WO2023090704A1 - Antibody specific for humanized cd22 and chimeric antigen receptor using same - Google Patents
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Definitions
- the present invention relates to a humanized CD22-specific antibody and a chimeric antigen receptor using the same, and more particularly, to an antibody that specifically binds to CD22 (4F5 antibody) and a humanized antibody thereof, comprising the antibody or the CD19xCD22 antibody It relates to a chimeric antigen receptor, a CAR-T cell expressing the chimeric antigen receptor, and a pharmaceutical composition for preventing or treating diseases mediated by B cells including the chimeric antigen receptor.
- CD22 is associated with 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.
- B-ALL acute lymphoblastic leukemia
- B-CLL chronic lymphocytic leukemia
- ANLL acute non-lymphocytic leukemia
- Antibodies specific to CD22 are being developed for the treatment or diagnosis of diseases related to CD22 expression.
- International Publication No. WO1998-041641 discloses a recombinant anti-CD22 antibody having cysteine residues at positions V H 44 and V L 100
- International Publication No. WO 1998-042378 discloses an antibody for the treatment of B-cell malignancies. -CD22 antibody is disclosed.
- mice As described above, for the production of antibodies for treatment, monoclonal antibodies are mainly produced using mice. However, since non-human antibodies such as mouse-derived monoclonal antibodies are regarded as foreign antigens in the human body, they induce an immune response and have a short half-life, so their therapeutic effect is limited.
- humanized antibodies have been developed in which the remaining parts of the antibody except for the antigen-binding portion are substituted with human antibodies.
- the most similar human antibody gene for the antibody to be substituted is selected, and only the CDR region of the mouse antibody is replaced with the human antibody CDR position by a method called CDR transplantation.
- Such humanized antibodies have the advantage of reducing the immune response in the human body because most of the genes are humanized.
- CD19 is generally a target in addition to the CD22 antigen.
- Clinical trials on CAR-T cells targeting these antigens are also in progress.
- there is a difference in the amount of target antigen expression depending on cells such as leukemia that do not express CD19, and single CAR or single CAR-T cell therapy that targets only one antigen loses the target antigen due to the immune evasion strategy of tumor cells problems may arise.
- CAR-T cells targeting dual or multiple antigens are being studied, and simultaneous targeting of two antigens can reduce the possibility of antigen-loss variants.
- an antibody that binds to CD22 was selected, and humanized anti-CD22 antibodies (4F5 (V4) and 4F5 (V11)) were prepared using the antibody.
- a humanized anti-CD22 antibody of the invention was used to generate chimeric antigen receptors and CAR-T cells targeting CD22.
- 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 prepared, and the CD22-CAR-T cell prepared in the present invention and the dual It was confirmed that the chimeric antigen receptor was normally expressed in specific CD19xCD22-CAR-T cells, and the present invention was completed.
- an object of the present invention is to provide an antibody specific for CD22 or 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 a chimeric antigen receptor comprising the CD22-specific antibody or CD22-specific humanized antibody, a polynucleotide encoding the chimeric antigen receptor, a vector comprising the polynucleotide, and the polynucleotide or It is to provide an immune effector cell expressing a chimeric antigen receptor containing a vector.
- Another object of the present invention is 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 the polynucleotide Or to provide an immune effector cell expressing a bispecific chimeric antigen receptor comprising a vector.
- Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases mediated by B cells, including the immune effector cells.
- the present invention relates to a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a CDR3 region represented by the amino acids of SEQ ID NO: 3, and a heavy chain variable region comprising the amino acids represented by SEQ ID NO: 4
- a CDR1 region represented by the amino acids of SEQ ID NO: 1 a CDR2 region represented by the amino acids of SEQ ID NO: 2
- a CDR3 region represented by the amino acids of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acids represented by SEQ ID NO: 4
- an antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region, a CDR2 region represented by the amino acids of SEQ ID NO: 5, and a CDR3 region represented by the amino acids of SEQ ID NO: 6.
- the antibody specifically binding to CD22 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.
- the present invention provides a polynucleotide encoding the antibody or fragment thereof that specifically binds to CD22.
- the present invention provides a vector comprising a polynucleotide encoding the antibody or fragment thereof that specifically binds to CD22.
- the present invention provides a recombinant cell that produces an antibody or fragment thereof that specifically binds to CD22 transformed with the vector.
- the present invention relates to 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 CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4
- An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6
- the antibody specifically binding to CD22 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.
- the transmembrane domain is a protein selected from the group consisting of CD8 ⁇ , CD4, CD28, CD137, CD80, CD86, CD152 and PD1, and the costimulatory domain is CD28, 4-1BB, It is a protein selected from the group consisting of OX-40 and ICOS, and the signaling domain may be derived from CD3 ⁇ .
- 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 CD8 ⁇ may be of origin.
- the present invention provides a polynucleotide encoding the chimeric antigen receptor.
- the present invention provides a vector comprising a polynucleotide encoding the chimeric antigen receptor.
- the present invention provides an immune effector cell comprising a polynucleotide encoding the chimeric antigen receptor or a vector containing the polynucleotide and expressing the chimeric antigen receptor.
- the present invention relates to a CD19-binding domain and a CD22-binding domain
- CAR bispecific chimeric antigen receptor
- the CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4
- An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6
- a bispecific chimeric antigen receptor targeting CD19 and CD22 Provided is a bispecific chimeric antigen receptor targeting CD19 and CD22.
- the CD19-binding domain and the CD22-binding domain are 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 - CD22
- the light chain variable region of an antibody that specifically binds to the heavy chain variable region of an antibody that specifically binds to CD19 may be linked in sequence.
- the light chain variable region of the antibody specifically binding to CD19 is a CDR1 region (QDISKY) represented by amino acids of SEQ ID NO: 44, a CDR2 region represented by amino acids of SEQ ID NO: 45 ( HTS) and a CDR3 region (QQGNTLPYT) represented by the amino acids of SEQ ID NO: 46, preferably represented by the amino acid sequence of SEQ ID NO: 48.
- QDISKY CDR1 region represented by amino acids of SEQ ID NO: 44
- HTS CDR2 region represented by amino acids of SEQ ID NO: 45
- QQGNTLPYT CDR3 region represented by the amino acids of SEQ ID NO: 46, preferably represented by the amino acid sequence of SEQ ID NO: 48.
- the heavy chain variable region of the antibody specifically binding to CD19 is the CDR1 region (GVSLPDYG) represented by the amino acids of SEQ ID NO: 41 and the CDR2 region represented by the amino acids of SEQ ID NO: 42.
- IWGSETT the CDR3 region
- AKHYYYGGSYAMDY the amino acids of SEQ ID NO: 43, preferably represented by the amino acid sequence of SEQ ID NO: 47.
- the antibody specifically binding to CD22 is CD22 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.
- the transmembrane domain is a protein selected from the group consisting of CD8 ⁇ , CD4, CD28, CD137, CD80, CD86, CD152 and PD1, and the costimulatory domain is CD28, 4-1BB , OX-40 and ICOS, and the signaling domain may be derived from CD3 ⁇ .
- 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 CD8 ⁇ may be of origin.
- the present invention also provides a polynucleotide encoding the bispecific chimeric antigen receptor.
- the present invention provides a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor.
- the present invention provides an immune effector cell comprising a polynucleotide encoding the bispecific chimeric antigen receptor or a vector containing the polynucleotide and expressing the bispecific chimeric antigen receptor.
- the present invention relates to an antibody or fragment thereof that specifically binds to the CD22;
- a pharmaceutical composition for preventing or treating a disease mediated by B cells including the immune effector cells is provided.
- the disease mediated by B cells is a tumor, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent delayed NHL, refractory 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.
- NHL non-Hogkins lymphoma
- aggressive NHL recurrent aggressive NHL
- recurrent delayed NHL recurrent delayed NHL
- refractory NHL refractory delayed NHL
- CLL chronic lymphocytic leukemia
- small lymphocytic lymphoma small lymphocytic lymphoma
- leukemia hairy cell leukemia
- HCL hairy cell leukemia
- ALL acute lymphocytic leukemia
- an antibody that specifically binds to CD22 and a humanized antibody thereof were prepared, and using them, a single CAR-T cell targeting CD22 and a bispecific CAR-T cell targeting CD19 and CD22 were prepared .
- CD22-CAR-T cells and the bispecific CD19xCD22-CAR-T cells prepared in the present invention effectively recognized the CD22 antigen and activated the CAR-T cells, and effectively killed the cells expressing CD22. Confirmed.
- the CD22-CAR-T cells based on the humanized antibody specifically binding to CD22 of the present invention and the bispecific CD19xCD22-CAR-T cells CAR-T cells can be usefully utilized as a composition for preventing or treating a disease related to CD22 (or CD19) expression or a disease related to B cells.
- Figure 1 is data confirming the binding ability to CD22 of the 4F5 antibody (mouse) and the humanized 4F5 antibodies (4F5 (V4) and 4F5 (V11)) selected in the present invention by FACS.
- FIG. 2 is a schematic diagram showing a chimeric antigen receptor (single CAR) targeting CD22.
- FIG. 3 is a schematic diagram showing a method for preparing CD22-CAR-T cells using a lentiviral vector expressing CD22-CAR.
- FIG. 4 is a schematic diagram showing (a) a method for preparing CD22-CART cells using peripheral blood mononuclear cells (PBMC) and (b) a method for confirming the CD22 peptide-binding ability of the prepared CD22-CAR-T cells.
- PBMC peripheral blood mononuclear cells
- Figure 5a is data confirming the CD22 binding ability of CD22-CAR-T cells prepared using the 4F5 antibody.
- 5B is data confirming the CD22 binding ability of CD22-CAR-T cells based on humanized anti-CD22 antibodies, 4F5 (V4) and 4F5 (V11).
- Figure 7a is data confirming the killing effect of U2932 cells (CD22 expressing cells) and K562 cells (CD22 non-expressing cells) by CD22-CAR-T cells prepared using the 4F5 antibody.
- Figure 7b is 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 4F5 (V4) and 4F5 (V11), which are humanized anti-CD22 antibodies.
- FIG. 8 is a schematic diagram showing a bispecific chimeric antigen receptor (Bispecific CAR) targeting CD19 and CD22.
- FIG. 9 is a schematic diagram showing a method for preparing CD19xCD22-CAR expressing cells using a lentivirus expressing a bispecific CD19xCD22-CAR targeting CD19 and CD22.
- FIG. 10 shows (a) a method for transfecting a HEK293 cell line with a lentiviral vector expressing CD19xCD22-CAR and (b) a method for confirming the CD19 peptide and CD22 peptide binding ability and CD19xCD22-CAR expression level of the transformed HEK293 cells It is a schematic diagram showing
- FIG. 11 is data confirming the expression levels of CD19x4F5-CAR, CD19x4F5(V4)-CAR and CD19x4F5(V11)-CAR in HEK293FT cells transfected with a lentiviral vector expressing CD19xCD22-CAR.
- FIG. 12 is a schematic diagram showing a method for preparing CD19xCD22-CAR-T cells using a lentiviral vector expressing CD19xCD22-CAR.
- FIG. 13 shows (a) a method for preparing CD19xCD22-CAR-T cells using peripheral blood mononuclear cells (PBMC) and (b) a method for confirming the CD19 peptide and CD22 peptide binding ability of the prepared CD19xCD22-CAR-T cells. It is also a model.
- PBMC peripheral blood mononuclear cells
- CD19xCD22-CAR-T cells is data confirming the activation of CD19xCD22-CAR-T cells, and it was confirmed that CD19xCD22-CAR-T cells simultaneously bind to CD22 peptide and CD19 peptide in CD3+ CD19xCD22-CAR-T cells.
- K562 cells CD19 and CD22 non-expressing cells
- K562-CD19 cells CD19 expressing cells
- K562-CD22 cells CD22 expressing cells
- K562-CD19/CD22 cells CD19/CD22 cells
- the present invention is a heavy chain variable region comprising a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a CDR3 region represented by the amino acids of SEQ ID NO: 3, and SEQ ID NO: 4.
- An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 will be.
- the antibody or fragment thereof that specifically binds to CD22 is specifically composed of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 12. a humanized antibody or fragment thereof that binds; or
- It may be a humanized antibody or fragment thereof that specifically binds to CD22 composed of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.
- the antibody may be a monoclonal antibody.
- the term "monoclonal antibody” is also called a monoclonal antibody or a monoclonal antibody, and is an antibody produced by a single antibody-forming cell, characterized by 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.
- the antibody of the present invention can be prepared as a humanized antibody with increased similarity with 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.
- the most common method for humanizing an antibody is a CDR-grafting method in which a CDR region of an animal antibody is grafted onto a human antibody, but is not limited thereto, and a method known in the art is used.
- Humanized antibodies can be prepared using
- CDR complementarity determining region
- antibody can be used not only in its complete form having two full-length light chains and two full-length heavy chains, but also fragments of antibody molecules.
- 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 , single domain, and the like.
- Fab has a structure having variable regions of light and heavy chains, constant regions of light chains, and a first constant region (CH1) of heavy chains, and has one antigen-binding site.
- Fab' is different 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.
- the F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'.
- Fv is a minimal antibody fragment that has only the heavy chain variable region and the light chain variable region.
- Double-chain Fv has a heavy chain variable region and light chain variable region connected by a disulfide bond
- 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 covalently linked via.
- the monoclonal antibody specifically binding to CD22 of the present invention can be prepared using all or a partial peptide of CD22 protein as an immunogen (or antigen). More specifically, first, as an immunogen, CD22 protein, a fusion protein containing CD22 protein, or a carrier containing CD22 protein is administered to humans together with an adjuvant (eg, Freund adjuvant) as an immune enhancer, if necessary. Immunization is performed by injecting once or more subcutaneously, intramuscularly, intravenously, balboloxal, or intraperitoneally in mammals other than those excluded.
- an adjuvant eg, Freund adjuvant
- Mammals other than humans are preferably mice, rats, hamsters, marmots, chickens, rabbits, cats, dogs, pigs, goats, sheep, donkeys, horses or cows (transgenic mice that produce human antibodies). (including transgenic animals engineered to produce antibodies derived from other animals such as), more preferably mice, rats, hamsters, marmots, chickens or rabbits.
- 1 to 4 immunizations are performed about every 1 to 21 days from the first immunization, and about 1 to 10 days after the final immunization, antibody-producing cells can be obtained from immunosensitized mammals. The number of immunizations and time intervals can be appropriately changed depending on the characteristics of the immunogen to be used.
- Preparation 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) and a method similar thereto. Any one selected from the group consisting of spleen, lymph node, bone marrow, or tonsil collected from animals other than humans immunosensitized as described above, preferably derived from a mammal that does not have the ability to produce an antibody and an antibody-producing cell contained in the spleen.
- a hybridoma can be prepared by cell fusion of myeloma cells.
- the mammal may be a mouse, rat, marmot, hamster, chicken, rabbit or human, preferably a mouse, rat, chicken or human.
- a fusion promoter such as polyethylene glycol or Sendai virus or a method using an electric pulse is used.
- a fusion medium containing a fusion promoter antibody-producing cells and mammalian-derived cells capable of immortal growth are used. is suspended 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.
- the fusion medium for example, MEM medium, RPMI1640 medium, and Iscove's Modified Dulbecco's Medium may be used, and serum types such as bovine serum are preferably excluded.
- the method for screening hybridoma clones producing the monoclonal antibody is, first, transfer the fused cells obtained as described above to a selection medium such as HAT medium, and incubate at about 30 to 40 ° C. for about 3 days to 3 weeks Cells other than hybridomas are killed. Subsequently, after culturing the hybridomas in 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 prepared as RIA (radioactive substance-marked immunoassay). antibody) or an immunoassay method such as ELISA (Enzyme-Linked Immunosorbent Assay). In addition, the clone producing the monoclonal antibody found above shows 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.
- a conventional method for culturing mammalian-derived cells is used, and for collecting a monoclonal antibody from a culture or the like, a conventional method in this field for purifying an antibody in general is used.
- a conventional method in this field for purifying an antibody in general is used.
- each method for example, salting out, dialysis, filtration, concentration, centrifugation, fractional precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, high-speed liquid chromatography, gel electrophoresis and isoelectric point electrophoresis. These are applied in combination as needed.
- the purified monoclonal antibody is then concentrated and dried to be in a liquid or solid state depending on the application.
- the monoclonal antibody of the present invention includes DNA encoding heavy chain and light chain variable regions, respectively, and known DNA encoding heavy and light chain constant regions (eg, Japan 2007-252372 (Refer to Publication No.) and each ligated gene are synthesized by the PCR method or chemical synthesis, and transplanted into a known expression vector (pcDNA 3.1 (sold by Invitrogen)) or the like that enables the expression of the gene to obtain a transformant. It can be obtained by preparing and expressing in a host such as CHO cells or Escherichia coli to produce an antibody, and purifying the antibody from this culture solution using a Protein A or G column or the like.
- pcDNA 3.1 sold by Invitrogen
- a hybridoma that produces an anti-CD22 antibody is prepared and screened to obtain an antibody (scFv) that specifically binds to CD22. It was selected and named as 4F5.
- the 4F5 antibody is a heavy chain variable comprising a CDR1 region (GFSLTIYG) represented by the amino acids of SEQ ID NO: 1, a CDR2 region (MWSGGST) represented by the amino acids of SEQ ID NO: 2, and a CDR3 region (ARNDGYYWFAY) represented by the amino acids of SEQ ID NO: 3. region and a CDR1 region (QSLVHNNGNTY) represented by the amino acids of SEQ ID NO: 4, a CDR2 region (KVS) represented by the amino acids of SEQ ID NO: 5, and a CDR3 region (SQSTHVPYS) represented by the amino acids of SEQ ID NO: 6. configuration was confirmed.
- the 4F5 antibody is composed of a heavy chain variable region represented by the amino acids of SEQ ID NO: 7 and a light chain variable region represented by the amino acids of SEQ ID NO: 8, wherein the heavy chain variable region has the nucleotide sequence of SEQ ID NO: 9, the light chain variable region was confirmed to be encoded by the nucleotide sequence of SEQ ID NO: 10.
- humanized antibodies were prepared by changing the anti-CD22 antibody 4F5 to a structure corresponding to human, which was named 4F5 (V4) antibody and 4F5 (V11) antibody did
- the CDRs of the heavy chain variable region and the CDRs of the light chain variable region of the 4F5 (V4) antibody and the 4F5 (V11) antibody were the same as those of 4F5, and the remaining portions except for the CDR were humanized.
- 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 is SEQ ID NO: 13
- the light chain variable region may be encoded by the nucleotide sequence of SEQ ID NO: 14.
- the 4F5 (V11) antibody is composed 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 4F5 (V11) antibody is SEQ ID NO: 17
- 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 a scFv (single chain variable fragment), which can be prepared 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 may be encoded by the nucleotide sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, but is not limited thereto.
- the 4F5 antibody When linked by 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, and the 4F5 (V4) antibody of SEQ ID NO: 25 It may be represented by the amino acid sequence 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.
- the present invention relates to a polynucleotide encoding an antibody or fragment thereof that specifically binds to CD22.
- polynucleotide generally refers to nucleic acid molecules, deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated of any length.
- the polynucleotides of the invention can be used for (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 produced through synthesis such as chemical synthesis, and preferably the isolated polynucleotide is produced by recombinant DNA technology.
- PCR polymerase chain reaction
- nucleic acids for encoding antibodies or antigen-binding fragments thereof are 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.
- the present invention relates to a vector comprising a polynucleotide encoding an antibody or fragment thereof that specifically binds to CD22, and a recombinant cell transformed with the vector.
- the term "expression vector” is a gene product containing essential regulatory elements such as a promoter so that a target gene can be expressed in an appropriate host cell.
- Vectors may be selected from one or more of plasmids, retroviral vectors and lentiviral vectors. Once transformed into a suitable host, the vector can replicate and function independently of the host genome or, in some cases, can integrate into the genome itself.
- vectors may contain expression control elements that allow for correct expression of the coding region in a suitable host.
- 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 includes 5' ratios that participate in transcription initiation and translation initiation, such as TATA boxes, capped sequences, CAAT sequences, etc., respectively. -contains a transcribed sequence, and a 5' or 3' non-translated sequence.
- a 5' non-transcribed expression control sequence can include a promoter region that can include promoter sequences for transcribing and regulating functionally linked nucleic acids.
- promoter refers to a minimal sequence sufficient to direct transcription.
- 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 such constructs may be located on the 5' or 3' portion of the gene. . Both conserved promoters and inducible promoters are included. Promoter sequences may be of prokaryotic, eukaryotic or viral origin.
- 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 preparing a transformant by introducing an expression vector into a host cell.
- 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, and the like.
- antibody protein When the transformant expressing the vector is cultured in a nutrient medium, antibody protein can be produced and isolated in large quantities.
- Media and culture conditions can be appropriately selected and used according to the host cell. Conditions such as temperature, medium pH, and incubation time should be appropriately adjusted so as to be suitable for cell growth and mass production of proteins during culture.
- the vector according to the present invention can be transformed into a host cell, preferably a mammalian cell, for antibody production.
- 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., and these cells are readily available, for example, from the American Type Culture Collection (ATCC, USA).
- COS-1 eg ATCC CRL 1650
- COS-7 eg ATCC CRL-1651
- HEK293, BHK21 eg ATCC CRL-10
- CHO eg ATCC CRL 1610
- BSC-1 eg
- a chimeric antigen receptor (CAR) containing an intracellular signal transduction domain containing an intracellular signal transduction domain
- the CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4
- An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 It relates to a chimeric antigen receptor targeting CD22.
- the antibody specifically binding to CD22 is a humanized antibody specifically binding to CD22 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. antibodies or fragments thereof; or
- It may be a humanized antibody or fragment thereof that specifically binds to CD22 composed of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.
- chimeric antigen receptor generally refers to a fusion protein containing an antigen and an extracellular domain that has 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 can include an antigen binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain.
- a CAR may be combined with a T cell receptor-activating intracellular domain based on the antibody's antigenic (eg CD22) specificity. Genetically modified CAR-expressing T cells can specifically identify and eliminate target antigen-expressing malignant cells.
- CD22-binding domain generally refers to a domain capable of specifically binding to the CD22 protein.
- 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 on B cells.
- binding domain includes “extracellular domain”, “extracellular binding domain”, “antigen-specific binding domain” and “Extracellular antigen-specific binding domain” may be used interchangeably and refers to a CAR domain or fragment that has the ability to specifically bind to a target antigen (eg CD22) do.
- target antigen eg CD22
- the anti-CD22 antibody or fragment thereof is the aforementioned anti-CD22 antibody, which is a monoclonal antibody, preferably a single chain variable fragment (scFv). Specifically, it can be prepared using the 4F5 (V4) or 4F5 (V11) antibody, which is a humanized antibody specific to CD22 of the present invention.
- the chimeric antigen receptor may be a bispecific chimeric antigen receptor further comprising a B cell surface marker-binding domain in addition to the CD22-binding domain, and the B cell surface markers are CD10, CD19, and CD20. , CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85 or CD86, preferably CD19.
- 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: 36 is preferably used in 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, and the hinge region is derived from CD8 ⁇ , preferably SEQ ID NO: 37 amino acid sequence.
- the "hinge region” generally refers to the junction region between an antigen-binding region and an immune cell Fc receptor (FcR)-binding region.
- 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 CD8 ⁇ , CD4, CD28, CD137, CD80, CD86, CD152 and PD1, and may preferably be represented by the amino acid sequence of SEQ ID NO: 38.
- costimulatory domain generally refers to an intracellular domain capable of providing immune stimulatory molecules, which are cell surface molecules required for effective response of lymphocytes to antigens.
- the costimulatory domain described above may include a costimulatory domain of CD28, and may include a costimulatory domain of the TNF receptor family, such as the costimulatory domains of OX40 and 4-1BB, preferably SEQ ID NO: It may be 4-1BB represented by the amino acid sequence of 39.
- intracellular signal transduction domain generally refers to a domain located inside a cell and capable of transmitting a signal.
- the intracellular signaling domain is an intracellular signaling domain of a chimeric antigen receptor.
- the intracellular signaling domain may be selected from the CD3 ⁇ intracellular domain, the CD28 intracellular domain, the CD28 intracellular domain, the 4-1BB intracellular domain and the OX40 intracellular domain, preferably comprising the amino acids of SEQ ID NO: 40 It may be CD3 ⁇ represented by the sequence.
- the present invention relates to a polynucleotide encoding the chimeric antigen receptor (CAR).
- CAR chimeric antigen receptor
- the polynucleotide encoding the chimeric antigen receptor (CAR) is a polynucleotide encoding a CD22-binding domain; polynucleotides encoding transmembrane domains; polynucleotides coating the co-stimulatory domain; And it may include a polynucleotide encoding an intracellular signaling domain.
- the polynucleotide encoding the CD22-binding domain may be a 4F5 antibody specific for CD22, a humanized 4F5(V4) antibody specific for CD22, or a 4F5(V11) antibody specific for CD22 of the present invention, and the light chain variable region and the heavy chain variable region are linkers.
- the specific nucleotide sequence is as described above.
- the polynucleotide encoding the chimeric antigen receptor (CAR) of the present invention is a 4F5 antibody represented by the nucleotide sequence of SEQ ID NO: 24, a 4F5 (V4) antibody represented by the nucleotide sequence of SEQ ID NO: 26, or SEQ ID NO: 28 4F5 (V11) antibody represented by the nucleotide sequence of;
- transmembrane domain represented by the nucleotide sequence of SEQ ID NO: 32;
- 4-1BB (co-stimulatory domain) represented by the nucleotide sequence of SEQ ID NO: 33;
- CD3 ⁇ intracellular signaling domain
- SEQ ID NO: 34 The nucleotide sequence of SEQ ID NO: 34.
- a signal peptide represented by the nucleotide sequence of SEQ ID NO: 30 may be additionally included.
- a polynucleotide encoding a hinge region may be further included, preferably a CD8 hinge region represented by the nucleotide sequence of SEQ ID NO: 31 can
- the present invention relates to a vector comprising a polynucleotide encoding the chimeric antigen receptor (CAR).
- CAR chimeric antigen receptor
- the vector is a recombinant viral vector, preferably a lentiviral vector, comprising an operably linked EF1 ⁇ promoter; polynucleotides encoding signal peptides; a polynucleotide encoding a CD22-binding domain; polynucleotides encoding transmembrane domains; 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).
- WPRE woodchuck hepatitis virus post-transcriptional regulatory element
- the EF1 ⁇ promoter may be represented by the nucleotide sequence of SEQ ID NO: 29, 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: 27 , or sequences that are at least 99% identical.
- the promoter is operably linked to drive expression of a CD22-binding domain, an anti-CD22 antibody (scFv).
- a lentiviral vector into which a polynucleotide coating CD22-CAR was inserted was prepared, and the prepared vector was transformed 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 method 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.
- colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
- Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (eg, artificial membrane vesicles).
- an exemplary delivery vehicle is a liposome.
- lipid formulations is contemplated for the introduction (in vitro, ex vivo or in vivo) of nucleic acids into host cells.
- a nucleic acid can be associated with a lipid.
- Nucleic acids associated with lipids may be encapsulated in the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules associated with both liposomes and oligonucleotides, entrapped within liposomes, complexed with liposomes, 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 a micelle, or otherwise associated with a lipid.
- the lipid, lipid/DNA or lipid/expression vector associated composition is not limited to any particular structure in solution.
- the present invention includes a polynucleotide encoding a chimeric antigen receptor (CAR) based on an antibody specific for the CD22 or a vector comprising a polynucleotide encoding the chimeric antigen receptor (CAR), wherein the CD22 It relates to immune effector cells expressing an antibody-based chimeric antigen receptor (CAR) specific for
- the immune effector cells may be mammalian-derived isolated cells, preferably T cells, B cells, natural killer (NK) cells, dendritic cells, bone marrow cells, monocytes, or macrophages, and more Preferably it may be a T cell.
- immune effector cells expressing the chimeric antigen receptor can be prepared by introducing the CAR vector of the present invention into immune effector cells, for example, T cells or NK cells.
- the CAR vector may be introduced into cells by a method known in the art, such as electroporation or lipofectamine (lipofectamine 2000, Invitrogen).
- immune effector cells can be transformed 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.
- a transposon can be used to introduce a CAR carrier plasmid (transposon) and a transposase carrier plasmid into a target cell.
- CAR molecules can be added to the genome by gene editing methods (eg, CRISPRCas9).
- Immune effector cells for producing 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) from which an immune response can be elicited.
- the “subject” is a living organism (eg, a mammal) from which an immune response can be elicited.
- subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
- T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.
- the T cells can be obtained from a unit of blood collected from a subject using any of a number of techniques known to those skilled in the art, such as FicollTM separation.
- Cells from blood are obtained by apheresis, and the apheresis product typically contains T cells, monocytes, granulocytes, lymphocytes including B cells, other nucleated leukocytes, red blood cells, and platelets.
- T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, eg, by centrifugation over a PERCOLLTM gradient or by countercurrent centrifugation.
- the CD22-CAR lentiviral vector is applied to the T cells.
- CD22-CAR-T cells were prepared by transduction, and specifically, CD22-CAR-T cells were prepared using a 4F5 antibody, a humanized 4F5 (V4) antibody, and a 4F5 (V11) antibody, respectively.
- CD3, CD4 or CD8-activated CD22-CAR-T cells were examined for their ability to bind to CD22 peptides. As shown in Figures 5a and 5b, it was confirmed that the CD22-CAR-T cells prepared in the present invention bind to the CD22 peptide.
- CD22-CAR-T cells are U2932 expressing CD22. It was confirmed that the cell-specific killing effect was shown.
- the anti-CD22 antibody of the present invention the 4F5 antibody, the 4F5(V4) antibody, or the 4F5(V11) antibody-based chimeric antigen receptor, and the CAR-T cell using the same are a composition for preventing or treating diseases related to B cell or CD22 expression can be put to good use.
