WO2022181992A1 - Cd22-specific humanized antibody and use thereof - Google Patents

Abstract

The present invention relates to a humanized antibody specific to CD22 and a chimeric antigen receptor using same and, more specifically, to a humanized antibody that specifically binds to CD22, a chimeric antigen receptor comprising the antibody, a CAR-T cell expressing the chimeric antigen receptor, and a pharmaceutical composition for preventing or treating a disease mediated by CD22-expressing cells, comprising the antibody, the receptor, and the cell. In the present invention, a humanized anti-CD22 antibody was prepared by humanizing a CD22-binding antibody to reduce immune responses in the human body, and CD22-targeting CAR-T cells were prepared using same. Humanized anti-CD22 antibody-based CD22-CAR-T cells prepared in the present invention were confirmed not only to effectively bind to CD22, but also to effectively kill CD22-expressing cells. Therefore, the humanized anti-CD22 antibody-based CD22-CAR-T cells of the present invention can be effectively used as a composition for preventing or treating CD22 expression-associated diseases.

Description

Humanized antibodies specific for CD22 and uses thereof

The present invention relates to a humanized antibody specific for CD22 and a chimeric antigen receptor using the same, and more particularly, to a humanized antibody specifically binding to CD22, a chimeric antigen receptor comprising the antibody, and a chimeric antigen receptor expressing the chimeric antigen receptor It relates to a pharmaceutical composition for preventing or treating diseases mediated by CAR-T cells and cells expressing CD22 including them.

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

For the treatment or diagnosis of such CD22 expression-related diseases, CD22-specific antibodies have been 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 antibiotic for the treatment of B-cell malignancies. -CD22 antibody is disclosed.

As described above, monoclonal antibodies are mainly produced using mice for the production of antibodies for treatment. However, since non-human antibodies such as mouse-derived monoclonal antibodies are considered foreign antigens in the human body, they induce an immune response and have a limited therapeutic effect because of their short half-life.

In order to solve the above problem, a humanized antibody has been developed in which the rest of the antibody except for the antigen-binding site is substituted with a human antibody. As a method of replacing a mouse antibody with a humanized antibody currently used, a human antibody gene most similar to the antibody to be replaced is selected, and only the CDR regions of a mouse antibody are replaced with human antibody CDR positions by a method called CDR grafting. These humanized antibodies have the advantage of reducing the immune response in the human body because most of the genes are humanized.

In the present invention, an antibody that binds to CD22 is selected in order to reduce the immune response in the human body, and a humanized anti-CD22 antibody was prepared using this, and a chimeric antigen targeting CD22 using the humanized anti-CD22 antibody of the present invention Receptor and CAR-T cells were prepared.

Accordingly, it is an object of the present invention to provide a humanized antibody specific for CD22.

Another object of the present invention is to provide 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 the humanized anti-CD22 antibody.

Another object of the present invention is to provide a polynucleotide encoding the chimeric antigen receptor, a vector comprising the same, and immune effector cells expressing the chimeric antigen receptor comprising the polynucleotide or vector.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases mediated by cells expressing CD22, including the immune effector cells.

Another object of the present invention is to provide a composition for diagnosing or monitoring a disease mediated by a cell expressing CD22 comprising the antibody.

In order to achieve the above object,

The present invention provides 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.

To achieve another object, the present invention provides a polynucleotide encoding the antibody that specifically binds to CD22.

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

In addition, the present invention provides a recombinant cell that produces an antibody or fragment thereof that specifically binds to CD22 transformed with the vector.

To achieve another object, the present invention provides a CD22-binding domain; transmembrane domain; costimulatory domain; and a chimeric antigen receptor (CAR) comprising 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

It may be 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 may be derived from a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

In another preferred embodiment of the present invention, the costimulatory domain may be derived from 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, it may further include a hinge region located between the C-terminus of the CD22-binding domain and the N-terminus of the transmembrane domain, wherein the hinge region is CD8α may be of origin.

To achieve another object, the present invention provides a polynucleotide encoding the chimeric antigen receptor (CAR).

The present invention also provides a vector comprising a polynucleotide encoding a chimeric antigen receptor (CAR).

In a preferred embodiment of the present invention, the vector may be a plasmid, a retroviral vector or a lentiviral vector.

In addition, the present invention provides an immune effector cell comprising the polynucleotide encoding the chimeric antigen receptor (CAR) or a polynucleotide encoding the chimeric antigen receptor (CAR), and expressing the chimeric antigen receptor (CAR). do.

In a preferred embodiment of the present invention, the immune effector cells may be T cells, B cells, NK cells, dendritic cells, bone marrow cells, mononuclear cells, or macrophages.

In order to achieve another object, the present invention provides an immune effector cell expressing the chimeric antigen receptor targeting CD22; Or it provides a pharmaceutical composition for preventing or treating a disease mediated by a cell expressing CD22, comprising a humanized antibody or fragment thereof that specifically binds to CD22.

In a preferred embodiment of the present invention, the disease mediated by cells expressing CD22 is lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed delayed NHL, refractory Sexual NHL, refractory delayed 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 another preferred embodiment of the present invention, the composition may include a therapeutic agent for a disease mediated by cells expressing CD22.

In the present invention, a humanized anti-CD22 antibody was prepared by humanizing an antibody that binds to CD22 in order to reduce the immune response in the human body, and CAR-T cells targeting CD22 were prepared using the humanized antibody. It was confirmed that the CD22-CAR-T cells prepared in the present invention not only effectively bound to CD22, but also activated the CD22-bound CAR-T cells, and it was confirmed that the CD22-expressing cells were effectively killed. Therefore, the humanized anti-CD22 antibody-based CD22-CAR-T cells of the present invention can be usefully used as a composition for preventing or treating diseases related to CD22 expression.

1 is data confirming the binding affinity to CD22 of 1C2 antibody, humanized 1C2-V9 antibody and 1C2-V12 antibody selected in the present invention by flow cytometry.

2 is a schematic diagram showing a chimeric antigen receptor (single CAR) targeting CD22.

3 is a schematic diagram illustrating a method for preparing CD22-CAR-expressing cells using a lentiviral vector expressing CD22-CAR.

4 is a schematic diagram showing (A) a process for preparing HEK293 cells transformed with a CD22-CAR-expressing lentiviral vector and (B) a method for confirming the antigen-binding ability of the transformed CD22-CAR-expressing HEK293 cells.

