes first transferring the fused cells obtained as described above to a selective medium such as HAT medium, and culturing them at about 30 to 40°C for about 3 days to 3 weeks to kill cells other than hybridomas. Next, the hybridomas are cultured in a microtiter plate or the like, and the reactivity between the immunogen used for the immune response of an animal other than a human described above and the culture supernatant is found to be increased using an immunoassay method such as RIA (radioactive substance-marked immuno antibody) or ELISA (Enzyme-Linked Immunosorbent Assay). And the clones producing the above monoclonal antibodies show specific binding affinity to the immunogen.

The monoclonal antibody of the present invention can be obtained by culturing such a hybridoma in and out of a living body. For culturing, a conventional method for culturing cells derived from mammals is used, and for collecting a monoclonal antibody from a culture, etc., a conventional method in this field for purifying antibodies in general is used. As each method, for example, salting out, dialysis, filtration, concentration, centrifugation, differential precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography, gel electrophoresis, and isoelectric focusing electrophoresis can be mentioned, and these are applied in combination as necessary. The purified monoclonal antibody is then concentrated and dried to be in a liquid or solid phase depending on the intended use.

In addition, the monoclonal antibody of the present invention can be obtained by synthesizing genes in which DNA encoding heavy chain and light chain variable regions are each linked to known DNA encoding heavy chain and light chain constant regions (see, for example, Japanese Publication No. 2007-252372), by the PCR method or chemical synthesis, transplanting the genes into a known expression vector (pcDNA 3.1 (sold by Invitrogen) or the like) that enables expression of the genes to produce a transformant, expressing the genes in a host such as CHO cells or Escherichia coli to produce antibodies, and purifying the antibodies from the culture solution using a Protein A or G column or the like.

In a specific embodiment of the present invention, in order to produce an antibody that specifically binds to CD22, a hybridoma producing an anti-CD22 antibody was produced and screened, and an antibody (scFv) that specifically binds to CD22 was selected, which was named 4F5.

It was confirmed that the above 4F5 antibody is composed of a heavy chain variable region including a CDR1 region (GFSLTIYG) represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region (MWSGGST) represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region (ARNDGYYWFAY) represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region including a CDR1 region (QSLVHNNGNTY) represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region (KVS) represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region (SQSTHVPYS) represented by an amino acid sequence of SEQ ID NO: 6.

Specifically, it was confirmed that the 4F5 antibody is composed of a heavy chain variable region represented by an amino acid sequence of SEQ ID NO: 7 and a light chain variable region represented by an amino acid sequence of SEQ ID NO: 8, and that the heavy chain variable region is coded by a base sequence of SEQ ID NO: 9 and the light chain variable region is coded by a base sequence of SEQ ID NO: 10.

In another specific embodiment of the present invention, humanized antibodies were prepared by changing the anti-CD22 antibody 4F5 into a structure corresponding to a human, and were named 4F5 (V4) antibody and 4F5 (V11) antibody.

The heavy chain variable region CDR and light chain variable region CDR of the above 4F5 (V4) antibody and 4F5 (V11) antibody are identical to those of 4F5, and the remaining portions except for the CDR portion are humanized. Preferably, the 4F5 (V4) antibody is composed of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12, and the heavy chain variable region of the 4F5 (V4) antibody can be coded by the base sequence of SEQ ID NO: 13, and the light chain variable region can be coded by the base sequence of SEQ ID NO: 14.

In addition, the 4F5 (V11) antibody is composed of a heavy chain variable region represented by an amino acid sequence of SEQ ID NO: 15 and a light chain variable region represented by an amino acid sequence of SEQ ID NO: 16, and the heavy chain variable region of the 4F5 (V11) antibody can be coded by a base sequence of SEQ ID NO: 17, and the light chain variable region can be coded by a base sequence of SEQ ID NO: 18.

The antibody specific for CD22 of the present invention is preferably a scFv (single chain variable fragment), and can be produced through genetic recombination technology so that the heavy chain variable region and the light chain variable region can be connected by a linker. The linker is preferably represented by the amino acid sequence of SEQ ID NO: 19, or can be encoded by the base sequence of SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, but is not limited thereto.

When linked by the light chain variable region-linker-heavy chain variable region, the 4F5 antibody (mouse antibody) may be represented by the amino acid sequence of SEQ ID NO: 23 or encoded by the nucleotide sequence of SEQ ID NO: 24, the 4F5 (V4) antibody may be represented by the amino acid sequence of SEQ ID NO: 25 or encoded by the nucleotide sequence of SEQ ID NO: 26, and the 4F5 (V11) antibody may be represented by the amino acid sequence of SEQ ID NO: 27 or encoded by the nucleotide sequence of SEQ ID NO: 28.

In another aspect, the present invention relates to a polynucleotide encoding an antibody that specifically binds to CD22.

In the present invention, the term "polynucleotide" generally refers to a nucleic acid molecule, deoxyribonucleotide or ribonucleotide, or an analogue thereof, separated into any length. The polynucleotide can be prepared by (1) in-vitro amplification such as polymerase chain reaction (PCR) amplification; (2) cloning and recombination; (3) purification such as digestion and gel electrophoresis separation; (4) synthesis such as chemical synthesis, and preferably, the separated polynucleotide can be prepared by recombinant DNA technology. In the present invention, a polynucleotide for encoding an antibody or an antigen-binding fragment thereof can be prepared by various methods known in the art, including but not limited to, restriction fragment operation of synthetic oligonucleotides or application of SOE PCR.

In another aspect, the present invention relates to a vector comprising a polynucleotide encoding an antibody that specifically binds to CD22, and a recombinant cell transformed with the vector.

In the present invention, the term "expression vector" is a genetic product containing essential regulatory elements such as a promoter so that a target gene can be expressed in a suitable host cell. The vector may be selected from one or more of a plasmid, a retroviral vector, and a lentiviral vector. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, can be integrated into the genome itself.

In addition, the vector may contain expression control elements that enable the coding region to be accurately expressed in a suitable host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome-binding sites, enhancers, and other control elements for controlling gene transcription or mRNA translation. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally contains a 5' non-transcribed sequence, such as a TATA box, a capped sequence, a CAAT sequence, etc., which participate in transcription initiation and translation initiation, respectively, and a 5' or 3' non-transcribed sequence. For example, the 5' non-transcribed expression control sequence may include a promoter region, which may include a promoter sequence for transcribing and controlling a functionally linked nucleic acid.

In the present invention, the term "promoter" means a minimal sequence sufficient to direct transcription. In addition, promoter structures sufficient to cause expression of a regulatable promoter-dependent gene that is induced by cell type-specific or external signals or agents may be included, and such structures may be located 5' or 3' of the gene. Both conserved promoters and inducible promoters are included. The promoter sequence may be derived from a prokaryote, a eukaryote, or a virus.

In the present invention, the term "transformant" means a cell transformed by introducing a vector having a polynucleotide encoding one or more target proteins into a host cell, and methods for producing a transformant by introducing an expression vector into a host cell include the calcium phosphate method or the calcium chloride/rubidium chloride method described in the literature (Sambrook, J., et al., Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1. 74, 1989), electroporation, electroinjection, chemical treatment methods such as PEG, methods using a gene gun, etc.

When the transformant expressing the above vector is cultured in a nutrient medium, antibody proteins can be produced and isolated in large quantities. The medium and culture conditions can be appropriately selected and used according to the host cell. During culture, conditions such as temperature, pH of the medium, and culture time must be appropriately adjusted to suit cell growth and mass production of proteins.

The vector according to the present invention can be transformed into a host cell, preferably a mammalian cell, for the production of an antibody. A number of suitable host cell lines capable of expressing fully glycosylated proteins have been developed in the art and include COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8653, SP2/0-Agl4, 293 cells, HeLa cells, etc., which are readily available from, for example, ATCC (American Type Culture Collection, USA). Preferred host cells include cells of lymphocytic origin, such as melanoma and lymphoma cells.

Chimeric antigen receptor targeting CD22

The present invention is from another perspective,

CD22-binding domain;

transmembrane domain;

costimulatory domain; and

A chimeric antigen receptor (CAR) containing an intracellular signal transduction domain,

The CD22-binding domain comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12; or

The present invention relates to a chimeric antigen receptor targeting CD22, characterized in that it is a humanized antibody or fragment thereof that specifically binds to CD22, comprising a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.

In the present invention, the term "chimeric antigen receptor (CAR)" generally refers to a fusion protein containing an extracellular domain having the ability to bind an antigen and one or more intracellular domains. The CAR is a core part of a chimeric antigen receptor T cell (CAR-T) and may include an antigen binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. The CAR can be combined with a T cell receptor-activating intracellular domain based on the antigen (e.g., CD22) specificity of an antibody. The genetically modified CAR-expressing T cells can specifically identify and eliminate target antigen-expressing malignant cells.

In the present invention, the term "CD22-binding domain" generally refers to a domain capable of specifically binding to a CD22 protein. For example, the CD22-binding domain may contain an anti-CD22 antibody or fragment thereof capable of specifically binding to a human CD22 polypeptide or fragment thereof expressed in a B cell.

In the present invention, the term "binding domain", "extracellular domain", "extracellular binding domain", "antigenspecific binding domain" and "extracellular antigen-specific biding domain" may be used interchangeably, and refers to a CAR domain or fragment having the ability to specifically bind to a target antigen (e.g., CD22).

