Dual antibody and car-t combination therapy

Abstract

The present invention relates to a pharmaceutical composition for combination therapy, and a cancer prevention and / or treatment use using same, the composition comprising an anti-CD20 / anti-CD55 bispecific antibody and immune cells expressing a chimeric antigen receptor (CAR) that includes a CD19 antigen-binding domain. According to the present invention, when a pharmaceutical composition for combination therapy, comprising a bispecific antibody and CAR-T, is used, cancer cell attack is maximized through a plurality of different targets such that anticancer activity can be significantly increased, and the problem of reduced anticancer activity of CAR-T caused by a reduction or loss of CD19 target factors in tumor cells can be overcome, and thus the present invention can be effectively used in various industrial fields, including the pharmaceutical industry, as an effective agent for cancer prevention and / or treatment.

Description

Combination therapy of dual antibodies and CAR-T

[0001] The present invention relates to a pharmaceutical composition for combination administration comprising an anti-CD20 / anti-CD55 bispecific antibody and an immune cell expressing a chimeric antigen receptor (CAR) comprising a CD19 antigen binding domain, and a use thereof for the prevention and / or treatment of cancer.

[0002] This application claims priority to Korean Patent Application No. 10-2024-0012317, filed January 26, 2024, the entire disclosure of which is incorporated herein by reference.

[0003]

[0004] The global anticancer drug market has grown at an average annual rate of 10-13% over the past decade and is expected to reach a total of $200 billion by 2022. As of 2020, one in five men and one in six women worldwide will experience at least one case of cancer in their lifetime, and one in eight men and one in 11 women will die from cancer, indicating a continued increase in the prevalence and mortality rates of cancer.

[0005] The global anticancer drug market is undergoing a paradigm shift, starting with first-generation chemotherapy, and progressing to second-generation targeted anticancer drugs and third-generation immunotherapy. Moving beyond the initial treatment methods that simply aimed to reduce or suppress cancer, active research is being conducted on targeted anticancer therapies that selectively attack cancer cells without damaging rapidly dividing cells. The development of these targeted anticancer therapies involves two stages: the selection of receptors specifically expressed in cancer cells, enabling selective targeting of cancer cells, and the development of targeting compounds that bind to these receptors.

[0006] Meanwhile, CD55 (decay-accelerating factor, DAF) is a receptor that suppresses complement immune mechanisms. It is highly expressed in various solid cancers, including colon, lung, stomach, breast, ovarian, leukemia, and head and neck cancer. It promotes cancer cell proliferation by inhibiting the body's complement immune system from killing cancer cells. Therefore, CD55 is being actively studied as a target molecule for targeted anticancer therapy.

[0007] In particular, solid tumors with high CD55 expression show low therapeutic response to various anticancer drugs that use complement-dependent cytotoxicity (CDC) as their mechanism of action. Therefore, there is a need for the development of superior antibody therapeutics that can bind to CD55 with higher affinity and specificity and efficiently inhibit its activity.

[0008] Furthermore, CAR-T cell therapy has garnered attention by demonstrating dramatic effects, particularly in clinical trials targeting hematological malignancies. Specifically, in the case of CAR-T cell therapy using antibodies that recognize CD19, a B-lymphocyte-derived hematological malignancy antigen, 90% of patients (27 out of 30) achieved complete remission within one month in an early clinical trial targeting patients with acute lymphoblastic leukemia who had not responded to any previous treatments, and the treatment showed an astonishing therapeutic effect, with an overall survival rate of 78% at six months (Maude SL, et al., N Engl J Med. 2014;371(16):1507-17). Based on these results, two CD19 CAR-T cell therapies were successfully commercialized under FDA approval at the end of 2017.

[0009] CAR proteins are designed in a form in which the variable region (single chain variable fragment; scFv) of an antibody that recognizes a tumor antigen is connected to an intracellular signaling domain through a backbone (Dotti G, et al., Immunol Rev. 2014;257(1):107-26). The intracellular signaling domain is mainly based on the intracellular signaling domain of the CD3 zeta (ζ) chain, a signaling subunit of the T cell receptor (first-generation CAR), and has evolved to add the intracellular signaling domain of a co-stimulatory molecule that promotes T cell growth and differentiation.

[0010] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0011]

[0012] The present inventors have completed the present invention by confirming that by co-administering a bispecific antibody targeting two different antigens, CD20 and CD55, and a chimeric antigen receptor T-cell (CAR-T) targeting CD19 antigen, anti-tumor activity is significantly enhanced and the problem of decreased anti-tumor activity of CAR-T due to a decrease or loss of CD19 targeting factor in tumor cells can be overcome.

[0013] One object of the present invention is to provide a pharmaceutical composition for combination administration for preventing or treating cancer, comprising as active ingredients (i) an anti-CD20 / anti-CD55 bispecific antibody comprising a first domain specifically binding to CD20 and a second domain specifically binding to CD55; and (ii) an immune cell expressing a chimeric antigen receptor (CAR) comprising a CD19 antigen binding domain.