FIG. 5 shows CD22-CAR expression and binding ability to CD22 in HEK293 cells transformed with a lentiviral vector expressing CD22-CAR based on humanized anti-CD22 antibodies (1C2-V9 and 1C2-V12). ) is the confirmed data.

6 is a schematic diagram illustrating a method for preparing CD22-CAR-expressing T cells using a lentiviral vector expressing CD22-CAR.

7 is a schematic diagram showing (A) a process for producing T cells using CD22-CAR and (B) a method for confirming the antigen-binding ability of the produced T cells.

Figure 8 shows the antigen (CD22) binding ability of CD22-CAR-T cells based on humanized anti-CD22 antibodies (1C2-V9 and 1C2-V12). CD3, CD4 and CD8 activated CD22-CAR-T cells, respectively. and the data confirming the binding ability of the CD22 peptide.

9 is data confirming the killing effect of U2932 cells (CD22 expressing cells) and K562 cells (CD22 non-expressing cells) by humanized anti-CD22 antibody (1C2-V9 and 1C2-V12)-based CD22-CAR-T cells. .

10 is data confirming the killing effect of NALM6 cells (CD22 expressing cells) and K562 cells (CD22 non-expressing cells) by humanized anti-CD22 antibody (1C2-V9 and 1C2-V12) based CD22-CAR-T cells. .

Hereinafter, the present invention will be described in detail.

Humanized Antibodies that Specific Binding to CD22

The present invention provides 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, which consists 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 term "humanized antibody" refers to an antibody with increased similarity to a human antibody by making the remaining parts except for the CDR region, which is a key part for antigen binding, an amino acid sequence corresponding to an antibody produced by humans. do. The most common method for humanizing an antibody (non-human antibody) is a CDR-grafting method in which the CDR region of an animal antibody is grafted into a human antibody, but is not limited thereto, and methods known in the art can be used. can be used to prepare humanized antibodies.

In the present invention, the antibody may be a monoclonal antibody. In the present invention, the term "monoclonal antibody" is also called a monoclonal antibody or monoclonal antibody, and is an antibody produced by a single antibody-forming cell, and has a uniform primary structure (amino acid sequence). It recognizes only one antigenic determinant and is generally produced by culturing a hybridoma cell in which cancer cells and antibody-producing cells are fused.

As used herein, the term "CDR", ie, "complementarity determining region", refers to a non-contiguous antigen binding site found within the variable region of both the heavy and light chain regions.

In the present invention, the term “antibody” can be used not only in a complete form having two full-length light chains and two full-length heavy chains, but also as a fragment of an antibody molecule. A fragment of an antibody molecule refers to a fragment having at least a peptide tag (epitope) binding function, and includes scFv, Fab, F(ab'), F(ab') ₂ , a single domain, and the like.

Among the antibody fragments, Fab has a structure having variable regions of light and heavy chains, a constant region of a light chain and a first constant region (CH1) of a heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C terminus of the heavy chain CH1 domain. The F(ab') ₂ antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. A double chain Fv (dsFv) has a heavy chain variable region and a light chain variable region connected by a disulfide bond, and a single chain Fv (scFv) is generally a peptide linker. The variable region of the heavy chain and the variable region of the light chain are linked by a covalent bond. Such antibody fragments may be obtained using proteolytic enzymes, or preferably produced through genetic recombination techniques.

The monoclonal antibody that specifically binds to CD22 of the present invention can be prepared by using all or part of the CD22 protein as an immunogen (or antigen). More specifically, first, as an immunogen, CD22, a fusion protein containing CD22 protein, or a carrier containing CD22 protein is prepared with an adjuvant (eg, Freund adjuvant) as an adjuvant (eg, Freund adjuvant), except for humans. Immunization is achieved by subcutaneous, intramuscular, intravenous, bolus or intraperitoneal injection of one or more injections in mammals. The mammals other than humans are preferably mice, rats, hamsters, malmots, chickens, rabbits, cats, dogs, pigs, goats, sheep, donkeys, horses or cattle (transgenic mice that produce human antibodies) (including transgenic animals engineered to produce antibodies from other animals such as From the first immunization, immunization is performed 1 to 4 times every 1 to 21 days, and antibody-producing cells can be obtained from the immune-sensitized mammal about 1 to 10 days after the final immunization. The number of times and time intervals for immunization can be appropriately changed depending on the characteristics of the immunogen to be used, and the like.

Preparation of a hybridoma secreting a monoclonal antibody can be carried out according to the method of Keira and Mirstein et al. (Nature, 1975, Vol. 256, p. 495-497) and a method similar thereto. Any one selected from the group consisting of spleen, lymph node, bone marrow, or tonsils collected from animals other than humans who have been immunosensitized as described above, preferably, antibody-producing cells contained in the spleen and derived from mammals without the ability to produce autoantibodies Hybridomas can be prepared by cell fusion of myeloma cells of The mammal may be a mouse, a rat, a guinea pig, a hamster, a chicken, a rabbit or a human, preferably a mouse, a rat, a chicken or a human.

For cell fusion, for example, a fusion promoter such as polyethylene glycol or Sendai virus, or a method by electric pulse is used. For example, an antibody-producing cell and a mammalian-derived cell that can proliferate indefinitely in a fusion medium containing a fusion promoter is used. is suspended in a ratio of about 1:1 to 1:10, and in this state, incubated at about 30 to 40° C. for about 1 to 5 minutes. As the fusion medium, for example, MEM medium, RPMI1640 medium, and Iscove's Modified Dulbecco's Medium may be used, and it is preferable to exclude sera such as bovine serum.

In the method for screening the hybridoma clones producing the monoclonal antibody, first, the fusion cells obtained as described above are transferred to a selection medium such as HAT medium, and cultured at about 30 to 40° C. for about 3 days to 3 weeks. It kills cells other than hybridomas. Then, after culturing the hybridoma on a microtiter plate, etc., the part with increased reactivity between the immunogen used for the immune response of animals other than humans described above and the culture supernatant was subjected to RIA (radioactive substance-marked immunotherapy). antibody) or ELISA (Enzyme-Linked Immunosorbent Assay). And the clone producing the monoclonal antibody found above shows a specific binding ability to the immunogen.

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

In a specific embodiment of the present invention, in order to prepare an antibody that specifically binds to CD22, a hybridoma producing an anti-CD22 antibody is prepared and screened to obtain an antibody (scFv) that specifically binds to CD22. was selected, and it was designated as 1C2.