In the present invention, the anti-CD22 antibody or fragment thereof is the above-described anti-CD22 antibody, a monoclonal antibody, preferably a single chain variable fragment (scFv). Specifically, it can be produced using 4F5 (V4) or 4F5 (V11) antibody, which is a humanized antibody specific for CD22 of the present invention.

In the present invention, the chimeric antigen receptor may be a bispecific chimeric antigen receptor additionally comprising a B cell surface marker-binding domain in addition to a CD22-binding domain, and the B cell surface marker may be CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85 or CD86, and preferably, CD19.

In the present invention, a signal peptide may be additionally included at the N-terminus of the CD22-binding domain, and the "signal peptide" generally refers to a peptide chain for guiding protein transport. The signal peptide may be a short peptide having a length of 5 to 30 amino acids, and in the present invention, the amino acid sequence of SEQ ID NO: 36 is preferably used.

In the present invention, a hinge region located between the C-terminus of the CD22-binding domain and the N-terminus of the transmembrane domain may be additionally included, and the hinge region may be derived from CD8α and preferably represented by the amino acid sequence of SEQ ID NO: 37. The "hinge region" generally refers to a connecting region between an antigen-binding region and an immune cell Fc receptor (FcR)-binding region.

In the present invention, the "transmembrane domain" generally refers to a domain of a CAR that passes through a cell membrane and is connected to an intracellular signaling domain to play a role in signal transduction. The transmembrane domain may be derived from a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1, and may preferably be represented by the amino acid sequence of SEQ ID NO: 38.

In the present invention, the "costimulatory domain" generally refers to an intracellular domain capable of providing an immunostimulatory molecule, which is a cell surface molecule necessary for an effective response of a lymphocyte to an antigen. The costimulatory domain described above may include a costimulatory domain of CD28, and may include a costimulatory domain of a TNF receptor family such as a costimulatory domain of OX40 and 4-1BB, and preferably may be 4-1BB represented by the amino acid sequence of SEQ ID NO: 39.

In the present invention, the "intracellular signal transduction domain" generally refers to a domain that is located inside a cell and can transduce a signal. In the present invention, the intracellular signal transduction domain is an intracellular signal transduction domain of a chimeric antigen receptor. For example, the intracellular signal transduction domain can be selected from a CD3ζ intracellular domain, a CD28 intracellular domain, a CD28 intracellular domain, a 4-1BB intracellular domain, and an OX40 intracellular domain, and preferably, it can be CD3ζ represented by the amino acid sequence of SEQ ID NO: 40.

Chimeric antigen receptor coding polynucleotide and chimeric antigen receptor expression vector

In another aspect, the present invention relates to a polynucleotide encoding the chimeric antigen receptor (CAR).

In the present invention, the polynucleotide encoding the chimeric antigen receptor (CAR) may include a polynucleotide encoding a CD22-binding domain; a polynucleotide encoding a transmembrane domain; a polynucleotide encoding a co-stimulatory domain; and a polynucleotide encoding an intracellular signaling domain.

The polynucleotide encoding the CD22-binding domain may be a humanized 4F5 (V4) antibody or 4F5 (V11) antibody specific for CD22 of the present invention, in the form of scFv in which the light chain variable region and the heavy chain variable region are connected by a linker, and the specific base sequence is as described above.

Preferably, the polynucleotide encoding the chimeric antigen receptor (CAR) of the present invention is a 4F5 (V4) antibody represented by the nucleotide sequence of SEQ ID NO: 26 or a 4F5 (V11) antibody represented by the nucleotide sequence of SEQ ID NO: 28;

A transmembrane domain represented by the nucleotide sequence of SEQ ID NO: 32;

4-1BB (co-stimulatory domain) represented by the base sequence of sequence number 33; and

It can be composed of CD3ζ (intracellular signaling domain) represented by the base sequence of sequence number 34.

When a signal peptide is included at the N-terminus of the CD22-binding domain, a signal peptide represented by the base sequence of SEQ ID NO: 30 may be additionally included. In addition, a polynucleotide encoding a hinge region may be additionally included between the polynucleotide encoding the CD22-binding domain and the transmembrane domain, and preferably, it may be a CD8 hinge region represented by the base sequence of SEQ ID NO: 31.

In another aspect, the present invention relates to a vector comprising a polynucleotide encoding the chimeric antigen receptor (CAR).

In the present invention, the vector is a recombinant viral vector, preferably a lentiviral vector, and comprises an operably linked EF1α promoter; a polynucleotide encoding a signal peptide; a polynucleotide encoding a CD22-binding domain; a polynucleotide encoding a transmembrane domain; and a polynucleotide encoding an intracellular signaling domain, and may further comprise a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to increase protein expression (Fig. 3).

The above EF1α promoter may be represented by the base sequence of SEQ ID NO: 29, and may include a sequence that is 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the base sequence of SEQ ID NO: 27, as needed.

Additionally, the promoter is operably linked to drive the expression of an anti-CD22 antibody (scFv), which is a CD22-binding domain.

In a specific embodiment of the present invention, a lentiviral vector having a polynucleotide coating CD22-CAR inserted therein was prepared, and the prepared vector was transfected into 293FT cells to prepare CD22-CAR expressing cells.

Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, and particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian, e.g. human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses, and adeno-associated viruses.

Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods for targeted delivery of nucleic acids using the latest technology are available, such as targeted nanoparticles or other suitable sub-micrometer sized delivery systems for polynucleotides.

When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introduction of nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped within the liposome, complexed with the liposome, dispersed in a lipid-containing solution, mixed with the lipid, combined with the lipid, contained as a suspension in the lipid, contained or complexed with micelles, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated composition is not limited to any particular structure in solution.

Chimeric antigen receptor (CAR) expressing immune effector cells

In another aspect, the present invention relates to an immune effector cell expressing the humanized antibody-based chimeric antigen receptor (CAR) specific for CD22, comprising a polynucleotide encoding the humanized antibody-based chimeric antigen receptor (CAR) or a vector comprising the polynucleotide encoding the chimeric antigen receptor (CAR).

In the present invention, the immune effector cell may be an isolated cell derived from a mammal, preferably a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a myeloid cell, a monocyte, or a macrophage, more preferably a T cell.

In the present invention, the immune effector cell expressing the chimeric antigen receptor (CAR) can be produced by introducing the CAR vector of the present invention into an immune effector cell, such as a T cell or NK cell.

Specifically, the CAR vector can be introduced into cells by methods known in the art, such as electroporation, lipofectamine (lipofectamine 2000, Invitrogen), and the like. For example, immune effector cells can be transfected with a lentiviral vector to integrate the viral genome carrying the CAR molecule into the host genome, thereby ensuring long-term and stable expression of the target gene. As another example, a transposon can be used to introduce the CAR carrying plasmid (transposon) and the transposase carrying plasmid into the target cell. As another example, the CAR molecule can be added to the genome by a gene editing method (e.g., CRISPR/Cas9).

Immune effector cells for producing immune effector cells expressing a chimeric antigen receptor (CAR) can be obtained from a subject, wherein the "subject" includes any living organism (e.g., a mammal) in which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic strains thereof. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.

The T cells may be obtained from a unit of blood collected from a subject using any of a number of techniques known to those of ordinary skill in the art, for example, Ficoll™ separation. Cells from blood are obtained by apheresis, and the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.

Cells collected by apheresis may be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps. T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or by countercurrent centrifugation.

In another specific embodiment of the present invention, as shown in FIG. 4, activated T cells were isolated from peripheral blood mononuclear cells (PBMCs), and CD22-CAR lentivirus was transduced into the T cells to produce CD22-CAR-T cells. Specifically, CD22-CAR-T cells were produced using humanized 4F5 (V4) antibody and 4F5 (V11) antibody, respectively.

To confirm the activity of the manufactured CD22-CAR-T cells, the binding ability of CD22-CAR-T cells activated with CD3, CD4 or CD8 to CD22 peptide was confirmed. As shown in Fig. 5, it was confirmed that the CD22-CAR-T cells manufactured in the present invention bind to CD22 peptide.

In another specific embodiment of the present invention, as a result of confirming the killing effect of target cells by CD22-CAR-T cells, as shown in FIG. 6, it was confirmed that CD22-CAR-T cells exhibited a killing effect specifically on U2932 cells expressing CD22.

That is, the humanized anti-CD22 antibody of the present invention, the 4F5(V4) antibody or the 4F5(V11) antibody-based chimeric antigen receptor, and the CAR-T cell using the same can be usefully utilized as a composition for preventing or treating diseases related to B cell or CD22 expression.

Bispecific chimeric antigen receptor (Bispecific CAR) targeting CD19 and CD22

The present invention from another perspective,

CD19-binding domain and CD22-binding domain;

transmembrane domain;

costimulatory domain; and

A bispecific chimeric antigen receptor (CAR) containing an intracellular signal transduction domain,

The above CD22-binding domain relates to a bispecific chimeric antigen receptor (CD19xCD22 bispecific CAR), characterized in that it is an antibody or a fragment thereof that specifically binds to CD22, comprising a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 6.

In the present invention, the antibody or fragment thereof that specifically binds to CD22 is an anti-CD22 antibody consisting of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 7 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 8, or

a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12; or

It may be a humanized anti-CD22 antibody consisting of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.