- Bispecific Chimeric Antigen Receptor Targeting CD19 and CD22
- CD19-binding domain and CD22-binding domain CD19-binding domain and CD22-binding domain
- CAR bispecific chimeric antigen receptor
- the CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4
- An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 It relates to a bispecific chimeric antigen receptor (CD19xCD22 bispecific CAR).
- the antibody specifically binding to CD22 is an anti-CD22 antibody composed 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;
- a humanized anti-CD22 antibody 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; or
- It may be a humanized anti-CD22 antibody composed 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.
- the bispecific (bispecific or bivalent) chimeric antigen receptor is a CAR capable of simultaneously binding two different types of antigens, and in the present invention, the bispecific chimeric antigen receptor preferably targets both CD19 and CD22 A receptor was prepared, and the CD19-binding domain may use a known anti-CD19 antibody sequence without limitation.
- the chimeric antigen receptor 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 were 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) - It may be linked in sequence to the heavy chain variable region (CD19VH) of an antibody that specifically binds to CD19.
- the light chain variable region of the antibody specifically binding to CD19 is represented by the amino acid sequence of SEQ ID NO: 48, and the heavy chain variable region of the antibody specifically binding to CD19 is represented by the amino acid sequence of SEQ ID NO: 47. .
- the CD19-binding domain and the CD22-binding domain can be prepared through genetic recombination technology so that they can be linked by a linker, and preferably CD19VL and CD22VH or CD22VL and CD19VH are linkers represented by the amino acid sequence of SEQ ID NO: 51 (linker in FIG. 8). 1, and CD22VH and CD22VL may be linked by a linker represented by the amino acid sequence of SEQ ID NO: 54 (linker 6 in FIG. 8), but is not limited thereto, and does not affect antibody activity Peptide consisting of any amino acid sequence can be used.
- the present invention relates to a polynucleotide encoding a bispecific chimeric antigen receptor targeting CD19 and CD22.
- the polynucleotide encoding the bispecific chimeric antigen receptor includes a polynucleotide encoding a CD19-binding domain and a polynucleotide encoding a CD22-binding domain; polynucleotides encoding transmembrane domains; polynucleotides coating the co-stimulatory domain; And it may include a polynucleotide encoding an intracellular signaling domain.
- the polynucleotide encoding the bispecific chimeric antigen receptor of the present invention is
- CD19VL Light chain variable region of antibody specifically binding to CD19 (CD19VL; SEQ ID NO: 50) - heavy chain variable region of antibody specifically binding to CD22 (CD22VH; SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 17) - CD22 Bispecific consisting of the light chain variable region of the antibody specifically binding (CD22VL; SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 18) - the heavy chain variable region of the antibody specifically binding to CD19 (CD19VH; SEQ ID NO: 49) antibodies;
- transmembrane domain represented by the nucleotide sequence of SEQ ID NO: 32;
- 4-1BB (co-stimulatory domain) represented by the nucleotide sequence of SEQ ID NO: 33;
- CD3 ⁇ intracellular signaling domain
- SEQ ID NO: 34 The nucleotide sequence of SEQ ID NO: 34.
- the polynucleotide for the linker may be represented by the nucleotide sequence of SEQ ID NO: 52 or SEQ ID NO: 53, ,
- the polynucleotide for the linker may be represented by the nucleotide sequence of SEQ ID NO: 55, but is not limited thereto and affects antibody activity. Polynucleotides encoding any amino acid sequence not specified may be used.
- a signal peptide represented by the nucleotide sequence of SEQ ID NO: 30 may be additionally included.
- a polynucleotide encoding a hinge region may be further included, preferably a CD8 hinge region represented by the nucleotide sequence of SEQ ID NO: 31 can
- the present invention relates to a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor.
- the vector is a recombinant viral vector, preferably a lentiviral vector, comprising an operably linked EF1 ⁇ promoter; polynucleotides encoding signal peptides; polynucleotides encoding a CD19-binding domain and a CD22-binding domain; polynucleotides encoding transmembrane domains; 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. 9).
- WPRE woodchuck hepatitis virus post-transcriptional regulatory element
- the EF1 ⁇ promoter may be represented by the nucleotide sequence of SEQ ID NO: 29, 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: 27 , or sequences that are at least 99% identical.
- the promoter is operably linked to induce expression of an anti-CD19/CD22 antibody (CD19VL-CD22VH-CD22VL-CD19VH), which is a CD19xCD22-binding domain, and details of the vector are the same as described above.
- a lentiviral vector into which a polynucleotide coating CD19xCD22-CAR was inserted was prepared, and the prepared vector was transformed into 293FT cells to express CD19xCD22-CAR cells were prepared.
- FIG. 11 it was confirmed that the bispecific chimeric antigen receptor targeting CD19 and CD22 was expressed in the prepared CD19xCD22-CAR expressing cells.
- the present invention includes a polynucleotide encoding the bispecific chimeric antigen receptor (CAR) or a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor (CAR), It relates to immune effector cells that express specific chimeric antigen receptors (CARs).
- CAR bispecific chimeric antigen receptor
- CARs immune effector cells that express specific chimeric antigen receptors
- the immune effector cells may be mammalian-derived isolated cells, preferably T cells, B cells, natural killer (NK) cells, dendritic cells, bone marrow cells, monocytes, or macrophages, and more Preferably it may be a T cell. Further, details of the immune effector cells expressing the chimeric antigen receptor are the same as described above.
- CD19xCD22-CAR lentiviral vector is applied to the T cells.
- CD19xCD22-CAR-T cells were prepared by transduction, and specifically, CD19xCD22-CAR-T cells were prepared using a 4F5 antibody (mouse), a humanized 4F5 (V4) antibody, and a 4F5 (V11) antibody, respectively.
- the binding ability of the CD3-activated CD19xCD22-CAR-T cells to the CD22 peptide and the CD19 peptide was confirmed. As shown in FIG. 14, it was confirmed that the CD19xCD22-CAR-T cells prepared in the present invention simultaneously bind to the CD22 peptide and the CD19 peptide.
- CD19xCD22-CAR-T cells are 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 or CD22 expression
- the present invention provides an antibody that specifically binds to CD22; humanized antibodies that specifically bind to CD22; immune effector cells expressing chimeric antigen receptors that target CD22; or a pharmaceutical composition for preventing or treating a disease mediated by B cells, comprising immune effector cells expressing a bispecific chimeric antigen receptor that specifically binds to CD19xCD22.
- the B cell may preferably be a cell expressing CD19 or CD22, and the disease is tumor/cancer, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent delayed NHL, refractory NHL, refractory delayed NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute It may be selected from the group consisting of acute lymphocytic leukemia (ALL), Burkitt's lymphoma and mantle cell lymphoma.
- NHL non-Hogkins lymphoma
- aggressive NHL recurrent aggressive NHL
- recurrent delayed NHL recurrent delayed NHL
- refractory NHL refractory delayed NHL
- CLL chronic lymphocytic leukemia
- small lymphocytic lymphoma small lymphocytic lymphoma
- leukemia hairy cell leukemia
- HCL hairy cell leuk
- the composition may include a therapeutic agent for a disease mediated by B cells, and the therapeutic agent is present in a state covalently bound to an antibody that specifically binds to CD19 or CD22, or the CD22- It can be administered in combination with CAR immune effector cells or CD19xCD22-CAR immune effector cells.
- the therapeutic agent includes a small molecule drug, a peptide drug, a toxin (eg, cytotoxin), and the like.
- the small molecule drug may be a compound that exhibits a pharmaceutical activity of interest and generally has a molecular weight of about 800 Da or less or 2000 Da or less.
- Inorganic small molecules refer to molecules containing no carbon atoms, whereas organic small molecules refer to compounds containing at least one carbon atom.
- the peptidic drug refers to an amino acid containing polymeric compound, which includes naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides and proteins, as well as peptide mimetics.
- the peptide drug may be obtained by chemical synthesis or generated 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, including, but not limited to, ricin, abrin, diphtheria toxin, Pseudomonas exotoxin (e.g., PE35, PE37, PE38, PE40, etc.), saporin, gelonin, orca.
- cytotoxin including, but not limited to, ricin, abrin, diphtheria toxin, Pseudomonas exotoxin (e.g., PE35, PE37, PE38, PE40, etc.), saporin, gelonin, orca.
- PAP antiviral protein
- botulinum toxin briodin
- momordin momordin
- bunin antiviral protein
- the therapeutic agent may be an anticancer agent.
- Anti-cancer agents reduce the proliferation of cancer cells and include non-peptidic (i.e., non-proteinaceous) compounds, including cytotoxic agents and cytostatic agents.
- Non-limiting examples of anticancer agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids and steroid hormones. Peptidic compounds may also be used.
- the humanized antibody, CD22-CAR immune effector cells or CD19xCD22-CAR immune effector cells that specifically bind to CD22 are the only active ingredients in the therapeutic or diagnostic composition, or, for example, anti-T cells, It can be used with other active ingredients including other antibody components such as anti-IFN ⁇ or anti-LPS antibodies, or non-antibody components such as xanthines.
- the pharmaceutical composition preferably comprises a therapeutically effective amount of an antibody of the present invention.
- therapeutically effective amount means the amount of a therapeutic agent required to treat, ameliorate, or prevent the target disease or condition, or to produce an appreciable therapeutic or prophylactic effect.
- the therapeutically effective dose can be determined initially 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. This information can be used to determine useful dosages and routes for human administration.
- an effective dosage is 0.01 to 50 mg/kg, preferably 0.1 to 20 mg/kg, more preferably about 15 mg/kg.
- compositions may be administered to a patient individually or in combination with other agents, drugs or hormones.
- the dosage at which an antibody of the invention is administered depends on the nature of the condition being treated, the grade of the malignant lymphoma or leukemia, and whether the antibody is being 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 persistence of the drug effect. If the antibody molecule has a short half-life (eg, 2-10 hours), it may be necessary to give 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 give a dose once daily, once a week, or once every 1 or 2 months.
- the pharmaceutical composition may contain a pharmaceutically acceptable carrier for administration of the antibody.
- the carrier must not itself induce the production of antibodies harmful to the subject to which the composition is administered, and must be non-toxic.
- Suitable carriers can be slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers and inactive viral particles.
- 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 substances may be present in these 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 forms suitable for parenteral administration, eg by injection or infusion (eg bolus injection or continuous infusion).
- parenteral administration eg by injection or infusion (eg bolus injection or continuous infusion).
- the product may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, which may contain such prescriptive agents as suspending agents, preservatives, stabilizing and/or dispersing agents.
- the antibody molecule may be in anhydrous form and reconstituted with an appropriate sterile solution prior to use.
- compositions of the present invention can be administered directly to a patient.
- the patients to be treated may be animals. However, it is preferred that the compositions are tailored for administration to human patients.
- the pharmaceutical composition of the present invention may be used, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (see eg WO 98/20734), subcutaneous, It may be administered by any route including intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes.
- a hypospray can be used to administer the pharmaceutical composition of the present invention.
- therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions.
- solid forms suitable for solution or suspension in liquid excipients may be prepared prior to injection.
- Direct delivery of the composition may generally be by injection, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, or may be delivered into the interstitial space of a tissue.
- the composition may be administered to a 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 can be susceptible to degradation within 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.
- Example 1 Production and screening of antibodies that specifically bind to CD22
- a hybridoma producing an antibody that binds to CD22 was prepared and the antibody was selected.
- splenocytes were isolated by immunization with CD22 protein (ACRObiosystems Inc., cat#CD2-H52H8, USA), and hybridoma cells were prepared through cell fusion with mouse myeloma cells.
- HGPRT HypoxanthineGuanidine-Phosphoribosyl-Transferase
- a limiting dilution method was used to select hybridomas producing antibodies that bind to CD22 among the proliferated hybridomas.
- the number of cells per 96 well was reduced to 1 or less, and then, whether the antibodies obtained from the clones proliferated from 1 cell were binding to CD22 was confirmed by ELISA, and clones binding to CD22 were selected. By repeating the above process three times, hybridomas producing antibodies binding to CD22 were selected. In this way, an antibody binding to CD22 was obtained.
- the antibody was named 4F5, and its base sequence and amino acid sequence were analyzed. Sequence information for 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 the underlined portion in Table 1 means the complementarity determining region (CDR).
- CDR complementarity determining region
- Sequence information of 4F5 antibody 4F5 sequence information sequence number heavy chain variable region CDR1 GFSLTIYG SEQ ID NO: 1 heavy chain variable region CDR2 MWSGGST SEQ ID NO: 2 heavy chain variable region CDR3 ARNDGYYWFAY SEQ ID NO: 3 light chain variable region CDR1 QSLVHNNGNTY SEQ ID NO: 4 light chain variable region CDR2 KVS SEQ ID NO: 5 light chain variable region CDR3 SQSTHVPYS SEQ ID NO: 6 heavy chain variable region amino acid sequence QVQLKESGPGLVQPSQSLSITCTVS GFSLTIYG VHWIRQSPGKGLEWLGV MWSGGST DYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYC ARNDGYYWFAY WGQGTLVTVSA SEQ ID NO: 7 light chain variable region amino acid sequence DVLMTQTPLSLPVSLGDQASISCRSS QSLVHNNGNTY LHWYLQKPGQ
- a humanized antibody was prepared by changing the 4F5 antibody selected in Example 1 to a structure corresponding to human.
- mouse 4F5 antibody is obtained by a CDR-grafting method in which the CDR of a mouse antibody that binds to CD22 is replaced with the CDR of a human antibody using the germline sequence of a human antibody as a frame.
- a humanized antibody was prepared.
- the humanized antibodies were named 4F5(V4) antibody and 4F5(V11) antibody, and the amino acid sequences were analyzed.
- Sequence information for the heavy chain variable region and the light chain variable region of the antibody according to the sequencing results are shown in Tables 2 and 3 below, and the underlined portions in Tables 2 and 3 are complementary determining regions (complementarity determining regions; CDRs).
- CD22-expressing non-cell lymphoma U2932 (B-cell lymphoma U2932 cell) was reacted with 1x10 6 cells and 1 ⁇ g of 4F5 antibody for 30 minutes, and then, after staining the surface with a secondary antibody, flow cytometry measured.
- PE-conjugated anti-CD22 antibody Biolegend Inc., cat# 302506, USA
- PE-conjugated anti-mouse IgG antibody PE-conjugated goat anti-mouse IgG; Biolegend Inc., cat# 405307, USA
- the 4F5 antibody, the humanized 4F5(V4) antibody, and the 4F5(V11) antibody specifically bind to cells expressing CD22.
- lentivirus expressing a chimeric antigen receptor (CAR) targeting CD22 A vector CD22-CAR lentiviral vector
- a polynucleotide encoding the CD22-binding domain (4F5 antibody represented by the nucleotide sequence of SEQ ID NO: 24, 4F5 (V4) antibody represented by the nucleotide sequence of SEQ ID NO: 26, or 4F5 (V11) represented by the nucleotide sequence of SEQ ID NO: 28 antibody);
- polynucleotide encoding 4-1BB (co-stimulatory domain) (SEQ ID NO: 33);
- CAR DNA consisting of a polynucleotide (SEQ ID NO: 35) encoding WPRE was synthesized in vitro and inserted into a third-generation lentiviral vector.
- Lentiviral vector DNA (0.5 ⁇ g) was transferred to HEK293FT cells (5 ⁇ 10 5 cells/500 ⁇ l), and 293HEK cells expressing the CD22-CAR gene were constructed.
- Lipofectamine 3000 transfection kit (Invitrogen, cat# L3000-015) was used to transfer the gene to 293HEK cells, and cultured in Opti-MEM (gibco, cat# 51985-034) medium for 4 hours.
- CD22-CAR-T cells based on 4F5 antibody, 4F5(V4) antibody and 4F5(V11) antibody were prepared by transfecting T cells with the CD22-CAR lentiviral vector prepared in Example 4 above.
- T cell activation beads (Miltenyl Biotec, cat# 130-091-441 ) was used to activate T cells.
- 4F5 antibody-based CD22-CAR-T cells, 4F5 (V4) antibody-based CD22-CAR-T cells, and 4F5 ( V11) antibody-based CD22-CAR-T cells were respectively prepared.
- CD22 peptide binding capacity of CD22-CAR-T cells was confirmed by flow cytometry.
- the 4F5 antibody, 4F5(V4) antibody-based CD22-CAR-T cells, and 4F5(V11) antibody-based CD22-CAR-T cells prepared above were subjected to CD3, anti-CD4, and anti-CD8 antibodies.
- CD4 or CD8 activated CD22-CAR-T cells were respectively sorted, reacted with FITC-CD22 peptide, and fluorescence intensity was measured using a FACS machine.
- CD3, CD4, or CD8-activated CD22-CAR-T cells all bind to the CD22 peptide.
- Example 6 Confirmation of the killing effect of CD22-CAR-T cells on CD22 expressing cells
- K562 cells human erythroleukemic cell line
- the degree of cell death was calculated using Equation 1 below with the measured value.
- Target Spontaneous Luminescence value derived from the target cell only medium
- Target Maximum Luminescence value derived from 100% lysis of target cells (using Lysis Reagent)
- the anti-CD22 antibody of the present invention the 4F5 antibody, the 4F5(V4) antibody or the 4F5(V11) antibody, the antibody-based chimeric antigen receptor, and the CAR-T cell using the same are used to prevent or prevent diseases associated with B cell or CD22 expression. It can be usefully utilized as a therapeutic composition.
- Example 7 Construction of a bispecific chimeric antigen receptor expression vector targeting CD19/CD22
- a lentiviral vector (CD19xCD22-CAR lentiviral vector) expressing a bispecific chimeric antigen receptor targeting CD19 and CD22 was prepared in the same manner as in Example 4 above.
- polynucleotide encoding 4-1BB (co-stimulatory domain) (SEQ ID NO: 33);
- CAR DNA consisting of a polynucleotide (SEQ ID NO: 35) encoding WPRE was synthesized in vitro and inserted into a third-generation lentiviral vector.
- the known anti-CD19 antibody (FMC63) was used for the CD19-binding domain
- the 4F5 (mouse) antibody, 4F5 (V4) antibody and 4F5 (V11) antibody of the present invention were used for the CD22-binding domain.
- the CD19xCD22-binding domain 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) - linked in sequence to the heavy chain variable region (CD19VH) of an antibody specifically binding to CD19 (LoopCAR), and sequence information for polynucleotides 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. 8) - CD22VH represented by the nucleotide sequence of SEQ ID NO: 9 - nucleotide sequence of SEQ ID NO: 55 Linker represented by sequence (linker 6 in FIG. 8) - CD22VL represented by nucleotide sequence of SEQ ID NO: 10 - Linker represented by nucleotide sequence of SEQ ID NO: 53 (linker 1 in FIG. 8) - represented by nucleotide sequence of SEQ ID NO: 49 becoming CD19VH;
- 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. 8) - CD22VH represented by the nucleotide sequence of SEQ ID NO: 13 - nucleotide sequence of SEQ ID NO: 55 Linker represented by sequence (linker 6 in FIG. 8) - CD22VL represented by nucleotide sequence of SEQ ID NO: 14 - Linker represented by nucleotide sequence of SEQ ID NO: 53 (linker 1 in FIG. 8) - represented by nucleotide sequence of SEQ ID NO: 49 becoming CD19VH;
- 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. 8) - CD22VH represented by the nucleotide sequence of SEQ ID NO: 17 - nucleotide sequence of SEQ ID NO: 55 Linker represented by sequence (linker 6 in FIG. 8) - CD22VL represented by nucleotide sequence of SEQ ID NO: 18 - Linker represented by nucleotide sequence of SEQ ID NO: 53 (linker 1 in FIG. 8) - represented by nucleotide sequence of SEQ ID NO: 49 being CD19VH.
- Lentiviral vector DNA (0.5 ⁇ g) was transferred to HEK293FT cells (5 ⁇ 10 5 cells/500 ⁇ l), and 293HEK cells expressing the CD19/CD22-CAR gene were constructed.
- Lipofectamine 3000 transfection kit (Invitrogen, cat# L3000-015) was used to transfer the gene to 293HEK cells, and cultured in Opti-MEM (gibco, cat# 51985-034) medium for 4 hours (FIGS. 9 and 10). ).
- CD19xCD22-CAR-T cells CD19x4F5, C19x4F5 (V4) and CD19x4F5 (V11), were transfected into T cells in the same manner as in Example 5 with the CD19xCD22-CAR lentiviral vector prepared in Example 7, respectively. was prepared (Figs. 12 and 13).
- CD19xCD22-CAR-T cells (CD19x4F5, CD19x 4F5 (V4), and CD19x4F5 (V11)) were sorted into CD3-activated CD19xCD22-CAR-T cells using an anti-CD3 antibody, and then PE-CD19 peptide and FITC After reacting with the -CD22 peptide, fluorescence intensity was measured using a flow cytometer.
- CD3-activated CD19xCD22-CAR-T cells simultaneously bind to CD19 peptide and CD22 peptide.
- Example 9 Confirmation of the killing effect of CD19xCD22-CAR-T cells on CD22 or CD19 expressing cells
- K562 cells (ATCC, cat# CCL-243) that do not express CD19 and CD22 were used to construct three cells K562-CD19, K562-CD22, and K562-CD19/CD22 that express CD19, CD22 or CD19xCD22. , The target cell killing effect by CD19xCD22-CAR-T cells was confirmed.
- the CD19x4F5-CAR-T cells, CD19x4F5(V4)-CAR-T cells and CD19x4F5(V11)-CAR-T cells prepared in the present invention express CD22 or CD19 or CD19/CD22. It was confirmed that the expressing cells were specifically killed.
- a humanized antibody that specifically binds to CD22 was prepared, and using this, single CAR-T cells targeting CD22 and bispecific CAR-T cells targeting CD19 and CD22 were prepared.
- CD22-CAR-T cells and the bispecific CD19xCD22-CAR-T cells prepared in the present invention effectively recognized the CD22 antigen, activated the CAR-T cells, and effectively killed cells expressing CD22.
- CD22 (or CD19) can be usefully used as a composition for preventing or treating a disease related to expression or a disease related to B cells.
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Abstract
The present invention relates to a CD22-specific antibody and a chimeric antigen receptor using same and, more specifically, to an antibody biding specifically to CD22 (4F5 antibody) and a humanized antibody thereof, a chimeric antigen receptor including the antibody or a CD19xCD22 antibody, a CAR-T cell expressing the chimeric antigen receptor, and a pharmaceutical composition comprising same for preventing or treating B cell-mediated diseases. In the present invention, an antibody specifically binding to CD22 and a humanized antibody thereof were prepared and a single CAR-T cell targeting CD22 and a bispecific CAR-T cell targeting CD19 and CD22 were prepared using same. The CD22-CAR-T cells and CD19xCD22-CAR-T cells prepared in the present invention were found to effectively recognize the CD22 antigen and effectively kill cells expressing CD22, and thus can be advantageously used as a composition for prevention or treatment of CD22 (or CD19) expression-related diseases.
Description
본 발명은 인간화된 CD22에 특이적인 항체 및 이를 이용한 키메라 항원 수용체에 관한 것으로, 보다 상세하게는 CD22에 특이적으로 결합하는 항체(4F5 항체) 및 이의 인간화된 항체, 상기 항체 또는 CD19xCD22 항체를 포함하는 키메라 항원 수용체, 상기 키메라 항원 수용체를 발현하는 CAR-T 세포, 및 이들을 포함하는 B 세포에 의해 매개되는 질환 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a humanized CD22-specific antibody and a chimeric antigen receptor using the same, and more particularly, to an antibody that specifically binds to CD22 (4F5 antibody) and a humanized antibody thereof, comprising the antibody or the CD19xCD22 antibody It relates to a chimeric antigen receptor, a CAR-T cell expressing the chimeric antigen receptor, and a pharmaceutical composition for preventing or treating diseases mediated by B cells including the chimeric antigen receptor.
CD22는 NHL, 급성 림프모구백혈병(acute lymphoblastic leukaemia: B-ALL), 만성 림프모구백혈병(chronic lymphocytic leukaemia: B-CLL) 및 특히 급성 비림프구 백혈병(acute non-lymphocytic leukaemia: ANLL)을 포함하는, 대부분의 B세포 백혈병 및 림프종에서 발현된다.CD22 is associated with 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.
이러한 CD22 발현과 관련된 질환 등의 치료 또는 진단을 위해 CD22에 특이적인 항체의 개발이 이루어지고 있다. 국제공개특허 WO1998-041641호에는 VH44및 VL100위치에 시스테인 잔기를 갖는 재조합 항-CD22 항체에 대해 개시되어 있으며, 국제공개특허 WO 1998-042378호에는 B세포 악성종양의 치료를 위한 항-CD22 항체에 대해 개시되어 있다.Antibodies specific to CD22 are being developed for the treatment or diagnosis of diseases related to CD22 expression. International Publication No. WO1998-041641 discloses a recombinant anti-CD22 antibody having cysteine residues at positions V H 44 and V L 100, and International Publication No. WO 1998-042378 discloses an antibody for the treatment of B-cell malignancies. -CD22 antibody is disclosed.
상기와 같이 치료를 위한 항체의 생산을 위해 주로 마우스를 이용하여 단일클론 항체(monoclonal antibody)를 생산하고 있다. 하지만 마우스 유래 단일클론 항체와 같은 비인간 항체들은 인체 내에서 외래 항원으로 간주되기 때문에 면역반응을 유발하고 반감기가 짧기 때문에 치료효과가 제한적인 문제점이 있다.As described above, for the production of antibodies for treatment, monoclonal antibodies are mainly produced using mice. However, since non-human antibodies such as mouse-derived monoclonal antibodies are regarded as foreign antigens in the human body, they induce an immune response and have a short half-life, so their therapeutic effect is limited.
상기 문제를 해결하기 위해 항체의 항원과 결합하는 부위만을 제외한 나머지 부분을 인간 항체로 치환한 인간화 항체가 개발되었다. 현재 사용되고 있는 마우스 항체의 인간화 항체로의 치환방법으로는 치환할 항체에 대한 가장 유사한 인간 항체 유전자를 선정하고, CDR 이식이라 불리는 방법으로 마우스 항체의 CDR 부위만을 인간 항체 CDR 위치로 치환하는 것이다. 이와 같은 인간화 항체는 유전자의 대부분을 인간화 하였으므로 인체 내에서의 면역반응을 줄일 수 있는 장점이 있다.In order to solve the above problems, humanized antibodies have been developed in which the remaining parts of the antibody except for the antigen-binding portion are substituted with human antibodies. As a currently used method of replacing a mouse antibody with a humanized antibody, the most similar human antibody gene for the antibody to be substituted is selected, and only the CDR region of the mouse antibody is replaced with the human antibody CDR position by a method called CDR transplantation. Such humanized antibodies have the advantage of reducing the immune response in the human body because most of the genes are humanized.
한편, B 세포 질환이나 질병, 자가면역 질환, 이식거부반응 등의 치료를 위해, 상기 다른 다양한 B 세포 표면 마커(항원)에 특이적인 항체가 개발되고 있으며, CD22 항원이외에 CD19는 일반적으로 표적으로 많이 사용되고 있는 항원으로 이를 표적으로 하는 CAR-T 세포에 대한 임상 역시 많이 진행되고 있다. 하지만, CD19를 발현하지 않은 백혈병등 세포에 따라 표적 항원 발현량에 차이가 있으며, 하나의 항원만을 표적하는 단일 CAR 또는 단일 CAR-T 세포 치료는, 종양세포의 면역회피 전략으로 인해 표적 항원이 손실되는 등의 문제가 발생할 수 있다. 실제로 B 세포 ALL(급성림프구백혈병) 환자의 경우 CD19가 발현되지 않은 CD19 음성 재발이 관찰되었으며(CD19 CAR-T 요법에 처음 반응한 B 세포 ALL 환자의 최대 25%), 이 현상은 CAR-T 세포 치료에 대한 종양 세포의 저항기전으로 밝혀졌다 (Maude, S.L., et al., N. Engl. J. Med., 378:439-448, 2018).On the other hand, for the treatment of B cell diseases or diseases, autoimmune diseases, transplant rejection, etc., antibodies specific to the various other B cell surface markers (antigens) have been developed, and CD19 is generally a target in addition to the CD22 antigen. Clinical trials on CAR-T cells targeting these antigens are also in progress. However, there is a difference in the amount of target antigen expression depending on cells such as leukemia that do not express CD19, and single CAR or single CAR-T cell therapy that targets only one antigen loses the target antigen due to the immune evasion strategy of tumor cells problems may arise. Indeed, in patients with B-cell acute lymphocytic leukemia (ALL), CD19-negative relapses with no CD19 expression have been observed (up to 25% of patients with B-cell ALL who initially respond to CD19 CAR-T therapy), a phenomenon associated with CAR-T cell It has been found to be a mechanism of resistance of tumor cells to treatment (Maude, SL, et al. , N. Engl. J. Med ., 378:439-448, 2018).
이를 해결하기 위해 이중 또는 다중 항원을 표적으로하는 CAR-T 세포가 연구되고 있으며, 두 개의 항원을 동시에 표적화하면 항원 손실 변이체의 가능성을 줄일 수 있다.To address this, CAR-T cells targeting dual or multiple antigens are being studied, and simultaneous targeting of two antigens can reduce the possibility of antigen-loss variants.
이에, 본 발명에서는 인체 내에서 면역반응을 줄이기 위해 CD22에 결합하는 항체(4F5 항체)를 선별하고, 이를 이용하여 인간화 항-CD22 항체(4F5(V4) 및 4F5(V11))를 제조하였으며, 본 발명의 인간화 항-CD22 항체를 이용하여 CD22를 표적으로 하는 키메라 항원 수용체 및 CAR-T 세포를 제조하였다.Therefore, in the present invention, in order to reduce the immune response in the human body, an antibody (4F5 antibody) that binds to CD22 was selected, and humanized anti-CD22 antibodies (4F5 (V4) and 4F5 (V11)) were prepared using the antibody. A humanized anti-CD22 antibody of the invention was used to generate chimeric antigen receptors and CAR-T cells targeting CD22.