The 1C2 antibody is a heavy chain variable comprising a CDR1 region represented by the amino acid of SEQ ID NO: 1 (GYTFTSYWMNW), a CDR2 region represented by the amino acid of SEQ ID NO: 2 (IDPSDSET) and a CDR3 region represented by the amino acid of SEQ ID NO: 3 (ARWGNYDYDVWAMDY) A light chain variable region comprising a region and a CDR1 region represented by the amino acid of SEQ ID NO: 4 (QNIVHLNGNTF), a CDR2 region represented by the amino acid of SEQ ID NO: 5 (KVS) and a CDR3 region represented by the amino acid of SEQ ID NO: 6 (FQGSHVPYT) configuration was confirmed.

Specifically, the 1C2 antibody is composed of a heavy chain variable region represented by the amino acid of SEQ ID NO: 7 and a light chain variable region represented by the amino acid of SEQ ID NO: 8, wherein the heavy chain variable region is the nucleotide sequence of SEQ ID NO: 9, and the light chain variable region is It is encoded by the nucleotide sequence of SEQ ID NO: 10.

In another specific embodiment of the present invention, a humanized antibody in which 1C2, an anti-CD22 antibody, was changed to a structure corresponding to a human was prepared, which was named 1C2-V9 and 1C2-V12.

The heavy chain variable region CDRs and light chain variable region CDRs of 1C2-V9 and 1C2-V12 are the same as in 1C2, except for the CDR regions, humanized. Preferably, 1C2-V9 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 1C2-V9 antibody is the base sequence of SEQ ID NO: 13 Thus, the light chain variable region may be encoded by the nucleotide sequence of SEQ ID NO: 14.

In addition, 1C2-V12 consists 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, and the heavy chain variable region of the 1C2-V12 antibody is the base sequence of SEQ ID NO: 17 Thus, the light chain variable region may be encoded by the nucleotide sequence of SEQ ID NO: 18.

The CD22-specific antibody of the present invention is preferably an 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 linked with a linker. The linker is preferably represented by the amino acid sequence of SEQ ID NO: 19, or may be encoded by the nucleotide sequence of SEQ ID NO: 20 or SEQ ID NO: 21, but is not limited thereto.

When linked by a light chain variable region-linker-heavy chain variable region, the 1C2-V9 antibody may be represented by the amino acid sequence of SEQ ID NO: 22 or encoded by the nucleotide sequence of SEQ ID NO: 23, and the 1C2-V12 antibody is the amino acid sequence of SEQ ID NO: 24 indicated or may be encoded by the nucleotide sequence of SEQ ID NO: 25.

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

As used herein, the term "polynucleotide" generally refers to a nucleic acid molecule, deoxyribonucleotide or ribonucleotide, or an analog thereof, separated by any length. In some embodiments, the polynucleotides of the present invention can be administered by (1) in-vitro amplification, such as polymerase chain reaction (PCR) amplification; (2) cloning and recombination; (3) purification such as digestion and gel electrophoretic separation; (4) It can be prepared through synthesis such as chemical synthesis, and preferably, the isolated polynucleotide is prepared by recombinant DNA technology. In the present invention, the nucleic acid for encoding the antibody or 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. can be manufactured.

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 "vector (expression vector)" refers to a gene preparation including essential regulatory elements such as a promoter so that a target gene can be expressed in an appropriate host cell. The vector may be selected from one or more of a plasmid, a retroviral vector and a lentiviral vector. Once transformed into an appropriate 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 allow the coding region to be accurately expressed in a suitable host. Such regulatory elements are well known to those skilled in the art and include, for example, promoters, ribosome-binding sites, enhancers and other regulatory elements for regulating gene transcription or mRNA translation. can do. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally 5' ratios participating in transcription initiation and translation initiation, respectively, such as TATA box, capped sequence, CAAT sequence, etc. - contains a transcribed sequence, and a 5' or 3' non-translated sequence. For example, a 5' non-transcriptional expression control sequence may include a promoter region that may include a promoter sequence for transcription and control of a functionally linked nucleic acid.

As used herein, the term "promoter" means a minimal sequence sufficient to direct transcription. In addition, promoter constructs sufficient to allow expression of a regulatable promoter-dependent gene induced by cell type-specific or external signals or agents may be included, and these constructs may be located in the 5' or 3' portion of the gene. . Both conservative and inducible promoters are included. Promoter sequences may be derived from prokaryotes, eukaryotes or viruses.

In the present invention, the term "transformant" refers to a cell transformed by introducing a vector having a polynucleotide encoding one or more target proteins into a host cell, and introducing the expression vector into the host cell to prepare a transformant As a method for this, 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) , an electroporation method, an electroinjection method, a chemical treatment method such as PEG, a method using a gene gun, or the like.

When the transformant expressing the vector is cultured in a nutrient medium, it is possible to manufacture and isolate antibody proteins in large quantities. Medium and culture conditions can be appropriately selected and used depending on the host cell. In culture, conditions such as temperature, pH of the medium, and incubation time should be appropriately adjusted to be suitable for 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. Suitable host cells capable of expressing fully glycosylated proteins include COS-1 (eg ATCC CRL 1650), COS-7 (eg ATCC CRL-1651), HEK293, BHK21 (eg ATCC CRL-10), CHO (eg ATCC CRL 1610) and BSC-1 (eg ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0 -Agl4, 293 cells, HeLa cells, etc., these cells are readily available from, for example, ATCC (American Type Culture Collection, USA).

Chimeric antigen receptor targeting CD22

The present invention from another point of view,

CD22-binding domain;

transmembrane domain;

costimulatory domain; and

A chimeric antigen receptor (CAR) comprising 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 chimera 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 It relates to antigen receptors.

As used herein, the term “chimeric antigen receptor (CAR)” generally refers to a fusion protein containing an antigen and an extracellular domain having the ability to bind one or more intracellular domains. A CAR is a key part of a chimeric antigen receptor T cell (CAR-T) and may include an antigen (eg, CD22) binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. A CAR can be combined with a T cell receptor-activating intracellular domain based on the antigen (eg, CD22) specificity of the antibody. 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 humanized 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" refers to "extracellular domain", "extracellular binding domain", "antigen-specific binding domain" and "Extracellular antigen-specific bidding domain" can be used interchangeably and refers to a CAR domain or fragment that has the ability to specifically bind a target antigen (eg CD22). do.

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, a chimeric antigen receptor targeting CD22 can be prepared using 1C2-V9 or 1C2-V12 antibodies, which are humanized antibodies specific for CD22 of the present invention.