In the present invention, the bispecific (or bivalent) chimeric antigen receptor is a CAR capable of simultaneously binding two different types of antigens. In the present invention, a bispecific chimeric antigen receptor targeting both CD19 and CD22 is preferably prepared, and the CD19-binding domain can use a known anti-CD19 antibody sequence without limitation.

The specific details of the chimeric antigen receptor are the same as described above, and the CD19-binding domain and the CD22-binding domain of the bispecific chimeric antigen receptor are connected in a loop (LoopCAR) form as shown in Fig. 7. That is, the light chain variable region of an antibody that specifically binds to CD19 (CD19VL) - the heavy chain variable region of an antibody that specifically binds to CD22 (CD22VH) - the light chain variable region of an antibody that specifically binds to CD22 (CD22VL) - the heavy chain variable region of an antibody that specifically binds to CD19 (CD19VH) can be connected in this order.

The light chain variable region of the antibody that specifically binds to the CD19 above may be represented by the amino acid sequence of SEQ ID NO: 48, and the heavy chain variable region of the antibody that specifically binds to the CD19 above may be represented by the amino acid sequence of SEQ ID NO: 47.

The above CD19-binding domain and CD22-binding domain can be produced through genetic recombination technology so as to be connected by a linker, and preferably, CD19VL and CD22VH or CD22VL and CD19VH can be connected by a linker represented by the amino acid sequence of SEQ ID NO: 51 (linker 1 of FIG. 7), and CD22VH and CD22VL can be connected by a linker represented by the amino acid sequence of SEQ ID NO: 54 (linker 6 of FIG. 7), but the present invention is not limited thereto, and a peptide composed of any amino acid sequence that does not affect antibody activity can be used.

Polynucleotide encoding a bispecific chimeric antigen receptor targeting CD19 and CD22 and expression vector of a bispecific chimeric antigen receptor targeting CD19/CD22

In another aspect, the present invention relates to a polynucleotide encoding a bispecific chimeric antigen receptor targeting CD19 and CD22.

In the present invention, the polynucleotide encoding the bispecific chimeric antigen receptor may include a polynucleotide encoding a CD19-binding domain and a polynucleotide encoding a CD22-binding domain; a polynucleotide encoding a transmembrane domain; a polynucleotide encoding a co-stimulatory domain; and a polynucleotide encoding an intracellular signaling domain.

Preferably, the polynucleotide encoding the bispecific chimeric antigen receptor of the present invention comprises:

A bispecific antibody consisting of a light chain variable region of an antibody that specifically binds to CD19 (CD19VL; SEQ ID NO: 50) - a heavy chain variable region of an antibody that specifically binds to CD22 (CD22VH; SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 17) - a light chain variable region of an antibody that specifically binds to CD22 (CD22VL; SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 18) - a heavy chain variable region of an antibody that specifically binds to CD19 (CD19VH; SEQ ID NO: 49);

A transmembrane domain represented by the nucleotide sequence of SEQ ID NO: 32;

4-1BB (co-stimulatory domain) represented by the base sequence of sequence number 33; and

It can be composed of CD3ζ (intracellular signaling domain) represented by the base sequence of sequence number 34.

When the above 'CD19VL and CD22VH' or 'CD22VL and CD19VH' are linked by a linker represented by the amino acid sequence of SEQ ID NO: 51, the polynucleotide for the linker may be represented by the base sequence of SEQ ID NO: 52 or SEQ ID NO: 53, and when 'CD22VH and CD22VL' are linked by a linker represented by the amino acid sequence of SEQ ID NO: 54, the polynucleotide for the linker may be represented by the base sequence of SEQ ID NO: 55, but is not limited thereto, and a polynucleotide encoding any amino acid sequence that does not affect antibody activity may be used.

When a signal peptide is included at the N-terminus of the CD19/CD22-binding domain, a signal peptide represented by the base sequence of SEQ ID NO: 30 may be additionally included. In addition, a polynucleotide encoding a hinge region may be additionally included between the polynucleotide encoding the CD22-binding domain and the transmembrane domain, and preferably, it may be a CD8 hinge region represented by the base sequence of SEQ ID NO: 31.

In another aspect, the present invention relates to a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor.

In the present invention, the vector is a recombinant viral vector, preferably a lentiviral vector, and comprises an operably linked EF1α promoter; a polynucleotide encoding a signal peptide; a polynucleotide encoding a CD19-binding domain and a CD22-binding domain; a polynucleotide encoding a transmembrane domain; and a polynucleotide encoding an intracellular signaling domain, and may further comprise a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to increase protein expression (Fig. 8).

The above EF1α promoter may be represented by the base sequence of SEQ ID NO: 29, and may include a sequence that is 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the base sequence of SEQ ID NO: 27, as needed.

In addition, the promoter is operably linked to drive the expression of an anti-CD19/CD22 antibody (CD19VL-CD22VH-CD22VL-CD19VH), which is a CD19xCD22-binding domain, and the specific details of the vector are the same as described above.

In a specific embodiment of the present invention, as shown in FIGS. 8 and 9, a lentiviral vector having a polynucleotide coating CD19xCD22-CAR was manufactured, and the manufactured vector was transfected into 293FT cells to manufacture CD19xCD22-CAR expressing cells. In addition, as shown in FIG. 10, it was confirmed that a bispecific chimeric antigen receptor targeting CD19 and CD22 was expressed in the manufactured CD19xCD22-CAR expressing cells.

Immune effector cells expressing bispecific chimeric antigen receptors targeting CD19 and CD22

In another aspect, the present invention relates to an immune effector cell expressing the bispecific chimeric antigen receptor (CAR), comprising a polynucleotide encoding the bispecific chimeric antigen receptor (CAR) or a vector comprising the polynucleotide encoding the bispecific chimeric antigen receptor (CAR).

In the present invention, the immune effector cell may be an isolated cell derived from a mammal, and preferably may be a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a myeloid cell, a monocyte, or a macrophage, and more preferably a T cell. In addition, the specific details of the chimeric antigen receptor expressing immune effector cell are the same as described above.

In a specific embodiment of the present invention, as shown in FIGS. 11 and 12, activated T cells were separated from peripheral blood mononuclear cells (PBMCs), and CD19xCD22-CAR lentivirus was transduced into the T cells to produce CD19xCD22-CAR-T cells. Specifically, CD19xCD22-CAR-T cells were produced using 4F5 antibody (mouse), humanized 4F5 (V4) antibody, and 4F5 (V11) antibody, respectively.

To confirm the activity of the manufactured CD19xCD22-CAR-T cells, the binding ability of CD3-activated CD19xCD22-CAR-T cells to CD22 peptide and CD19 peptide was confirmed. As shown in Fig. 13, it was confirmed that the CD19xCD22-CAR-T cells manufactured in the present invention simultaneously bind to CD22 peptide and CD19 peptide.

In another specific embodiment of the present invention, the killing effect of target cells by CD19xCD22-CAR-T cells was confirmed. As shown in FIG. 14, it was confirmed that CD19xCD22-CAR-T cells exhibited a killing effect on K562-CD19 cells (CD19 expressing cells), K562-CD22 cells (CD22 expressing cells), and K562-CD19/CD22 cells (CD19 and CD22 expressing cells).

Composition for preventing or treating diseases mediated by B cells or diseases associated with CD19 and CD22 expression

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating a disease mediated by B cells, comprising a humanized antibody that specifically binds to CD22; an immune effector cell expressing a chimeric antigen receptor that targets CD22; or an immune effector cell expressing a bispecific chimeric antigen receptor that specifically binds to CD19xCD22.

In the present invention, the B cell may preferably be a cell expressing CD19 or CD22, and the disease may be selected from the group consisting of tumor/cancer, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt's lymphoma, and mantle cell lymphoma.

In the present invention, the composition may include a therapeutic agent for a disease mediated by B cells, and the therapeutic agent may be covalently bound to an antibody that specifically binds to CD19 or CD22, or may be administered in combination with the CD22-CAR immune effector cells or CD19xCD22-CAR immune effector cells of the present invention.

The above therapeutic agents include small molecule drugs, peptide drugs, toxins (e.g., cytotoxins), etc.

The above small molecule drugs exhibit the pharmaceutical activity of interest and can generally be compounds having a molecular weight of about 800 Da or less or 2000 Da or less. Inorganic small molecules refer to molecules that do not contain any carbon atoms, whereas organic small molecules refer to compounds that contain at least one carbon atom.

The above peptide drugs refer to amino acid containing polymeric compounds, which include naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides and proteins, as well as peptide mimetics. The peptide drugs can be obtained by chemical synthesis or produced from genetically encoded sources (e.g., recombinant sources). The molecular weight of the peptide drugs can range from 200 Da to 10 kDa or more.

The above toxin is preferably a cytotoxin, which includes, but is not limited to, ricin, abrin, diphtheria toxin, Pseudomonas exotoxin (e.g., PE35, PE37, PE38, PE40, etc.), saporin, gelonin, pore-forming antiviral protein (PAP), botulinum toxin, bryodin, momordin, and buganin.

Additionally, the therapeutic agent may be an anticancer agent. Anticancer agents include non-peptide (i.e., non-protein-based) compounds that reduce the proliferation of cancer cells and encompass cytotoxic agents and cytostatic agents. Non-limiting examples of anticancer agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptide compounds may also be used.