나아가, CD22뿐만 아니라 CD19를 표적으로 하는 이중특이적 키메라 항원 수용체(Bivalent CAR 또는 Bispecific CAR) 및 이를 이용한 이중특이적 CAR-T 세포를 제조하였으며, 본 발명에서 제조한 CD22-CAR-T 세포 및 이중특이적 CD19xCD22-CAR-T 세포에서 키메라 항원 수용체가 정상적으로 발현하는 것을 확인하고, 본 발명을 완성하였다.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 prepared, and the CD22-CAR-T cell prepared in the present invention and the dual It was confirmed that the chimeric antigen receptor was normally expressed in specific CD19xCD22-CAR-T cells, and the present invention was completed.
따라서, 본 발명의 목적은, CD22에 특이적인 항체 또는 CD22에 특이적인 인간화 항체, 상기 항체를 코딩하는 폴리뉴클레오타이드, 상기 항체를 발현하는 벡터, 상기 벡터로 형질전환된 재조합 세포를 제공하는 데 있다.Accordingly, an object of the present invention is to provide an antibody specific for CD22 or a humanized antibody specific for CD22, a polynucleotide encoding the antibody, a vector expressing the antibody, and a recombinant cell transformed with the vector.
본 발명의 다른 목적은, 상기 CD22에 특이적인 항체 또는 CD22에 특이적인 인간화 항체를 포함하는 키메라 항원 수용체, 상기 키메라 항원 수용체를 코딩하는 폴리뉴클레오타이드, 상기 폴리뉴클레오타이드를 포함하는 벡터, 및 상기 폴리뉴클레오타이드 또는 벡터를 포함하는 키메라 항원 수용체 발현하는 면역 이펙터 세포를 제공하는 데 있다. Another object of the present invention is a chimeric antigen receptor comprising the CD22-specific antibody or CD22-specific humanized antibody, a polynucleotide encoding the chimeric antigen receptor, a vector comprising the polynucleotide, and the polynucleotide or It is to provide an immune effector cell expressing a chimeric antigen receptor containing a vector.
본 발명의 또 다른 목적은, CD19 및 CD22에 특이적인 항체를 포함하는 이중특이적 키메라 항원 수용체, 상기 이중특이적 키메라 항원 수용체를 코딩하는 폴리뉴클레오타이드, 상기 폴리뉴클레오타이드를 포함하는 벡터, 및 상기 폴리뉴클레오타이드 또는 벡터를 포함하는 이중특이적 키메라 항원 수용체 발현하는 면역 이펙터 세포를 제공하는 데 있다. Another object of the present invention is 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 the polynucleotide Or to provide an immune effector cell expressing a bispecific chimeric antigen receptor comprising a vector.
본 발명의 또 다른 목적은 상기 면역 이펙터 세포를 포함하는, B 세포에 의해 매개되는 질환의 예방 또는 치료용 약학적 조성물을 제공하는 데 있다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases mediated by B cells, including the immune effector cells.
상술한 목적을 달성하기 위해, In order to achieve the above purpose,
본 발명은 서열번호 1의 아미노산으로 표시되는 CDR1 영역, 서열번호 2의 아미노산으로 표시되는 CDR2 영역 및 서열번호 3의 아미노산으로 표시되는 CDR3 영역을 포함하는 중쇄 가변부위 및 서열번호 4의 아미노산으로 표시되는 CDR1 영역, 서열번호 5의 아미노산으로 표시되는 CDR2 영역 및 서열번호 6의 아미노산으로 표시되는 CDR3 영역을 포함하는 경쇄 가변 부위를 포함하는 CD22에 특이적으로 결합하는 항체 또는 이의 단편을 제공한다. The present invention relates to a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a CDR3 region represented by the amino acids of SEQ ID NO: 3, and a heavy chain variable region comprising the amino acids represented by SEQ ID NO: 4 Provided is an antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region, a CDR2 region represented by the amino acids of SEQ ID NO: 5, and a CDR3 region represented by the amino acids of SEQ ID NO: 6.
본 발명의 바람직한 일실시예에 있어서, 상기 CD22에 특이적으로 결합하는 항체는 서열번호 11의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 12의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체; 또는 In a preferred embodiment of the present invention, the antibody specifically binding to CD22 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. humanized antibodies that bind antagonistically; or
서열번호 15의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 16의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체일 수 있다.It may be a humanized antibody that specifically binds to CD22 composed of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.
또한, 본 발명은 상기 CD22에 특이적으로 결합하는 항체 또는 이의 단편을 코딩하는 폴리뉴클레오타이드를 제공한다.In addition, the present invention provides a polynucleotide encoding the antibody or fragment thereof that specifically binds to CD22.
또한, 본 발명은 상기 CD22에 특이적으로 결합하는 항체 또는 이의 단편을 코딩하는 폴리뉴클레오타이드를 포함하는 벡터를 제공한다. In addition, the present invention provides a vector comprising a polynucleotide encoding the antibody or fragment thereof that specifically binds to CD22.
또한, 본 발명은 상기 벡터로 형질전환된 CD22에 특이적으로 결합하는 항체 또는 이의 단편을 생산하는 재조합 세포를 제공한다. 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.
다른 목적을 달성하기 위해, to achieve other purposes,
본 발명은 CD22-결합 도메인; 막관통 도메인(transmembrane domain); 공동자극 도메인(costimulatory domain); 및 세포 내 신호전달 도메인(intracellular signal transduction domain)을 포함하는 키메릭 항원 수용체(chimeric antigen receptor: CAR)로,The present invention relates to a CD22-binding domain; transmembrane domain; costimulatory domain; And a chimeric antigen receptor (CAR) comprising an intracellular signal transduction domain,
상기 CD22-결합 도메인은 서열번호 1의 아미노산으로 표시되는 CDR1 영역, 서열번호 2의 아미노산으로 표시되는 CDR2 영역 및 서열번호 3의 아미노산으로 표시되는 CDR3 영역을 포함하는 중쇄 가변부위 및 서열번호 4의 아미노산으로 표시되는 CDR1 영역, 서열번호 5의 아미노산으로 표시되는 CDR2 영역 및 서열번호 6의 아미노산으로 표시되는 CDR3 영역을 포함하는 경쇄 가변 부위를 포함하는 CD22에 특이적으로 결합하는 항체 또는 이의 단편인 것을 특징으로 하는, CD22를 표적으로 하는 키메라 항원 수용체를 제공한다.The CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4 An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 To provide a chimeric antigen receptor targeting CD22.
본 발명의 바람직한 일실시예에 있어서, 상기 CD22에 특이적으로 결합하는 항체는 서열번호 11의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 12의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체; 또는 In a preferred embodiment of the present invention, the antibody specifically binding to CD22 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. humanized antibodies that bind antagonistically; or
서열번호 15의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 16의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체일 수 있다.It may be a humanized antibody that specifically binds to CD22 composed of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.
본 발명의 바람직한 다른 일실시예에 있어서, 상기 막관통 도메인은 CD8α, CD4, CD28, CD137, CD80, CD86, CD152 및 PD1로 구성된 군에서 선택되는 단백질이며, 공동자극 도메인은 CD28, 4-1BB, OX-40 및 ICOS로 구성된 군에서 선택되는 단백질이고, 상기 신호전달 도메인은 CD3ζ 유래일 수 있다.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, and the costimulatory domain is CD28, 4-1BB, It is a protein selected from the group consisting of OX-40 and ICOS, and the signaling domain may be derived from CD3ζ.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 CD22-결합 도메인의 C 말단 및 막경유 도메인의 N 말단 사이에 위치된 힌지 부위(hinge region)를 추가로 포함할 수 있으며, 상기 힌지 부위는 CD8α 유래일 수 있다. 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.
또한, 본 발명은 상기 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드를 제공한다.In addition, the present invention provides a polynucleotide encoding the chimeric antigen receptor.
또한, 본 발명은 상기 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터를 제공한다. In addition, the present invention provides a vector comprising a polynucleotide encoding the chimeric antigen receptor.
또한, 본 발명은 상기 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드 또는 상기 폴리뉴클레오타이드를 포함하는 벡터를 포함하고, 상기 키메릭 항원 수용체를 발현하는 면역 이펙터 세포를 제공한다. In addition, the present invention provides an immune effector cell comprising a polynucleotide encoding the chimeric antigen receptor or a vector containing the polynucleotide and expressing the chimeric antigen receptor.
또 다른 목적을 달성하기 위해, To achieve another purpose,
본 발명은 CD19-결합 도메인 및 CD22-결합 도메인; The present invention relates to a CD19-binding domain and a CD22-binding domain;
막관통 도메인(transmembrane domain); transmembrane domain;
공동자극 도메인(costimulatory domain); 및 costimulatory domain; and
세포 내 신호전달 도메인(intracellular signal transduction domain)을 포함하는 이중특이적 키메릭 항원 수용체(chimeric antigen receptor: CAR)로,A bispecific chimeric antigen receptor (CAR) containing an intracellular signal transduction domain,
상기 CD22-결합 도메인은 서열번호 1의 아미노산으로 표시되는 CDR1 영역, 서열번호 2의 아미노산으로 표시되는 CDR2 영역 및 서열번호 3의 아미노산으로 표시되는 CDR3 영역을 포함하는 중쇄 가변부위 및 서열번호 4의 아미노산으로 표시되는 CDR1 영역, 서열번호 5의 아미노산으로 표시되는 CDR2 영역 및 서열번호 6의 아미노산으로 표시되는 CDR3 영역을 포함하는 경쇄 가변부위를 포함하는 CD22에 특이적으로 결합하는 항체 또는 이의 단편인 것을 특징으로 하는, CD19 및 CD22를 표적으로 하는 이중특이적 키메라 항원 수용체를 제공한다.The CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4 An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 Provided is a bispecific chimeric antigen receptor targeting CD19 and CD22.
본 발명의 바람직한 일실시예에 있어서, 상기 CD19-결합 도메인 및 CD22-결합 도메인은, CD19에 특이적으로 결합하는 항체의 경쇄 가변부위 - CD22에 특이적으로 결합하는 항체의 중쇄 가변부위 - CD22에 특이적으로 결합하는 항체의 경쇄 가변부위 - CD19에 특이적으로 결합하는 항체의 중쇄 가변부위 순서로 연결될 수 있다.In a preferred embodiment of the present invention, the CD19-binding domain and the CD22-binding domain are 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 - CD22 The light chain variable region of an antibody that specifically binds to the heavy chain variable region of an antibody that specifically binds to CD19 may be linked in sequence.
본 발명의 바람직한 다른 일실시예에 있어서, 상기 CD19에 특이적으로 결합하는 항체의 경쇄 가변부위는 서열번호 44의 아미노산으로 표시되는 CDR1 영역(QDISKY), 서열번호 45의 아미노산으로 표시되는 CDR2 영역(HTS) 및 서열번호 46의 아미노산으로 표시되는 CDR3 영역(QQGNTLPYT)을 포함할 수 있으며, 바람직하게는 서열번호 48의 아미노산 서열로 표시될 수 있다.In another preferred embodiment of the present invention, the light chain variable region of the antibody specifically binding to CD19 is a CDR1 region (QDISKY) represented by amino acids of SEQ ID NO: 44, a CDR2 region represented by amino acids of SEQ ID NO: 45 ( HTS) and a CDR3 region (QQGNTLPYT) represented by the amino acids of SEQ ID NO: 46, preferably represented by the amino acid sequence of SEQ ID NO: 48.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 CD19에 특이적으로 결합하는 항체의 중쇄 가변부위는 서열번호 41의 아미노산으로 표시되는 CDR1 영역(GVSLPDYG), 서열번호 42의 아미노산으로 표시되는 CDR2 영역(IWGSETT) 및 서열번호 43의 아미노산으로 표시되는 CDR3 영역(AKHYYYGGSYAMDY)을 포함할 수 있으며, 바람직하게는 서열번호 47의 아미노산 서열로 표시될 수 있다.In another preferred embodiment of the present invention, the heavy chain variable region of the antibody specifically binding to CD19 is the CDR1 region (GVSLPDYG) represented by the amino acids of SEQ ID NO: 41 and the CDR2 region represented by the amino acids of SEQ ID NO: 42. (IWGSETT) and the CDR3 region (AKHYYYGGSYAMDY) represented by the amino acids of SEQ ID NO: 43, preferably represented by the amino acid sequence of SEQ ID NO: 47.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 CD22에 특이적으로 결합하는 항체는 서열번호 11의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 12의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체; 또는 In another preferred embodiment of the present invention, the antibody specifically binding to CD22 is CD22 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. A humanized antibody that specifically binds to; or
서열번호 15의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 16의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체일 수 있다.It may be a humanized antibody that specifically binds to CD22 composed of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 막관통 도메인은 CD8α, CD4, CD28, CD137, CD80, CD86, CD152 및 PD1로 구성된 군에서 선택되는 단백질이며, 공동자극 도메인은 CD28, 4-1BB, OX-40 및 ICOS로 구성된 군에서 선택되는 단백질이고, 상기 신호전달 도메인은 CD3ζ 유래일 수 있다.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, and the costimulatory domain is CD28, 4-1BB , OX-40 and ICOS, and the signaling domain may be derived from CD3ζ.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 CD22-결합 도메인의 C 말단 및 막경유 도메인의 N 말단 사이에 위치된 힌지 부위(hinge region)를 추가로 포함할 수 있으며, 상기 힌지 부위는 CD8α 유래일 수 있다. 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.
또한, 본 발명은 상기 이중특이적 키메라 항원 수용체를 코딩하는 폴리뉴클레오타이드를 제공한다.The present invention also provides a polynucleotide encoding the bispecific chimeric antigen receptor.
또한, 본 발명은 상기 이중특이적 키메라 항원 수용체를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터를 제공한다. In addition, the present invention provides a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor.
또한, 본 발명은 상기 이중특이적 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드 또는 상기 폴리뉴클레오타이드를 포함하는 벡터를 포함하고, 상기 이중특이적 키메릭 항원 수용체를 발현하는 면역 이펙터 세포를 제공한다. In addition, the present invention provides an immune effector cell comprising a polynucleotide encoding the bispecific chimeric antigen receptor or a vector containing the polynucleotide and expressing the bispecific chimeric antigen receptor.
또 다른 목적을 달성하기 위해, To achieve another purpose,
본 발명은 상기 CD22에 특이적으로 결합하는 항체 또는 이의 단편; 또는 상기 면역 이펙터 세포를 포함하는 B 세포에 의해 매개되는 질환의 예방 또는 치료용 약학적 조성물을 제공한다. The present invention relates to an antibody or fragment thereof that specifically binds to the CD22; Alternatively, a pharmaceutical composition for preventing or treating a disease mediated by B cells including the immune effector cells is provided.
본 발명의 바람직한 일실시예에 있어서, B 세포에 의해 매개되는 질환은 종양, 림프종, 비호치킨 림프종(non-Hogkins lymphoma: NHL), 공격적 NHL, 재발성 공격적 NHL, 재발성 지연성 NHL, 불응성 NHL, 불응성 지연성 NHL, 만성 림프성 백혈병(chronic lymphocytic leukemia: CLL), 소형 림프성 림프종, 백혈병, 모발성 세포 백혈병(hairy cell leukemia: HCL), 급성 림프성 백혈병(acute lymphocytic leukemia: ALL), 버킷트 림프종 및 외투 세포 림프종로 구성된 군에서 선택될 수 있다. In a preferred embodiment of the present invention, the disease mediated by B cells is a tumor, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent delayed NHL, refractory 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.
본 발명에서는 CD22에 특이적으로 결합하는 항체 및 이의 인간화 항체를 제조하였으며, 이를 이용하여 CD22를 표적으로 하는 단일 CAR-T 세포 및 CD19 및 CD22를 표적으로 하는 이중특이적 CAR-T 세포를 제조하였다. In the present invention, an antibody that specifically binds to CD22 and a humanized antibody thereof were prepared, and using them, a single CAR-T cell targeting CD22 and a bispecific CAR-T cell targeting CD19 and CD22 were prepared .
본 발명에서 제조한 CD22-CAR-T 세포 및 이중특이적 CD19xCD22-CAR-T 세포는 CD22 항원을 효과적으로 인식하여 CAR-T 세포의 활성화가 이루어진 것을 확인하였으며, CD22를 발현하는 세포를 효과적으로 사멸시키는 것을 확인하였다. It was confirmed that the CD22-CAR-T cells and the bispecific CD19xCD22-CAR-T cells prepared in the present invention effectively recognized the CD22 antigen and activated the CAR-T cells, and effectively killed the cells expressing CD22. Confirmed.
나아가, 이중특이적 CD19xCD22-CAR-T 세포는 동물모델에서 우수한 항종양 효과를 보이는 것을 확인하였으므로, 본 발명의 CD22에 특이적으로 결합하는 인간화 항체 기반 CD22-CAR-T 세포 및 이중특이적 CD19xCD22-CAR-T 세포는 CD22(또는 CD19) 발현과 관련된 질환 또는 B 세포와 관련된 질환의 예방 또는 치료용 조성물로 유용하게 활용할 수 있다. Furthermore, since it was confirmed that the bispecific CD19xCD22-CAR-T cells showed excellent antitumor effects in animal models, the CD22-CAR-T cells based on the humanized antibody specifically binding to CD22 of the present invention and the bispecific CD19xCD22-CAR-T cells CAR-T cells can be usefully utilized as a composition for preventing or treating a disease related to CD22 (or CD19) expression or a disease related to B cells.
도 1은 본 발명에서 선별한 4F5 항체(mouse) 및 인간화된 4F5 항체(4F5(V4) 및 4F5(V11))의 CD22에 대한 결합력을 FACS로 확인한 데이터이다.Figure 1 is data confirming the binding ability to CD22 of the 4F5 antibody (mouse) and the humanized 4F5 antibodies (4F5 (V4) and 4F5 (V11)) selected in the present invention by FACS.
도 2는 CD22를 표적으로 하는 키메라 항원 수용체(single CAR)를 나타낸 모식도이다.2 is a schematic diagram showing a chimeric antigen receptor (single CAR) targeting CD22.
도 3은 CD22-CAR를 발현하는 렌티바이러스 벡터를 이용한 CD22-CAR-T 세포 제조방법을 나타낸 모식도이다.3 is a schematic diagram showing a method for preparing CD22-CAR-T cells using a lentiviral vector expressing CD22-CAR.
도 4는 (a) 말초 혈액 단핵세포(PBMC)를 이용한 CD22-CART 세포 제조방법 및 (b) 제조된 CD22-CAR-T 세포의 CD22 펩타이드에 대한 결합능을 확인하는 방법을 나타낸 모식도이다.4 is a schematic diagram showing (a) a method for preparing CD22-CART cells using peripheral blood mononuclear cells (PBMC) and (b) a method for confirming the CD22 peptide-binding ability of the prepared CD22-CAR-T cells.
도 5a는 4F5 항체를 이용하여 제조한 CD22-CAR-T 세포의 CD22 결합능을 확인한 데이터이다.Figure 5a is data confirming the CD22 binding ability of CD22-CAR-T cells prepared using the 4F5 antibody.
도 5b는 인간화된 항-CD22 항체인 4F5(V4) 및 4F5(V11) 기반 CD22-CAR-T 세포의 CD22 결합능을 확인한 데이터이다.5B is data confirming the CD22 binding ability of CD22-CAR-T cells based on humanized anti-CD22 antibodies, 4F5 (V4) and 4F5 (V11).
도 6은 4F5 항체를 이용하여 제조한 CD22-CAR-T 세포의 활성화를 확인하기 위해, 표적 세포의 존재하에 CD22-CAR-T 세포에 의한 IFNγ 발현 정도를 확인한 데이터이다.6 is data confirming the degree of IFNγ expression by CD22-CAR-T cells in the presence of target cells in order to confirm the activation of CD22-CAR-T cells prepared using the 4F5 antibody.
도 7a은 4F5 항체를 이용하여 제조한 CD22-CAR-T 세포에 의한 U2932 세포(CD22 발현 세포) 및 K562 세포(CD22 미발현 세포)의 사멸효과를 확인한 데이터이다.Figure 7a is data confirming the killing effect of U2932 cells (CD22 expressing cells) and K562 cells (CD22 non-expressing cells) by CD22-CAR-T cells prepared using the 4F5 antibody.
도 7b는 인간화된 항-CD22 항체인 4F5(V4) 및 4F5(V11) 기반 CD22-CAR-T 세포에 의한 U2932 세포(CD22 발현 세포) 및 K562 세포(CD22 미발현 세포)의 사멸효과를 확인한 데이터이다.Figure 7b is 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 4F5 (V4) and 4F5 (V11), which are humanized anti-CD22 antibodies. am.
도 8은 CD19 및 CD22를 표적으로 하는 이중특이적 키메라 항원 수용체(Bispecific CAR)를 나타낸 모식도이다.8 is a schematic diagram showing a bispecific chimeric antigen receptor (Bispecific CAR) targeting CD19 and CD22.
도 9는 CD19 및 CD22를 표적으로 하는 이중특이적 CD19xCD22-CAR를 발현하는 렌티바이러스를 이용한 CD19xCD22-CAR 발현 세포 제조방법을 나타낸 모식도이다. 9 is a schematic diagram showing a method for preparing CD19xCD22-CAR expressing cells using a lentivirus expressing a bispecific CD19xCD22-CAR targeting CD19 and CD22.
도 10은 (a) HEK293 세포주에 CD19xCD22-CAR 발현 렌티바이러스 벡터를 형질전환시키는 방법 및 (b) 형질전환된 HEK293 세포의 CD19 펩타이드 및 CD22 펩타이드에 대한 결합능, 및 CD19xCD22-CAR 발현정도를 확인하는 방법을 나타낸 모식도이다.10 shows (a) a method for transfecting a HEK293 cell line with a lentiviral vector expressing CD19xCD22-CAR and (b) a method for confirming the CD19 peptide and CD22 peptide binding ability and CD19xCD22-CAR expression level of the transformed HEK293 cells It is a schematic diagram showing
도 11은 CD19xCD22-CAR를 발현하는 렌티바이러스 벡터로 형질전환된 HEK293FT 세포에서 CD19x4F5-CAR, CD19x4F5(V4)-CAR 및 CD19x4F5(V11)-CAR 발현정도를 확인한 데이터이다.11 is data confirming the expression levels of CD19x4F5-CAR, CD19x4F5(V4)-CAR and CD19x4F5(V11)-CAR in HEK293FT cells transfected with a lentiviral vector expressing CD19xCD22-CAR.
도 12는 CD19xCD22-CAR를 발현하는 렌티바이러스 벡터를 이용한 CD19xCD22-CAR-T 세포 제조방법을 나타낸 모식도이다.12 is a schematic diagram showing a method for preparing CD19xCD22-CAR-T cells using a lentiviral vector expressing CD19xCD22-CAR.
도 13은 (a) 말초 혈액 단핵세포(PBMC)를 이용한 CD19xCD22-CAR-T 세포 제조방법 및 (b) 제조된 CD19xCD22-CAR-T 세포의 CD19 펩타이드 및 CD22 펩타이드에 대한 결합능을 확인하는 방법을 나타낸 모식도이다.13 shows (a) a method for preparing CD19xCD22-CAR-T cells using peripheral blood mononuclear cells (PBMC) and (b) a method for confirming the CD19 peptide and CD22 peptide binding ability of the prepared CD19xCD22-CAR-T cells. It is also a model.
도 14는 CD19xCD22-CAR-T 세포의 활성화를 확인한 데이터로, CD3+ CD19xCD22-CAR-T 세포에서 CD19xCD22-CAR-T 세포가 CD22 펩타이드 및 CD19 펩타이드와 동시에 결합하는 것을 확인하였다.14 is data confirming the activation of CD19xCD22-CAR-T cells, and it was confirmed that CD19xCD22-CAR-T cells simultaneously bind to CD22 peptide and CD19 peptide in CD3+ CD19xCD22-CAR-T cells.
도 15는 CD19xCD22-CAR-T 세포에 의한 K562 세포 (CD19과 CD22 미발현 세포), K562-CD19 세포(CD19 발현 세포), K562-CD22 세포(CD22 발현 세포) 및 K562-CD19/CD22 세포(CD19과 CD22 발현 세포)의 사멸효과를 확인한 데이터이다.15 shows 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/CD22 cells) by CD19xCD22-CAR-T cells. and CD22 expressing cells) confirming the killing effect.
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
CD22에 특이적으로 결합하는 항체 및 이의 인간화된 항체Antibodies that specifically bind to CD22 and humanized antibodies thereof
본 발명은 일관점에서, 서열번호 1의 아미노산으로 표시되는 CDR1 영역, 서열번호 2의 아미노산으로 표시되는 CDR2 영역 및 서열번호 3의 아미노산으로 표시되는 CDR3 영역을 포함하는 중쇄 가변부위 및 서열번호 4의 아미노산으로 표시되는 CDR1 영역, 서열번호 5의 아미노산으로 표시되는 CDR2 영역 및 서열번호 6의 아미노산으로 표시되는 CDR3 영역을 포함하는 경쇄 가변 부위를 포함하는 CD22에 특이적으로 결합하는 항체 또는 이의 단편에 관한 것이다.In one aspect, the present invention is a heavy chain variable region comprising a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a CDR3 region represented by the amino acids of SEQ ID NO: 3, and SEQ ID NO: 4. An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 will be.
본 발명에 있어서, 상기 CD22에 특이적으로 결합하는 항체 또는 이의 단편은 서열번호 11의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 12의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체 또는 이의 단편; 또는 In the present invention, the antibody or fragment thereof that specifically binds to CD22 is specifically composed of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 11 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 12. a humanized antibody or fragment thereof that binds; or
서열번호 15의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 16의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체 또는 이의 단편일 수 있다.It may be a humanized antibody or fragment thereof that specifically binds to CD22 composed of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.
본 발명에서, 상기 항체는 단클론 항체(monoclonal antibody)일 수 있다. 본 발명에서, 용어 "단클론 항체(monoclonal antibody)"는 모노클로날 항체 또는 단일클론항체라고도 불리며, 단일 항체 형성세포가 생성하는 항체로, 1차 구조(아미노산 배열)가 균일한 특징이 있다. 오직 하나의 항원 결정기만을 인식하며, 일반적으로 암세포와 항체생산세포를 융합한 하이브리도마(hybridoma cell)을 배양하여 생산된다.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 a monoclonal antibody, and is an antibody produced by a single antibody-forming cell, characterized by 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.
본 발명의 항체는 항원 결합에 핵심적인 부분인 CDR 부위를 제외한 나머지 부분을 인간에 의해 생산된 항체와 상응하는 아미노산 서열이 되도록 하여, 보다 인간 항체와의 유사성이 증가된 인간화 항체로 제조할 수 있다. 항체(non-human antibody)를 인간화하는 가장 일반적인 방법으로는 동물항체의 CDR 부위를 인간 항체에 이식하는 CDR-그라프팅(CDR-grafting) 방법이 있으며, 이에 한정되지 않고 당업계에 공지된 방법을 이용하여 인간화 항체를 제조할 수 있다. The antibody of the present invention can be prepared as a humanized antibody with increased similarity with 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. . The most common method for humanizing an antibody (non-human antibody) is a CDR-grafting method in which a CDR region of an animal antibody is grafted onto a human antibody, but is not limited thereto, and a method known in the art is used. Humanized antibodies can be prepared using
본 발명에서, 용어 "CDR", 즉 "상보성 결정 영역"은 중쇄 및 경쇄 부위 모두의 가변 영역 내에서 발견되는 비근접(non-contiguous) 항원 결합 부위를 의미하는 것이다.In the present invention, the term "CDR", ie "complementarity determining region", refers to a non-contiguous antigen binding site found within the variable regions of both heavy and light chain regions.
본 발명에서, 용어 "항체"는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라 항체 분자의 단편도 사용될 수 있다. 항체 분자의 단편이란 적어도 펩타이드 태그(에피토프) 결합 기능을 보유하고 있는 단편을 뜻하며 scFv, Fab, F(ab'), F(ab')2, 단일 도메인(single domain) 등을 포함한다. In the present invention, the term "antibody" can be used not only in its complete form having two full-length light chains and two full-length heavy chains, but also fragments of antibody molecules. 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 , single domain, and the like.
항체 단편 중 Fab는 경쇄 및 중쇄의 가변영역과 경쇄의 불변 영역 및 중쇄의 첫 번째 불변 영역(CH1)을 가지는 구조로 1개의 항원 결합 부위를 가진다. Fab'는 중쇄 CH1 도메인의 C 말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 부위(hinge region)를 가진다는 점에서 Fab와 차이가 있다. F(ab')2 항체는 Fab'의 힌지 부위의 시스테인 잔기가 디설파이드 결합을 이루면서 생성된다. Fv는 중쇄 가변부위 및 경쇄 가변부위만을 가지고 있는 최소한의 항체조각으로, 이중쇄 Fv(dsFv)는 디설파이드 결합으로 중쇄 가변부위와 경쇄 가변부위가 연결되어 있고, 단쇄 Fv(scFv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변 영역과 경쇄의 가변 영역이 공유 결합으로 연결되어 있다. 이러한 항체 단편은 단백질 가수 분해 효소를 이용해서 수득하거나, 바람직하게는 유전자 재조합 기술을 통하여 제작할 수 있다. Among antibody fragments, Fab has a structure having variable regions of light and heavy chains, constant regions of light chains, and a first constant region (CH1) of heavy chains, and has one antigen-binding site. Fab' is different 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. The F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv is a minimal antibody fragment that has only the heavy chain variable region and the light chain variable region. Double-chain Fv (dsFv) has a heavy chain variable region and light chain variable region connected by a disulfide bond, and 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 covalently linked via. These antibody fragments can be obtained using proteolytic enzymes or, preferably, can be produced through genetic recombination technology.