In the present invention, a signal peptide may be further included at the N-terminus of the CD22-binding domain, and the "signal peptide" generally refers to a peptide chain for guiding protein transduction. . The signal peptide may be a short peptide having a length of 5 to 30 amino acids, and the amino acid sequence of SEQ ID NO: 33 is preferably used in the present invention.

In the present invention, it may further comprise a hinge region located between the C terminus of the CD22-binding domain and the N terminus of the transmembrane domain, wherein the hinge region is derived from CD8α, preferably SEQ ID NO: It can be represented by the amino acid sequence of 34. The "hinge region" generally refers to the linking region between the antigen-binding region and the immune cell Fc receptor (FcR)-binding region.

In the present invention, "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 signaling role. 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: 35.

In the present invention, "costimulatory domain" generally refers to an intracellular domain capable of providing immune-stimulatory molecules, which are cell surface molecules necessary for an effective response of lymphocytes to antigens. The costimulatory domain described above may comprise a costimulatory domain of CD28, and may comprise a costimulatory domain of the TNF receptor family, such as the costimulatory domain of OX40 and 4-1BB, preferably SEQ ID NO: It may be 4-1BB represented by the amino acid sequence of 36.

In the present invention, "intracellular signal transduction domain" generally refers to a domain located inside a cell and capable of transmitting a signal. In the present invention, the intracellular signaling domain is the intracellular signaling domain of the chimeric antigen receptor. For example, the intracellular signaling domain may be selected from CD3ζ intracellular domain, CD28 intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain and OX40 intracellular domain, preferably the amino acid of SEQ ID NO: 37 It may be CD3ζ represented by the sequence.

Chimeric Antigen Receptor Encoding Polynucleotides and Chimeric Antigen Receptor Expression Vectors

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) comprises 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 1C2-V9 or 1C2-V12 antibody specific for CD22 of the present invention, in the form of an scFv in which the light chain variable region and the heavy chain variable region are linked by a linker, and the specific nucleotide sequence is As described above.

Preferably, the polynucleotide encoding the chimeric antigen receptor (CAR) of the present invention comprises: a signal peptide represented by the nucleotide sequence of SEQ ID NO: 27;

1C2-V9 antibody represented by the nucleotide sequence of SEQ ID NO: 23 or 1C2-V12 antibody represented by the nucleotide sequence of SEQ ID NO: 25;

a transmembrane domain represented by the nucleotide sequence of SEQ ID NO: 28;

4-1BB (costimulatory domain) represented by the nucleotide sequence of SEQ ID NO: 29; and

It may be composed of CD3ζ (intracellular signaling domain) represented by the nucleotide sequence of SEQ ID NO: 30.

In addition, between the polynucleotide encoding the CD22-binding domain and the transmembrane domain, a polynucleotide encoding a hinge region may be further included, preferably the CD8 hinge region represented by the nucleotide sequence of SEQ ID NO: 28 can

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, comprising an operably linked EF1α promoter; a polynucleotide encoding a signal peptide; a polynucleotide encoding a CD22-binding domain; a polynucleotide encoding a transmembrane domain; It includes a polynucleotide encoding an intracellular signaling domain, and may further include a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to increase protein expression ( FIG. 3 ).

The EF1α promoter may be represented by the nucleotide sequence of SEQ ID NO: 26, and if necessary, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more of the nucleotide sequence of SEQ ID NO: 26 , or sequences that are at least 99% identical.

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

In a specific embodiment of the present invention, as shown in FIG. 3 , a lentiviral vector into which a polynucleotide encoding CD22-CAR was inserted was prepared, and the prepared vector was transformed into 293FT cells to prepare CD22-CAR expressing cells. did. In addition, as shown in FIG. 5 , it was confirmed that the chimeric antigen receptor targeting CD22 was expressed in the prepared CD22-CAR expressing cells.

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

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. include An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle). Other methods are available for state-of-the-art targeted delivery of nucleic acids, such as delivery of polynucleotides using targeted nanoparticles or other suitable sub-micron sized delivery systems.

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

Chimeric Antigen Receptor (CAR) Expression Immune Effector Cells

In another aspect, the present invention includes a vector comprising a polynucleotide encoding the chimeric antigen receptor (CAR) or a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor (CAR) It relates to immune effector cells expressing

In the present invention, the immune effector cells may be mammalian-derived cells, preferably T cells, B cells, natural killer (NK) cells, dendritic cells, bone marrow cells, mononuclear cells, or macrophages, more preferably may be T cells.

In the present invention, the immune effector cells expressing the chimeric antigen receptor (CAR) can be prepared by introducing the CAR vector of the present invention into immune effector cells, for example, T cells or NK cells.

Specifically, the CAR vector may be introduced into cells by methods known in the art, such as electroporation, 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 to ensure long-term and stable expression of the target gene. For another example, a transposon can be used to introduce a CAR transport plasmid and a transferase transport plasmid into a target cell. For another example, a CAR molecule can be added to the genome by a gene editing method (eg, CRISPR Cas9).

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

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

Cells collected by apheresis can be washed to remove the plasma fraction and place the cells 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 a specific embodiment of the present invention, as shown in FIG. 7 , activated T cells are isolated from peripheral blood mononuclear cells (PBMCs), and then the CD22-CAR lentiviral vector is transfected into the T cells. introduced to prepare CD22-CAR-T cells. As a result of confirming the CD22-peptide binding ability of the prepared CD22-CAR-T cells, it was confirmed that all of the CD22-CAR-T cells in which CD3, CD4 or CD8 were activated effectively bind to CD22-peptide, as shown in FIG. 8 .

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. 9 , CD22-CAR-T cells are CD22-expressing U2932 cells and NALM6 cells. It was confirmed that the cell-specific killing effect was shown.

That is, the humanized anti-CD22 antibody (1C2-V9 and 1C2-V12)-based chimeric antigen receptor of the present invention and CAR-T cells using the same can be usefully utilized as a composition for preventing or treating diseases related to B cells or CD22 expression. have.

Composition for preventing or treating diseases mediated by CD22

In another aspect, the present invention provides for the prevention or treatment of diseases mediated by cells expressing CD22, including immune effector cells expressing a humanized antibody that specifically binds to CD22 or a chimeric antigen receptor targeting CD22 It relates to a pharmaceutical composition.

In the present invention, the disease mediated by cells expressing CD22 is lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed delayed NHL, refractory NHL, refractory Delayed NHL, chronic lymphocytic leukemia (CLL), small 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 cells expressing CD22, and the therapeutic agent is present in a state covalently bound to the heavy chain and/or light chain of an antibody that specifically binds to CD22. Alternatively, it may be administered in combination with the CD22-CAR immune effector cells of the present invention.