In the pharmaceutical composition, the humanized antibody that specifically binds to CD22, CD22-CAR immune effector cells or CD19xCD22-CAR immune effector cells may be the sole active ingredient in the therapeutic or diagnostic composition, or may be used together with other active ingredients, including, for example, other antibody components, such as anti-T cell, anti-IFNγ or anti-LPS antibodies, or non-antibody components, such as xanthine.

The pharmaceutical composition preferably comprises a therapeutically effective amount of an antibody of the invention. The term "therapeutically effective amount" as used herein means the amount of a therapeutic agent required to treat, ameliorate, or prevent a target disease or condition, or the amount of a therapeutic agent required to exhibit a detectable therapeutic or preventive effect. For any antibody, the therapeutically effective dose can be initially determined by cell culture assays or by animal models, typically rodents, rabbits, dogs, pigs, or primates. The animal models can also be used to determine appropriate concentration ranges and routes of administration. This information can be used to determine useful doses and routes for human administration.

The precise effective dose for a human patient may vary depending on the severity of the disease state, the patient's general health, the patient's age, weight and sex, diet, administration time, administration frequency, drug composition, response sensitivity and tolerance/response to treatment. The amount can be determined by routine experimentation and is within the range of the clinician's judgment. Generally, the effective dosage is 0.01 to 50 mg/kg, preferably 0.1 to 20 mg/kg, and more preferably about 15 mg/kg.

The composition may be administered individually to a patient or in combination with other agents, drugs or hormones.

The dosage at which the antibody of the present invention is administered will vary depending on the nature of the condition being treated, the grade of the malignant lymphoma or leukemia, and whether the antibody is being used for disease prevention or to treat an existing condition.

The frequency of administration depends on the half-life of the antibody molecule and the duration of the drug effect. If the antibody molecule has a short half-life (e.g., 2 to 10 hours), it may be necessary to administer one or more doses per day. Alternatively, if the antibody molecule has a long half-life (e.g., 2 to 15 days), it may be necessary to administer one dose per day, once a week, or once every month or every two months.

Additionally, the pharmaceutical composition may contain a pharmaceutically acceptable carrier for administration of the antibody. The carrier must not itself induce the production of antibodies that are harmful to the subject receiving the composition and must be nontoxic. Suitable carriers may be slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, and inactivated virus particles.

Pharmaceutically acceptable salts may be used, for example, mineral salts such as hydrochlorides, hydrobromates, phosphates and sulfates, or salts of organic acids such as acetic, propionic, malonic and benzoic acids.

Pharmaceutically acceptable carriers in the therapeutic composition may additionally include liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances such as wetting agents, emulsifiers or pH buffering agents may be present in the composition. The carrier may be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for ingestion of the pharmaceutical composition by a patient.

Preferred forms for administration include those suitable for parenteral administration, for example, by injection or infusion (e.g., bolus injection or continuous infusion). When the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or water-soluble vehicle, which may contain preservatives such as suspending agents, preservatives, stabilizers and/or dispersing agents. Alternatively, the antibody molecule may be in anhydrous form and may be reconstituted with a suitable sterile solution before use.

Once formulated, the composition of the present invention may be administered directly to a patient. The patients to be treated may be animals. However, it is preferred that the composition be adapted for administration to human patients.

The pharmaceutical composition of the present invention may be administered by any route including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (see, e.g., WO 98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal. A hypospray may be used to administer the pharmaceutical composition of the present invention. Typically, the therapeutic composition may be prepared as an injectable material, either as a liquid solution or suspension. In addition, a solid form suitable for solution or suspension in a liquid excipient prior to injection may be prepared.

Direct delivery of the composition can generally be by injection, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, or can be delivered to the interstitial space of the tissue. In addition, the composition can be administered to the wound site. Dosage treatment can be a single dose schedule or a multiple dose schedule.

The active ingredient in the composition may be an antibody molecule. As such, it may be susceptible to degradation in the gastrointestinal tract. Therefore, if the composition is administered by a route that uses the gastrointestinal tract, the composition will need to contain an agent that protects the antibody from degradation, but releases the antibody once absorbed from the gastrointestinal tract.

A complete discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ, 1991).

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

Production and selection of antibodies that specifically bind to CD22

To select antibodies specific for the CD22 peptide, hybridomas producing antibodies that bind to CD22 were prepared and the antibodies were screened.

First, splenocytes were extracted by immunization with CD22 protein (ACRObiosystems Inc., cat#CD2-H52H8, USA), and hybridoma cells were produced through cell fusion with mouse myeloma cells.

Mouse myeloma cells used for cell fusion do not have HGPRT (HypoxanthineGuanidine-Phosphoribosyl-Transferase) and therefore cannot survive in HAT medium, but hybridomas can survive in HAT medium by fusing with spleen cells. Since this allows only hybridomas to proliferate, they are usually proliferated in HAT medium until hybridomas are established.

The limiting dilution method was used to select hybridomas that produce antibodies that bind to CD22 among the proliferated hybridomas. First, the number of cells per 96 wells was reduced to 1 or less, and then the antibody obtained from the clone proliferated from 1 cell was confirmed to bind to CD22 by ELISA, and the clones that bind to CD22 were selected. The above process was repeated 3 times to select hybridomas that produce antibodies that bind to CD22. An antibody that binds to CD22 was obtained in this way.

The above antibody was named 4F5, and its base sequence and amino acid sequence were analyzed. The sequence information for the heavy chain variable region and light chain variable region of each antibody according to the sequence analysis results is shown in Table 1 below, and the underlined part in Table 1 indicates the complementarity determining region (CDR).

Sequence information of 4F5 antibody 4F5 Sequence information Sequence number Heavy chain variable region CDR1 GFSLTIYG Sequence number 1 Heavy chain variable region CDR2 MWSGGST Sequence number 2 Heavy chain variable region CDR3 ARNDGYYWFAY Sequence number 3 Light chain variable region CDR1 QSLVHNNGNTY Sequence number 4 Light chain variable region CDR2 KVS Sequence number 5 Light chain variable region CDR3 SQSTHVPYS Sequence number 6 Heavy chain variable region
Amino acid sequence QVQLKESGPGLVQPSQSLSITCTVS GFSLTIYG VHWIRQSPGKGLEWLGV MWSGGST DYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYC ARNDGYYWFAY WGQGTLVTVSA Sequence number 7 Light chain variable region
Amino acid sequence DVLMTQTPLSLPVSLGDQASISCRSS QSLVHNNGNTY LHWYLQKPGQSPKLLIY KVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPYS FGGGTKLEIK Sequence number 8 Heavy chain variable region
Base sequence CAGGTGCAGCTGAAGGAGAGCGGCCCCGGCCTGGTGCAGCCCAGCCAGAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAGCCTGACCATCTACGGGCGTGCACTGGATCAGGCAGAGCCCCGGCAAGGGCCTGGAGTGGCTGGGCGTGATGTGGAGCGGCGGCAGCACCGACT ACAACGCCGCCTTCATCAGCAGGCTGAGCATCAGCAAGGACAACAGCAAGAGCCAGGTTGTTCTTCAAGATGAACAGCCTGCAGGCCAACGACACCGCCATCTACTACTGCGCCAGGAACGACGGCTACTACTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC Sequence number 9 Light chain variable region
Base sequence GACGTGCTGATGACCCAGACCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGGTGCACAACAACGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTG AGCAACAGGTTCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTTCTGCAGCCAGAGCACCCACGTGCCCTACAGCTTCGGCGGCGGCACCAAGCTGGAGATCAAG Sequence number 10

4F5 Antibody-Based Humanized Antibody Manufacturing

A humanized antibody was prepared by changing the 4F5 antibody selected in Example 1 above into a structure corresponding to a human.

Specifically, a humanized mouse 4F5 antibody was produced by the CDR grafting method, which replaces the CDR of a mouse antibody that binds to CD22 with the CDR of a human antibody in the frame of the germline sequence of the human antibody. The humanized antibodies were named 4F5 (V4) antibody and 4F5 (V11) antibody, and their amino acid sequences were analyzed. The sequence information for the heavy chain variable region and the light chain variable region of the antibodies according to the sequence analysis results are shown in Tables 2 and 3 below, and the underlined parts in Tables 2 and 3 indicate the complementarity determining region (CDR).