본 발명의 CD22에 특이적으로 결합하는 단클론 항체는 CD22 단백질 전체 또는 일부 펩타이드를 면역원(또는 항원)으로 이용하여 제조할 수 있다. 보다 상세하게는, 우선 면역원으로서 CD22 단백질, CD22 단백질을 포함하는 융합 단백질 또는 CD22 단백질을 포함하는 캐리어(carrier)를 필요에 따라서 면역증강제인 아주반트(adjuvant)(예, Freund adjuvant)와 함께 인간을 제외한 포유동물의 피하, 근육, 정맥, 발볼록살 또는 복강 내에 1회 내지 그 이상 주사하는 것으로써 면역감작(immunization)을 시킨다. 상기 인간을 제외한 포유동물은 바람직하게는, 마우스, 래트, 햄스터, 몰모트, 닭, 토끼, 고양이, 개, 돼지, 염소, 양, 당나귀, 말 또는 소(인간 항체를 생산하는 형질 전환(transgenic) 마우스와 같은 다른 동물 유래의 항체를 생산하도록 조작된 형질 전환(transgenic) 동물을 포함한다.)이며, 보다 바람직하게는, 마우스, 래트, 햄스터, 몰모트, 닭 또는 토끼이다. 첫 번째 면역으로부터 약 1~21일 마다 1~4회 면역을 실시하여, 최종 면역으로부터 약 1~10일 후에 면역 감작 된 포유동물로부터 항체 생산하는 세포를 수득할 수 있다. 면역을 시키는 회수 및 시간적 간격은 사용하는 면역원의 특징 등에 의하여 적당히 변경할 수 있다.The monoclonal antibody specifically binding to CD22 of the present invention can be prepared using all or a partial peptide of CD22 protein as an immunogen (or antigen). More specifically, first, as an immunogen, CD22 protein, a fusion protein containing CD22 protein, or a carrier containing CD22 protein is administered to humans together with an adjuvant (eg, Freund adjuvant) as an immune enhancer, if necessary. Immunization is performed by injecting once or more subcutaneously, intramuscularly, intravenously, balboloxal, or intraperitoneally in mammals other than those excluded. Mammals other than humans are preferably mice, rats, hamsters, marmots, chickens, rabbits, cats, dogs, pigs, goats, sheep, donkeys, horses or cows (transgenic mice that produce human antibodies). (including transgenic animals engineered to produce antibodies derived from other animals such as), more preferably mice, rats, hamsters, marmots, chickens or rabbits. 1 to 4 immunizations are performed about every 1 to 21 days from the first immunization, and about 1 to 10 days after the final immunization, antibody-producing cells can be obtained from immunosensitized mammals. The number of immunizations and time intervals can be appropriately changed depending on the characteristics of the immunogen to be used.
단클론 항체를 분비하는 하이브리도마(hybridoma)의 제조는 케이라 및 미르슈타인 등의 방법(Nature, 1975, Vol. 256, p. 495-497) 및 이에 준하는 방법에 따라 실시할 수 있다. 상기와 같이 면역 감작된 인간을 제외한 동물로부터 채취한 비장, 림프절, 골수 또는 편도로 이루어지는 군으로부터 선택되는 어느 하나, 바람직하게는 비장에 포함되는 항체 생산하는 세포와 자가 항체 생산 능력이 없는 포유동물 유래의 골수종 세포(myeloma cells)를 세포 융합시키는 것에 의해 하이브리도마(hybridoma)를 제조할 수 있다. 상기 포유동물은 마우스, 래트, 몰모트, 햄스터, 닭, 토끼 또는 인간일 수 있고, 바람직하게는 마우스, 래트, 닭 또는 인간일 수 있다.Preparation 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) and a method similar thereto. Any one selected from the group consisting of spleen, lymph node, bone marrow, or tonsil collected from animals other than humans immunosensitized as described above, preferably derived from a mammal that does not have the ability to produce an antibody and an antibody-producing cell contained in the spleen. A hybridoma can be prepared by cell fusion of myeloma cells. The mammal may be a mouse, rat, marmot, hamster, chicken, rabbit or human, preferably a mouse, rat, chicken or human.
세포 융합은, 예를 들면, 폴리에틸렌글리콜이나 센다이 바이러스를 비롯한 융합 촉진제나 전기 펄스에 의한 방법이 이용되고, 일례를 들면, 융합 촉진제를 함유하는 융합 배지에 항체 생산 세포와 무한 증식 가능한 포유류 유래의 세포를 약 1:1 내지 1:10의 비율로 부유시켜, 이 상태로, 약 30 내지 40℃로 약 1 내지 5분간 배양한다. 융합 배지에는, 예를 들면, MEM 배지, RPMI1640 배지 및 이스코브 변형 둘베코 배지(Iscove's Modified Dulbecco's Medium)를 비롯한 통상의 일반적인 것을 이용하면 좋고, 소 혈청 등의 혈청류는 제외해 두는 것이 바람직하다.For cell fusion, for example, a fusion promoter such as polyethylene glycol or Sendai virus or a method using an electric pulse is used. For example, in a fusion medium containing a fusion promoter, antibody-producing cells and mammalian-derived cells capable of immortal growth are used. is suspended 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, for example, MEM medium, RPMI1640 medium, and Iscove's Modified Dulbecco's Medium may be used, and serum types such as bovine serum are preferably excluded.
상기 단클론 항체를 생산하는 하이브리도마 클론을 스크리닝하는 방법은 우선, 상기한 바와 같이 획득한 융합 세포를 HAT 배지 등의 선택용 배지에 옮기고, 약 30 내지 40℃로 약 3일 내지 3주일 배양해서 하이브리도마 이외의 세포를 사멸시킨다. 이어서, 마이크로타이터 플레이트(microtiter plate) 등에서 하이브리도마를 배양한 후, 위에서 기술한 인간을 제외한 동물의 면역반응에 사용한 면역원과 배양 상청액과의 반응성이 증가된 부분을 RIA(radioactive substance-marked immuno antibody) 또는 ELISA(Enzyme-Linked Immunosorbent Assay)같은 면역분석방법을 통하여 찾는 방법을 통해 수행할 수 있다. 그리고 상기에서 찾은 단클론 항체를 생산하는 클론은 상기 면역원에 대하여 특이적인 결합력을 보여준다.The method for screening hybridoma clones producing the monoclonal antibody is, first, transfer the fused cells obtained as described above to a selection medium such as HAT medium, and incubate at about 30 to 40 ° C. for about 3 days to 3 weeks Cells other than hybridomas are killed. Subsequently, after culturing the hybridomas in 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 prepared as RIA (radioactive substance-marked immunoassay). antibody) or an immunoassay method such as ELISA (Enzyme-Linked Immunosorbent Assay). In addition, the clone producing the monoclonal antibody found above shows specific binding ability to the immunogen.
본 발명의 단클론 항체는, 이와 같은 하이브리도마를 생체 내외에서 배양함으로써 얻을 수 있다. 배양에는, 포유동물 유래의 세포를 배양하기 위한 통상의 방법이 이용되며, 배양물 등으로부터 단클론 항체를 채취하기 위해서는, 항체 일반을 정제하기 위한 이 분야에서의 통상의 방법이 이용된다. 각각의 방법으로서는, 예를들면, 염석(鹽析), 투석, 여과, 농축, 원심분리, 분별 침전, 겔 여과 크로마토그래피, 이온 교환 크로마토그래피, 어피니티 크로마토그래피, 고속액체 크로마토그래피, 겔 전기영동 및 등전점 전기영동 등을 들 수 있고, 이들은 필요에 따라서 조합해서 적용된다. 정제한 단클론 항체는, 그 후, 농축, 건조하여, 용도에 따라서 액상 또는 고상으로 한다.The monoclonal antibody of the present invention can be obtained by culturing such a hybridoma in vitro or in vivo. For culturing, a conventional method for culturing mammalian-derived cells is used, and for collecting a monoclonal antibody from a culture or the like, a conventional method in this field for purifying an antibody 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-speed liquid chromatography, gel electrophoresis and isoelectric point electrophoresis. These are applied in combination as needed. The purified monoclonal antibody is then concentrated and dried to be in a liquid or solid state depending on the application.
또한, 본 발명의 단클론 항체는, 중쇄(重鎖) 및 경쇄(輕鎖) 가변영역을 코딩하는 DNA를 각각, 중쇄 및 경쇄의 정상영역을 코딩하는 기지의 DNA(예를 들면, 일본 2007-252372호 공보 참조)와 각각 연결한 유전자를, PCR법, 또는, 화학 합성에 의해 합성하고, 그 유전자의 발현을 가능하게 하는 공지의 발현 벡터(pcDNA 3.1(Invitrogen 사 판매) 등에 이식하여 형질 전환체를 제조하고, CHO 세포나 대장균 등의 숙주 중에서 발현시킴으로써 항체를 생산하고, 이러한 배양액으로부터, 프로테인 A 또는 G(Protein A 또는 G) 컬럼 등을 이용해서 항체를 정제함으로써 얻을 수 있다.In addition, the monoclonal antibody of the present invention includes DNA encoding heavy chain and light chain variable regions, respectively, and known DNA encoding heavy and light chain constant regions (eg, Japan 2007-252372 (Refer to Publication No.) and each ligated gene are synthesized by the PCR method or chemical synthesis, and transplanted into a known expression vector (pcDNA 3.1 (sold by Invitrogen)) or the like that enables the expression of the gene to obtain a transformant. It can be obtained by preparing and expressing in a host such as CHO cells or Escherichia coli to produce an antibody, and purifying the antibody from this culture solution using a Protein A or G column or the like.
본 발명의 구체적인 일실시예에서는, CD22에 특이적으로 결합하는 항체를 제조하기 위해, 항-CD22 항체를 생산하는 하이브리도마를 제조 및 스크리닝 하여, CD22에 특이적으로 결합하는 항체(scFv)를 선별하였으며, 이를 4F5로 명명하였다.In a specific embodiment of the present invention, in order to prepare an antibody that specifically binds to CD22, a hybridoma that produces an anti-CD22 antibody is prepared and screened to obtain an antibody (scFv) that specifically binds to CD22. It was selected and named as 4F5.
상기 4F5 항체는 서열번호 1의 아미노산으로 표시되는 CDR1 영역(GFSLTIYG), 서열번호 2의 아미노산으로 표시되는 CDR2 영역(MWSGGST) 및 서열번호 3의 아미노산으로 표시되는 CDR3 영역(ARNDGYYWFAY)을 포함하는 중쇄 가변부위 및 서열번호 4의 아미노산으로 표시되는 CDR1 영역(QSLVHNNGNTY), 서열번호 5의 아미노산으로 표시되는 CDR2 영역(KVS) 및 서열번호 6의 아미노산으로 표시되는 CDR3 영역(SQSTHVPYS)을 포함하는 경쇄 가변 부위로 구성되는 것을 확인하였다. The 4F5 antibody is a heavy chain variable comprising a CDR1 region (GFSLTIYG) represented by the amino acids of SEQ ID NO: 1, a CDR2 region (MWSGGST) represented by the amino acids of SEQ ID NO: 2, and a CDR3 region (ARNDGYYWFAY) represented by the amino acids of SEQ ID NO: 3. region and a CDR1 region (QSLVHNNGNTY) represented by the amino acids of SEQ ID NO: 4, a CDR2 region (KVS) represented by the amino acids of SEQ ID NO: 5, and a CDR3 region (SQSTHVPYS) represented by the amino acids of SEQ ID NO: 6. configuration was confirmed.
구체적으로, 4F5 항체는 서열번호 7의 아미노산으로 표시되는 중쇄 가변부위 및 서열번호 8의 아미노산으로 표시되는 경쇄 가변부위로로 구성되며, 상기 중쇄 가변 부위는 서열번호 9의 염기서열로, 경쇄 가변 부위는 서열번호 10의 염기서열로 코딩되는 것을 확인하였다. Specifically, the 4F5 antibody is composed of a heavy chain variable region represented by the amino acids of SEQ ID NO: 7 and a light chain variable region represented by the amino acids of SEQ ID NO: 8, wherein the heavy chain variable region has the nucleotide sequence of SEQ ID NO: 9, the light chain variable region was confirmed to be encoded by the nucleotide sequence of SEQ ID NO: 10.
본 발명의 구체적인 다른 일실시예에서, 항-CD22 항체인 4F5를 인간에 대응하는 구조로 변경한 인간화된 항체(humanized antibody)를 제조하였으며, 이를 4F5(V4) 항체 및 4F5(V11) 항체로 명명하였다. In another specific embodiment of the present invention, humanized antibodies were prepared by changing the anti-CD22 antibody 4F5 to a structure corresponding to human, which was named 4F5 (V4) antibody and 4F5 (V11) antibody did
상기 4F5(V4) 항체 및 4F5(V11) 항체의 중쇄 가변부위 CDR과 경쇄 가변부위 CDR은 4F5와 동일한 것으로 CDR 부분을 제외한 나머지 부분을 인간화하였다. 바람직하게 4F5(V4) 항체는 서열번호 11의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 12의 아미노산 서열로 표시되는 경쇄 가변부위로 구성되며, 4F5(V4) 항체의 중쇄 가변부위는 서열번호 13의 염기서열로, 경쇄 가변부위는 서열번호 14의 염기서열로 코딩될 수 있다.The CDRs of the heavy chain variable region and the CDRs of the light chain variable region of the 4F5 (V4) antibody and the 4F5 (V11) antibody were the same as those of 4F5, and the remaining portions except for the CDR were 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 is SEQ ID NO: 13 As the nucleotide sequence of, the light chain variable region may be encoded by the nucleotide sequence of SEQ ID NO: 14.
또한, 4F5(V11) 항체는 서열번호 15의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 16의 아미노산 서열로 표시되는 경쇄 가변부위로 구성되며, 4F5(V11)항체의 중쇄 가변부위는 서열번호 17의 염기서열로, 경쇄 가변부위는 서열번호 18의 염기서열로 코딩될 수 있다.In addition, the 4F5 (V11) antibody is composed 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 4F5 (V11) antibody is SEQ ID NO: 17 As the nucleotide sequence of, the light chain variable region may be encoded by the nucleotide sequence of SEQ ID NO: 18.
본 발명의 CD22에 특이적인 항체는 바람직하게 scFv(single chain variable fragment)로, 중쇄 가변부위 및 경쇄 가변부위가 링커로 연결될 수 있도록 유전자 재조합 기술을 통하여 제작할 수 있다. 상기 링커는 바람직하게 서열번호 19의 아미노산 서열로 표시되거나, 서열번호 20, 서열번호 21 또는 서열번호 22의 염기서열로 코딩될 수 있으나, 이에 한정되지는 않는다.The CD22-specific antibody of the present invention is preferably a scFv (single chain variable fragment), which can be prepared 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 may be encoded by the nucleotide sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, but is not limited thereto.
경쇄 가변부위-링커-중쇄 가변부위로 연결된 경우 4F5 항체(마우스 항체)는 서열번호 23의 아미노산 서열로 표시되거나 서열번호 24의 염기서열로 코딩될 수 있으며, 4F5(V4) 항체는 서열번호 25의 아미노산 서열로 표시되거나 서열번호 26의 염기서열로 코딩될 수 있고, 4F5(V11) 항체는 서열번호 27의 아미노산 서열로 표시되거나 서열번호 28의 염기서열로 코딩될 수 있다.When linked by 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, and the 4F5 (V4) antibody of SEQ ID NO: 25 It may be represented by the amino acid sequence 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.
본 발명은 다른 관점에서, 상기 CD22에 특이적으로 결합하는 항체 또는 이의 단편을 코딩하는 폴리뉴클레오타이드에 관한 것이다.In another aspect, the present invention relates to a polynucleotide encoding an antibody or fragment thereof that specifically binds to CD22.
본 발명에서, 용어 "폴리뉴클레오타이드"는 일반적으로 임의의 길이로 분리된 핵산 분자(nucleic acid molecule), 데옥시리보뉴클레오티드 또는 리보뉴클레오티드, 또는 그의 유사체를 지칭한다. 일부 구현예에서, 본 발명의 폴리뉴클레오타이드는 (1) 중합효소 연쇄반응(PCR) 증폭과 같은 in-vitro 증폭; (2) 클로닝 및 재조합; (3) 절단(digestion) 및 겔 전기영동 분리와 같은 정제; (4) 화학 합성과 같은 합성을 통해 제조될 수 있으며, 바람직하게 분리된 폴리뉴클레오타이드는 재조합 DNA 기술에 의해 제조된다. 본 발명에서, 항체 또는 이의 항원 결합 단편을 코딩하기 위한 핵산은 합성 올리고뉴클레오티드의 제한 단편 조작(restriction fragment operation) 또는 SOE PCR의 적용을 포함하지만 이에 제한하지 않고, 당업계에 공지된 다양한 방법에 의해 제조될 수 있다.In the present invention, the term “polynucleotide” generally refers to nucleic acid molecules, deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated of any length. In some embodiments, the polynucleotides of the invention can be used for (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 produced through synthesis such as chemical synthesis, and preferably the isolated polynucleotide is produced by recombinant DNA technology. In the present invention, nucleic acids for encoding antibodies or antigen-binding fragments thereof are 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.
본 발명은 또 다른 관점에서, 상기 CD22에 특이적으로 결합하는 항체 또는 이의 단편을 코딩하는 폴리뉴클레오타이드를 포함하는 벡터, 및 상기 벡터로 형질전환된 재조합 세포에 관한 것이다.In another aspect, the present invention relates to a vector comprising a polynucleotide encoding an antibody or fragment thereof that specifically binds to CD22, and a recombinant cell transformed with the vector.
본 발명에서, 용어 "벡터(expression vector)"는 적당한 숙주세포 내에서 목적 유전자가 발현할 수 있도록 프로모터 등의 필수적인 조절 요소를 포함하는 유전자 제조물이다. 벡터는 플라스미드, 레트로바이러스(retroviral) 벡터 및 렌티바이러스(lentiviral) 벡터 중 하나 이상으로부터 선택될 수 있다. 적당한 숙주로 형질전환되면, 벡터는 숙주 게놈과 무관하게 복제하고 기능할 수 있거나, 또는 일부 경우에 게놈 그 자체에 통합될 수 있다. In the present invention, the term "expression vector" is a gene product containing essential regulatory elements such as a promoter so that a target gene can be expressed in an appropriate host cell. Vectors may be selected from one or more of plasmids, retroviral vectors and lentiviral vectors. Once transformed into a suitable host, the vector can replicate and function independently of the host genome or, in some cases, can integrate into the genome itself.
또한, 벡터는 코딩 영역이 적합한 숙주에서 정확하게 발현될 수 있게 하는 발현 제어 요소를 포함할 수 있다. 이러한 조절 요소는 당업자에게 잘 알려져 있으며, 예를 들어 프로모터, 리보솜 결합 부위(ribosome-binding site), 인핸서(enhancer) 및 유전자 전사(transcription) 또는 mRNA 번역(translation)을 조절하기 위한 다른 조절 요소를 포함할 수 있다. 발현 조절 서열의 특정 구조는 종 또는 세포 유형의 기능에 따라 달라질 수 있으나, 일반적으로 TATA 박스(box), 캡핑된(capped) 서열, CAAT 서열 등과 같은 전사 개시 및 번역 개시에 각각 참여하는 5' 비-전사 서열, 및 5' 또는 3' 비-번역 서열을 함유한다. 예를 들어, 5' 비-전사 발현 조절 서열은 기능적으로 연결된 핵산을 전사 및 조절하기 위한 프로모터 서열을 포함할 수 있는 프로모터 영역을 포함할 수 있다. In addition, vectors may contain expression control elements that allow for correct expression of the coding region 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 includes 5' ratios that participate in transcription initiation and translation initiation, such as TATA boxes, capped sequences, CAAT sequences, etc., respectively. -contains a transcribed sequence, and a 5' or 3' non-translated sequence. For example, a 5' non-transcribed expression control sequence can include a promoter region that can include promoter sequences for transcribing and regulating functionally linked nucleic acids.
본 발명에서, 용어 "프로모터"는 전사를 지시하기에 충분한 최소 서열을 의미한다. 또한, 세포 유형 특이적 또는 외부의 신호 또는 제제에 의해 유도되는 조절 가능한 프로모터 의존적 유전자를 발현하도록 하는 데 충분한 프로모터 구성이 포함될 수 있으며, 이러한 구성들은 유전자의 5' 또는 3' 부분에 위치할 수 있다. 보존적 프로모터 및 유도적 프로모터 둘 다 포함된다. 프로모터 서열은 원핵생물, 진핵생물 또는 바이러스로부터 유래될 수 있다.In the present invention, the term "promoter" refers to 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 such constructs may be located on the 5' or 3' portion of the gene. . Both conserved promoters and inducible promoters are included. Promoter sequences may be of prokaryotic, eukaryotic or viral origin.
본 발명에서, 용어 "형질전환체"는 하나 이상의 목적 단백질을 암호화하는 폴리뉴클레오타이드를 갖는 벡터가 숙주세포에 도입되어 형질전환된 세포를 의미하고, 발현 벡터를 숙주세포에 도입하여 형질전환체를 제조하기 위한 방법으로는 문헌(Sambrook, J., et al., Molecular Cloning, A Laboratory Manual(2판), Cold Spring Harbor Laboratory, 1. 74, 1989)에 기재된 인산칼슘법 또는 염화캄슘/염화루비듐법, 일렉트로포레이션법(electroporation), 전기주입법(electroinjection), PEG 등의 화학적 처리방법, 유전자 총(gene gun) 등을 이용하는 방법 등이 있다. 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 preparing a transformant by introducing an expression vector into a host cell. As a method for doing 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) , electroporation, electroinjection, chemical treatment methods such as PEG, methods using a gene gun, and the like.
상기 벡터가 발현되는 형질전환체를 영양배지에서 배양하면 항체 단백질을 대량으로 제조, 분리 가능하다. 배지와 배양조건은 숙주 세포에 따라 관용되는 것을 적절히 선택하여 이용할 수 있다. 배양시 세포의 생육과 단백질의 대량 생산에 적합하도록 온도, 배지의 pH 및 배양시간 등의 조건들을 적절하게 조절하여야 한다. When the transformant expressing the vector is cultured in a nutrient medium, antibody protein can be produced and isolated in large quantities. Media and culture conditions can be appropriately selected and used according to the host cell. Conditions such as temperature, medium pH, and incubation time should be appropriately adjusted so as to be suitable for cell growth and mass production of proteins during culture.
본 발명에 따른 벡터는 항체의 생산을 위해 숙주세포, 바람직하게는 포유동물 세포에 형질전환 시킬 수 있다. 완벽한 글리코실화된 단백질을 발현할 수 있는 적합한 숙주 세포는 COS-1(예를 들면, ATCC CRL 1650), COS-7(예를 들면, ATCC CRL-1651), HEK293, BHK21(예를 들면, ATCC CRL-10), CHO(예를 들면, ATCC CRL 1610) 및 BSC-1(예를 들면, ATCC CRL-26) 세포주, Cos-7 세포, CHO 세포, hep G2 세포, P3X63Ag8.653, SP2/0-Agl4, 293 세포, HeLa 세포 등을 포함하며, 이들 세포는 예를 들면, ATCC(American Type Culture Collection, 미국)으로부터 용이하게 이용가능하다.The vector according to the present invention can be transformed into a host cell, preferably a mammalian cell, for antibody production. 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., and these cells are readily available, for example, from the American Type Culture Collection (ATCC, USA).
CD22를 표적으로 하는 키메라 항원 수용체(Chimeric antigen receptor)Chimeric antigen receptor that targets CD22
본 발명은 다른 관점에서, From another point of view, the present invention
CD22-결합 도메인; CD22-binding domain;
막관통 도메인(transmembrane domain); transmembrane domain;
공동자극 도메인(costimulatory domain); 및 costimulatory domain; and
세포 내 신호전달 도메인(intracellular signal transduction domain)을 포함하는 키메릭 항원 수용체(chimeric antigen receptor: CAR)로,A chimeric antigen receptor (CAR) containing an intracellular signal transduction domain,
상기 CD22-결합 도메인은 서열번호 1의 아미노산으로 표시되는 CDR1 영역, 서열번호 2의 아미노산으로 표시되는 CDR2 영역 및 서열번호 3의 아미노산으로 표시되는 CDR3 영역을 포함하는 중쇄 가변부위 및 서열번호 4의 아미노산으로 표시되는 CDR1 영역, 서열번호 5의 아미노산으로 표시되는 CDR2 영역 및 서열번호 6의 아미노산으로 표시되는 CDR3 영역을 포함하는 경쇄 가변 부위를 포함하는 CD22에 특이적으로 결합하는 항체 또는 이의 단편인 것을 특징으로 하는, CD22를 표적으로 하는 키메라 항원 수용체에 관한 것이다.The CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4 An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 It relates to a chimeric antigen receptor targeting CD22.
본 발명에 있어서, 상기 CD22에 특이적으로 결합하는 항체는 서열번호 11의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 12의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체 또는 이의 단편; 또는 In the present invention, the antibody specifically binding to CD22 is a humanized antibody specifically binding to CD22 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. antibodies or fragments thereof; or
서열번호 15의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 16의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체 또는 이의 단편일 수 있다.It may be a humanized antibody or fragment thereof that specifically binds to CD22 composed of the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.
본 발명에서, 용어 "키메릭 항원 수용체 (CAR)"는 일반적으로 항원 및 하나 이상의 세포 내 도메인과 결합하는 능력을 갖는 세포 외 도메인을 함유하는 융합 단백질을 지칭한다. CAR는 키메릭 항원 수용체 T 세포(CAR-T)의 핵심 부분이며, 항원 결합 도메인, 막 관통 도메인, 공동 자극 도메인 및 세포 내 신호전달 도메인을 포함할 수 있다. CAR는 항체의 항원(예를 들어 CD22) 특이성에 기초하여 T 세포 수용체-활성화 세포 내 도메인과 조합될 수 있다. 유전자가 변형된 CAR-발현 T 세포는 표적 항원-발현 악성 세포를 특이적으로 식별하고 제거할 수 있다. In the present invention, the term "chimeric antigen receptor (CAR)" generally refers to a fusion protein containing an antigen and an extracellular domain that has 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 can include an antigen binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain. A CAR may be combined with a T cell receptor-activating intracellular domain based on the antibody's antigenic (eg CD22) specificity. Genetically modified CAR-expressing T cells can specifically identify and eliminate target antigen-expressing malignant cells.
본 발명에서, 용어 "CD22-결합 도메인(CD22-binding domain)"은 일반적으로 CD22 단백질에 특이적으로 결합할 수 있는 도메인을 지칭한다. 예를 들어, CD22-결합 도메인은 B 세포에서 발현된 인간 CD22 폴리펩타이드 또는 이의 단편에 특이적으로 결합할 수 있는 항-CD22 항체 또는 이의 단편을 함유할 수 있다. In the present invention, the term "CD22-binding domain" generally refers to a domain capable of specifically binding to the 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 on B cells.
본 발명에서, 용어 "결합 도메인(binding domain)"은 "세포 외 도메인(extracellular domain)", "세포 외 결합 도메인(extracellular binding domain)", "항원-특이적 결합 도메인(antigenspecific binding domain)" 및 "세포 외 항원-특이적 결합 도메인(extracellular antigen-specific biding domain)"은 상호 교환적으로 사용될 수 있으며, 표적 항원(예를 들어 CD22)에 특이적으로 결합하는 능력을 갖는 CAR 도메인 또는 단편을 지칭한다. In the present invention, the term "binding domain" includes "extracellular domain", "extracellular binding domain", "antigen-specific binding domain" and "Extracellular antigen-specific binding domain" may be used interchangeably and refers to a CAR domain or fragment that has the ability to specifically bind to a target antigen (eg CD22) do.
본 발명에 있어서, 상기 항-CD22 항체 또는 이의 단편은 상술한 항-CD22 항체로, 단클론 항체(monoclonal antibody), 바람직하게는 scFv(single chain variable fragment)이다. 구체적으로, 본 발명의 CD22에 특이적인 인간화된 항체인 4F5(V4) 또는 4F5(V11) 항체를 이용하여 제조할 수 있다.In the present invention, the anti-CD22 antibody or fragment thereof is the aforementioned anti-CD22 antibody, which is a monoclonal antibody, preferably a single chain variable fragment (scFv). Specifically, it can be prepared using the 4F5 (V4) or 4F5 (V11) antibody, which is a humanized antibody specific to CD22 of the present invention.
본 발명에 있어서, 상기 키메릭 항원 수용체는 CD22-결합 도메인 이외에 B 세포 표면 마커-결합 도메인이 추가로 포함된 이중특이적 키메릭 항원 수용체일 수 있으며, 상기 B 세포 표면 마커는 CD1O, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85 또는 CD86일 수 있으며, 바람직하게는 CD19일 수 있다.In the present invention, the chimeric antigen receptor may be a bispecific chimeric antigen receptor further comprising a B cell surface marker-binding domain in addition to the CD22-binding domain, and the B cell surface markers are CD10, CD19, and CD20. , CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85 or CD86, preferably CD19.
본 발명에 있어서, CD22-결합 도메인의 N 말단에 신호 펩타이드(signal peptide)를 추가로 포함할 수 있으며, 상기 "신호 펩타이드(signal peptide)"는 일반적으로 단백질 전달을 안내하기 위한 펩타이드 사슬을 지칭한다. 신호 펩타이드는 5 내지 30 개의 아미노산 길이를 갖는 짧은 펩타이드일 수 있으며, 본 발명에서는 바람직하게 서열번호 36의 아미노산 서열을 이용하였다.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: 36 is preferably used in the present invention.