The therapeutic agent includes a small molecule drug, a peptide drug, a toxin (eg, a cytotoxin), and the like.

The low molecular weight drug exhibits a pharmaceutical activity of interest, and generally may be a compound having a molecular weight of about 800 Da or less or 2000 Da or less. A small inorganic molecule refers to a molecule containing no carbon atoms, while an organic small molecule refers to a compound containing at least one carbon atom.

The peptidic drug refers to amino acids containing polymeric compounds, including naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides and proteins, as well as peptide mimics. The peptide drug may be obtained by chemical synthesis or produced from a genetically encoded source (eg, a recombinant source). The molecular weight of the peptide drug may range from 200 Da to 10 kDa or more.

The toxin is preferably a cytotoxin, and includes, but is not limited to, ricin, abrin, diphtheria toxin, Pseudomonas exotoxin (eg, PE35, PE37, PE38, PE40, etc.), saporin, gelonin, USA. antiviral protein (PAP), botulinum toxin, bryodin, momordin and buganin.

In addition, the therapeutic agent may be an anticancer agent. Anticancer agents reduce the proliferation of cancer cells and include non-peptidyl (ie, non-proteinaceous) compounds, including cytotoxic agents and cytostatic agents. Non-limiting examples of anticancer agents include alkylating agents, nitrosourea, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptide compounds may also be used.

In the pharmaceutical composition, the humanized antibody or CD22-CAR immune effector cell that specifically binds to CD22 is the only active ingredient in the composition for treatment or diagnosis, or, for example, anti-T cell, anti-IFNγ or anti-LPS It can be used in combination with other active ingredients, including other antibody components, such as antibodies, or non-antibody components, such as xanthine.

The pharmaceutical composition preferably comprises a therapeutically effective amount of an antibody of the invention. As used herein, the term "therapeutically effective amount" means an 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 prophylactic effect. means For any antibody, a therapeutically effective dose can be initially determined by cell culture assays or animal models, usually rodents, rabbits, dogs, pigs, or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can be used to determine useful dosages and routes for administration in humans.

The precise effective amount for a human patient will 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 resistance/response to treatment. can The amount can be determined by routine experimentation and is within the judgment of the clinician. In general, an effective dosage is 0.01-50 mg/kg, preferably 0.1-20 mg/kg, more preferably about 15 mg/kg.

The compositions may be administered to the patient individually or in combination with other agents, agents, or hormones.

The dosage at which the antibody of the present invention is administered depends on the nature of the condition to be treated, the grade of malignant lymphoma or leukemia, and whether the antibody is used to prevent disease 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's effect. If the antibody molecule has a short half-life (eg 2-10 hours), it may be necessary to provide one or more doses per day. Alternatively, if the antibody molecule has a long half-life (eg, 2-15 days), it may be necessary to provide a dose once a day, once a week, or once every 1 or 2 months.

In addition, the pharmaceutical composition may contain a pharmaceutically acceptable carrier for administration of the antibody. The carrier itself must not cause the production of antibodies that are deleterious to the individual receiving the composition, and must be non-toxic. Suitable carriers may be slowly metabolized macromolecules, such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers and inactive viral particles.

Pharmaceutically acceptable salts are, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate and sulfate, or acetic acid, propionic acid. Salts of organic acids such as malonic acid and benzoic acid may be used.

Pharmaceutically acceptable carriers in therapeutic compositions may additionally include liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances such as wetting agents, emulsifying agents or pH buffering agents may be present in such compositions. 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 (eg, bolus injection or continuous infusion). When the product is intended for infusion or injection, it may take the form of suspensions, solutions or emulsions in oil or water-soluble excipients, which may contain prescription agents such as suspending, preservative, stabilizing and/or dispersing agents. Alternatively, the antibody molecule may be in anhydrous form and reconstituted with an appropriate sterile solution prior to use.

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

The pharmaceutical composition of the present invention is not limited, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (see, e.g., WO 98/20734), subcutaneous, Administration may be by any route, including intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes. A hypospray may be used to administer the pharmaceutical composition of the present invention. Typically, therapeutic compositions may be prepared as injectables as liquid solutions or suspensions. In addition, solid forms suitable for solution or suspension in liquid excipients prior to injection may be prepared.

Direct delivery of the composition may generally be achieved by injection, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, or may be delivered to the interstitial space of a tissue. In addition, the composition may be administered to the wound site. Dosage treatment may 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. Thus, if the composition is administered by a route using 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).

Diagnosis or monitoring of diseases mediated by cells expressing CD22

In another aspect, the present invention relates to a composition for diagnosing or monitoring a disease mediated by cells expressing CD22, comprising a humanized antibody that specifically binds to CD22.

The humanized antibody that specifically binds to CD22 may be directly or indirectly labeled. Indirect labels include secondary antibodies comprising a detectable label, wherein the secondary antibody binds to a humanized antibody that specifically binds to CD22. Other indirect labels include biotin, wherein a humanized antibody that specifically binds to biotinylated CD22 can be detected using avidin or streptavidin comprising a detectable label.

Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable labels include, but are not limited to, magnetic beads, fluorescent dyes (eg, fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.), radioactive labels (e.g., For example, ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C or ³² P), enzymes (eg, mustard radish peroxidase, alkaline phosphatase, luciferase and enzyme-linked immunosorbent assay (ELISA)) commonly used) and colorimetric labels such as colloidal gold or colored glass or plastic (eg polystyrene, polypropylene, latex, etc.) beads.

In addition, for diagnosis or monitoring, the humanized antibody may be labeled with a fluorescent protein and may contain a contrast agent or radioisotope.

When the humanized antibody that specifically binds to CD22 of the present invention is used in a diagnostic kit, the humanized antibody is immobilized on a support, and the support may be a microplate, microarray, chip, glass, bead or particle, or a membrane. have.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1: Preparation and selection of antibodies that specifically bind to CD22

In order to select the CD22 peptide-specific antibody, a hybridoma producing an antibody binding to CD22 was prepared and the antibody was selected.

First, splenocytes were extracted by immunization with CD22 protein (ACRObiosystems Inc., cat. NO CD2-H52H8, USA), and hybridoma cells were prepared through cell fusion with mouse myeloma cells.

Mouse myeloma cells used for cell fusion cannot survive in HAT medium because they do not have HGPRT (HypoxanthineGuanidine-Phosphoribosyl-Transferase), but hybridomas can survive in HAT medium by fusion with splenocytes. Since only hybridomas can be propagated using this, it is usually grown in HAT medium until hybridomas are established.