Sequence information of 4F5(V4) antibody 4F5(V4) Sequence information Sequence number Heavy chain variable region CDR1 GFSLTIYG Sequence number 1 Heavy chain variable region CDR2 MWSGGST Sequence number 2 Heavy chain variable region CDR3 ARNDGYYWFAY Sequence number 3 Light chain variable region CDR1 QSLVHNNGNTY Sequence number 4 Light chain variable region CDR2 KVS Sequence number 5 Light chain variable region CDR3 SQSTHVPYS Sequence number 6 Heavy chain variable region
Amino acid sequence QVQLQESGPGLVKPSQTLSLTCTVS GFSLTIYG VHWIRQPPGKGLEWLGV MWSGGST DYNAALKSRVTISKDNSKSQVSLKLSSVTAADTAVYYC ARNDGYYWFAY WGQGTLVTVSS Sequence number 11 Light chain variable region
Amino acid sequence DVVMTQSPDSLAVSLGERATINCKSS QSLVHNNGNTY LHWYQQKPGQPPKLLIY KVS NRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC SQSTHVPYS FGGGTKLEIK Sequence number 12 Heavy chain variable region
Base sequence CAGGTGCAGCTTCAGGAGAGCGGACCCGGTCTCGTGAAACCCAGCCAGACTTTGTCCCTGACCTGCACTGTCAGCGGCTTTTCCCTAACCATTTACGGCGTCCATTGGATCCGCCAGCCACCTGGCAAGGGGCTGGAATGGCTGGGCGTGATGTGGTCTGGGGGATCCACCGACT ATAACGCGGCTCTGAAGTCCCGGGTGACCATCTCCAAGGACAACAGCAAGAGTCAAGTCAGCCTTAAACTGAGCTCCGTTACAGCCGCGGACACCGCTGTCTACTACTGTGCGCGCAATGACGGCTATTACTGGTTCGCCTACTGGGGCCAGGGCACTCTGGTGACCGTGTCCTCG Sequence number 13 Light chain variable region
Base sequence GATGTGGTGATGACCCAGAGTCCCGATTCTCTGGCAGTTTCTTTAGGCGAGCGTGCCACCATTAACTGCAAAAGCTCCCAGTCTCTGGTGCACAACAACGGCAATACCTACCTGCACTGGTACCAGCAGAAGCCGGGGCAGCCACCTAAGCTGCTGATCTACAAGGTG TCCAACCGCTTCTCTGGTGTCCCCGACAGGTTTTCTGGCTCAGGCAGCGGCACAGACTTCACCCTCACGATCTCCTCCCTCCAGGCCGAGGACGTGGCCGTGTACTACTGTTCACAGTCGACCCACGTACCGTATTCCTTCGGAGGAGGCACGAAGTTGGAGATCAAG Sequence number 14

Sequence information of 4F5(V11) antibody 4F5(V11) Sequence information Sequence number Heavy chain variable region CDR1 GFSLTIYG Sequence number 1 Heavy chain variable region CDR2 MWSGGST Sequence number 2 Heavy chain variable region CDR3 ARNDGYYWFAY Sequence number 3 Light chain variable region CDR1 QSLVHNNGNTY Sequence number 4 Light chain variable region CDR2 KVS Sequence number 5 Light chain variable region CDR3 SQSTHVPYS Sequence number 6 Heavy chain variable region
Amino acid sequence QVQLKESGPVLVKPTETLTTLTCTVS GFSLTIYG VHWIRQPPGKALEWLGV MWSGGST DYNAALKSRLTISKDNSKSQVVLTMTNMDPVDTATYYC ARNDGYYWFAY WGQGTLVTVSS Sequence number 15 Light chain variable region
Amino acid sequence DVVMTQSPDSLAVSLGERATINCKSS QSLVHNNGNTY LHWYQQKPGQPPKLLIY KVS NRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFC SQSTHVPYS FGGGTKLEIK Sequence number 16 Heavy chain variable region
Base sequence CAGGTGCAGCTGAAGGAGAGTGGTCCTGTCCTGGTGAAGCCTACCGAGACTCTTACACTGACCTGCACCGTGTCCGGCTTCTCTCTCACCATCTACGGGCGTCCACTGGATCCGCCAGCCACCCGGCAAGGCCCTGGAATGGCTAGGCGTGATGTGGTCTGGCGGCTCCACCGACT ACAACGCTGCGCTCAAGTCCCGGCTGACGATCTCTAAGGACAACTCCAAGTCGCAGGTCGTGCTCACTATGACTAACATGGATCCAGTGGACACCGCCACCTACTACTGTGCTCGCAACGACGGGTACTATTGGTTCGCCTATTGGGGCCAGGGCACCTTGGTCACCGTCTCCTCC Sequence number 17 Light chain variable region
Base sequence GATGTGGTGATGACCCAGAGCCCCGACAGCCTGGCCGTATCTCTGGGCGAGCGTGCAACCATTAACTGCAAAAGCTCCCAGAGCTTGGTGCACAACAATGGCAACACGTACCTGCATTGGTACCAGCAGAAGCCGGGACAGCCGCCTAAGCTGCTGATTTACAAGGTG TCAAATAGGTTTTCAGGAGTTCCCGACCGCTTCTCCGGGTCGGGTTCTGGAACAGATTTCACGCTTACCATCTCCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTTTTGTAGTCAATCGACCCACGTTCCCTACAGCTTCGGAGGCGGTACTAAACTGGAGATCAAG Sequence number 18

Confirmation of the specificity of the selected antibodies to CD22

In the present invention, to confirm the specificity of the 4F5 antibody (mouse) of Example 1, the humanized 4F5 (V4) antibody and the 4F5 (V11) antibody of Example 2 for CD22, flow cytometry analysis was performed.

First, ^1x106 B-cell lymphoma U2932 cells expressing CD22 were reacted with 1 ㎍ of 4F5 antibody for 30 minutes, and then the surface was stained with a secondary antibody, and then measured using a flow cytometer.

PE-conjugated anti-CD22 antibody (PE-conjugated anti-CD22 antibody; Biolegend Inc., cat# 302506, USA) was used as a positive control, and PE-conjugated anti-mouse IgG antibody (PE-conjugated goat anti-mouse IgG; Biolegend Inc., cat# 405307, USA) was used as a secondary antibody.

As a result, as shown in Fig. 1, it was confirmed that all 4F5 antibodies, humanized 4F5(V4) antibodies, and 4F5(V11) antibodies specifically bind to cells expressing CD22.

Production of a chimeric antigen receptor (CD22-CAR) expression vector targeting CD22

In the present invention, a lentiviral vector (CD22-CAR lentivirus) expressing a chimeric antigen receptor (CAR) targeting CD22 was produced using the humanized 4F5 (V4) antibody and 4F5 (V11) antibody produced in Example 2.

As shown in the schematic diagram of Fig. 3,

EF1α promoter (SEQ ID NO: 29);

A polynucleotide encoding a signal peptide (SEQ ID NO: 30);

A polynucleotide encoding a CD22-binding domain (4F5(V4) antibody represented by the nucleotide sequence of SEQ ID NO: 26 or 4F5(V11) antibody represented by the nucleotide sequence of SEQ ID NO: 28);

A polynucleotide encoding a CD8 hinge region (SEQ ID NO: 31);

A polynucleotide encoding a transmembrane domain (SEQ ID NO: 32);

A polynucleotide encoding 4-1BB (co-stimulatory domain) (SEQ ID NO: 33);

A polynucleotide encoding CD3ζ (intracellular signaling domain) (SEQ ID NO: 34); and

CAR DNA consisting of a polynucleotide encoding WPRE (SEQ ID NO: 35) was synthesized in vitro and inserted into a third-generation lentiviral vector.

CD22-CAR lentivirus was produced by transfecting HEK293FT cells with lentiviral vector. For infection, Lipofectamine 3000 transfection kit (Invitrogen, cat# L3000-015) and Opti-MEM+GlutaMAX (gibco, cat# 51985-034) medium were used, and the vector and HEK293FT cells were incubated for 4 hours.

CD22-CAR-T cell manufacturing

In the present invention, the CD22-CAR lentiviral vector manufactured in Example 4 was used to transfect T cells to manufacture humanized anti-CD22 antibodies, 4F5 (V4) antibody and 4F5 (V11) antibody-based CD22-CAR-T cells, respectively.

Specifically, as shown in the schematic diagram in Fig. 4, peripheral blood mononuclear cells (PBMCs) were separated from blood, and then T cells were activated using T cell activation beads (T cell activation beads; Miltenyl Biotec, cat# 130-091-441). The CD22-CAR lentivirus manufactured in Example 4 was transduced into the activated T cells to manufacture 4F5 (V4) antibody-based CD22-CAR-T cells and 4F5 (V11) antibody-based CD22-CAR-T cells.

The CD22 peptide binding ability of CD22-CAR-T cells was confirmed by flow cytometry. The 4F5 (V4) antibody-based CD22-CAR-T cells and 4F5 (V11) antibody-based CD22-CAR-T cells manufactured above were classified into CD3, CD4 or CD8-activated CD22-CAR-T cells using anti-CD3, anti-CD4 and anti-CD8 antibodies, respectively, and then reacted with FITC-CD22 peptide, and the fluorescence intensity was measured using a FACS machine.

As a result, as shown in Fig. 5, it was confirmed that all CD22-CAR-T cells activated by CD3, CD4, or CD8 bound to the CD22 peptide.

Confirmation of the killing effect of CD22-CAR-T cells on CD22 expressing cells

In the present invention, the target cell killing effect by humanized 4F5 (V4) antibody-based CD22-CAR-T cells and 4F5 (V11) antibody-based CD22-CAR-T cells was confirmed.

K562 cells (human erythroleukemic cell line) that do not express CD22 and U2932 cells (B cell lymphoma) that express CD22 were used as target cells, and were mixed with CD22-CAR-T cells at ratios of 1:4, 1:2, 1:1, 1:0.5, and 1:0.25, respectively, and cultured for 8 hours, and then luminescence (CytoTox-Glo Cytotoxicity Assay, Promega, cat. NO G9291) was measured. The degree of cell death was calculated using the following mathematical equation 1 with the measured value.

[Mathematical formula 1]

% Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)]

Experimental: Luminescence values derived from the medium of target cell and CAR-T cell complex cultures

Effector Spontaneous: Luminescence derived from CAR-T cell-only medium

Target Spontaneous: Luminescence value derived from the medium of target cells only

Target Maximum: Luminescence value derived from 100% lysis of target cells (using lysis reagent)

As a result, as shown in Fig. 6, it was confirmed that humanized 4F5 (V4) antibody-based CD22-CAR-T cells and 4F5 (V11) antibody-based CD22-CAR-T cells specifically killed U2932 cells expressing CD22.