본 발명에 있어서, CD22-결합 도메인의 C 말단 및 막관통 도메인의 N 말단 사이에 위치된 힌지 부위(hinge region)를 추가로 포함할 수 있으며, 상기 힌지 부위는 CD8α 유래로, 바람직하게는 서열번호 37의 아미노산 서열로 표시될 수 있다. 상기 "힌지 부위(hinge region)"는 일반적으로 항원-결합 영역과 면역 세포 Fc 수용체 (FcR)-결합영역 사이의 연결 영역을 지칭한다.In 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, and the hinge region is derived from CD8α, preferably SEQ ID NO: 37 amino acid sequence. The "hinge region" generally refers to the junction region between an antigen-binding region and an immune cell Fc receptor (FcR)-binding region.
본 발명에 있어서, "막관통 도메인(transmembrane domain)"은 일반적으로 세포막을 통과하고 세포 내 신호전달 도메인에 연결되어 신호전달의 역할을 하는 CAR의 도메인을 지칭한다. 상기 막관통 도메인은 CD8α, CD4, CD28, CD137, CD80, CD86, CD152 및 PD1로 구성된 군에서 선택되는 단백질로부터 유래될 수 있으며, 바람직하게는 서열번호 38의 아미노산 서열로 표시될 수 있다. 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 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.
본 발명에 있어서, "공동 자극 도메인(costimulatory domain)"은 일반적으로 림프구의 항원에 대한 효과적인 반응에 필요한 세포 표면 분자인 면역 자극 분자를 제공할 수 있는 세포 내 도메인을 지칭한다. 상기 기재된 공동자극 도메인(costimulatory domain)은 CD28의 공동 자극 도메인을 포함할 수 있고, OX40 및 4-1BB의 공동 자극 도메인과 같은 TNF 수용체 패밀리의 공동 자극 도메인을 포함할 수 있으며, 바람직하게는 서열번호 39의 아미노산 서열로 표시되는 4-1BB일 수 있다.In the present invention, "costimulatory domain" generally refers to an intracellular domain capable of providing immune stimulatory molecules, which are cell surface molecules required for effective response of lymphocytes to antigens. The costimulatory domain described above may include a costimulatory domain of CD28, and may include a costimulatory domain of the TNF receptor family, such as the costimulatory domains of OX40 and 4-1BB, preferably SEQ ID NO: It may be 4-1BB represented by the amino acid sequence of 39.
본 발명에 있어서, "세포 내 신호전달 도메인(intracellular signal transduction domain)"은 일반적으로 세포 내부에 위치하고 신호를 전달할 수 있는 도메인을 지칭한다. 본 발명에서, 세포 내 신호전달 도메인은 키메라 항원 수용체의 세포 내 신호전달 도메인이다. 예를 들어, 세포 내 신호전달 도메인은 CD3ζ 세포 내 도메인, CD28 세포 내 도메인, CD28 세포 내 도메인, 4-1BB 세포 내 도메인 및 OX40 세포 내 도메인으로부터 선택될 수 있으며, 바람직하게는 서열번호 40의 아미노산 서열로 표시되는 CD3ζ일 수 있다.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 an intracellular signaling domain of a chimeric antigen receptor. For example, the intracellular signaling domain may be selected from the CD3ζ intracellular domain, the CD28 intracellular domain, the CD28 intracellular domain, the 4-1BB intracellular domain and the OX40 intracellular domain, preferably comprising the amino acids of SEQ ID NO: 40 It may be CD3ζ represented by the sequence.
키메릭 항원 수용체 코딩 폴리뉴클레오타이드 및 키메릭 항원 수용체 발현 벡터Chimeric Antigen Receptor Encoding Polynucleotides and Chimeric Antigen Receptor Expression Vectors
본 발명은 또 다른 관점에서, 상기 키메릭 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드에 관한 것이다.In another aspect, the present invention relates to a polynucleotide encoding the chimeric antigen receptor (CAR).
본 발명에 있어서, 상기 키메릭 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드는 CD22-결합 도메인를 코딩하는 폴리뉴클레오타이드; 막관통 도메인을 코딩하는 폴리뉴클레오타이드; 공동 자극 도메인을 코팅하는 폴리뉴클레오타이드; 및 세포 내 신호전달 도메인을 코딩하는 폴리뉴클레오타이드를 포함할 수 있다. In the present invention, the polynucleotide encoding the chimeric antigen receptor (CAR) is a polynucleotide encoding a CD22-binding domain; polynucleotides encoding transmembrane domains; polynucleotides coating the co-stimulatory domain; And it may include a polynucleotide encoding an intracellular signaling domain.
상기 CD22-결합 도메인를 코딩하는 폴리뉴클레오타이드는 본 발명의 CD22에 특이적인 4F5 항체, CD22에 특이적인 인간화된 4F5(V4) 항체 또는 4F5(V11) 항체일 수 있으며, 경쇄 가변부위 및 중쇄 가변부위가 링커로 연결된 scFv 형태로, 구체적인 염기서열은 상술한 바와 같다.The polynucleotide encoding the CD22-binding domain may be a 4F5 antibody specific for CD22, a humanized 4F5(V4) antibody specific for CD22, or a 4F5(V11) antibody specific for CD22 of the present invention, and the light chain variable region and the heavy chain variable region are linkers. In the form of scFv linked to, the specific nucleotide sequence is as described above.
바람직하게, 본 발명의 키메릭 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드는, 서열번호 24의 염기서열로 표시되는 4F5 항체, 서열번호 26의 염기서열로 표시되는 4F5(V4) 항체 또는 서열번호 28의 염기서열로 표시되는 4F5(V11) 항체;Preferably, the polynucleotide encoding the chimeric antigen receptor (CAR) of the present invention is a 4F5 antibody represented by the nucleotide sequence of SEQ ID NO: 24, a 4F5 (V4) antibody represented by the nucleotide sequence of SEQ ID NO: 26, or SEQ ID NO: 28 4F5 (V11) antibody represented by the nucleotide sequence of;
서열번호 32의 염기서열로 표시되는 막관통 도메인; a transmembrane domain represented by the nucleotide sequence of SEQ ID NO: 32;
서열번호 33의 염기서열로 표시되는 4-1BB(공동자극 도메인); 및 4-1BB (co-stimulatory domain) represented by the nucleotide sequence of SEQ ID NO: 33; and
서열번호 34의 염기서열로 표시되는 CD3ζ(세포 내 신호전달 도메인)로 구성될 수 있다.It may be composed of CD3ζ (intracellular signaling domain) represented by the nucleotide sequence of SEQ ID NO: 34.
CD22-결합 도메인의 N 말단에 신호 펩타이드(signal peptide)가 포함된 경우, 서열번호 30의 염기서열로 표시되는 신호 펩타이드가 추가로 포함될 수 있다. 또한, CD22-결합 도메인를 코딩하는 폴리뉴클레오타이드 및 막관통 도메인 사이에, 힌지 부위(hinge region)를 코딩하는 폴리뉴클레오타이드가 추가로 포함될 수 있으며, 바람직하게 서열번호 31의 염기서열로 표시되는 CD8 힌지 부위일 수 있다.When a signal peptide is included at the N-terminus of the CD22-binding domain, a signal peptide represented by the nucleotide sequence of SEQ ID NO: 30 may be additionally included. 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 a CD8 hinge region represented by the nucleotide sequence of SEQ ID NO: 31 can
본 발명은 또 다른 관점에서, 상기 키메릭 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터에 관한 것이다. In another aspect, the present invention relates to a vector comprising a polynucleotide encoding the chimeric antigen receptor (CAR).
본 발명에서, 상기 벡터는 재조합 바이러스 벡터로, 바람직하게는 렌티바이러스 벡터이며, 작동가능하게 연결된 EF1α 프로모터; 시그널 펩타이드를 코딩하는 폴리뉴클레오타이드; CD22-결합 도메인을 코딩하는 폴리뉴클레오타이드; 막관통 도메인을 코딩하는 폴리뉴클레오타이드; 세포 내 신호전달 도메인을 코딩하는 폴리뉴클레오타이드를 포함하며, 단백질 발현을 증가시키기 위해 WPRE(woodchuck hepatitis virus post-transcriptional regulatory element)를 추가로 포함할 수 있다 (도 3). In the present invention, the vector is a recombinant viral vector, preferably a lentiviral vector, comprising an operably linked EF1α promoter; polynucleotides encoding signal peptides; a polynucleotide encoding a CD22-binding domain; polynucleotides encoding transmembrane domains; 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).
상기 EF1α 프로모터는 서열번호 29의 염기서열로 표시될 수 있으며, 필요에 따라 상기 서열번호 27의 염기서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함할 수 있다.The EF1α promoter may be represented by the nucleotide sequence of SEQ ID NO: 29, 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: 27 , or sequences that are at least 99% identical.
또한, 상기 프로모터는 CD22-결합 도메인인 항-CD22 항체(scFv)의 발현을 유도하도록 작동 가능하게 연결되어 있다.In addition, the promoter is operably linked to drive expression of a CD22-binding domain, an anti-CD22 antibody (scFv).
본 발명의 구체적인 일구현예에서, CD22-CAR를 코팅하는 폴리뉴클레오타이드가 삽입된 렌티바이러스 벡터를 제조하였으며, 제조된 벡터를 293FT 세포에 형질전환 시켜 CD22-CAR 발현 세포를 제조하였다.In a specific embodiment of the present invention, a lentiviral vector into which a polynucleotide coating CD22-CAR was inserted was prepared, and the prepared vector was transformed into 293FT cells to prepare CD22-CAR expressing cells.
숙주 세포 내로 폴리뉴클레오티드를 도입하기 위한 생물학적 방법은 DNA 및 RNA 벡터의 사용을 포함한다. 바이러스 벡터, 및 특히 레트로바이러스 벡터는 유전자를 포유동물, 예를 들어 인간 세포 내로 삽입하기 위해 가장 널리 사용되는 방법이 되었다. 다른 바이러스 벡터는 렌티바이러스, 폭스바이러스, 단순 포진 바이러스, 아데노바이러스 및 아데노-연관 바이러스 등으로부터 유래될 수 있다. 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 method 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.
숙주 세포 내로 폴리뉴클레오티드를 도입하기 위한 화학적 수단은 콜로이드성 분산액 시스템, 예컨대 거대분자 복합체, 나노캡슐, 마이크로구체, 비드, 및 수중유 에멀젼, 미셀, 혼합된 미셀, 및 리포솜을 비롯한 지질-기반 시스템을 포함한다. 시험관 내 및 생체 내에서 전달 비히클로서 사용하기 위한 예시적인 콜로이드성 시스템은 리포솜(예를 들어, 인공 막 소포)이다.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 Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (eg, artificial membrane vesicles).
비-바이러스 전달 시스템이 이용되는 경우에, 예시적인 전달 비히클은 리포솜이다. 지질 제제의 사용은 숙주세포 내로의 핵산의 도입(시험관 내, 생체 외 또는 생체 내)을 위해 고려된다. 또 다른 측면에서, 핵산은 지질과 회합될 수 있다. 지질과 회합된 핵산은 리포솜의 수성 내부에 캡슐화되거나, 리포솜의 지질 이중층 내에 점재되거나, 리포솜 및 올리고뉴클레오티드 둘 다와 회합된 연결 분자를 통해 리포솜에 부착되거나, 리포솜 내에 포획되거나, 리포솜과 복합체화되거나, 지질 함유 용액 중에 분산되거나, 지질과 혼합되거나, 지질과 조합되거나, 지질 내에 현탁액으로서 함유되거나, 미셀과 함께 함유 또는 복합체화되거나, 또는 지질과 달리 회합될 수 있다. 지질, 지질/DNA 또는 지질/발현 벡터 회합 조성물은 용액 중의 임의의 특정한 구조로 제한되지 않는다.When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction (in vitro, ex vivo or in vivo) of nucleic acids into host cells. In another aspect, a nucleic acid can be associated with a lipid. Nucleic acids associated with lipids may be encapsulated in the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules associated with both liposomes and oligonucleotides, entrapped within liposomes, complexed with liposomes, 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 a micelle, or otherwise associated with a lipid. The lipid, lipid/DNA or lipid/expression vector associated composition is not limited to any particular structure in solution.
키메릭 항원 수용체(CAR) 발현 면역 이펙터 세포Chimeric antigen receptor (CAR) expressing immune effector cells
본 발명은 또 다른 관점에서, 상기 CD22에 특이적인 항체 기반 키메릭 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드 또는 키메릭 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터를 포함하고, 상기 CD22에 특이적인 항체 기반 키메릭 항원 수용체(CAR)를 발현하는 면역 이펙터 세포에 관한 것이다.In another aspect, the present invention includes a polynucleotide encoding a chimeric antigen receptor (CAR) based on an antibody specific for the CD22 or a vector comprising a polynucleotide encoding the chimeric antigen receptor (CAR), wherein the CD22 It relates to immune effector cells expressing an antibody-based chimeric antigen receptor (CAR) specific for
본 발명에 있어서, 상기 면역 이펙터 세포는 포유동물 유래 단리된 세포 일 수 있으며, 바람직하게는 T 세포, B 세포, 자연 살해(NK) 세포, 수지상 세포, 골수 세포, 단핵세포, 또는 대식세포, 더 바람직하게는 T 세포일 수 있다.In the present invention, the immune effector cells may be mammalian-derived isolated cells, preferably T cells, B cells, natural killer (NK) cells, dendritic cells, bone marrow cells, monocytes, or macrophages, and more Preferably it may be a T cell.
본 발명에 있어서, 상기 키메릭 항원 수용체(CAR)를 발현하는 면역 이펙터 세포는 본 발명의 CAR 벡터를 면역 이펙터 세포, 예를 들어 T 세포 또는 NK 세포 내로 도입시켜 제조할 수 있다. In the present invention, 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.
구체적으로, CAR 벡터는 전기천공법, 리포펙타민(lipofectamine 2000, Invitrogen) 등과 같은 당업계에 공지된 방법에 의해 세포 내로 도입될 수 있다. 예를 들어, 면역 이펙터 세포는 렌티바이러스 벡터에 의해 형질전환되어 CAR 분자를 운반하는 바이러스 게놈을 숙주 게놈에 통합시켜 표적 유전자의 장기적이고 안정적인 발현을 보장할 수 있다. 다른 예를 들어, 전이인자(transposon)는 CAR 운반 플라스미드(transposon) 및 전이효소 운반 플라스미드를 표적 세포 내로 도입하는데 이용될 수 있다. 다른 예를 들어, CAR 분자는 유전자 편집방법(예를 들어, CRISPRCas9)에 의해 게놈에 첨가될 수 있다.Specifically, the CAR vector may be introduced into cells by a method known in the art, such as electroporation or lipofectamine (lipofectamine 2000, Invitrogen). For example, immune effector cells can be transformed 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. In another example, a transposon can be used to introduce a CAR carrier plasmid (transposon) and a transposase carrier plasmid into a target cell. In another example, CAR molecules can be added to the genome by gene editing methods (eg, CRISPRCas9).
키메릭 항원 수용체(CAR)를 발현하는 면역 이펙터 세포 제조를 위한 면역 이펙터 세포는 대상체로부터 수득할 수 있으며, 상기 "대상체"는 면역 반응이 도출될 수 있는 살아있는 유기체 (예를 들어, 포유동물)를 포함한다. 대상체의 예는 인간, 개, 고양이, 마우스, 래트, 및 그의 트랜스제닉 종을 포함한다. T 세포는 말초 혈액 단핵 세포, 골수, 림프절 조직, 제대혈, 흉선 조직, 감염 부위로부터의 조직, 복수, 흉막 삼출, 비장 조직, 및 종양을 비롯한 수많은 공급원으로부터 수득될 수 있다.Immune effector cells for producing 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) from which an immune response can be elicited. include Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.
상기 T 세포는 통상의 기술자에게 공지된 임의의 많은 기술, 예를 들면, 피콜(Ficoll)™ 분리를 사용하여 대상체로부터 수집된 혈액 단위로부터 수득될 수 있다. 혈액으로부터 세포는 분리반출술에 의해 수득되며, 분리반출술 생성물은 전형적으로 T 세포, 단핵구, 과립구, B 세포를 비롯한 림프구, 다른 유핵 백혈구, 적혈구, 및 혈소판을 함유한다. The T cells can be obtained from a unit of blood collected from a subject using any of a number of techniques known to those skilled in the art, such as Ficoll™ separation. Cells from blood are obtained by apheresis, and the apheresis product typically contains T cells, monocytes, granulocytes, lymphocytes including B cells, other nucleated leukocytes, red blood cells, and platelets.
분리반출술에 의해 수집된 세포는 혈장 분획을 제거하고 세포를 후속 프로세싱 단계를 위해 적절한 완충제 또는 배지에 두기 위해 세척될 수 있다. T 세포는 적혈구를 용해시키고, 예를 들어 퍼콜(PERCOLL)™ 구배를 통한 원심분리에 의해 또는 역류 원심 분리에 의해 단핵구를 고갈시킴으로써 말초 혈액 림프구로부터 단리된다.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, eg, by centrifugation over a PERCOLL™ gradient or by countercurrent centrifugation.
본 발명의 구체적인 다른 일구현예에서, 도 4에 나타난 바와 같이, 말초 혈액 단핵세포(peripheral blood mononuclear cell, PBMC)로 부터 활성화된 T 세포를 분리한 다음, CD22-CAR 렌티바이러스 벡터를 T 세포에 형질도입시켜 CD22-CAR-T 세포를 제조하였으며, 구체적으로 4F5 항체, 인간화된 4F5(V4) 항체 및 4F5(V11) 항체를 이용하여 CD22-CAR-T 세포를 각각 제조하였다.In another specific embodiment of the present invention, as shown in FIG. 4, after isolating activated T cells from peripheral blood mononuclear cells (PBMC), the CD22-CAR lentiviral vector is applied to the T cells. CD22-CAR-T cells were prepared by transduction, and specifically, CD22-CAR-T cells were prepared using a 4F5 antibody, a humanized 4F5 (V4) antibody, and a 4F5 (V11) antibody, respectively.
제조된 CD22-CAR-T 세포의 활성을 확인하기 위해, CD3, CD4 또는 CD8이 활성화된 CD22-CAR-T 세포에 대한 CD22 펩타이드에 대한 결합능을 확인하였다. 도 5a 및 도 5b에 나타난 바와 같이, 본 발명에서 제조한 CD22-CAR-T 세포가 CD22 펩타이드와 결합하는 것을 확인하였다.In order to confirm the activity of the prepared CD22-CAR-T cells, CD3, CD4 or CD8-activated CD22-CAR-T cells were examined for their ability to bind to CD22 peptides. As shown in Figures 5a and 5b, it was confirmed that the CD22-CAR-T cells prepared in the present invention bind to the CD22 peptide.
본 발명의 구체적인 또 다른 일구현예에서, CD22-CAR-T 세포에 의한 표적 세포의 사멸효과를 확인한 결과, 도 7a 및 도 7b에 나타난 바와 같이, CD22-CAR-T 세포는 CD22를 발현하는 U2932 세포 특이적으로 사멸효과를 보이는 것을 확인하였다.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 Figures 7a and 7b, CD22-CAR-T cells are U2932 expressing CD22. It was confirmed that the cell-specific killing effect was shown.
즉, 본 발명의 항-CD22 항체인 4F5 항체, 4F5(V4) 항체 또는 4F5(V11) 항체 기반 키메라 항원 수용체, 및 이를 이용한 CAR-T 세포는 B 세포 또는 CD22 발현과 관련된 질환 예방 또는 치료용 조성물로 유용하게 활용할 수 있다. That is, the anti-CD22 antibody of the present invention, the 4F5 antibody, the 4F5(V4) antibody, or the 4F5(V11) antibody-based chimeric antigen receptor, and the CAR-T cell using the same are a composition for preventing or treating diseases related to B cell or CD22 expression can be put to good use.
CD19 및 CD22를 표적으로 하는 이중특이적 키메라 항원 수용체(Bispecific CAR)Bispecific Chimeric Antigen Receptor (Bispecific CAR) Targeting CD19 and CD22
본 발명은 또 다른 관점에서, In another aspect, the present invention
CD19-결합 도메인 및 CD22-결합 도메인; CD19-binding domain and CD22-binding domain;
막관통 도메인(transmembrane domain); transmembrane domain;
공동자극 도메인(costimulatory domain); 및 costimulatory domain; and
세포 내 신호전달 도메인(intracellular signal transduction domain)을 포함하는 이중특이적 키메릭 항원 수용체(chimeric antigen receptor: CAR)로,A bispecific chimeric antigen receptor (CAR) containing an intracellular signal transduction domain,
상기 CD22-결합 도메인은 서열번호 1의 아미노산으로 표시되는 CDR1 영역, 서열번호 2의 아미노산으로 표시되는 CDR2 영역 및 서열번호 3의 아미노산으로 표시되는 CDR3 영역을 포함하는 중쇄 가변부위 및 서열번호 4의 아미노산으로 표시되는 CDR1 영역, 서열번호 5의 아미노산으로 표시되는 CDR2 영역 및 서열번호 6의 아미노산으로 표시되는 CDR3 영역을 포함하는 경쇄 가변부위를 포함하는 CD22에 특이적으로 결합하는 항체 또는 이의 단편인 것을 특징으로 하는이중특이적 키메라 항원 수용체(CD19xCD22 bispecific CAR)에 관한 것이다.The CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4 An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 It relates to a bispecific chimeric antigen receptor (CD19xCD22 bispecific CAR).
본 발명에 있어서, 상기 CD22에 특이적으로 결합하는 항체는 서열번호 7의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 8의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 항-CD22 항체이거나,In the present invention, the antibody specifically binding to CD22 is an anti-CD22 antibody composed 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;
서열번호 11의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 12의 아미노산 서열로 표시되는 경쇄 가변부위 구성된 인간화된 항-CD22 항체; 또는 a humanized anti-CD22 antibody 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; or
서열번호 15의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 16의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 인간화된 항-CD22 항체일 수 있다.It may be a humanized anti-CD22 antibody composed 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.
본 발명에서, 상기 이중특이적(bispecific 또는 bivalent) 키메라 항원 수용체는 두 가지 다른 유형의 항원을 동시에 결합할 수 있는 CAR로, 본 발명에서는 바람직하게 CD19 및 CD22 모두를 표적으로 하는 이중특이적 키메라 항원 수용체를 제조하였으며, 상기 CD19-결합 도메인은 공지된 항-CD19 항체 서열을 제한없이 사용할 수 있다.In the present invention, the bispecific (bispecific or bivalent) chimeric antigen receptor is a CAR capable of simultaneously binding two different types of antigens, and in the present invention, the bispecific chimeric antigen receptor preferably targets both CD19 and CD22 A receptor was prepared, and the CD19-binding domain may use a known anti-CD19 antibody sequence without limitation.
키메라 항원 수용체에 대한 구체적인 내용은 상술한 바와 동일하며, 상기 이중특이적 키메라 항원 수용체의 CD19-결합 도메인 및 CD22-결합 도메인은 도 7에 나타난 바와 같이 루프(LoopCAR) 형태로 연결하였다. 즉, CD19에 특이적으로 결합하는 항체의 경쇄 가변부위(CD19VL) - CD22에 특이적으로 결합하는 항체의 중쇄 가변부위(CD22VH) - CD22에 특이적으로 결합하는 항체의 경쇄 가변부위(CD22VL) - CD19에 특이적으로 결합하는 항체의 중쇄 가변부위(CD19VH) 순서로 연결될 수 있다.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 were 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) - It may be linked in sequence to the heavy chain variable region (CD19VH) of an antibody that specifically binds to CD19.
상기 상기 CD19에 특이적으로 결합하는 항체의 경쇄 가변부위는 서열번호 48의 아미노산 서열로 표시되며, 상기 CD19에 특이적으로 결합하는 항체의 중쇄 가변부위는 서열번호 47의 아미노산 서열로 표시될 수 있다.The light chain variable region of the antibody specifically binding to CD19 is represented by the amino acid sequence of SEQ ID NO: 48, and the heavy chain variable region of the antibody specifically binding to CD19 is represented by the amino acid sequence of SEQ ID NO: 47. .
상기 CD19-결합 도메인 및 CD22-결합 도메인은 링커로 연결될 수 있도록 유전자 재조합 기술을 통하여 제작할 수 있으며, 바람직하게 CD19VL 및 CD22VH 또는 CD22VL 및 CD19VH는 서열번호 51의 아미노산 서열로 표시되는 링커(도 8의 linker 1로 연결될 수 있으며, CD22VH 및 CD22VL는 서열번호 54의 아미노산 서열로 표시되는 링커(도 8의 linker 6)로 연결될 수 있으나, 이에 한정되지 않고 항체 활성에 영향을 미치지 않은 임의의 아미노산 서열로 구성된 펩타이드를 사용할 수 있다.The CD19-binding domain and the CD22-binding domain can be prepared through genetic recombination technology so that they can be linked by a linker, and preferably CD19VL and CD22VH or CD22VL and CD19VH are linkers represented by the amino acid sequence of SEQ ID NO: 51 (linker in FIG. 8). 1, and CD22VH and CD22VL may be linked by a linker represented by the amino acid sequence of SEQ ID NO: 54 (linker 6 in FIG. 8), but is not limited thereto, and does not affect antibody activity Peptide consisting of any amino acid sequence can be used.
CD19 및 CD22를 표적으로 하는 이중특이적 키메릭 항원 수용체를 코딩 폴리뉴클레오타이드 및 CD19/CD22를 표적으로 하는 이중특이적 키메릭 항원 수용체 발현 벡터A polynucleotide encoding a bispecific chimeric antigen receptor targeting CD19 and CD22 and a bispecific chimeric antigen receptor expression vector targeting CD19/CD22
본 발명은 또 다른 관점에서, 상기 CD19 및 CD22를 표적으로 하는 이중특이적 키메라 항원 수용체를 코딩하는 폴리뉴클레오타이드에 관한 것이다.In another aspect, the present invention relates to a polynucleotide encoding a bispecific chimeric antigen receptor targeting CD19 and CD22.
본 발명에 있어서, 상기 이중특이적 키메라 항원 수용체를 코딩하는 폴리뉴클레오타이드는, CD19-결합 도메인을 코딩하는 폴리뉴클레오타이드 및 CD22-결합 도메인를 코딩하는 폴리뉴클레오타이드; 막관통 도메인을 코딩하는 폴리뉴클레오타이드; 공동 자극 도메인을 코팅하는 폴리뉴클레오타이드; 및 세포 내 신호전달 도메인을 코딩하는 폴리뉴클레오타이드를 포함할 수 있다. In the present invention, the polynucleotide encoding the bispecific chimeric antigen receptor includes a polynucleotide encoding a CD19-binding domain and a polynucleotide encoding a CD22-binding domain; polynucleotides encoding transmembrane domains; polynucleotides coating the co-stimulatory domain; And it may include a polynucleotide encoding an intracellular signaling domain.
바람직하게, 본 발명의 이중특이적 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드는, Preferably, the polynucleotide encoding the bispecific chimeric antigen receptor of the present invention is
CD19에 특이적으로 결합하는 항체의 경쇄 가변부위(CD19VL; 서열번호 50) - CD22에 특이적으로 결합하는 항체의 중쇄 가변부위(CD22VH; 서열번호 9, 서열번호 13 또는 서열번호 17) - CD22에 특이적으로 결합하는 항체의 경쇄 가변부위(CD22VL; 서열번호 10, 서열번호 14 또는 서열번호 18) - CD19에 특이적으로 결합하는 항체의 중쇄 가변부위(CD19VH; 서열번호 49)로 구성된 이중특이적 항체;Light chain variable region of antibody specifically binding to CD19 (CD19VL; SEQ ID NO: 50) - heavy chain variable region of antibody specifically binding to CD22 (CD22VH; SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 17) - CD22 Bispecific consisting of the light chain variable region of the antibody specifically binding (CD22VL; SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 18) - the heavy chain variable region of the antibody specifically binding to CD19 (CD19VH; SEQ ID NO: 49) antibodies;
서열번호 32의 염기서열로 표시되는 막관통 도메인; a transmembrane domain represented by the nucleotide sequence of SEQ ID NO: 32;
서열번호 33의 염기서열로 표시되는 4-1BB(공동자극 도메인); 및 4-1BB (co-stimulatory domain) represented by the nucleotide sequence of SEQ ID NO: 33; and
서열번호 34의 염기서열로 표시되는 CD3ζ(세포 내 신호전달 도메인)로 구성될 수 있다.It may be composed of CD3ζ (intracellular signaling domain) represented by the nucleotide sequence of SEQ ID NO: 34.
상기 'CD19VL 및 CD22VH' 또는 'CD22VL 및 CD19VH'가 서열번호 51의 아미노산 서열로 표시되는 링커로 연결되는 경우, 상기 링커에 대한 폴리뉴클레오타이드는 서열번호 52 또는 서열번호 53의 염기서열로 표시될 수 있으며, 'CD22VH 및 CD22VL'가 서열번호 54의 아미노산 서열로 표시되는 링커로 연결되는 경우, 상기 링커에 대한 폴리뉴클레오타이드는 서열번호 55의 염기서열로 표시될 수 있으나, 이에 한정되지 않고 항체 활성에 영향을 미치지 않은 임의의 아미노산 서열을 코딩하는 폴리뉴클레오타이드를 사용할 수 있다.When the '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 nucleotide sequence of SEQ ID NO: 52 or SEQ ID NO: 53, , 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 nucleotide sequence of SEQ ID NO: 55, but is not limited thereto and affects antibody activity. Polynucleotides encoding any amino acid sequence not specified may be used.
CD19/CD22-결합 도메인의 N 말단에 신호 펩타이드(signal peptide)가 포함된 경우, 서열번호 30의 염기서열로 표시되는 신호 펩타이드가 추가로 포함될 수 있다. 또한, CD22-결합 도메인를 코딩하는 폴리뉴클레오타이드 및 막관통 도메인 사이에, 힌지 부위(hinge region)를 코딩하는 폴리뉴클레오타이드가 추가로 포함될 수 있으며, 바람직하게 서열번호 31의 염기서열로 표시되는 CD8 힌지 부위일 수 있다.When a signal peptide is included at the N-terminus of the CD19/CD22-binding domain, a signal peptide represented by the nucleotide sequence of SEQ ID NO: 30 may be additionally included. 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 a CD8 hinge region represented by the nucleotide sequence of SEQ ID NO: 31 can
본 발명은 또 다른 관점에서, 상기 이중특이적 키메라 항원 수용체를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터에 관한 것이다. In another aspect, the present invention relates to a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor.