The limiting dilution method was used to select hybridomas producing an antibody binding to CD22 from among the proliferated hybridomas. First, it was made to be less than one cell per 96 well, and then, it was confirmed by ELISA whether the antibody obtained from the clones proliferated from one cell binds to CD22, and clones that bind to CD22 were selected. The above process was repeated three times to select hybridomas producing an antibody binding to CD22. In this way, an antibody binding to CD22 was obtained.

The antibody was named 1C2, and their base and amino acid sequences were analyzed. Sequence information on the heavy chain variable region and the light chain variable region of each antibody according to the sequencing results is shown in Table 1 below, and underlined parts in Table 1 mean the complementarity determining region (CDR).

Sequence information of 1C2 antibody

1C2	sequence information	SEQ ID NO:
Heavy chain variable region CDR1	GYTFTSYWMNW	SEQ ID NO: 1
Heavy chain variable region CDR2	IDPSDSET	SEQ ID NO: 2
Heavy chain variable region CDR3	ARWGNYDYDVWAMDY	SEQ ID NO: 3
Light chain variable region CDR1	QNIVHLNGNTF	SEQ ID NO: 4
Light chain variable region CDR2	KVS	SEQ ID NO: 5
Light chain variable region CDR3	FQGSHVPYT	SEQ ID NO: 6
heavy chain variable region amino acid sequence	QVQLQQPGAELVRPGASVKLSCKAS GYTFTSYWMNW VKQRPGQGLEWIGM IDPSDSET HYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYC ARWGNYDYDVWAMDY WGQGTSVTVSS	SEQ ID NO: 7
light chain variable region amino acid sequence	DVLMTQTPLSLPVSLGDQASISCRSS QNIVHLNGNTF LEWFLQKPGQSPKLLIY KVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPYT FGGGTKLEIK	SEQ ID NO: 8
heavy chain variable region base sequence	CAGGTGCAGCTGCAGCAGCCCGGCGCCGAGCTGGTGAGGCCCGGCGCCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTGGATGAACTGGGTGAAGCAGAGGCCCGGCCAGGGCCTGGAGTGGATCGGCATGATCGACCCCAGCGACAGCGAGACCCACTACAACCAGATGTTCAAGGACAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGTGGGGCAACTACGACTACGACGTGTGGGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC	SEQ ID NO: 9
light chain variable region base sequence	GACGTGCTGATGACCCAGACCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGGAGCAGCCAGAACATCGTGCACCTGAACGGCAACACCTTCCTGGAGTGGTTCCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTGAGCAACAGGTTCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTACTGCTTCCAGGGCAGCCACGTGCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG	SEQ ID NO: 10

Example 2: Confirmation of specificity for CD22 of the selected antibody

2-1: ELISA analysis

In the present invention, in order to confirm the specificity of the 1C2 antibody established in Example 1 for CD22, ELISA analysis was performed.

First, in order to encode the CD22 peptide, CD22-His tag (CD22 extracellular domain; ACRObiosystems Inc., cat. NO CD2-H52H8) was dispensed in a 96-well plate at a concentration of 100 ng/well, and then overnight at 4°C. reacted. Then, after treatment with 1 X PBST containing 3% BSA, blocking at room temperature for 30 minutes.

1C2 antibody was treated in each well, and then reacted at room temperature for 2 hours, and then washed 3 times with 1 X PBST. Secondary antibody (anti-HRP, 1:10,000) was treated and reacted at room temperature for 30 minutes, washed 3 times with 1 X PBST, and then treated with TMB for color development and reacted at room temperature for 5 minutes. Finally, the reaction was terminated by treatment with a stop solution of 1N H ₂ SO ₄ , and then the absorbance was measured at 450 nm.

ELISA Experimental Conditions

Immobilization	Corning (cat. No 3690)
Antigen	CD22-His tag, 100 ng/well
Primary Ab	0.4 μg/ml
Secondary Ab-HRP	1:10,000
TMB substrate incubation	5 min
Measurement filter	450nm
plate reader	Infinite F50

ELISA test results

Antibody type	OD450 measurements
1C2	2.4289

As a result, as shown in Table 3, it was confirmed that the antibody selected in the present invention specifically binds to CD22.

2-2: flow cytometer

In the present invention, in order to confirm the specificity of the 1C2 antibody established in Example 1 for CD22, flow cytometer was performed.

First, 1x10 ⁶ B-cell lymphoma U2932 cells expressing CD22 were reacted with 2 μg of 1C2 antibody for 30 minutes, and then the surface was stained with a secondary antibody, followed by measurement by flow cytometry. did.

As a positive control, a PE-conjugated anti-CD22 antibody (PE-conjugated anti-CD22 antibody; Biolegend Inc., cat. NO 302506, USA) was used, and as a secondary antibody, PE-conjugated anti-mouse IgG was used. Antibody (PE-conjugated goat anti-mouse IgG; Biolegend Inc., cat. NO 405307, USA) was used.

As a result, as shown in FIG. 1 , it was confirmed that the 1C2 antibody specifically binds to cells expressing CD22.

That is, since the antibody selected in the present invention specifically recognized cells expressing CD22, it can be usefully used in various fields such as diagnosis as well as compositions for preventing or treating diseases mediated by CD22-expressing cells.

Example 3: Preparation of 1C2 antibody-based humanized antibody and confirmation of specificity for CD22

3-1: humanized antibody production

A humanized antibody was prepared in which the 1C2 antibody selected in Example 1 was changed to a structure corresponding to a human.