In the present invention, it was confirmed through the above experiments that humanized 4F5 (V4) antibody-based CD22-CAR-T cells and 4F5 (V11) antibody-based CD22-CAR-T cells specifically kill U2932 cells derived from diffuse large B-cell lymphoma.

That is, the humanized anti-CD22 antibodies of the present invention, 4F5(V4) antibody and 4F5(V11) antibody, the antibody-based chimeric antigen receptor, and CAR-T cells using the same can be usefully utilized as a composition for preventing or treating diseases related to B cell or CD22 expression.

Construction of a bispecific chimeric antigen receptor expression vector targeting CD19/CD22

A lentiviral vector (CD19xCD22-CAR lentivirus) expressing a bispecific chimeric antigen receptor targeting CD19 and CD22 was prepared using the same method as in Example 4 above.

As shown in the schematic diagram of Fig. 7,

EF1α promoter (SEQ ID NO: 29);

A polynucleotide encoding a signal peptide (SEQ ID NO: 30);

A polynucleotide encoding a CD19/CD22-binding domain;

A polynucleotide encoding a CD8 hinge region (SEQ ID NO: 31);

A polynucleotide encoding a transmembrane domain (SEQ ID NO: 32);

A polynucleotide encoding 4-1BB (co-stimulatory domain) (SEQ ID NO: 33);

A polynucleotide encoding CD3ζ (intracellular signaling domain) (SEQ ID NO: 34); and

CAR DNA consisting of a polynucleotide encoding WPRE (SEQ ID NO: 35) was synthesized in vitro and inserted into a third-generation lentiviral vector.

In the present invention, the CD19-binding domain used a known anti-CD19 antibody (FMC63), and the CD22-binding domain used the 4F5 (mouse) antibody, 4F5 (V4) antibody, and 4F5 (V11) antibody of the present invention.

The above CD19xCD22-binding domain is sequentially connected to a light chain variable region of an antibody that specifically binds to CD19 (CD19VL) - a heavy chain variable region of an antibody that specifically binds to CD22 (CD22VH) - a light chain variable region of an antibody that specifically binds to CD22 (CD22VL) - a heavy chain variable region of an antibody that specifically binds to CD19 (CD19VH) (LoopCAR), and the sequence information for the polynucleotide encoding them is as follows:

CD19x4F5 (mouse): CD19VL represented by the nucleotide sequence of SEQ ID NO: 50 - Linker represented by the nucleotide sequence of SEQ ID NO: 52 (linker 1 in FIG. 7) - CD22VH represented by the nucleotide sequence of SEQ ID NO: 9 - Linker represented by the nucleotide sequence of SEQ ID NO: 55 (linker 6 in FIG. 7) - CD22VL represented by the nucleotide sequence of SEQ ID NO: 10 - Linker represented by the nucleotide sequence of SEQ ID NO: 53 (linker 1 in FIG. 7) - CD19VH represented by the nucleotide sequence of SEQ ID NO: 49;

CD19x4F5(V4): CD19VL represented by the nucleotide sequence of SEQ ID NO: 50 - Linker represented by the nucleotide sequence of SEQ ID NO: 52 (linker 1 in FIG. 7) - CD22VH represented by the nucleotide sequence of SEQ ID NO: 13 - Linker represented by the nucleotide sequence of SEQ ID NO: 55 (linker 6 in FIG. 7) - CD22VL represented by the nucleotide sequence of SEQ ID NO: 14 - Linker represented by the nucleotide sequence of SEQ ID NO: 53 (linker 1 in FIG. 7) - CD19VH represented by the nucleotide sequence of SEQ ID NO: 49;

CD19x4F5(V11): CD19VL represented by the nucleotide sequence of SEQ ID NO: 50 - Linker represented by the nucleotide sequence of SEQ ID NO: 52 (linker 1 in FIG. 7) - CD22VH represented by the nucleotide sequence of SEQ ID NO: 17 - Linker represented by the nucleotide sequence of SEQ ID NO: 55 (linker 6 in FIG. 7) - CD22VL represented by the nucleotide sequence of SEQ ID NO: 18 - Linker represented by the nucleotide sequence of SEQ ID NO: 53 (linker 1 in FIG. 7) - CD19VH represented by the nucleotide sequence of SEQ ID NO: 49.

CD19/CD22-CAR lentivirus was produced by transfecting HEK293FT cells with lentiviral vector. For infection, Lipofectamine 3000 transfection kit (Invitrogen, cat# L3000-015) and Opti-MEM+GlutaMAX (gibco, cat# 51985-034) medium were used, and the vector and HEK293FT cells were incubated for 4 hours (Fig. 8 and Fig. 9).

As a result of confirming whether CD19xCD22-specific CAR was expressed in HEK293FT transfected with a lentiviral vector, it was confirmed that anti-CD19/anti-CD22 antibody expression occurred normally, as shown in Fig. 10.

CD19xCD22-CAR-T cell manufacturing

In the present invention, CD19xCD22-CAR lentiviral vector manufactured in Example 7 was used to transfect T cells in the same manner as in Example 5 to manufacture CD19xCD22-CAR-T cells, CD19x4F5, C19x4F5 (V4), and CD19x4F5 (V11), respectively (Figs. 11 and 12).

The CD22 peptide binding ability of CD19xCD22-CAR-T cells was confirmed by flow cytometry. CD19xCD22-CAR-T cells (CD19x4F5, CD19x 4F5 (V4), and CD19x4F5 (V11)) were each sorted into CD3-activated CD19xCD22-CAR-T cells using anti-CD3 antibody, and then reacted with PE-CD19 peptide and FITC-CD22 peptide, and the fluorescence intensity was measured using a flow cytometry machine.

As a result, as shown in Fig. 13, it was confirmed that both CD3-activated CD19xCD22-CAR-T cells simultaneously bound to CD19 peptide and CD22 peptide.

Confirmation of the killing effect of CD19xCD22-CAR-T cells on CD22 or CD19 expressing cells

In the present invention, three types of cells, K562-CD19, K562-CD22, and K562-CD19/CD22, expressing CD19, CD22, or CD19xCD22 were produced from K562 cells (ATCC, cat#CCL-243) that do not express CD19 and CD22, and the target cell killing effect by CD19xCD22-CAR-T cells was confirmed.

The target cells and CD19xCD22-CAR-T cells, CD19x4F5-CAR-T cells, CD19x4F5(V4)-CAR-T cells, and CD19x4F5(V11)-CAR-T cells were mixed in a ratio of 1:4, 1:2, 1:1, 1:0.5, and 1:0.25, respectively, and cultured for 8 hours, and then luminescence (CytoTox-Glo Cytotoxicity Assay, Promega, cat# G9291) was measured. The degree of cell death was calculated using the mathematical equation 1 of Example 6 with the measured value.

As a result, as shown in Fig. 14, it was confirmed that CD19x4F5-CAR-T cells, CD19x4F5(V4)-CAR-T cells, and CD19x4F5(V11)-CAR-T cells manufactured in the present invention specifically kill cells expressing CD22 or CD19 or CD19/CD22.