본 발명에서, 상기 벡터는 재조합 바이러스 벡터로, 바람직하게는 렌티바이러스 벡터이며, 작동 가능하게 연결된 EF1α 프로모터; 시그널 펩타이드를 코딩하는 폴리뉴클레오타이드; CD19-결합 도메인 및 CD22-결합 도메인을 코딩하는 폴리뉴클레오타이드; 막관통 도메인을 코딩하는 폴리뉴클레오타이드; 세포 내 신호전달 도메인을 코딩하는 폴리뉴클레오타이드를 포함하며, 단백질 발현을 증가시키기 위해 WPRE(woodchuck hepatitis virus post-transcriptional regulatory element)를 추가로 포함할 수 있다 (도 9). In the present invention, the vector is a recombinant viral vector, preferably a lentiviral vector, comprising an operably linked EF1α promoter; polynucleotides encoding signal peptides; polynucleotides encoding a CD19-binding domain and a CD22-binding domain; polynucleotides encoding transmembrane domains; 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. 9).
상기 EF1α 프로모터는 서열번호 29의 염기서열로 표시될 수 있으며, 필요에 따라 상기 서열번호 27의 염기서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함할 수 있다.The EF1α promoter may be represented by the nucleotide sequence of SEQ ID NO: 29, 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: 27 , or sequences that are at least 99% identical.
또한, 상기 프로모터는 CD19xCD22-결합 도메인인 항-CD19/CD22 항체(CD19VL-CD22VH-CD22VL-CD19VH)의 발현을 유도하도록 작동 가능하게 연결되어 있으며, 벡터에 대한 구체적인 내용은 상술한 바와 동일하다.In addition, the promoter is operably linked to induce expression of an anti-CD19/CD22 antibody (CD19VL-CD22VH-CD22VL-CD19VH), which is a CD19xCD22-binding domain, and details of the vector are the same as described above.
본 발명의 구체적인 일실시예에서는, 도 9 및 도 10에 나타난 바와 같이 CD19xCD22-CAR를 코팅하는 폴리뉴클레오타이드가 삽입된 렌티바이러스 벡터를 제조하였으며, 제조된 벡터를 293FT 세포에 형질전환 시켜 CD19xCD22-CAR 발현 세포를 제조하였다. 또한 도 11에 나타난 바와 같이, 제조된 CD19xCD22-CAR 발현 세포에서 CD19 및 CD22를 표적으로 하는 이중특이적 키메라 항원 수용체가 발현하는 것을 확인하였다.In a specific embodiment of the present invention, as shown in FIGS. 9 and 10, a lentiviral vector into which a polynucleotide coating CD19xCD22-CAR was inserted was prepared, and the prepared vector was transformed into 293FT cells to express CD19xCD22-CAR cells were prepared. In addition, as shown in FIG. 11 , it was confirmed that the bispecific chimeric antigen receptor targeting CD19 and CD22 was expressed in the prepared CD19xCD22-CAR expressing cells.
CD19 및 CD22를 표적으로 하는 이중특이적 키메릭 항원 수용체 발현 면역 이펙터 세포Immune effector cells expressing bispecific chimeric antigen receptors targeting CD19 and CD22
본 발명은 또 다른 관점에서, 상기 이중특이적 키메릭 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드 또는 이중특이적 키메릭 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터를 포함하고, 상기 이중특이적 키메릭 항원 수용체(CAR)를 발현하는 면역 이펙터 세포에 관한 것이다.In another aspect, the present invention includes a polynucleotide encoding the bispecific chimeric antigen receptor (CAR) or a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor (CAR), It relates to immune effector cells that express specific chimeric antigen receptors (CARs).
본 발명에 있어서, 상기 면역 이펙터 세포는 포유동물 유래 단리된 세포 일 수 있으며, 바람직하게는 T 세포, B 세포, 자연 살해(NK) 세포, 수지상 세포, 골수 세포, 단핵세포, 또는 대식세포, 더 바람직하게는 T 세포일 수 있다. 또한, 키메릭 항원 수용체 발현 면역 이펙터 세포에 대한 구체적인 내용은 상술한 바와 동일하다.In the present invention, the immune effector cells may be mammalian-derived isolated cells, preferably T cells, B cells, natural killer (NK) cells, dendritic cells, bone marrow cells, monocytes, or macrophages, and more Preferably it may be a T cell. Further, details of the immune effector cells expressing the chimeric antigen receptor are the same as described above.
본 발명의 구체적인 일구현예에서, 도 12 및 도 13과 같이, 말초 혈액 단핵세포(peripheral blood mononuclear cell, PBMC)로 부터 활성화된 T 세포를 분리한 다음, CD19xCD22-CAR 렌티바이러스 벡터를 T 세포에 형질도입시켜 CD19xCD22-CAR-T 세포를 제조하였으며, 구체적으로 4F5 항체(mouse), 인간화된 4F5(V4) 항체 및 4F5(V11) 항체를 이용하여 CD19xCD22-CAR-T 세포를 각각 제조하였다.In a specific embodiment of the present invention, as shown in FIGS. 12 and 13, after isolating activated T cells from peripheral blood mononuclear cells (PBMC), the CD19xCD22-CAR lentiviral vector is applied to the T cells. CD19xCD22-CAR-T cells were prepared by transduction, and specifically, CD19xCD22-CAR-T cells were prepared using a 4F5 antibody (mouse), a humanized 4F5 (V4) antibody, and a 4F5 (V11) antibody, respectively.
제조된 CD19xCD22-CAR-T 세포의 활성을 확인하기 위해, CD3이 활성화된 CD19xCD22-CAR-T 세포에 대한 CD22 펩타이드 및 CD19 펩타이드에 대한 결합능을 확인하였다. 도 14에 나타난 바와 같이, 본 발명에서 제조한 CD19xCD22-CAR-T 세포가 CD22 펩타이드 및 CD19 펩타이드와 동시에 결합하는 것을 확인하였다.In order to confirm the activity of the prepared CD19xCD22-CAR-T cells, the binding ability of the CD3-activated CD19xCD22-CAR-T cells to the CD22 peptide and the CD19 peptide was confirmed. As shown in FIG. 14, it was confirmed that the CD19xCD22-CAR-T cells prepared in the present invention simultaneously bind to the CD22 peptide and the CD19 peptide.
본 발명의 구체적인 또 다른 일구현예에서, CD19xCD22-CAR-T 세포에 의한 표적 세포의 사멸효과를 확인한 결과, 도 15에 나타난 바와 같이, CD19xCD22-CAR-T 세포는 K562-CD19 세포(CD19 발현 세포), K562-CD22 세포(CD22 발현 세포) 및 K562-CD19/CD22 세포(CD19과 CD22 발현 세포)에 대한 사멸 효과를 보이는 것을 확인하였다. In another specific embodiment of the present invention, as a result of confirming the killing effect of target cells by CD19xCD22-CAR-T cells, as shown in FIG. 15, CD19xCD22-CAR-T cells are K562-CD19 cells (CD19 expressing cells). ), K562-CD22 cells (CD22 expressing cells) and K562-CD19/CD22 cells (CD19 and CD22 expressing cells).
B 세포에 의해 매개되는 질환, 또는 CD19 또는 CD22 발현과 관련된 질환 예방 또는 치료용 조성물Composition for preventing or treating diseases mediated by B cells or diseases associated with CD19 or CD22 expression
본 발명은 또 다른 관점에서, CD22에 특이적으로 결합하는 항체; CD22에 특이적으로 결합하는 인간화 항체; CD22를 표적하는 키메라 항원 수용체를 발현하는 면역 이펙터 세포; 또는 CD19xCD22에 특이적으로 결합하는 이중특이적 키메라 항원 수용체를 발현하는 면역 이펙터 세포를 포함하는, B 세포에 의해 매개되는 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다. In another aspect, the present invention provides an antibody that specifically binds to CD22; humanized antibodies that specifically bind to CD22; immune effector cells expressing chimeric antigen receptors that target CD22; or a pharmaceutical composition for preventing or treating a disease mediated by B cells, comprising immune effector cells expressing a bispecific chimeric antigen receptor that specifically binds to CD19xCD22.
본 발명에 있어서, 상기 B 세포는 바람직하게, CD19 또는 CD22를 발현하는 세포일 수 있으며, 상기 질환은 종양/암, 림프종, 비호치킨 림프종 (non-Hogkins lymphoma: NHL), 공격적 NHL, 재발성 공격적 NHL, 재발성 지연성 NHL, 불응성 NHL, 불응성 지연성 NHL, 만성 림프성 백혈병(chronic lymphocytic leukemia: CLL), 소형 림프성 림프종, 백혈병, 모발성 세포 백혈병(hairy cell leukemia: HCL), 급성 림프성 백혈병(acute lymphocytic leukemia: ALL), 버킷트 림프종 및 외투 세포 림프종로 구성된 군에서 선택될 수 있다. In the present invention, the B cell may preferably be a cell expressing CD19 or CD22, and the disease is tumor/cancer, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent delayed NHL, refractory NHL, refractory delayed NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute It may be selected from the group consisting of acute lymphocytic leukemia (ALL), Burkitt's lymphoma and mantle cell lymphoma.
본 발명에 있어서, 상기 조성물은 B 세포에 의해 매개되는 질환의 치료제를 포함할 수 있으며, 상기 치료제는 CD19 또는 CD22에 특이적으로 결합하는 항체에 공유결합된 상태로 존재하거나, 본 발명의 CD22-CAR 면역 이펙터 세포 또는 CD19xCD22-CAR 면역 이펙터 세포와 병용투여할 수 있다.In the present invention, the composition may include a therapeutic agent for a disease mediated by B cells, and the therapeutic agent is present in a state covalently bound to an antibody that specifically binds to CD19 or CD22, or the CD22- It can be administered in combination with CAR immune effector cells or CD19xCD22-CAR immune effector cells.
상기 치료제는 저분자 약물, 펩타이드성 약물, 독소(예를 들어, 세포독소) 등을 포함한다.The therapeutic agent includes a small molecule drug, a peptide drug, a toxin (eg, cytotoxin), and the like.
상기 저분자 약물는 관심대상의 약제학적 활성을 나타내고, 일반적으로 분자량이 약 800 Da 이하또는 2000 Da 이하의 화합물일 수 있다. 무기 저분자는 탄소원자를 하나도 포함하지 않은 분자를 가리키고, 반면 유기저분자는 최소한 하나의 탄소원자를 함유하는 화합물을 가리킨다.The small molecule drug may be a compound that exhibits a pharmaceutical activity of interest and generally has a molecular weight of about 800 Da or less or 2000 Da or less. Inorganic small molecules refer to molecules containing no carbon atoms, whereas organic small molecules refer to compounds containing at least one carbon atom.
상기 펩타이드성 약물은 중합체성 화합물을 함유하는 아미노산을 가리키고, 이것은 자연발생 및 비-자연발생 펩타이드, 올리고펩타이드, 환형 펩타이드, 폴리펩타이드 및 단백질, 뿐만 아니라 펩타이드 모방물을 포함한다. 상기 펩타이드 약물은 화학적 합성에 의해 획득되거나 또는 유전적으로 인코딩된 공급원(예를 들어, 재조합공급원)에서 생성될 수 있다. 펩타이드 약물의 분자량의 범위는 200 Da 내지 10 kDa 또는그 이상일 수 있다.The peptidic drug refers to an amino acid containing polymeric compound, which includes naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides and proteins, as well as peptide mimetics. The peptide drug may be obtained by chemical synthesis or generated 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.
상기 독소는 바람직하게 세포독소이며, 세포독소에는 비제한적으로, 리신, 아브린, 디프테리아 독소, 슈도모나스 균체외독소(예를 들어, PE35, PE37, PE38, PE40 등), 사포린, 겔로닌, 미국자리공 항바이러스 단백질(PAP), 보툴리눔 독소, 브리오딘, 모모르딘 및 부가닌이 포함된다.The toxin is preferably a cytotoxin, including, but not limited to, ricin, abrin, diphtheria toxin, Pseudomonas exotoxin (e.g., PE35, PE37, PE38, PE40, etc.), saporin, gelonin, orca. These include antiviral protein (PAP), botulinum toxin, briodin, momordin and bunin.
또한, 상기 치료제는 항암제일 수 있다. 항암제는 암 세포의 증식을 감소시키고, 세포독성 약제 및 세포 증식 억제제를 아우르는 비-펩타이드성(즉, 비-단백질계) 화합물을 포함한다. 항암제의 비제한적인 예는 알킬화제, 니트로소요소, 항대사물질, 항종양 항생물질, 식물(빈카) 알칼로이드 및 스테로이드 호르몬을 포함한다. 펩타이드성 화합물 또한 사용될 수 있다.In addition, the therapeutic agent may be an anticancer agent. Anti-cancer agents reduce the proliferation of cancer cells and include non-peptidic (i.e., non-proteinaceous) compounds, including cytotoxic agents and cytostatic agents. Non-limiting examples of anticancer agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids and steroid hormones. Peptidic compounds may also be used.
상기 약학적 조성물에서 CD22에 특이적으로 결합하는 인간화 항체, CD22-CAR 면역 이펙터 세포 또는 CD19xCD22-CAR 면역 이펙터 세포는 치료 또는 진단용 조성물 내에서 유일한 활성성분이거나, 또는 예를 들면, 항-T 세포, 항-IFNγ 또는 항-LPS 항체와 같은 다른 항체성분들, 또는 크산틴과 같은 비항체 성분들을 포함하는 다른 활성성분들과 함께 사용 가능하다.In the pharmaceutical composition, the humanized antibody, CD22-CAR immune effector cells or CD19xCD22-CAR immune effector cells that specifically bind to CD22 are the only active ingredients in the therapeutic or diagnostic composition, or, for example, anti-T cells, It can be used with other active ingredients including other antibody components such as anti-IFNγ or anti-LPS antibodies, or non-antibody components such as xanthines.
약제 조성물은 치료적 유효량의 본 발명의 항체를 포함하는 것이 바람직하다. 여기에서 사용된 용어 "치료적 유효량"은 목표 질환 또는 상태를 치료, 개선 또는 예방하는 데 필요한 치료제의 양을 의미하고, 또는 감지할 수 있는 정도의 치료 또는 예방효과를 나타내는 데 필요한 치료제의 양을 뜻한다. 어떤 항체에 대하여, 치료적 유효 투여량은 세포배양 분석법 또는 보통 설치류, 토끼, 개, 돼지 또는 영장류와 같은 동물 모델로 최초로 결정될 수 있다. 동물 모델은 또한 적절한 농도범위와 투여루트를 결정하는 데 사용될 수 있다. 이러한 정보는 인간의 투약을 위해 유용한 투여량 및 루트를 결정하는 데 사용될 수 있다.The pharmaceutical composition preferably comprises a therapeutically effective amount of an antibody of the present invention. As used herein, the term "therapeutically effective amount" means the amount of a therapeutic agent required to treat, ameliorate, or prevent the target disease or condition, or to produce an appreciable therapeutic or prophylactic effect. means For any antibody, the therapeutically effective dose can be determined initially 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. This information can be used to determine useful dosages and routes for human administration.
인간환자를 위한 정밀한 유효량은 질환상태의 심각도, 환자의 일반적 건강 상태, 환자의 나이, 체중 및 성별, 식이요법, 투여시간, 투여빈도, 약제조성, 반응감도 및 치료에 대한 내성/반응에 따라 달라질 수 있다. 상기 양은 통상적인 실험에 의해 결정될 수 있고, 임상의사의 판단의 범위 내에 있다. 일반적으로, 유효 투여량은 0.01~50mg/kg, 바람직하게는 0.1~20mg/kg, 더욱 바람직하게는 약 15mg/kg이다.The precise effective amount for a human patient will depend on the severity of the disease state, the patient's general state of health, the patient's age, weight and sex, diet, administration time, frequency of administration, pharmaceutical composition, response sensitivity and tolerance/response to treatment. can Such amounts can be determined by routine experimentation and are within the judgment of the clinician. Generally, an effective dosage is 0.01 to 50 mg/kg, preferably 0.1 to 20 mg/kg, more preferably about 15 mg/kg.
조성물은 환자에게 개별적으로 투여되거나, 또는 다른 제제, 약제 또는 호르몬과 조합하여 투여될 수 있다.Compositions may be administered to a patient individually or in combination with other agents, drugs or hormones.
본 발명의 항체가 투여되는 투여량은 치료될 상태의 성질, 악성 림프종 또는 백혈병의 등급, 및 항체가 질환 예방 차원에서 사용되는지 또는 현존하는 상태를 치료하기 위해 사용되는지에 따라 달라진다.The dosage at which an antibody of the invention is administered depends on the nature of the condition being treated, the grade of the malignant lymphoma or leukemia, and whether the antibody is being used to prevent disease or to treat an existing condition.
투여빈도는 항체분자의 반감기, 약 효과의 지속성에 따라 달라진다. 만약 항체분자가 짧은 반감기(예, 2~10시간)를 가지면, 하루당 1회 또는 그 이상의 투여량을 제공할 필요가 있다. 또는, 항체분자가 긴 반감기(예, 2~15일)를 가지면, 하루에 한번, 일주일에 한차례, 또는 매 1개월 또는 2개월당 한차례의 투여량을 제공할 필요가 있다.The frequency of administration depends on the half-life of the antibody molecule and the persistence of the drug effect. If the antibody molecule has a short half-life (eg, 2-10 hours), it may be necessary to give 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 give a dose once daily, once a week, or once every 1 or 2 months.
또한, 약제 조성물은 항체의 투여를 위하여 약제학적으로 허용가능한 담체를 함유할 수 있다. 담체는 그 자신이 조성물을 투여받는 개체에 유해한 항체의 생성을 유발해서는 안되고, 독성이 없어야만 한다. 적당한 담체로는 단백질, 폴리펩타이드, 리포오좀, 다당류, 폴리락틱산, 폴리글리콜산, 아미노산 중합체, 아미노산 공중합체 및 비활성 바이러스 입자들과 같은, 서서히 물질대사되는 거대분자일 수 있다.In addition, the pharmaceutical composition may contain a pharmaceutically acceptable carrier for administration of the antibody. The carrier must not itself induce the production of antibodies harmful to the subject to which the composition is administered, and must be non-toxic. Suitable carriers can be slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers and inactive viral particles.
약제학적으로 허용가능한 염들은, 예를 들면, 염화수소산염, 브롬화수소산염, 인산염 및 황산염과 같은 미네랄산염들, 또는 아세트산, 프로피온산. 말론산 및 벤조산 같은 유기산의 염들이 사용될 수 있다.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.
치료 조성물내의 약제학적으로 허용가능한 담체는 부가적으로, 물, 식염수, 글리세롤 및 에탄올과 같은 액체들을 포함할 수 있다. 부가적으로, 습윤제, 유화제 또는 pH 완충물질과 같은 보조 물질들이 이러한 조성물 내에 존재할 수 있다. 상기 담체는 환자에 의한 약제 조성물 섭취를 위해, 정제, 환약, 당의정, 캡슐, 액체, 겔, 시럽, 슬러리 및 현탁제로서 제제화될 수 있다.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 substances may be present in these 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.
투여를 위한 바람직한 형태는, 예로써 주사(injection) 또는 주입(infusion)(예를 들면, 환괴(bolus) 주사 또는 연속적 주입)에 의한 비경구적 투약에 적합한 형태를 포함한다. 생성물이 주입 또는 주사용일 경우에는, 오일 또는 수용성 부형제내의 현탁제, 용액 또는 에멀젼의 형태를 취할 수 있고, 이는 현탁제, 방부제, 안정화제 및/또는 분산제와 같은 처방제들을 포함할 수 있다. 또는, 항체분자는 무수형태일 수 있고, 사용전에 적절한 멸균액으로 재구성될 수 있다.Preferred forms for administration include forms suitable for parenteral administration, eg by injection or infusion (eg bolus injection or continuous infusion). When the product is for infusion or injection, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, which may contain such prescriptive agents as suspending agents, preservatives, stabilizing and/or dispersing agents. Alternatively, the antibody molecule may be in anhydrous form and reconstituted with an appropriate sterile solution prior to use.
일단 제제화된 경우, 본 발명의 조성물은 환자에게 직접 투여될 수 있다. 치료받을 환자들은 동물일 수 있다. 그러나, 조성물은 인간 환자 투여를 위해 맞추는 것이 바람직하다.Once formulated, the compositions of the present invention can be administered directly to a patient. The patients to be treated may be animals. However, it is preferred that the compositions are tailored for administration to human patients.
본 발명의 약제 조성물은 제한은 없지만, 경구, 정맥, 근육내, 동맥내, 골수내, 척추강내, 심실내, 경피(transdermal), 경피(transcutaneous)(예, WO 98/20734 참조), 피하, 복강내, 비강내, 장내, 국소, 혀밑, 질내 또는 직장 경로를 포함하는 어떤 경로에 의해 투여될 수 있다. 본 발명의 약제 조성물을 투여하는 데 하이포스프레이 (hypospray)가 사용될 수 있다. 전형적으로, 치료 조성물은 액체 용액 또는 현탁액으로서 주사가능한 물질로서 제조될 수 있다. 또한, 주입전에 액체 부형제내용액 또는 현탁액에 적합한 고체 형태가 제조될 수 있다.The pharmaceutical composition of the present invention may be used, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (see eg WO 98/20734), subcutaneous, It may be administered by any route including intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes. A hypospray can be used to administer the pharmaceutical composition of the present invention. Typically, therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions. In addition, solid forms suitable for solution or suspension in liquid excipients may be prepared prior to injection.
조성물의 직접적인 전달은 일반적으로 주사, 피하주사, 복강내주사, 정맥내주사, 근육내주사에 의해 이루어질 수 있거나, 또는 조직의 간질(interstitial) 공간으로 전달될 수도 있다. 또한, 조성물은 상처부위로 투여될 수 있다. 투여량 처리는 단일 복용 스케쥴 또는 다중 복용 스케쥴일 수 있다.Direct delivery of the composition may generally be by injection, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, or may be delivered into the interstitial space of a tissue. In addition, the composition may be administered to a wound site. Dosage treatment can be a single dose schedule or a multiple dose schedule.
조성물내의 활성성분은 항체분자일 수 있다. 그 자체로, 위장관내에서 분해에 민감할 수 있다. 따라서, 조성물이 위장관을 사용하는 경로에 의해 투여되면, 조성물은, 분해로부터 항체를 보호하지만 일단 위장관으로부터 흡수된 항체를 방출시키는 제제를 함유할 필요가 있을 것이다.The active ingredient in the composition may be an antibody molecule. As such, it can be susceptible to degradation within 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.
약제학적으로 허용가능한 담체의 완벽한 논의는 레밍톤 약제학지(Remington's Pharmaceutical Sciences)(Mack Publishing Company, NJ, 1991)를 이용할 수 있다.A complete discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ, 1991).
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.
실시예 1 : CD22에 특이적으로 결합하는 항체 제조 및 선별Example 1: Production and screening of antibodies that specifically bind to CD22
CD22 펩타이드 특이적인 항체를 선별하기위해, CD22와 결합하는 항체를 생산하는 하이브리도마를 제조하여 항체를 선별하였다. In order to select a CD22 peptide-specific antibody, a hybridoma producing an antibody that binds to CD22 was prepared and the antibody was selected.
먼저, CD22 단백질 (ACRObiosystems Inc., cat#CD2-H52H8, 미국)을 면역하여 비장세포를 적출하고 마우스 골수종세포와 세포 융합을 통하여 하이브리도마 세포를 제작하였다. First, splenocytes were isolated by immunization with CD22 protein (ACRObiosystems Inc., cat#CD2-H52H8, USA), and hybridoma cells were prepared through cell fusion with mouse myeloma cells.
세포 융합에 이용하는 마우스 골수종 세포는 HGPRT(HypoxanthineGuanidine-Phosphoribosyl-Transferase)를 가지고 있지 않기 때문에 HAT 배지에서는 생존할 수 없으나, 하이브리도마는 비장세포와 융합함으로써 HAT 배지에서 생존할 수 있다. 이를 이용하면 하이브리도마만을 증식시킬 수 있으므로, 통상 하이브리도마를 확립시킬때까지 HAT 배지에서 증식시켰다.Mouse myeloma cells used for cell fusion cannot survive in HAT medium because they do not have HypoxanthineGuanidine-Phosphoribosyl-Transferase (HGPRT), but hybridomas can survive in HAT medium by fusing with splenocytes. Since only hybridomas can be grown using this, they were usually grown in HAT medium until hybridomas were established.
증식된 하이브리도마 중에서 CD22와 결합하는 항체를 생산하는 하이브리도마를 선별하기 위해 한계희석법을 사용하였다. 우선 96웰당 1개 세포 이하가 되도록 한 다음, 1개의 세포로부터 증식된 클론에서 얻어진 항체가 CD22와 결합하는지를 ELISA로 확인하고 CD22와 결합하는 클론을 선별하였다. 상기과정을 3회 반복하여 CD22와 결합하는 항체를 생산하는 하이브리도마를 선별하였다. 이와 같은 방법으로 CD22에 결합하는 항체를 수득하였다.A limiting dilution method was used to select hybridomas producing antibodies that bind to CD22 among the proliferated hybridomas. First, the number of cells per 96 well was reduced to 1 or less, and then, whether the antibodies obtained from the clones proliferated from 1 cell were binding to CD22 was confirmed by ELISA, and clones binding to CD22 were selected. By repeating the above process three times, hybridomas producing antibodies binding to CD22 were selected. In this way, an antibody binding to CD22 was obtained.
상기 항체는 4F5로 명명하였으며, 이들의 염기서열과 아미노산 서열을 분석하였다. 서열분석 결과에 따른 각 항체의 중쇄 가변부위 및 경쇄 가변부위에 대한 서열정보는 하기 표 1에 나타내었으며, 표 1에서 밑줄 친 부분은 상보적 결정 부위(complementarity determining region; CDR)를 의미한다.The antibody was named 4F5, and its base sequence and amino acid sequence were analyzed. Sequence information for 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 the underlined portion in Table 1 means the complementarity determining region (CDR).
4F54F5 | 서열정보sequence information | 서열번호sequence number |
중쇄가변부위 CDR1heavy chain variable region CDR1 | GFSLTIYGGFSLTIYG | 서열번호 1SEQ ID NO: 1 |
중쇄가변부위 CDR2heavy chain variable region CDR2 | MWSGGSTMWSGGST | 서열번호 2SEQ ID NO: 2 |
중쇄가변부위 CDR3heavy chain variable region CDR3 | ARNDGYYWFAYARNDGYYWFAY | 서열번호 3SEQ ID NO: 3 |
경쇄가변부위 CDR1light chain variable region CDR1 | QSLVHNNGNTYQSLVHNNGNTY | 서열번호 4SEQ ID NO: 4 |
경쇄가변부위 CDR2light chain variable region CDR2 | KVSKVS | 서열번호 5SEQ ID NO: 5 |
경쇄가변부위 CDR3light chain variable region CDR3 | SQSTHVPYSSQSTHVPYS | 서열번호 6SEQ ID NO: 6 |
중쇄가변부위 아미노산서열heavy chain variable region amino acid sequence |
QVQLKESGPGLVQPSQSLSITCTVS GFSLTIYG VHWIRQSPGKGLEWLGV MWSGGST DYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYC ARNDGYYWFAY WGQGTLVTVSAQVQLKESGPGLVQPSQSLSITCTVS GFSLTIYG VHWIRQSPGKGLEWLGV MWSGGST DYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYC ARNDGYYWFAY WGQGTLVTVSA | 서열번호 7SEQ ID NO: 7 |
경쇄가변부위 아미노산서열light chain variable region amino acid sequence |
DVLMTQTPLSLPVSLGDQASISCRSS QSLVHNNGNTY LHWYLQKPGQSPKLLIY KVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPYS FGGGTKLEIKDVLMTQTPLSLPVSLGDQASISCRSS QSLVHNNGNTY LHWYLQKPGQSPKLLIY KVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPYS FGGGTKLEIK | 서열번호 8SEQ ID NO: 8 |
중쇄가변부위 염기서열heavy chain variable region base sequence |
CAGGTGCAGCTGAAGGAGAGCGGCCCCGGCCTGGTGCAGCCCAGCCAGAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAGCCTGACCATCTACGGCGTGCACTGGATCAGGCAGAGCCCCGGCAAGGGCCTGGAGTGGCTGGGCGTGATGTGGAGCGGCGGCAGCACCGACTACAACGCCGCCTTCATCAGCAGGCTGAGCATCAGCAAGGACAACAGCAAGAGCCAGGTGTTCTTCAAGATGAACAGCCTGCAGGCCAACGACACCGCCATCTACTACTGCGCCAGGAACGACGGCTACTACTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC CAGGTGCAGCTGAAGGAGAGCGGCCCCGGCCTGGTGCAGCCCAGCCAGAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAGCCTGACCATCTACGGCGTGCACTGGATCAGGCAGAGCCCCGGCAAGGGCCTGGAGTGGCTGGGCGTGATGTGGAGCGGCGGCAGCACCGACTACAACGCCGCCTTCATCAGCAGGCTGAGCATCAGCAAGGACAACAGCAAGAGCCAGGTTCTT CAAGATGAACAGCCTGCAGGCCAACGACACCGCCATCTACTACTGCGCCAGGAACGACGGCTACTACTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC | 서열번호 9SEQ ID NO: 9 |
경쇄가변부위 염기서열light chain variable region base sequence |
GACGTGCTGATGACCCAGACCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGGTGCACAACAACGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTGAGCAACAGGTTCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTTCTGCAGCCAGAGCACCCACGTGCCCTACAGCTTCGGCGGCGGCACCAAGCTGGAGATCAAG GACGTGCTGATGACCCAGACCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGGTGCACAACAACGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTGAGCAACAGGTTCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGAT CAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTTCTGCAGCCAGAGCACCCACGTGCCCTACAGCTTCGGCGGCGGCACCAAGCTGGAGATCAAG | 서열번호 10SEQ ID NO: 10 |
실시예 2 : 4F5 항체 기반 인간화 항체 제조Example 2: Preparation of humanized antibody based on 4F5 antibody
상기 실시예 1에서 선별한 4F5 항체를 인간에 대응하는 구조로 변경한 인간화된 항체(humanized antibody)를 제조하였다.A humanized antibody was prepared by changing the 4F5 antibody selected in Example 1 to a structure corresponding to human.