Specifically, a mouse 1C2 antibody by a CDR-grafting method that replaces the CDRs of a human antibody with the CDRs of a mouse antibody that binds to CD22 using the germline sequence of a human antibody as a frame. A humanized antibody was prepared. Humanized antibodies were named 1C2-V9 and 1C2-V12, and amino acid sequences were analyzed. The sequence information on the heavy chain variable region and the light chain variable region of the antibody according to the sequencing results is shown in Tables 4 and 5 below, and the underlined portions in Tables 4 and 5 are complementarity determining regions (CDRs). means

Sequence information of 1C2-V9 antibody

1C2-V9	sequence information	SEQ ID NO:
Heavy chain variable region CDR1	GYTFTSYWMNW	SEQ ID NO: 1
Heavy chain variable region CDR2	IDPSDSET	SEQ ID NO: 2
Heavy chain variable region CDR3	ARWGNYDYDVWAMDY	SEQ ID NO: 3
Light chain variable region CDR1	QNIVHLNGNTF	SEQ ID NO: 4
Light chain variable region CDR2	KVS	SEQ ID NO: 5
Light chain variable region CDR3	FQGSHVPYT	SEQ ID NO: 6
heavy chain variable region amino acid sequence	QVQLVQSGSELKKPGASVKVSCKAS GYTFTSYWMNW VRQAPGQGLEWIGM IDPSDSET HYNQGFTGRFVLSVDKSVSTAYLQISSLKAEDTAVYYC ARWGNYDYDVWAMDY WGQGTTVTVSS	SEQ ID NO: 11
light chain variable region amino acid sequence	DVVMTQSPLSLPVTLGQPASISCRSS QNIVHLNGNTF LEWFQQRPGQSPRLLIY KVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHVPYT FGGGTKLEIK	SEQ ID NO: 12
heavy chain variable region base sequence	CAGGTGCAGCTGGTGCAGAGTGGCTCCGAGCTGAAGAAGCCCGGGGCTTCCGTAAAAGTGTCCTGTAAGGCTTCTGGGTACACCTTCACCAGCTATTGGATGAATTGGGTCCGCCAGGCCCCTGGTCAGGGCCTTGAGTGGATCGGTATGATTGACCCGTCCGACTCTGAGACTCATTACAACCAGGGCTTCACCGGCCGCTTCGTCCTGAGCGTCGACAAATCCGTGTCCACGGCCTACCTGCAAATTTCTTCCCTAAAGGCGGAAGACACCGCGGTGTACTACTGCGCTCGTTGGGGCAACTACGACTACGACGTGTGGGCCATGGATTATTGGGGCCAGGGGACAACTGTCACAGTTTCGTCA	SEQ ID NO: 13
light chain variable region base sequence	GATGTGGTGATGACCCAGAGCCCCCTGAGCCTCCCAGTCACTTTGGGCCAGCCGGCATCGATCTCGTGTCGGTCCTCTCAGAACATCGTGCACCTCAACGGCAACACGTTCCTGGAGTGGTTCCAGCAGAGACCAGGTCAGTCTCCTCGCCTGCTTATCTACAAGGTAAGCAACCGCTTCTCGGGGGTGCCCGACAGGTTTTCAGGGTCCGGCTCCGGCACCGACTTTACCCTGAAGATCTCCCGCGTGGAGGCCGAGGATGTGGGCGTGTACTACTGCTTTCAGGGAAGTCACGTTCCCTATACCTTCGGTGGTGGCACCAAGCTGGAGATCAAG	SEQ ID NO: 14

Sequence information of 1C2-V12 antibody

1C2-V12	sequence information	SEQ ID NO:
Heavy chain variable region CDR1	GYTFTSYWMNW	SEQ ID NO: 1
Heavy chain variable region CDR2	IDPSDSET	SEQ ID NO: 2
Heavy chain variable region CDR3	ARWGNYDYDVWAMDY	SEQ ID NO: 3
Light chain variable region CDR1	QNIVHLNGNTF	SEQ ID NO: 4
Light chain variable region CDR2	KVS	SEQ ID NO: 5
Light chain variable region CDR3	FQGSHVPYT	SEQ ID NO: 6
heavy chain variable region amino acid sequence	QVQLVQSGSELKKPGASVKVSCKAS GYTFTSYWMNW VRQAPGQGLEWIGM IDPSDSET HYNQGFTGRFVLSVDKSVSTAYLQISSLKAEDTAVYYC ARWGNYDYDVWAMDY WGQGTTVTVSS	SEQ ID NO: 15
light chain variable region amino acid sequence	DVQMTQSPSSLSASVGDRVTITCRSS QNIVHLNGNTF LEWYQQKPGKVPKLLIY KVS NRFSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC FQGSHVPYT FGGGTKLEIK	SEQ ID NO: 16
heavy chain variable region base sequence	CAGGTCCAGCTGGTGCAGTCTGGCTCCGAGCTGAAGAAGCCTGGCGCGTCCGTGAAGGTGTCATGTAAGGCTTCCGGCTACACATTCACCAGCTATTGGATGAATTGGGTGCGCCAGGCCCCTGGACAGGGCCTGGAGTGGATCGGTATGATCGACCCGTCCGACAGCGAGACTCATTACAACCAGGGCTTCACTGGCCGCTTCGTCCTGTCCGTAGACAAATCCGTTTCGACTGCGTACCTGCAAATTTCGAGTCTAAAAGCAGAAGACACCGCCGTGTACTACTGTGCTCGTTGGGGCAACTACGACTACGATGTATGGGCCATGGATTATTGGGGCCAGGGGACAACCGTCACCGTGTCCTCT	SEQ ID NO: 17
light chain variable region base sequence	GACGTGCAGATGACCCAGAGCCCCTCTTCGTTGTCCGCCTCCGTGGGTGATCGGGTCACCATCACCTGCCGATCGAGCCAGAACATCGTGCACCTCAACGGCAACACGTTCCTGGAGTGGTACCAGCAGAAGCCCGGCAAAGTCCCCAAGTTGCTTATCTACAAGGTGAGCAACCGCTTTAGCGGTGTGCCATCCCGCTTTTCAGGTTCTGGTAGTGGGACCGACTTCACCCTGACTATTTCTTCTCTGCAGCCGGAGGACGTGGCCACGTACTACTGCTTTCAGGGGAGCCACGTTCCCTATACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG	SEQ ID NO: 18

3-2: Confirmation of specificity for CD22

In order to confirm the specificity of the humanized 1C2-V9 antibody and 1C2-V12 antibody prepared in Example 3-1 for CD22, flow cytometry was performed in the same manner as in Example 2-2.

First, 2 μg of 1C2-V9 antibody or 1C2-V12 antibody was reacted with 1x10 ⁶ B-cell lymphoma U2932 cells expressing CD22 for 30 minutes, and then the surface was stained with a secondary antibody. After that, it was measured by flow cytometry.

As a positive control, PE-conjugated anti-CD22 antibody (3 μl, PE-conjugated anti-human CD22 antibody; Biolegend Inc., cat. NO 302506, USA) was used, and as a secondary antibody, PE-conjugated antibody was used. Anti-human IgG antibody (5 μl, PE anti-human IgG Fc Ab; Biolegend Inc., cat. NO 409304, USA) was used.

As a result, as shown in FIG. 1 , it was confirmed that both the 1C2-V9 antibody and the 1C2-V12 antibody specifically reacted with CD22.