<110> InnoBation Bio Co., Ltd. <120> Humanized antibody specific for CD22 and chimeric antigen receptor using the same <130> - <160> 55 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 4F5_VH_CDR 1 <400> 1 Gly Phe Ser Leu Thr Ile Tyr Gly 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 4F5_VH_CDR 2 <400> 2 Met Trp Ser Gly Gly Ser Thr 1 5 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 4F5_VH_CDR 3 <400> 3 Ala Arg Asn Asp Gly Tyr Tyr Trp Phe Ala Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 4F5_VL_CDR 1 <400> 4 Gln Ser Leu Val His Asn Asn Gly Asn Thr Tyr 1 5 10 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 4F5_VL_CDR 2 <400> 5 Lys Val Ser 1 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 4F5_VL_CDR 3 <400> 6 Ser Gln Ser Thr His Val Pro Tyr Ser 1 5 <210> 7 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 4F5_VH <400> 7 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Val 85 90 95 Pro His Tyr Tyr Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 8 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 4F5_VL <400> 8 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Asn 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 9 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> 4F5_VH <400> 9 caggtgcagc tgaaggagag cggccccggc ctggtgcagc ccagccagag cctgagcatc 60 acctgcaccg tgagcggctt cagcctgacc atctacggcg tgcactggat caggcagagc 120 cccggcaagg gcctggagtg gctgggcgtg atgtggagcg gcggcagcac cgactacaac 180 gccgccttca tcagcaggct gagcatcagc aaggacaaca gcaagagcca ggtgttcttc 240 aagatgaaca gcctgcaggc caacgacacc gccatctact actgcgccag gaacgacggc 300 tactactggt tcgcctactg gggccagggc accctggtga ccgtgagcgc c 351 <210> 10 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 4F5_VL <400> 10 gacgtgctga tgacccagac ccccctgagc ctgcccgtga gcctgggcga ccaggccagc 60 atcagctgca ggagcagcca gagcctggtg cacaacaacg gcaacaccta cctgcactgg 120 tacctgcaga agcccggcca gagccccaag ctgctgatct acaaggtgag caacaggttc 180 agcggcgtgc ccgacaggtt cagcggcagc ggcagcggca ccgacttcac cctgaagatc 240 agcagggtgg aggccgagga cctgggcgtg tacttctgca gccagagcac ccacgtgccc 300 tacagcttcg gcggcggcac caagctggag atcaag 336 <210> 11 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 4F5(V4)_VH <400> 11 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ile Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Asp Gly Tyr Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 12 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 4F5(V4)_VL <400> 12 Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Val His Asn 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 13 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> 4F5(V4)_VH <400> 13 caggtgcagc ttcaggagag cggacccggt ctcgtgaaac ccagccagac tttgtccctg 60 acctgcactg tcagcggctt ttccctaacc atttacggcg tccattggat ccgccagcca 120 cctggcaagg ggctggaatg gctgggcgtg atgtggtctg ggggatccac cgactataac 180 gcggctctga agtcccgggt gaccatctcc aaggacaaca gcaagagtca agtcagcctt 240 aaactgagct ccgttacagc cgcgggacacc gctgtctact actgtgcgcg caatgacggc 300 tattactggt tcgcctactg gggccagggc actctggtga ccgtgtcctc g 351 <210> 14 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 4F5(V4)_VL <400> 14 gatgtggtga tgacccagag tcccgattct ctggcagttt ctttaggcga gcgtgccacc 60 attaactgca aaagctccca gtctctggtg cacaacaacg gcaataccta cctgcactgg 120 taccagcaga agccggggca gccacctaag ctgctgatct acaaggtgtc caaccgcttc 180 tctggtgtcc ccgacaggtt ttctggctca ggcagcggca cagacttcac cctcacgatc 240 tcctccctcc aggccgagga cgtggccgtg tactactgtt cacagtcgac ccacgtaccg 300 tattccttcg gaggaggcac gaagttggag atcaag 336 <210> 15 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 4F5(V11)_VH <400> 15 Gln Val Gln Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ile Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Val Leu 65 70 75 80 Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Asn Asp Gly Tyr Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 16 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 4F5(V11)_VL <400> 16 Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Val His Asn 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 17 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> 4F5(V11)_VH <400> 17 caggtgcagc tgaaggagag tggtcctgtc ctggtgaagc ctaccgagac tcttacactg 60 acctgcaccg tgtccggctt ctctctcacc atctacggcg tccactggat ccgccagcca 120 cccggcaagg ccctggaatg gctaggcgtg atgtggtctg gcggctccac cgactacaac 180 gctgcgctca agtcccggct gacgatctct aaggacaact ccaagtcgca ggtcgtgctc 240 actatgacta acatggatcc agtggacacc gccacctact actgtgctcg caacgacggg 300 tactattggt tcgcctattg gggccagggc accttggtca ccgtctcctc c 351 <210> 18 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 4F5(V11)_VL <400> 18 gatgtggtga tgacccagag ccccgacagc ctggccgtat ctctgggcga gcgtgcaacc 60 attaactgca aaagctccca gagcttggtg cacaacaatg gcaacacgta cctgcattgg 120 taccagcaga agccgggaca gccgcctaag ctgctgattt acaaggtgtc aaataggttt 180 tcaggagttc ccgaccgctt ctccgggtcg ggttctggaa cagatttcac gcttaccatc 240 tccagcctgc aggccgagga cgtggccgtg tacttttgta gtcaatcgac ccacgttccc 300 tacagcttcg gaggcggtac taaactggag atcaag 336 <210> 19 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> scfv linker <400> 19 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 20 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> scfv linker <400> 20 ggtggtggtg gttcgggtgg tggtggttcg ggtggtggtg gttcg 45 <210> 21 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> scfv linker <400> 21 ggtggcgggg ggtctggcgg tggtggatcc ggtggtggcg gctcg 45 <210> 22 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> scfv linker <400> 22 ggcggtggcg ggagcggcgg gggtgggtcc ggtggcggcg gctct 45 <210> 23 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> 4F5 scfv <400> 23 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Asn 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser 130 135 140 Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ile Tyr Gly 145 150 155 160 Val His Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly 165 170 175 Val Met Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile Ser 180 185 190 Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys 195 200 205 Met Asn Ser Leu Gln Ala Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Arg 210 215 220 Asn Asp Gly Tyr Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ala <210> 24 <211> 732 <212> DNA <213> Artificial Sequence <220> <223> 4F5 scfv <400> 24 gacgtgctga tgacccagac ccccctgagc ctgcccgtga gcctgggcga ccaggccagc 60 atcagctgca ggagcagcca gagcctggtg cacaacaacg gcaacaccta cctgcactgg 120 tacctgcaga agcccggcca gagccccaag ctgctgatct acaaggtgag caacaggttc 180 agcggcgtgc ccgacaggtt cagcggcagc ggcagcggca ccgacttcac cctgaagatc 240 agcagggtgg aggccgagga cctgggcgtg tacttctgca gccagagcac ccacgtgccc 300 tacagcttcg gcggcggcac caagctggag atcaagggtg gtggtggttc gggtggtggt 360 ggttcgggtg gtggtggttc gcaggtgcag ctgaaggaga gcggccccgg cctggtgcag 420 cccagccaga gcctgagcat cacctgcacc gtgagcggct tcagcctgac catctacggc 480 gtgcactgga tcaggcagag ccccggcaag ggcctggagt ggctgggcgt gatgtggagc 540 ggcggcagca ccgactacaa cgccgccttc atcagcaggc tgagcatcag caaggacaac 600 agcaagagcc aggtgttctt caagatgaac agcctgcagg ccaacgacac cgccatctac 660 tactgcgcca ggaacgacgg ctactactgg ttcgcctact ggggccaggg caccctggtg 720 accgtgagcg cc 732 <210> 25 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> 4F5(V4) scfv <400> 25 Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Val His Asn 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr 130 135 140 Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ile Tyr Gly 145 150 155 160 Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly 165 170 175 Val Met Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Leu Lys Ser 180 185 190 Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu Lys 195 200 205 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Asn Asp Gly Tyr Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser <210> 26 <211> 732 <212> DNA <213> Artificial Sequence <220> <223> 4F5(V4) scfv <400> 26 gatgtggtga tgacccagag tcccgattct ctggcagttt ctttaggcga gcgtgccacc 60 attaactgca aaagctccca gtctctggtg cacaacaacg gcaataccta cctgcactgg 120 taccagcaga agccggggca gccacctaag ctgctgatct acaaggtgtc caaccgcttc 180 tctggtgtcc ccgacaggtt ttctggctca ggcagcggca cagacttcac cctcacgatc 240 tcctccctcc aggccgagga cgtggccgtg tactactgtt cacagtcgac ccacgtaccg 300 tattccttcg gaggaggcac gaagttggag atcaagggtg gcggggggtc tggcggtggt 360 ggatccggtg gtggcggctc gcaggtgcag cttcaggaga gcggacccgg tctcgtgaaa 420 cccagccaga ctttgtccct gacctgcact gtcagcggct tttccctaac catttacggc 480 gtccattgga tccgccagcc acctggcaag gggctggaat ggctgggcgt gatgtggtct 540 gggggatcca ccgactataa cgcggctctg aagtcccggg tgaccatctc caaggacaac 600 agcaagagtc aagtcagcct taaactgagc tccgttacag ccgcggacac cgctgtctac 660 tactgtgcgc gcaatgacgg ctattactgg ttcgcctact ggggccaggg cactctggtg 720 accgtgtcct cg 732 <210> 27 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> 4F5(V11) scfv <400> 27 Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Val His Asn 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr 130 135 140 Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ile Tyr Gly 145 150 155 160 Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly 165 170 175 Val Met Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Leu Lys Ser 180 185 190 Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu Lys 195 200 205 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Asn Asp Gly Tyr Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser <210> 28 <211> 732 <212> DNA <213> Artificial Sequence <220> <223> 4F5(V11) scfv <400> 28 gatgtggtga tgacccagag tcccgattct ctggcagttt ctttaggcga gcgtgccacc 60 attaactgca aaagctccca gtctctggtg cacaacaacg gcaataccta cctgcactgg 120 taccagcaga agccggggca gccacctaag ctgctgatct acaaggtgtc caaccgcttc 180 tctggtgtcc ccgacaggtt ttctggctca ggcagcggca cagacttcac cctcacgatc 240 tcctccctcc aggccgagga cgtggccgtg tactactgtt cacagtcgac ccacgtaccg 300 tattccttcg gaggaggcac gaagttggag atcaagggtg gcggggggtc tggcggtggt 360 ggatccggtg gtggcggctc gcaggtgcag cttcaggaga gcggacccgg tctcgtgaaa 420 cccagccaga ctttgtccct gacctgcact gtcagcggct tttccctaac catttacggc 480 gtccattgga tccgccagcc acctggcaag gggctggaat ggctgggcgt gatgtggtct 540 gggggatcca ccgactataa cgcggctctg aagtcccggg tgaccatctc caaggacaac 600 agcaagagtc aagtcagcct taaactgagc tccgttacag ccgcggacac cgctgtctac 660 tactgtgcgc gcaatgacgg ctattactgg ttcgcctact ggggccaggg cactctggtg 720 accgtgtcct cg 732 <210> 29 <211> 1178 <212> DNA <213> Artificial Sequence <220> <223> EF1 promoter <400> 29 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900 cctcagccgt cgcttcatgt gactccactg agtaccgggc gccgtccagg cacctcgatt 960 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140 tggttcaaag tttttttctt ccatttcagg tgtcgtga 1178 <210> 30 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> signal peptide <400> 30 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 31 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> CD8 Hinge <400> 31 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 32 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> transmembrane domain <400> 32 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 accctttact gc 72 <210> 33 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> 4-1BB <400> 33 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 34 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> CD3 zeta <400> 34 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 35 <211> 591 <212> DNA <213> Artificial Sequence <220> <223> WPRE <400> 35 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc 60 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta 120 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt 180 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg 240 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta 300 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt 360 tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg 420 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca 480 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc 540 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct g 591 <210> 36 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> signal peptide <400> 36 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 37 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> CD8 Hinge <400> 37 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 38 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> transmembrane domain <400> 38 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 39 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> 4-1BB <400> 39 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 40 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD3 zeta <400> 40 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 41 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD19_VH_CDR1 <400> 41 Gly Val Ser Leu Pro Asp Tyr Gly 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CD19_VH_CDR2 <400> 42 Ile Trp Gly Ser Glu Thr Thr 1 5 <210> 43 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CD19_VH_CDR3 <400> 43 Ala Lys His Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 1 5 10 <210> 44 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CD19_VL_CDR1 <400> 44 Gln Asp Ile Ser Lys Tyr 1 5 <210> 45 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CD19_VL_CDR2 <400> 45 His Thr Ser 1 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CD19_VL_CDR3 <400> 46 Gln Gln Gly Asn Thr Leu Pro Tyr Thr 1 5 <210> 47 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> CD19_VH <400> 47 Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 48 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CD19_VL <400> 48 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 100 105 <210> 49 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> CD19_VH <400> 49 gaggtgaaac tccaggagag cggccccggg ctggtggctc ctagtcagtc tttgtcagtg 60 acttgcaccg tgtccggagt gtcgttgcct gattacggtg tcagctggat ccgccagccg 120 ccgcgcaagg gtttggaatg gctcggcgtt atatggggct cggagaccac atactacaac 180 tccgcgctga agtcccgtct gaccatcatc aaggacaact ccaagagcca ggtgttcctg 240 aagatgaaca gcctgcagac agatgacacg gccatctatt actgtgccaa gcattactac 300 tacggcggca gttacgccat ggattattgg ggccagggta catccgtcac cgtgtcgagc 360 360 <210> 50 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> CD19_VL <400> 50 gacatccaga tgacccagac gaccagctcc ttaagcgcat cccttggaga cagggtcacc 60 atctcatgcc gagcgtcaca ggacatctcc aagtacctca actggtacca gcagaagccc 120 gacggcaccg tgaagctgct gatttaccat acttcccgcc tgcactcggg cgtgccatcc 180 cgcttctcgg gctcggggtc gggtaccgac tactctctca ccatttccaa cctggaacag 240 gaggatatcg ccacctattt ctgtcaacag ggcaacacgc taccctatac cttcggcggt 300 ggcaccaaac tggagattac c 321 <210> 51 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Bi-specific CART linker <400> 51 Gly Gly Gly Gly Ser 1 5 <210> 52 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Bi-specific CART linker <400> 52 ggcggggggag gctct 15 <210> 53 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Bi-specific CART linker <400> 53 ggcggagggg gctcc 15 <210> 54 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Bi-specific CART linker <400> 54 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly <210> 55 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Bi-specific CART linker<400> 55 ggctccacca gtggttctgg gaaacctggt agtggggagg gctcgacgaa gggg 54