구체적으로, 인간 항체의 생식계열 염기서열(germline sequence)를 프레임(frame)으로하여 CD22와 결합하는 마우스 항체의 CDR를 인간 항체의 CDR를 교체하는 CDR-그라프팅(CDR grafting) 방법으로 마우스 4F5 항체를 인간화한 항체를 제작하였다. 인간화한 항체는 4F5(V4) 항체 및 4F5(V11) 항체로 명명하였으며, 아미노산 서열을 분석하였다. 서열분석 결과에 따른 항체의 중쇄 가변부위 및 경쇄 가변부위에 대한 서열정보는 하기 표 2 및 표 3에 나타내었으며, 표 2 및 표 3에서 밑줄 친 부분은 상보적 결정 부위(complementarity determining region; CDR)를 의미한다.Specifically, mouse 4F5 antibody is obtained by a CDR-grafting method in which the CDR of a mouse antibody that binds to CD22 is replaced with the CDR of a human antibody using the germline sequence of a human antibody as a frame. A humanized antibody was prepared. The humanized antibodies were named 4F5(V4) antibody and 4F5(V11) antibody, and the amino acid sequences were analyzed. Sequence information for the heavy chain variable region and the light chain variable region of the antibody according to the sequencing results are shown in Tables 2 and 3 below, and the underlined portions in Tables 2 and 3 are complementary determining regions (complementarity determining regions; CDRs). means
4F5(V4)4F5 (V4) | 서열정보sequence information | 서열번호sequence number |
중쇄가변부위 CDR1heavy chain variable region CDR1 | GFSLTIYGGFSLTIYG | 서열번호 1SEQ ID NO: 1 |
중쇄가변부위 CDR2heavy chain variable region CDR2 | MWSGGSTMWSGGST | 서열번호 2SEQ ID NO: 2 |
중쇄가변부위 CDR3heavy chain variable region CDR3 | ARNDGYYWFAYARNDGYYWFAY | 서열번호 3SEQ ID NO: 3 |
경쇄가변부위 CDR1light chain variable region CDR1 | QSLVHNNGNTYQSLVHNNGNTY | 서열번호 4SEQ ID NO: 4 |
경쇄가변부위 CDR2light chain variable region CDR2 | KVSKVS | 서열번호 5SEQ ID NO: 5 |
경쇄가변부위 CDR3light chain variable region CDR3 | SQSTHVPYSSQSTHVPYS | 서열번호 6SEQ ID NO: 6 |
중쇄가변부위 아미노산서열heavy chain variable region amino acid sequence |
QVQLQESGPGLVKPSQTLSLTCTVS GFSLTIYG VHWIRQPPGKGLEWLGV MWSGGST DYNAALKSRVTISKDNSKSQVSLKLSSVTAADTAVYYC ARNDGYYWFAY WGQGTLVTVSS QVQLQESGPGLVKPSQTLSLTCTVS GFSLTIYG VHWIRQPPGKGLEWLGV MWSGGST DYNAALKSRVTISKDNSKSQVSLKLSSVTAADTAVYYC ARNDGYYWFAY WGQGTLVTVSS | 서열번호 11SEQ ID NO: 11 |
경쇄가변부위 아미노산서열light chain variable region amino acid sequence |
DVVMTQSPDSLAVSLGERATINCKSS QSLVHNNGNTY LHWYQQKPGQPPKLLIY KVS NRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC SQSTHVPYS FGGGTKLEIKDVVMTQSPDSLAVSLGERATINCKSS QSLVHNNGNTY LHWYQQKPGQPPKLLIY KVS NRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC SQSTHVPYS FGGGTKLEIK | 서열번호 12SEQ ID NO: 12 |
중쇄가변부위 염기서열heavy chain variable region base sequence |
CAGGTGCAGCTTCAGGAGAGCGGACCCGGTCTCGTGAAACCCAGCCAGACTTTGTCCCTGACCTGCACTGTCAGCGGCTTTTCCCTAACCATTTACGGCGTCCATTGGATCCGCCAGCCACCTGGCAAGGGGCTGGAATGGCTGGGCGTGATGTGGTCTGGGGGATCCACCGACTATAACGCGGCTCTGAAGTCCCGGGTGACCATCTCCAAGGACAACAGCAAGAGTCAAGTCAGCCTTAAACTGAGCTCCGTTACAGCCGCGGACACCGCTGTCTACTACTGTGCGCGCAATGACGGCTATTACTGGTTCGCCTACTGGGGCCAGGGCACTCTGGTGACCGTGTCCTCGCAGGTGCAGCTTCAGGAGAGCGGACCCGGTCTCGTGAAACCCAGCCAGACTTTGTCCCTGACCTGCACTGTCAGCGGCTTTTCCCTAACCATTTACGGCGTCCATTGGATCCGCCAGCCACCTGGCAAGGGGCTGGAATGGCTGGGCGTGATGTGGTCTGGGGGATCCACCGACTATAACGCGGCTCTGAAGTCCCGGGTGACCATCTCCAAGGACAACAGCAAGAGTCAAGTCAGCCTTAA ACTGAGCTCCGTTACAGCCGCGGACACCGCTGTCTACTACTGTGCGCGCAATGACGGCTATTACTGGTTCGCCTACTGGGGCCAGGGCACTCTGGTGACCTGTGTCCTCG | 서열번호 13SEQ ID NO: 13 |
경쇄가변부위 염기서열light chain variable region base sequence |
GATGTGGTGATGACCCAGAGTCCCGATTCTCTGGCAGTTTCTTTAGGCGAGCGTGCCACCATTAACTGCAAAAGCTCCCAGTCTCTGGTGCACAACAACGGCAATACCTACCTGCACTGGTACCAGCAGAAGCCGGGGCAGCCACCTAAGCTGCTGATCTACAAGGTGTCCAACCGCTTCTCTGGTGTCCCCGACAGGTTTTCTGGCTCAGGCAGCGGCACAGACTTCACCCTCACGATCTCCTCCCTCCAGGCCGAGGACGTGGCCGTGTACTACTGTTCACAGTCGACCCACGTACCGTATTCCTTCGGAGGAGGCACGAAGTTGGAGATCAAGGATGTGGTGATGACCCAGAGTCCCGATTCTCTGGCAGTTTCTTTAGGCGAGCGTGCCACCATTAACTGCAAAAGCTCCCAGTCTCTGGTGCACAACAACGGCAATACCTACCTGCACTGGTACCAGCAGAAGCCGGGGCAGCCACCTAAGCTGCTGATCTACAAGGTGTCCAACCGCTTCTCTGGTGTCCCCGACAGGTTTTCTGGCTCAGGCAGCGGCACAGACTTCACCCTCACGATCTC CTCCCTCCAGGCCGAGGACGTGGCCGTGTACTACTGTTCACAGTCGACCCACGTACCGTATTCCTTCGGAGGAGGCACGAAGTTGGAGATCAAG | 서열번호 14SEQ ID NO: 14 |
4F5(V11)4F5 (V11) | 서열정보sequence information | 서열번호sequence number |
중쇄가변부위 CDR1heavy chain variable region CDR1 | GFSLTIYGGFSLTIYG | 서열번호 1SEQ ID NO: 1 |
중쇄가변부위 CDR2heavy chain variable region CDR2 | MWSGGSTMWSGGST | 서열번호 2SEQ ID NO: 2 |
중쇄가변부위 CDR3heavy chain variable region CDR3 | ARNDGYYWFAYARNDGYYWFAY | 서열번호 3SEQ ID NO: 3 |
경쇄가변부위 CDR1light chain variable region CDR1 | QSLVHNNGNTYQSLVHNNGNTY | 서열번호 4SEQ ID NO: 4 |
경쇄가변부위 CDR2light chain variable region CDR2 | KVSKVS | 서열번호 5SEQ ID NO: 5 |
경쇄가변부위 CDR3light chain variable region CDR3 | SQSTHVPYSSQSTHVPYS | 서열번호 6SEQ ID NO: 6 |
중쇄가변부위 아미노산서열heavy chain variable region amino acid sequence |
QVQLKESGPVLVKPTETLTLTCTVS GFSLTIYG VHWIRQPPGKALEWLGV MWSGGST DYNAALKSRLTISKDNSKSQVVLTMTNMDPVDTATYYC ARNDGYYWFAY WGQGTLVTVSSQVQLKESGPVLVKPTETLTLTCTVS GFSLTIYG VHWIRQPPGKALEWLGV MWSGGST DYNAALKSRLTISKDNSKSQVVVLTMTNMDPVDTATYYC ARNDGYYWFAY WGQGTLVTVSS | 서열번호 15SEQ ID NO: 15 |
경쇄가변부위 아미노산서열light chain variable region amino acid sequence |
DVVMTQSPDSLAVSLGERATINCKSS QSLVHNNGNTY LHWYQQKPGQPPKLLIY KVS NRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFC SQSTHVPYS FGGGTKLEIKDVVMTQSPDSLAVSLGERATINCKSS QSLVHNNGNTY LHWYQQKPGQPPKLLIY KVS NRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFC SQSTHVPYS FGGGTKLEIK | 서열번호 16SEQ ID NO: 16 |
중쇄가변부위 염기서열heavy chain variable region base sequence |
CAGGTGCAGCTGAAGGAGAGTGGTCCTGTCCTGGTGAAGCCTACCGAGACTCTTACACTGACCTGCACCGTGTCCGGCTTCTCTCTCACCATCTACGGCGTCCACTGGATCCGCCAGCCACCCGGCAAGGCCCTGGAATGGCTAGGCGTGATGTGGTCTGGCGGCTCCACCGACTACAACGCTGCGCTCAAGTCCCGGCTGACGATCTCTAAGGACAACTCCAAGTCGCAGGTCGTGCTCACTATGACTAACATGGATCCAGTGGACACCGCCACCTACTACTGTGCTCGCAACGACGGGTACTATTGGTTCGCCTATTGGGGCCAGGGCACCTTGGTCACCGTCTCCTCCCAGGTGCAGCTGAAGGAGAGTGGTCCTGTCCTGGTGAAGCCTACCGAGACTCTTACACTGACCTGCACCGTGTCCGGCTTCTCTCTCACCATCTACGGCGTCCACTGGATCCGCCAGCCACCCGGCAAGGCCCTGGAATGGCTAGGCGTGATGTGGTCTGGCGGCTCCACCGACTACAACGCTGCGCTCAAGTCCCGGCTGACGATCTCTAAGGACAACTCCAAGTCGCAGGTCGT GCTCACTATGACTAACATGGATCCAGTGGACACCGCCACCTACTACTGTGCTCGCAACGACGGGTACTATTGGTTCGCCTATTGGGGCCAGGGCACCTTGGTCACCGTCTCCTCC | 서열번호 17SEQ ID NO: 17 |
경쇄가변부위 염기서열light chain variable region base sequence |
GATGTGGTGATGACCCAGAGCCCCGACAGCCTGGCCGTATCTCTGGGCGAGCGTGCAACCATTAACTGCAAAAGCTCCCAGAGCTTGGTGCACAACAATGGCAACACGTACCTGCATTGGTACCAGCAGAAGCCGGGACAGCCGCCTAAGCTGCTGATTTACAAGGTGTCAAATAGGTTTTCAGGAGTTCCCGACCGCTTCTCCGGGTCGGGTTCTGGAACAGATTTCACGCTTACCATCTCCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTTTTGTAGTCAATCGACCCACGTTCCCTACAGCTTCGGAGGCGGTACTAAACTGGAGATCAAGGATGTGGTGATGACCCAGAGCCCCGACAGCCTGGCCGTATCTCTGGGCGAGCGTGCAACCATTAACTGCAAAAGCTCCCAGAGCTTGGTGCACAACAATGGCAACACGTACCTGCATTGGTACCAGCAGAAGCCGGGACAGCCGCCTAAGCTGCTGATTTACAAGGTGTCAAATAGGTTTTCAAGGAGTTCCCGACCGCTTCTCCGGGTCGGGTTCTGGAACAGATTTCACGCTTACCAT CTCCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTTTTGTAGTCAATCGACCCACGTTCCCTACAGCTTCGGAGGCGGTACTAAACTGGAGATCAAG | 서열번호 18SEQ ID NO: 18 |
실시예 3 : 선별한 항체의 CD22에 대한 특이성 확인Example 3: Confirmation of specificity of selected antibodies to CD22
본 발명에서는 상기 실시예 1의 4F5 항체(mouse), 실시예 2의 인간화된 4F5(V4) 항체 및 4F5(V11) 항체의 CD22에 대한 특이성을 확인하기 위해, 유세포 분석(flow cytometer)을 수행하였다. In the present invention, in order 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 to CD22, flow cytometer analysis was performed. .
먼저, CD22를 발현하는 비세포림프종 U2932(B-cell lymphoma U2932 cell)이 1x106개와 4F5 항체 1 ㎍ 각각을 30분간 반응시킨 다음, 2차 항체로 표면(surface)을 염색한 후, 유세포분석기로 측정하였다.First, CD22-expressing non-cell lymphoma U2932 (B-cell lymphoma U2932 cell) was reacted with 1x10 6 cells and 1 μg of 4F5 antibody for 30 minutes, and then, after staining the surface with a secondary antibody, flow cytometry measured.
양성대조군으로 PE-컨쥬게이션된 항-CD22 항체(PE-conjugated anti-CD22 antibody; Biolegend Inc., cat# 302506, 미국)를 사용하였으며, 2차 항체로는 PE-컨쥬게이션된 항-마우스 IgG 항체(PE-conjugated goat anti-mouse IgG; Biolegend Inc., cat# 405307, 미국)를 사용하였다. As a positive control, PE-conjugated anti-CD22 antibody (Biolegend Inc., cat# 302506, USA) was used, and as a secondary antibody, PE-conjugated anti-mouse IgG antibody (PE-conjugated goat anti-mouse IgG; Biolegend Inc., cat# 405307, USA) was used.
그 결과, 도 1에 나타난 바와 같이 4F5 항체, 인간화된 4F5(V4) 항체 및 4F5(V11) 항체 모두 CD22를 발현하는 세포와 특이적으로 결합하는 것을 확인하였다.As a result, as shown in FIG. 1 , it was confirmed that the 4F5 antibody, the humanized 4F5(V4) antibody, and the 4F5(V11) antibody specifically bind to cells expressing CD22.
실시예 4 : CD22를 표적으로 하는 키메라 항원 수용체(CD22-CAR) 발현 벡터 제작Example 4: Construction of Chimeric Antigen Receptor (CD22-CAR) Expression Vector Targeting CD22
본 발명에서는 상기 실시예 1 및 실시예 2에서 제조한 4F5 항체, 인간화된 4F5(V4) 항체 및 4F5(V11) 항체를 이용하여, CD22를 표적으로 하는 키메라 항원수용체(CAR)를 발현하는 렌티바이러스 벡터(CD22-CAR 렌티바이러스 벡터)를 제조하였다. In the present invention, using the 4F5 antibody, the humanized 4F5 (V4) antibody, and the 4F5 (V11) antibody prepared in Examples 1 and 2 above, lentivirus expressing a chimeric antigen receptor (CAR) targeting CD22 A vector (CD22-CAR lentiviral vector) was prepared.
도 3의 모식도에 나타난 바와 같이, As shown in the schematic diagram of Figure 3,
EF1α 프로모터(서열번호 29); EF1α promoter (SEQ ID NO: 29);
시그널 펩타이드를 코딩하는 폴리뉴클레오타이드(서열번호 30); a polynucleotide encoding a signal peptide (SEQ ID NO: 30);
CD22-결합 도메인을 코딩하는 폴리뉴클레오타이드(서열번호 24의 염기서열로 표시되는 4F5 항체, 서열번호 26의 염기서열로 표시되는 4F5(V4) 항체 또는 서열번호 28의 염기서열로 표시되는 4F5(V11) 항체); A polynucleotide encoding the CD22-binding domain (4F5 antibody represented by the nucleotide sequence of SEQ ID NO: 24, 4F5 (V4) antibody represented by the nucleotide sequence of SEQ ID NO: 26, or 4F5 (V11) represented by the nucleotide sequence of SEQ ID NO: 28 antibody);
CD8 힌지 부위를 코딩하는 폴리뉴클레오타이드(서열번호 31); a polynucleotide encoding the CD8 hinge region (SEQ ID NO: 31);
막관통 도메인을 코딩하는 폴리뉴클레오타이드(서열번호 32); a polynucleotide encoding a transmembrane domain (SEQ ID NO: 32);
4-1BB(공동자극도메인)을 코딩하는 폴리뉴클레오타이드(서열번호 33);polynucleotide encoding 4-1BB (co-stimulatory domain) (SEQ ID NO: 33);
CD3ζ(세포 내 신호전달 도메인)을 코딩하는 폴리뉴클레오타이드(서열번호 34); 및 polynucleotide encoding CD3ζ (intracellular signaling domain) (SEQ ID NO: 34); and
WPRE를 코딩하는 폴리뉴클레오타이드(서열번호 35)로 구성된 CAR DNA를 생체외 (in vitro)에서 합성하여 3세대 렌티바이러스 벡터에 삽입하였다.CAR DNA consisting of a polynucleotide (SEQ ID NO: 35) encoding WPRE was synthesized in vitro and inserted into a third-generation lentiviral vector.
렌티바이러스 벡터 DNA(0.5 ㎍)를 HEK293FT 세포(5×105cells/500㎕)로 전달하고, CD22-CAR 유전자를 발현하는 293HEK 세포를 제작하였다. 293HEK 세포로 유전자를 전달하기 위하여 Lipofectamine 3000 transfection kit(Invitrogen, cat# L3000-015)를 사용하였으며, Opti-MEM (gibco, cat# 51985-034) 배지에서 4시간 동안 배양하였다.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 constructed. Lipofectamine 3000 transfection kit (Invitrogen, cat# L3000-015) was used to transfer the gene to 293HEK cells, and cultured in Opti-MEM (gibco, cat# 51985-034) medium for 4 hours.
실시예 5 : CD22-CAR-T 세포 제조Example 5: Preparation of CD22-CAR-T cells
본 발명에서는 상기 실시예 4에서 제조한 CD22-CAR 렌티바이러스 벡터를 T 세포에 형질전환시켜 4F5 항체, 4F5(V4) 항체 및 4F5(V11) 항체 기반 CD22-CAR-T 세포를 각각 제조하였다. In the present invention, CD22-CAR-T cells based on 4F5 antibody, 4F5(V4) antibody and 4F5(V11) antibody were prepared by transfecting T cells with the CD22-CAR lentiviral vector prepared in Example 4 above.
구체적으로, 도 4에 나타낸 모식도와 같이, 혈액에서 말초혈액단핵세포(peripheral blood mononuclear cell, PBMC)를 분리한 다음, T 세포활성화비드(T cell activation bead; Miltenyl Biotec, cat# 130-091-441)를 사용해 T 세포를 활성화시켰다. 활성화된 T 세포에 상기 실시예 4에서 제조한 CD22-CAR 렌티바이러스 벡터를 T 세포에 형질 도입시켜 4F5 항체 기반 CD22-CAR-T 세포, 4F5(V4) 항체 기반 CD22-CAR-T 세포 및 4F5(V11) 항체 기반 CD22-CAR-T 세포를 각각 제조하였다.Specifically, as shown in the schematic diagram shown in FIG. 4, after isolating peripheral blood mononuclear cells (PBMC) from blood, T cell activation beads (Miltenyl Biotec, cat# 130-091-441 ) was used to activate T cells. 4F5 antibody-based CD22-CAR-T cells, 4F5 (V4) antibody-based CD22-CAR-T cells, and 4F5 ( V11) antibody-based CD22-CAR-T cells were respectively prepared.
CD22-CAR-T 세포의 CD22 펩타이드 결합능은 유세포분석(Flow Cytometry) 방법을 통해 확인하였다. 상기에서 제조한 4F5 항체, 4F5(V4) 항체 기반 CD22-CAR-T 세포 및 4F5(V11) 항체 기반 CD22-CAR-T 세포를 항-CD3, 항-CD4, 항-CD8 항체를 이용하여 CD3, CD4 또는 CD8이 활성화된 CD22-CAR-T 세포로 각각 분류한 다음, FITC-CD22 펩타이드와 반응시킨 후, FACS 기계를 이용해 형광 세기를 측정하였다.The CD22 peptide binding capacity of CD22-CAR-T cells was confirmed by flow cytometry. The 4F5 antibody, 4F5(V4) antibody-based CD22-CAR-T cells, and 4F5(V11) antibody-based CD22-CAR-T cells prepared above were subjected to CD3, anti-CD4, and anti-CD8 antibodies. CD4 or CD8 activated CD22-CAR-T cells were respectively sorted, reacted with FITC-CD22 peptide, and fluorescence intensity was measured using a FACS machine.
그 결과, 도 5a 및 도 5b에 나타난 바와 같이, CD3, CD4 또는 CD8이 활성화된 CD22-CAR-T 세포 모두 CD22 펩타이드와 결합하는 것을 확인하였다.As a result, as shown in FIGS. 5A and 5B , it was confirmed that CD3, CD4, or CD8-activated CD22-CAR-T cells all bind to the CD22 peptide.
실시예 6 : CD22 발현 세포에 대한 CD22-CAR-T 세포의 사멸 효과 확인Example 6: Confirmation of the killing effect of CD22-CAR-T cells on CD22 expressing cells
본발명에서는 4F5 항체 기반 CD22-CAR-T 세포, 인간화된 4F5(V4) 항체 기반 CD22-CAR-T 세포 및 4F5(V11) 항체 기반 CD22-CAR-T 세포 각각에 대한 표적세포의 사멸효과를 확인하였다. In the present invention, the target cell killing effect on 4F5 antibody-based CD22-CAR-T cells, humanized 4F5 (V4) antibody-based CD22-CAR-T cells, and 4F5 (V11) antibody-based CD22-CAR-T cells, respectively, was confirmed. did
표적세포로 CD22를 발현하지 않는 K562 세포(human erythroleukemic cell line)와 CD22를 발현하는 U2932 세포(B cell lymphoma)를 이용하였으며, CD22-CAR-T 세포와 1:4, 1:2, 1:1, 1:0.5 및 1:0.25 비율이 되도록 각각 혼합하여 8시간 동안 배양한 다음, 루미네센스 (CytoTox-Glo Cytotoxicity Assay, Promega, cat. NO G9291)를 측정하였다. 측정한 값으로 하기 수학식 1을 이용하여 세포 사멸 정도를 계산하였다. As target cells, K562 cells (human erythroleukemic cell line) that do not express CD22 and U2932 cells (B cell lymphoma) that express CD22 were used, and CD22-CAR-T cells and 1:4, 1:2, 1:1 , 1:0.5 and 1:0.25 ratio, respectively, and incubated for 8 hours, and then luminescence (CytoTox-Glo Cytotoxicity Assay, Promega, cat. NO G9291) was measured. The degree of cell death was calculated using Equation 1 below with the measured value.
[수학식 1][Equation 1]
% Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)] X 100% Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)] X 100
Experimental: 표적세포 및 CAR-T 세포 복합 배양의 배지로부터 도출된 발광(Luminescence)값Experimental: Luminescence value derived from medium of target cell and CAR-T cell complex culture
Effector Spontaneous: CAR-T 세포만의 배지로부터 도출된 발광값Effector Spontaneous: Luminescence value derived from the medium of only CAR-T cells
Target Spontaneous: 표적세포만의 배지로부터 도출된 발광값Target Spontaneous: Luminescence value derived from the target cell only medium
Target Maximum: 표적세포의 100% 용해(용해시약 (Lysis Reagent) 이용)로부터 도출된 발광값Target Maximum: Luminescence value derived from 100% lysis of target cells (using Lysis Reagent)
그 결과, 도 7a 및 도 7b에 나타난 바와 같이, 4F5 항체 기반 CD22-CAR-T 세포, 4F5(V4) 항체 기반 CD22-CAR-T 세포 및 4F5(V11) 항체 기반 CD22-CAR-T 세포는 CD22를 발현하는 U2932 세포를 특이적으로 사멸시키는 것을 확인하였다.As a result, as shown in FIGS. 7a and 7b, the 4F5 antibody-based CD22-CAR-T cells, the 4F5 (V4) antibody-based CD22-CAR-T cells, and the 4F5 (V11) antibody-based CD22-CAR-T cells It was confirmed that the specific killing of U2932 cells expressing .
본 발명에서는 상기 실험을 통해 4F5 항체 기반 CD22-CAR-T 세포, 4F5(V4) 항체 기반 CD22-CAR-T 세포 및 4F5(V11) 항체 기반 CD22-CAR-T 세포가 미만성 거대 B 세포 림프종(Diffuse Large B-cell Lymphoma) 유래 U2932 세포를 특이적으로 사멸시키는 것을 확인하였다. In the present invention, through the above experiments, 4F5 antibody-based CD22-CAR-T cells, 4F5 (V4) antibody-based CD22-CAR-T cells, and 4F5 (V11) antibody-based CD22-CAR-T cells were tested for diffuse large B-cell lymphoma (Diffuse Large B-cell Lymphoma)-derived U2932 cells were confirmed to be specifically killed.
즉, 본 발명의 항-CD22 항체인 4F5 항체, 4F5(V4) 항체 또는 4F5(V11) 항체, 상기 항체 기반 키메라 항원 수용체, 및 이를 이용한 CAR-T 세포는 B 세포 또는 CD22 발현과 관련된 질환 예방 또는 치료용 조성물로 유용하게 활용할 수 있다. That is, the anti-CD22 antibody of the present invention, the 4F5 antibody, the 4F5(V4) antibody or the 4F5(V11) antibody, the antibody-based chimeric antigen receptor, and the CAR-T cell using the same are used to prevent or prevent diseases associated with B cell or CD22 expression. It can be usefully utilized as a therapeutic composition.
실시예 7 : CD19/CD22를 표적으로 하는 이중특이적 키메라 항원 수용체 발현 벡터 제작Example 7: Construction of a bispecific chimeric antigen receptor expression vector targeting CD19/CD22
상기 실시예 4와 동일한 방법으로 CD19 및 CD22를 표적으로 하는 이중특이적 키메라 항원수용체를 발현하는 렌티바이러스 벡터(CD19xCD22-CAR 렌티바이러스 벡터)를 제조하였다. A lentiviral vector (CD19xCD22-CAR lentiviral vector) expressing a bispecific chimeric antigen receptor targeting CD19 and CD22 was prepared in the same manner as in Example 4 above.
도 8의 모식도에 나타난 바와 같이, As shown in the schematic diagram of Figure 8,
EF1α 프로모터(서열번호 29); EF1α promoter (SEQ ID NO: 29);
시그널 펩타이드를 코딩하는 폴리뉴클레오타이드(서열번호 30); a polynucleotide encoding a signal peptide (SEQ ID NO: 30);
CD19/CD22-결합 도메인을 코딩하는 폴리뉴클레오타이드;a polynucleotide encoding a CD19/CD22-binding domain;
CD8 힌지 부위를 코딩하는 폴리뉴클레오타이드(서열번호 31); a polynucleotide encoding the CD8 hinge region (SEQ ID NO: 31);
막관통 도메인을 코딩하는 폴리뉴클레오타이드(서열번호 32); a polynucleotide encoding a transmembrane domain (SEQ ID NO: 32);
4-1BB(공동자극도메인)을 코딩하는 폴리뉴클레오타이드(서열번호 33);polynucleotide encoding 4-1BB (co-stimulatory domain) (SEQ ID NO: 33);
CD3ζ(세포 내 신호전달 도메인)을 코딩하는 폴리뉴클레오타이드(서열번호 34); 및 polynucleotide encoding CD3ζ (intracellular signaling domain) (SEQ ID NO: 34); and
WPRE를 코딩하는 폴리뉴클레오타이드(서열번호 35)로 구성된 CAR DNA를 생체외 (in vitro)에서 합성하여 3세대 렌티바이러스 벡터에 삽입하였다.CAR DNA consisting of a polynucleotide (SEQ ID NO: 35) encoding WPRE was synthesized in vitro and inserted into a third-generation lentiviral vector.
본 발명에서는 CD19-결합 도메인은 공지된 항-CD19 항체(FMC63)를 이용하였으며, CD22-결합 도메인은 본 발명의 4F5(mouse) 항체, 4F5(V4) 항체 및 4F5(V11) 항체를 이용하였다. In the present invention, the known anti-CD19 antibody (FMC63) was used for the CD19-binding domain, and the 4F5 (mouse) antibody, 4F5 (V4) antibody and 4F5 (V11) antibody of the present invention were used for the CD22-binding domain.