Example 4: Construction of a chimeric antigen receptor (CAR) expression vector targeting CD22

In the present invention, a lentiviral vector expressing a chimeric antigen receptor (CAR) targeting CD22 (CD22-CAR lentiviral vector) was prepared using the humanized 1C2-V9 and 1C2-V12 antibodies prepared in Example 3 above. prepared.

As shown in the schematic diagram of Figure 3, EF1α promoter (SEQ ID NO: 26); a polynucleotide encoding a signal peptide (SEQ ID NO: 27); a polynucleotide encoding a CD22-binding domain (1C2-V9 represented by SEQ ID NO: 23 or 1C2-V12 represented by SEQ ID NO: 25); a polynucleotide encoding the CD8 hinge region (SEQ ID NO: 28); a polynucleotide encoding a transmembrane domain (SEQ ID NO: 29); a polynucleotide encoding 4-1BB (costimulatory domain) (SEQ ID NO: 30); a polynucleotide encoding CD3ζ intracellular signaling domain (SEQ ID NO: 31); And CAR DNA consisting of a polynucleotide encoding WPRE (SEQ ID NO: 32) was synthesized in vitro and inserted into a third-generation lentiviral vector.

Lentiviral vector DNA (0.5 μg) was transferred to HEK293FT cells (5×10 ⁵ cells/500 μl), and 293HEK cells expressing the CD22-CAR gene were prepared. Lipofectamine 3000 transfection kit (Invitrogen, cat# L3000-015) was used to transfer genes into 293HEK cells, and cultured in Opti-MEM (gibco, cat# 51985-034) medium for 4 hours.

CD22-specific CAR is normally expressed in HEK293FT transformed with lentiviral vector DNA, and it is confirmed by flow cytometry (Flow Cytometry) whether it binds to CD22 peptide (FIG. 4B), as shown in FIG. 5, CD22-CAR was normally expressed and confirmed to bind to the CD22 peptide.

Example 5: Preparation of CD22-CAR-T cells

In the present invention, the CD22-CAR lentiviral vector prepared in Example 4 was transformed into T cells to prepare CD22-CAR-T cells.

Specifically, as shown in FIG. 7A , peripheral blood mononuclear cells (PBMCs) were isolated from blood, and then T cell activation beads (T cell activation bead; Miltenyl Biotec, cat. NO 130-091-441) ) was used to activate T cells. CD22-CAR-T cells were prepared by transducing the activated T cells with the CD22-CAR lentiviral vector prepared in Example 4, and Lenti-boost-p was used to increase the transduction efficiency.

CD22 peptide binding capacity of CD22-CAR-T cells was confirmed by flow cytometry (FIG. 7B). The CD22-CAR-T cells prepared above were sorted into CD19/CD22-CAR-T cells in which CD3, CD4 or CD8 was activated using anti-CD3, anti-CD4, and anti-CD8 antibodies, respectively, and then FITC- After reacting with the CD22 peptide, the fluorescence intensity was measured using a flow cytometer.

As a result, as shown in FIG. 8 , it was confirmed that all of the CD22-CAR-T cells in which CD3, CD4 or CD8 were activated bind to the CD22 peptide.

Example 6: Confirmation of the killing effect of CD22-CAR-T cells on CD22-expressing cells

In the present invention, the killing effect of target cells by humanized anti-CD22 antibodies (1C2-V9 and 1C2-V12) based CD22-CAR-T cells was confirmed.

As target cells, K562 cells that do not express CD22 (human erythroleukemic cell line) and U2932 cells that express CD22 (B cell lymphoma) and NALM6 cells (human B cell precursor leukemia) were used, and CD22-CAR-T cells and 1 :4, 1:2, 1:1, 1:0.5 and 1:0.25 were mixed and cultured for 8 hours, and then luminescence (CytoTox-Glo Cytotoxicity Assay, Promega, cat. NO G9291) was measured. did. The degree of cell death was calculated using Equation 1 below as the measured value.

[Equation 1]

% Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)] X 100

Experimental: Luminescence value derived from the medium of the target cell and CAR-T cell complex culture

Effector Spontaneous: Luminescence value derived from the medium of CAR-T cells only

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 FIGS. 9 and 10 , CD22-CAR-T cells based on humanized anti-CD22 antibodies (1C2-V9 and 1C2-V12) specifically killed CD22-expressing U2932 cells and NALM6 cells. confirmed to do.

Since it was confirmed that the humanized anti-CD22 antibody (1C2-V9 and 1C2-V12)-based chimeric antigen receptor of the present invention and CAR-T cells using the same effectively kill CD22-expressing cells as well as effectively bind to CD22. , it can be usefully used as a composition for preventing or treating diseases related to CD22 expression.

Claims (12)

1. 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 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.
2. A polynucleotide encoding an antibody that specifically binds to the CD22 of claim 1.
3. A vector comprising a polynucleotide encoding an antibody that specifically binds to the CD22 of claim 2.
4. A recombinant cell producing an antibody or fragment thereof that specifically binds to CD22 transformed with the vector of claim 3 .
5. CD22-binding domain;

   transmembrane domain;

   costimulatory domain; and

   A chimeric antigen receptor (CAR) comprising 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

   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 Chimeric antigen receptor.
6. The method of claim 5, wherein 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,

   The chimeric antigen receptor targeting CD22, characterized in that the signaling domain is CD3ζ.
7. The chimeric antigen receptor targeting CD22 according to claim 5, further comprising a hinge region between the C terminus of the binding domain and the N terminus of the transmembrane domain.
8. A polynucleotide encoding a chimeric antigen receptor targeting the CD22 of any one of claims 5-7.
9. A vector comprising a polynucleotide encoding a chimeric antigen receptor targeting CD22 according to any one of claims 5 to 7.
10. 8. An immune effector cell comprising a polynucleotide encoding a chimeric antigen receptor targeting CD22 according to any one of claims 5 to 7, or a vector comprising the polynucleotide.
11. A humanized antibody or fragment thereof that specifically binds to the CD22 of claim 1; Or a pharmaceutical composition for preventing or treating a disease mediated by a cell expressing CD22, including the immune effector cell of claim 10.
12. 12. The method of claim 11, wherein the disease mediated by cells expressing CD22 is lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed delayed NHL, refractory NHL, Refractory delayed NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt A pharmaceutical composition for the prophylaxis or treatment of a disease mediated by CD22-expressing cells, characterized in that it is selected from the group consisting of tree lymphoma and mantle cell lymphoma.

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