Claims (24)

An antibody or fragment thereof that specifically binds to CD22, comprising a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 6.
An antibody or fragment thereof that specifically binds to CD22, characterized in that in claim 1, the antibody that specifically binds to CD22 is composed of a heavy chain variable region represented by an amino acid sequence number 7 and a light chain variable region represented by an amino acid sequence number 8.
In the first paragraph, the antibody specifically binding to CD22 is a humanized antibody specifically binding to CD22 consisting of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12; or
An antibody or fragment thereof that specifically binds to CD22, characterized in that it is a humanized antibody that specifically binds to CD22, comprising a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.
A polynucleotide encoding an antibody or fragment thereof that specifically binds to CD22 of any one of claims 1 to 3.
A vector comprising a polynucleotide encoding an antibody or fragment thereof that specifically binds to CD22 of any one of claims 1 to 3.
A recombinant cell producing an antibody or fragment thereof that specifically binds to CD22 transformed with the vector of claim 5.
CD22-binding domain;
transmembrane domain;
costimulatory domain; and
A chimeric antigen receptor (CAR) containing an intracellular signal transduction domain,
A chimeric antigen receptor targeting CD22, characterized in that the CD22-binding domain is an antibody or a fragment thereof that specifically binds to CD22, comprising a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 6.
In the seventh paragraph, the antibody that specifically binds to CD22
(a) an antibody that specifically binds to CD22, comprising a heavy chain variable region represented by an amino acid sequence of SEQ ID NO: 7 and a light chain variable region represented by an amino acid sequence of SEQ ID NO: 8;
(b) a humanized antibody that specifically binds to CD22, comprising a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12; or
(c) A chimeric antigen receptor targeting CD22, characterized in that it is a humanized antibody that specifically binds to CD22, comprising a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.
In claim 7, the transmembrane domain is a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.
The co-stimulatory domain is a protein selected from the group consisting of CD28, 4-1BB, OX-40, and ICOS.
A chimeric antigen receptor targeting CD22, characterized in that the signaling domain is CD3ζ.
A chimeric antigen receptor targeting CD22, characterized in that in claim 7, a hinge region is additionally included between the C-terminus of the binding domain and the N-terminus of the transmembrane domain.
A polynucleotide encoding a chimeric antigen receptor according to any one of claims 7 to 10.
A vector comprising a polynucleotide encoding a chimeric antigen receptor according to any one of claims 7 to 10.
A polynucleotide encoding a chimeric antigen receptor according to any one of claims 7 to 10; or
An immune effector cell comprising a vector comprising a polynucleotide encoding a chimeric antigen receptor according to any one of claims 7 to 10.
CD19-binding domain and CD22-binding domain;
transmembrane domain;
costimulatory domain; and
A bispecific chimeric antigen receptor (CAR) containing an intracellular signal transduction domain,
A bispecific chimeric antigen receptor targeting CD19 and CD22, characterized in that the CD22-binding domain is an antibody or a fragment thereof that specifically binds to CD22, comprising a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 6.
In claim 14, the CD19-binding domain and the CD22-binding domain are
A bispecific chimeric antigen receptor targeting CD19 and CD22, characterized in that the light chain variable region of an antibody that specifically binds to CD19 - the heavy chain variable region of an antibody that specifically binds to CD22 - the light chain variable region of an antibody that specifically binds to CD22 - the heavy chain variable region of an antibody that specifically binds to CD19 are linked in this order.
In claim 15, the light chain variable region of the antibody specifically binding to CD19 is represented by the amino acid sequence of SEQ ID NO: 48,
A bispecific chimeric antigen receptor targeting CD19 and CD22, characterized in that the heavy chain variable region of the antibody specifically binding to CD19 is represented by the amino acid sequence of SEQ ID NO: 47.
In claim 14, the transmembrane domain is a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.
The co-stimulatory domain is a protein selected from the group consisting of CD28, 4-1BB, OX-40, and ICOS.
A bispecific chimeric antigen receptor targeting CD19 and CD22, characterized in that the signaling domain is CD3ζ.
A bispecific chimeric antigen receptor targeting CD19 and CD22, characterized in that a hinge region is additionally included between the C-terminus of the binding domain and the N-terminus of the transmembrane domain in claim 14.
A polynucleotide encoding a bispecific chimeric antigen receptor according to any one of claims 14 to 18.
A vector comprising a polynucleotide encoding a bispecific chimeric antigen receptor according to any one of claims 14 to 18.
A polynucleotide encoding a bispecific chimeric antigen receptor according to any one of claims 14 to 18; or
An immune effector cell comprising a vector comprising a polynucleotide encoding a bispecific chimeric antigen receptor according to any one of claims 14 to 18.
A pharmaceutical composition for preventing or treating a disease mediated by B cells, comprising an antibody or a fragment thereof that specifically binds to CD22 of the first clause,
A pharmaceutical composition for preventing or treating a disease mediated by B cells, characterized in that the disease mediated by B cells is selected from the group consisting of tumor, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt's lymphoma, and mantle cell lymphoma.
A polynucleotide encoding a chimeric antigen receptor according to any one of claims 7 to 10; or
A pharmaceutical composition for the prevention or treatment of a disease mediated by B cells comprising immune effector cells, comprising a vector comprising a polynucleotide encoding a chimeric antigen receptor according to any one of claims 7 to 10,
A pharmaceutical composition for preventing or treating a disease mediated by B cells, characterized in that the disease mediated by B cells is selected from the group consisting of tumor, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt's lymphoma, and mantle cell lymphoma.
A polynucleotide encoding a bispecific chimeric antigen receptor according to any one of claims 14 to 18; or
A pharmaceutical composition for the prevention or treatment of a disease mediated by B cells comprising immune effector cells, comprising a vector comprising a polynucleotide encoding a bispecific chimeric antigen receptor according to any one of claims 14 to 18,
A pharmaceutical composition for preventing or treating a disease mediated by B cells, characterized in that the disease mediated by B cells is selected from the group consisting of tumor, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt's lymphoma, and mantle cell lymphoma.