상기 CD19xCD22-결합 도메인은 CD19에 특이적으로 결합하는 항체의 경쇄 가변부위(CD19VL) - CD22에 특이적으로 결합하는 항체의 중쇄 가변부위(CD22VH) - CD22에 특이적으로 결합하는 항체의 경쇄 가변부위 (CD22VL) - CD19에 특이적으로 결합하는 항체의 중쇄 가변부위(CD19VH)로 순서로 연결하였으며 (LoopCAR), 이들을 코딩하는 폴리뉴클레오타이드에 대한 서열정보는 하기와 같다:The CD19xCD22-binding domain 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) - linked in sequence to the heavy chain variable region (CD19VH) of an antibody specifically binding to CD19 (LoopCAR), and sequence information for polynucleotides encoding them is as follows:
CD19x4F5(mouse): 서열번호 50의 염기서열로 표시되는 CD19VL - 서열번호 52의 염기서열로 표시되는 링커(도 8의 linker 1) - 서열번호 9의 염기서열로 표시되는 CD22VH - 서열번호 55의 염기서열로 표시되는 링커(도 8의 linker 6) - 서열번호 10의 염기서열로 표시되는 CD22VL - 서열번호 53의 염기서열로 표시되는 링커(도 8의 linker 1) - 서열번호 49의 염기서열로 표시되는 CD19VH;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. 8) - CD22VH represented by the nucleotide sequence of SEQ ID NO: 9 - nucleotide sequence of SEQ ID NO: 55 Linker represented by sequence (linker 6 in FIG. 8) - CD22VL represented by nucleotide sequence of SEQ ID NO: 10 - Linker represented by nucleotide sequence of SEQ ID NO: 53 (linker 1 in FIG. 8) - represented by nucleotide sequence of SEQ ID NO: 49 becoming CD19VH;
CD19x4F5(V4): 서열번호 50의 염기서열로 표시되는 CD19VL - 서열번호 52의 염기서열로 표시되는 링커(도 8의 linker 1) - 서열번호 13의 염기서열로 표시되는 CD22VH - 서열번호 55의 염기서열로 표시되는 링커(도 8의 linker 6) - 서열번호 14의 염기서열로 표시되는 CD22VL - 서열번호 53의 염기서열로 표시되는 링커(도 8의 linker 1) - 서열번호 49의 염기서열로 표시되는 CD19VH;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. 8) - CD22VH represented by the nucleotide sequence of SEQ ID NO: 13 - nucleotide sequence of SEQ ID NO: 55 Linker represented by sequence (linker 6 in FIG. 8) - CD22VL represented by nucleotide sequence of SEQ ID NO: 14 - Linker represented by nucleotide sequence of SEQ ID NO: 53 (linker 1 in FIG. 8) - represented by nucleotide sequence of SEQ ID NO: 49 becoming CD19VH;
CD19x4F5(V11): 서열번호 50의 염기서열로 표시되는 CD19VL - 서열번호 52의 염기서열로 표시되는 링커(도 8의 linker 1) - 서열번호 17의 염기서열로 표시되는 CD22VH - 서열번호 55의 염기서열로 표시되는 링커(도 8의 linker 6) - 서열번호 18의 염기서열로 표시되는 CD22VL - 서열번호 53의 염기서열로 표시되는 링커(도 8의 linker 1) - 서열번호 49의 염기서열로 표시되는 CD19VH.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. 8) - CD22VH represented by the nucleotide sequence of SEQ ID NO: 17 - nucleotide sequence of SEQ ID NO: 55 Linker represented by sequence (linker 6 in FIG. 8) - CD22VL represented by nucleotide sequence of SEQ ID NO: 18 - Linker represented by nucleotide sequence of SEQ ID NO: 53 (linker 1 in FIG. 8) - represented by nucleotide sequence of SEQ ID NO: 49 being CD19VH.
렌티바이러스 벡터 DNA(0.5 ㎍)를 HEK293FT 세포(5×105cells/500㎕)로 전달하고, CD19/CD22-CAR 유전자를 발현하는 293HEK 세포를 제작하였다. 293HEK 세포로 유전자를 전달하기 위하여 Lipofectamine 3000 transfection kit(Invitrogen, cat# L3000-015)를 사용하였으며, Opti-MEM (gibco, cat# 51985-034) 배지에서 4시간 동안 배양하였다(도 9 및 도 10).Lentiviral vector DNA (0.5 μg) was transferred to HEK293FT cells (5×10 5 cells/500 μl), and 293HEK cells expressing the CD19/CD22-CAR gene were constructed. Lipofectamine 3000 transfection kit (Invitrogen, cat# L3000-015) was used to transfer the gene to 293HEK cells, and cultured in Opti-MEM (gibco, cat# 51985-034) medium for 4 hours (FIGS. 9 and 10). ).
렌티바이러스 벡터로 형질감염된 HEK293FT에서 CD19xCD22 특이적인 CAR가 발현이 되는지 확인한 결과, 도 11에 나타난 바와 같이, 항-CD19/항-CD22 항체 발현이 정상적으로 이루어지는 것을 확인하였다.As a result of confirming whether CD19xCD22-specific CAR was expressed in HEK293FT transfected with the lentiviral vector, it was confirmed that anti-CD19/anti-CD22 antibody was normally expressed as shown in FIG. 11 .
실시예 8 : CD19xCD22-CAR-T 세포 제조Example 8: Preparation of CD19xCD22-CAR-T cells
본 발명에서는 상기 실시예 7에서 제조한 CD19xCD22-CAR 렌티바이러스 벡터를 실시예 5와 동일한 방법으로 T 세포에 형질전환시켜 CD19xCD22-CAR-T 세포인 CD19x4F5, C19x4F5(V4) 및 CD19x4F5(V11)를 각각 제조하였다 (도 12 및 도 13). In the present invention, CD19xCD22-CAR-T cells, CD19x4F5, C19x4F5 (V4) and CD19x4F5 (V11), were transfected into T cells in the same manner as in Example 5 with the CD19xCD22-CAR lentiviral vector prepared in Example 7, respectively. was prepared (Figs. 12 and 13).
CD19xCD22-CAR-T 세포의 CD22 펩타이드 결합능은 유세포분석(Flow Cytometry) 방법을 통해 확인하였다. CD19xCD22-CAR-T세포(CD19x4F5, CD19x 4F5(V4) 및 CD19x4F5(V11))를 항-CD3 항체를 이용하여 CD3이 활성화된 CD19xCD22-CAR-T 세포로 각각 분류한 다음, PE-CD19 펩타이드 및 FITC-CD22 펩타이드와 반응시킨 후, 유세포분석기 기계를 이용해 형광 세기를 측정하였다.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 sorted into CD3-activated CD19xCD22-CAR-T cells using an anti-CD3 antibody, and then PE-CD19 peptide and FITC After reacting with the -CD22 peptide, fluorescence intensity was measured using a flow cytometer.
그 결과, 도 14에 나타난 바와 같이, CD3이 활성화된 CD19xCD22-CAR-T 세포 모두 CD19 펩타이드 및 CD22 펩타이드와 동시에 결합하는 것을 확인하였다.As a result, as shown in FIG. 14 , it was confirmed that CD3-activated CD19xCD22-CAR-T cells simultaneously bind to CD19 peptide and CD22 peptide.
실시예 9 : CD22 혹은 CD19 발현 세포에 대한 CD19xCD22-CAR-T 세포의 사멸 효과 확인Example 9: Confirmation of the killing effect of CD19xCD22-CAR-T cells on CD22 or CD19 expressing cells
본 발명에서는 CD19과 CD22를 발현하지 않는 K562 세포(ATCC, cat#CCL-243)를 CD19, CD22 또는 CD19xCD22를 발현하는 세 가지의 세포 K562-CD19, K562-CD22, K562-CD19/CD22를 제작하고, CD19xCD22-CAR-T 세포에 의한 표적세포의 사멸효과를 확인하였다. In the present invention, K562 cells (ATCC, cat# CCL-243) that do not express CD19 and CD22 were used to construct three cells K562-CD19, K562-CD22, and K562-CD19/CD22 that express CD19, CD22 or CD19xCD22. , The target cell killing effect by CD19xCD22-CAR-T cells was confirmed.
상기 표적 세포와 CD19xCD22-CAR-T세포인, CD19x4F5-CAR-T 세포, CD19x4F5(V4)-CAR-T 세포 및 CD19x4F5(V11)-CAR-T 세포 각각을 1:4, 1:2, 1:1, 1:0.5 및 1:0.25 비율이 되도록 혼합하여 8시간 동안 배양한 다음, 루미네센스 (CytoTox-Glo Cytotoxicity Assay, Promega, cat# G9291)를 측정하였다. 측정한 값으로 실시예 6의 수학식 1을 이용하여 세포 사멸 정도를 계산하였다. The target cells and CD19xCD22-CAR-T cells, CD19x4F5-CAR-T cells, CD19x4F5 (V4)-CAR-T cells, and CD19x4F5 (V11)-CAR-T cells, respectively, were 1:4, 1:2, 1: They were mixed at ratios of 1, 1:0.5 and 1:0.25 and incubated for 8 hours, and then luminescence (CytoTox-Glo Cytotoxicity Assay, Promega, cat# G9291) was measured. The degree of cell death was calculated using Equation 1 of Example 6 with the measured value.
그 결과, 도 15에 나타난 바와 같이, 본 발명에서 제조한 CD19x4F5-CAR-T 세포, CD19x4F5(V4)-CAR-T 세포 및 CD19x4F5(V11)-CAR-T 세포는 CD22 혹은 CD19 혹은 CD19/CD22를 발현하는 세포를 특이적으로 사멸시키는 것을 확인하였다.As a result, as shown in FIG. 15, the CD19x4F5-CAR-T cells, CD19x4F5(V4)-CAR-T cells and CD19x4F5(V11)-CAR-T cells prepared in the present invention express CD22 or CD19 or CD19/CD22. It was confirmed that the expressing cells were specifically killed.
본 발명에서는 CD22에 특이적으로 결합하는 인간화 항체를 제조하였으며, 이를 이용하여 CD22를 표적으로 하는 단일 CAR-T 세포 및 CD19 및 CD22를 표적으로 하는 이중특이적 CAR-T 세포를 제조하였다. In the present invention, a humanized antibody that specifically binds to CD22 was prepared, and using this, single CAR-T cells targeting CD22 and bispecific CAR-T cells targeting CD19 and CD22 were prepared.
본 발명에서 제조한 CD22-CAR-T 세포 및 이중특이적 CD19xCD22-CAR-T 세포는 CD22 항원을 효과적으로 인식하여 CAR-T 세포의 활성화가 이루어지며, CD22를 발현하는 세포를 효과적으로 사멸시키는 것을 확인하였으므로, CD22(또는 CD19) 발현과 관련된 질환 또는 B 세포와 관련된 질환의 예방 또는 치료용 조성물로 유용하게 활용할 수 있다. It was confirmed that the CD22-CAR-T cells and the bispecific CD19xCD22-CAR-T cells prepared in the present invention effectively recognized the CD22 antigen, activated the CAR-T cells, and effectively killed cells expressing CD22. , CD22 (or CD19) can be usefully used as a composition for preventing or treating a disease related to expression or a disease related to B cells.
Claims (24)
- 서열번호 1의 아미노산으로 표시되는 CDR1 영역, 서열번호 2의 아미노산으로 표시되는 CDR2 영역 및 서열번호 3의 아미노산으로 표시되는 CDR3 영역을 포함하는 중쇄 가변부위 및 서열번호 4의 아미노산으로 표시되는 CDR1 영역, 서열번호 5의 아미노산으로 표시되는 CDR2 영역 및 서열번호 6의 아미노산으로 표시되는 CDR3 영역을 포함하는 경쇄 가변 부위를 포함하는, CD22에 특이적으로 결합하는 항체 또는 이의 단편.The CDR1 region represented by the amino acids of SEQ ID NO: 1, the CDR2 region represented by the amino acids of SEQ ID NO: 2, and the heavy chain variable region including the CDR3 region represented by the amino acids of SEQ ID NO: 3 and the CDR1 region represented by the amino acids of SEQ ID NO: 4, An antibody or fragment thereof that specifically binds to CD22, comprising a light chain variable region comprising a CDR2 region represented by the amino acids of SEQ ID NO: 5 and a CDR3 region represented by the amino acids of SEQ ID NO: 6.
- 제1항에 있어서, 상기 CD22에 특이적으로 결합하는 항체는 서열번호 7의 아미노산으로 표시되는 중쇄 가변부위 및 서열번호 8의 아미노산으로 표시되는 경쇄 가변부위로 구성되는 것을 특징으로 하는, CD22에 특이적으로 결합하는 항체 또는 이의 단편.The method of claim 1, wherein the antibody specifically binding to CD22 is composed of a heavy chain variable region represented by amino acids of SEQ ID NO: 7 and a light chain variable region represented by amino acids of SEQ ID NO: 8. Antibodies or fragments thereof that bind antagonistically.
- 제1항에 있어서, 상기 CD22에 특이적으로 결합하는 항체는 서열번호 11의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 12의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체; 또는 The method of claim 1, wherein the antibody specifically binding to CD22 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. humanized antibodies; or서열번호 15의 아미노산 서열로 표시되는 중쇄 가변부위 및 서열번호 16의 아미노산 서열로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체인 것을 특징으로 하는, CD22에 특이적으로 결합하는 항체 또는 이의 단편.An antibody that specifically binds to CD22, characterized in that it is a humanized antibody that specifically binds to CD22 composed 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 or a fragment thereof.
- 제1항 내지 3항 중 어느 한항의 CD22에 특이적으로 결합하는 항체 또는 이의 단편을 코딩하는, 폴리뉴클레오타이드.A polynucleotide encoding an antibody or fragment thereof that specifically binds to CD22 according to any one of claims 1 to 3.
- 제1항 내지 3항 중 어느 한항의 CD22에 특이적으로 결합하는 항체 또는 이의 단편을 코딩하는 폴리뉴클레오타이드를 포함하는, 벡터.A vector comprising a polynucleotide encoding an antibody or fragment thereof that specifically binds to CD22 according to any one of claims 1 to 3.
- 제5항의 벡터로 형질전환된 CD22에 특이적으로 결합하는 항체 또는 이의 단편을 생산하는, 재조합 세포.A recombinant cell that produces an antibody or fragment thereof that specifically binds to CD22 transformed with the vector of claim 5.
- CD22-결합 도메인; CD22-binding domain;막관통 도메인(transmembrane domain); transmembrane domain;공동자극 도메인(costimulatory domain); 및 costimulatory domain; and세포 내 신호전달 도메인(intracellular signal transduction domain)을 포함하는 키메릭 항원 수용체(chimeric antigen receptor: CAR)로,A chimeric antigen receptor (CAR) containing an intracellular signal transduction domain,상기 CD22-결합 도메인은 서열번호 1의 아미노산으로 표시되는 CDR1 영역, 서열번호 2의 아미노산으로 표시되는 CDR2 영역 및 서열번호 3의 아미노산으로 표시되는 CDR3 영역을 포함하는 중쇄 가변부위 및 서열번호 4의 아미노산으로 표시되는 CDR1 영역, 서열번호 5의 아미노산으로 표시되는 CDR2 영역 및 서열번호 6의 아미노산으로 표시되는 CDR3 영역을 포함하는 경쇄 가변 부위를 포함하는 CD22에 특이적으로 결합하는 항체 또는 이의 단편인 것을 특징으로 하는, CD22를 표적으로 하는 키메릭 항원 수용체.The CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4 An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 , a chimeric antigen receptor targeting CD22.
- 제7항에 있어서, 상기 CD22에 특이적으로 결합하는 항체는 The method of claim 7, wherein the antibody specifically binds to CD22(a) 서열번호 7의 아미노산으로 표시되는 중쇄 가변부위 및 서열번호 8의 아미노산으로 표시되는 경쇄 가변부위로 구성된 CD22에 특이적으로 결합하는 항체;(a) an antibody that specifically binds to CD22 composed of a heavy chain variable region represented by amino acids of SEQ ID NO: 7 and a light chain variable region represented by amino acids of SEQ ID NO: 8;(b) 서열번호 11의 아미노산 서열로 표시되는 중쇄 가변 부위 및 서열번호 12의 아미노산 서열로 표시되는 경쇄 가변 부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체; 또는 (b) a humanized antibody that specifically binds to CD22 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; or(c) 서열번호 15의 아미노산 서열로 표시되는 중쇄 가변 부위 및 서열번호 16의 아미노산 서열로 표시되는 경쇄 가변 부위로 구성된 CD22에 특이적으로 결합하는 인간화 항체인 것을 특징으로 하는, CD22를 표적으로 하는 키메릭 항원 수용체.(c) a humanized antibody that specifically binds to CD22, which is composed 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, targeting CD22 Chimeric Antigen Receptors.
- 제7항에 있어서, 상기 막관통 도메인은 CD8α, CD4, CD28, CD137, CD80, CD86, CD152 및 PD1로 구성된 군에서 선택되는 단백질이며,The method of claim 7, wherein the transmembrane domain is a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1,공동자극 도메인은 CD28, 4-1BB, OX-40 및 ICOS로 구성된 군에서 선택되는 단백질이고, the costimulatory domain is a protein selected from the group consisting of CD28, 4-1BB, OX-40 and ICOS;상기 신호전달 도메인은 CD3ζ인 것을 특징으로 하는, CD22를 표적으로 하는 키메릭 항원 수용체. A chimeric antigen receptor targeting CD22, characterized in that the signaling domain is CD3ζ.
- 제7항에 있어서, 상기 결합 도메인의 C 말단 및 막관통 도메인의 N 말단 사이에 힌지 부위(hinge region)가 추가로 포함되는 것을 특징으로 하는, CD22를 표적으로 하는 키메릭 항원 수용체.The chimeric antigen receptor targeting CD22 according to claim 7, wherein a hinge region is further included between the C-terminus of the binding domain and the N-terminus of the transmembrane domain.
- 제7항 내지 제10항 중 어느 한 항의 키메릭 항원 수용체를 코딩하는, 폴리뉴클레오타이드.A polynucleotide encoding the chimeric antigen receptor according to any one of claims 7 to 10.
- 제7항 내지 제10항 중 어느 한 항의 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드를 포함하는, 벡터.A vector comprising a polynucleotide encoding the chimeric antigen receptor of any one of claims 7 to 10.
- 제7항 내지 제10항 중 어느 한 항의 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드; 또는A polynucleotide encoding the chimeric antigen receptor of any one of claims 7 to 10; or제7항 내지 제10항 중 어느 한 항의 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터를 포함하는, 면역 이펙터 세포.An immune effector cell comprising a vector comprising a polynucleotide encoding the chimeric antigen receptor of any one of claims 7 to 10.
- CD19-결합 도메인 및 CD22-결합 도메인; CD19-binding domain and CD22-binding domain;막관통 도메인(transmembrane domain); transmembrane domain;공동자극 도메인(costimulatory domain); 및 costimulatory domain; and세포 내 신호전달 도메인(intracellular signal transduction domain)을 포함하는 이중특이적 키메릭 항원 수용체(chimeric antigen receptor: CAR)로,A bispecific chimeric antigen receptor (CAR) containing an intracellular signal transduction domain,상기 CD22-결합 도메인은 서열번호 1의 아미노산으로 표시되는 CDR1 영역, 서열번호 2의 아미노산으로 표시되는 CDR2 영역 및 서열번호 3의 아미노산으로 표시되는 CDR3 영역을 포함하는 중쇄 가변부위 및 서열번호 4의 아미노산으로 표시되는 CDR1 영역, 서열번호 5의 아미노산으로 표시되는 CDR2 영역 및 서열번호 6의 아미노산으로 표시되는 CDR3 영역을 포함하는 경쇄 가변부위를 포함하는 CD22에 특이적으로 결합하는 항체 또는 이의 단편인 것을 특징으로 하는, CD19 및 CD22를 표적으로 하는 이중특이적 키메라 항원 수용체.The CD22-binding domain comprises a CDR1 region represented by the amino acids of SEQ ID NO: 1, a CDR2 region represented by the amino acids of SEQ ID NO: 2, and a heavy chain variable region comprising the CDR3 region represented by the amino acids of SEQ ID NO: 3 and amino acids of SEQ ID NO: 4 An antibody or fragment thereof that specifically binds to CD22 comprising a light chain variable region comprising a CDR1 region represented by amino acids of SEQ ID NO: 5, a CDR2 region represented by amino acids of SEQ ID NO: 5, and a CDR3 region represented by amino acids of SEQ ID NO: 6 A bispecific chimeric antigen receptor targeting CD19 and CD22.
- 제14항에 있어서, 상기 CD19-결합 도메인 및 CD22-결합 도메인은,The method of claim 14, wherein the CD19-binding domain and CD22-binding domain,CD19에 특이적으로 결합하는 항체의 경쇄 가변부위 - CD22에 특이적으로 결합하는 항체의 중쇄 가변부위 - CD22에 특이적으로 결합하는 항체의 경쇄 가변부위 - CD19에 특이적으로 결합하는 항체의 중쇄 가변부위 순서로 연결된 것을 특징으로 하는, CD19 및 CD22를 표적으로 하는 이중특이적 키메라 항원 수용체.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 A bispecific chimeric antigen receptor targeting CD19 and CD22, characterized in that they are linked in site order.
- 제15항에 있어서, 상기 CD19에 특이적으로 결합하는 항체의 경쇄 가변부위는 서열번호 48의 아미노산 서열로 표시되며, The method of claim 15, wherein the light chain variable region of the antibody specifically binding to CD19 is represented by the amino acid sequence of SEQ ID NO: 48,상기 CD19에 특이적으로 결합하는 항체의 중쇄 가변부위는 서열번호 47의 아미노산 서열로 표시되는 것을 특징으로 하는, CD19 및 CD22를 표적으로 하는 이중특이적 키메라 항원 수용체.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.
- 제14항에 있어서, 상기 막관통 도메인은 CD8α, CD4, CD28, CD137, CD80, CD86, CD152 및 PD1로 구성된 군에서 선택되는 단백질이며,15. The method of claim 14, wherein the transmembrane domain is a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1,공동자극 도메인은 CD28, 4-1BB, OX-40 및 ICOS로 구성된 군에서 선택되는 단백질이고, the costimulatory domain is a protein selected from the group consisting of CD28, 4-1BB, OX-40 and ICOS;상기 신호전달 도메인은 CD3ζ인 것을 특징으로 하는, CD19 및 CD22를 표적으로 하는 이중특이적 키메라 항원 수용체. A bispecific chimeric antigen receptor targeting CD19 and CD22, characterized in that the signaling domain is CD3ζ.
- 제14항에 있어서, 상기 결합 도메인의 C 말단 및 막관통 도메인의 N 말단 사이에 힌지 부위(hinge region)가 추가로 포함되는 것을 특징으로 하는, CD19 및 CD22를 표적으로 하는 이중특이적 키메라 항원 수용체.The bispecific chimeric antigen receptor targeting CD19 and CD22 according to claim 14, characterized in that a hinge region is further included between the C-terminus of the binding domain and the N-terminus of the transmembrane domain. .
- 제14항 내지 제18항 중 어느 한 항의 이중특이적 키메릭 항원 수용체를 코딩하는, 폴리뉴클레오타이드.A polynucleotide encoding the bispecific chimeric antigen receptor of any one of claims 14 to 18.
- 제14항 내지 제18항 중 어느 한 항의 이중특이적 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드를 포함하는, 벡터.A vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor of any one of claims 14-18.
- 제14항 내지 제18항 중 어느 한 항의 이중특이적 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드; 또는 A polynucleotide encoding the bispecific chimeric antigen receptor of any one of claims 14 to 18; or제14항 내지 제18항 중 어느 한 항의 이중특이적 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터를 포함하는, 면역 이펙터 세포.An immune effector cell comprising a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor of any one of claims 14-18.
- 제1항의 CD22에 특이적으로 결합하는 항체 또는 이의 단편을 포함하는 B 세포에 의해 매개되는 질환의 예방 또는 치료용 약학적 조성물로, A pharmaceutical composition for preventing or treating diseases mediated by B cells comprising the antibody or fragment thereof that specifically binds to CD22 of claim 1,상기 B 세포에 의해 매개되는 질환은 종양, 림프종, 비호치킨 림프종(non-Hogkins lymphoma: NHL), 공격적 NHL, 재발성 공격적 NHL, 재발성 지연성 NHL, 불응성 NHL, 불응성 지연성 NHL, 만성 림프성 백혈병(chronic lymphocytic leukemia: CLL), 소형 림프성 림프종, 백혈병, 모발성 세포 백혈병(hairy cell leukemia: HCL), 급성 림프성 백혈병(acute lymphocytic leukemia: ALL), 버킷트 림프종 및 외투 세포 림프종로 구성된 군에서 선택되는 것을 특징으로 하는, B 세포에 의해 매개되는 질환의 예방 또는 치료용 약학적 조성물.Diseases mediated by the B cells include tumor, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent delayed NHL, refractory NHL, refractory delayed NHL, chronic chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt's lymphoma and mantle cell lymphoma. Characterized in that selected from the group consisting of, a pharmaceutical composition for the prevention or treatment of diseases mediated by B cells.
- 제7항 내지 제10항 중 어느 한 항의 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드; 또는A polynucleotide encoding the chimeric antigen receptor of any one of claims 7 to 10; or제7항 내지 제10항 중 어느 한 항의 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터를 포함하는 면역 이펙터 세포를 포함하는 B 세포에 의해 매개되는 질환의 예방 또는 치료용 약학적 조성물로, A pharmaceutical composition for preventing or treating diseases mediated by B cells, including immune effector cells containing a vector containing a polynucleotide encoding the chimeric antigen receptor of any one of claims 7 to 10,상기 B 세포에 의해 매개되는 질환은 종양, 림프종, 비호치킨 림프종(non-Hogkins lymphoma: NHL), 공격적 NHL, 재발성 공격적 NHL, 재발성 지연성 NHL, 불응성 NHL, 불응성 지연성 NHL, 만성 림프성 백혈병(chronic lymphocytic leukemia: CLL), 소형 림프성 림프종, 백혈병, 모발성 세포 백혈병(hairy cell leukemia: HCL), 급성 림프성 백혈병(acute lymphocytic leukemia: ALL), 버킷트 림프종 및 외투 세포 림프종로 구성된 군에서 선택되는 것을 특징으로 하는, B 세포에 의해 매개되는 질환의 예방 또는 치료용 약학적 조성물.Diseases mediated by the B cells include tumor, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent delayed NHL, refractory NHL, refractory delayed NHL, chronic chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt's lymphoma and mantle cell lymphoma. Characterized in that selected from the group consisting of, a pharmaceutical composition for the prevention or treatment of diseases mediated by B cells.
- 제14항 내지 제18항 중 어느 한 항의 이중특이적 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드; 또는 A polynucleotide encoding the bispecific chimeric antigen receptor of any one of claims 14 to 18; or제14항 내지 제18항 중 어느 한 항의 이중특이적 키메릭 항원 수용체를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터를 포함하는 면역 이펙터 세포를 포함하는 B 세포에 의해 매개되는 질환의 예방 또는 치료용 약학적 조성물로, A pharmaceutical composition for the prevention or treatment of diseases mediated by B cells, including immune effector cells, comprising a vector comprising a polynucleotide encoding the bispecific chimeric antigen receptor of any one of claims 14 to 18. as a composition,상기 B 세포에 의해 매개되는 질환은 종양, 림프종, 비호치킨 림프종(non-Hogkins lymphoma: NHL), 공격적 NHL, 재발성 공격적 NHL, 재발성 지연성 NHL, 불응성 NHL, 불응성 지연성 NHL, 만성 림프성 백혈병(chronic lymphocytic leukemia: CLL), 소형 림프성 림프종, 백혈병, 모발성 세포 백혈병(hairy cell leukemia: HCL), 급성 림프성 백혈병(acute lymphocytic leukemia: ALL), 버킷트 림프종 및 외투 세포 림프종로 구성된 군에서 선택되는 것을 특징으로 하는, B 세포에 의해 매개되는 질환의 예방 또는 치료용 약학적 조성물.Diseases mediated by the B cells include tumor, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent delayed NHL, refractory NHL, refractory delayed NHL, chronic chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt's lymphoma and mantle cell lymphoma. Characterized in that selected from the group consisting of, a pharmaceutical composition for the prevention or treatment of diseases mediated by B cells.
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WO2016149578A1 (en) * | 2015-03-19 | 2016-09-22 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Dual specific anti-cd22-anti-cd19 chimeric antigen receptors |
US20170369550A1 (en) * | 2014-12-24 | 2017-12-28 | Ucl Business Plc | Cell |
CN110869046A (en) * | 2017-03-31 | 2020-03-06 | 塞勒克提斯公司 | Universal anti-CD22 chimeric antigen receptor engineered immune cells |
US20200087396A1 (en) * | 2018-09-26 | 2020-03-19 | LentigenTechnology, Inc. | Compositions and Methods for Treating Cancer with Anti-CD19/CD22 Immunotherapy |
WO2020108644A1 (en) * | 2018-11-30 | 2020-06-04 | Beijing Meikang Geno-Immune Biotechnology Co., Ltd. | Cd19-and cd22-based combined car-t immunotherapy |
WO2021235696A1 (en) * | 2020-05-19 | 2021-11-25 | 주식회사 이노베이션바이오 | Cd22-specific antibody and use thereof |
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US20170369550A1 (en) * | 2014-12-24 | 2017-12-28 | Ucl Business Plc | Cell |
WO2016149578A1 (en) * | 2015-03-19 | 2016-09-22 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Dual specific anti-cd22-anti-cd19 chimeric antigen receptors |
CN110869046A (en) * | 2017-03-31 | 2020-03-06 | 塞勒克提斯公司 | Universal anti-CD22 chimeric antigen receptor engineered immune cells |
US20200087396A1 (en) * | 2018-09-26 | 2020-03-19 | LentigenTechnology, Inc. | Compositions and Methods for Treating Cancer with Anti-CD19/CD22 Immunotherapy |
WO2020108644A1 (en) * | 2018-11-30 | 2020-06-04 | Beijing Meikang Geno-Immune Biotechnology Co., Ltd. | Cd19-and cd22-based combined car-t immunotherapy |
WO2021235696A1 (en) * | 2020-05-19 | 2021-11-25 | 주식회사 이노베이션바이오 | Cd22-specific antibody and use thereof |
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