[0014] Other objects of the present invention are more clearly explained by the detailed description of the invention, claims and drawings described below.

[0015]

[0016] Unless otherwise defined, scientific and technical terms used in connection with the present invention have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms include plurals, and plural terms include the singular. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization disclosed herein are well known and commonly used in the art.

[0017] To achieve the above-described object, one embodiment of the present invention provides a pharmaceutical composition for combination administration for preventing or treating cancer, comprising as active ingredients (i) an anti-CD20 / anti-CD55 bispecific antibody comprising a first domain specifically binding to CD20 and a second domain specifically binding to CD55; and (ii) an immune cell expressing a chimeric antigen receptor (CAR) comprising a CD19 antigen binding domain.

[0018] The term "antibody" as used herein refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically has reactivity with a specific antigen, and includes both polyclonal antibodies and monoclonal antibodies.

[0019] Antibodies include both full-length antibody forms and antigen-binding fragments of antibody molecules (antibody fragments). A complete antibody has two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond. The heavy chain constant regions are of the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The light chain constant regions are of the kappa (κ) and lambda (λ) types.

[0020] The term "antigen-binding fragment of an antibody" as used herein means a fragment within a whole antibody molecule that specifically recognizes an antigen and possesses antigen-antibody binding function, and includes single-domain antibodies (sdAb), single-chain antibodies (scFv), Fab, F(ab'), F(ab')2, and Fv. Examples of the fragments include a monovalent fragment (Fab fragment) consisting of VL, VH, CL, and CH1 domains; a bivalent fragment (F(ab')2 fragment) comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of one arm of an antibody, and a disulfide-linked Fv (sdFv); a dAb fragment consisting of a VH domain; and a combination of two or more separate complementarity determining regions (CDRs) that can be optionally joined by a linker. Additionally, scFv can be connected by a linker to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule. Such single-chain antibodies are also included in antibody fragments. Furthermore, the antibodies or antibody fragments include tetrameric antibodies comprising two heavy chain molecules and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers, antibody heavy chain dimers; intrabodies; monovalent antibodies; camelid antibodies; and single-domain antibodies (sdAbs).

[0021] Fv is the minimum antibody fragment that has only a heavy chain variable region and a light chain variable region, and recombinant technology for producing Fv fragments is disclosed in PCT International Patent Publication Nos. WO 88 / 10649, WO 88 / 106630, WO 88 / 07085, WO 88 / 07086, and WO 88 / 09344. A two-chain Fv has a heavy chain variable region and a light chain variable region linked non-covalently, and a single-chain Fv has a heavy chain variable region and a single chain variable region covalently linked through a peptide linker or directly linked at the C-terminus, so that they can form a dimer-like structure like a two-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, restriction digestion of whole antibodies with papain yields Fab fragments, and digestion with pepsin yields F(ab')2 fragments), or they can be produced using genetic recombination techniques.

[0022] As used herein, the term "heavy chain" refers to a full-length heavy chain and fragments thereof, which comprise a variable domain VH and three constant domains CH1, CH2 and CH3, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. In addition, the term "light chain" as used herein refers to a full-length light chain and fragments thereof, which comprise a variable domain VL and a constant domain CL, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.

[0023] As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulins (Kabat et al. Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chain (HCDR11, HCDR2, and HCDR3) and the light chain (LCDR1, LCDR2, and LCDR3) each contain three CDRs, which provide key contact residues for antibody binding to an antigen or epitope.

[0024] The scope of the antibody or antibody fragment of the present invention includes variants having conservative amino acid substitutions in the CDR region, and may include variants with respect to the amino acid sequences set forth in the attached sequence listing within the scope capable of specifically recognizing CD55. For example, in addition to the binding affinity of the antibody, additional changes may be made to the amino acid sequence of the antibody to further improve its half-life, biocompatibility, and other biological properties. Considering such variants having biologically equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding the same is interpreted to also include sequences that exhibit substantial identity with the sequences set forth in the sequence listing. The substantial identity refers to a sequence that exhibits at least 61% homology, in one specific example, 70% homology, in another specific example, 80% homology, and in yet another specific example, 90% homology, when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequences are analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are well known in the art. Various alignment methods and algorithms are described in Smith and Waterman, Adv. Appl. Math. (1981) 2:482; Needleman and Wunsch, J. Mol. Bio. (1970) 48:443; Pearson and Lipman, Methods in Mol. Biol. (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73:237-44; Higgins and Sharp, CABIOS (1989) 5:151-3; Corpet et al. Nuc. Acids Res. (1988) 16:10881-90; Huang et al. Comp. Appl. BioSci. (1992) 8:155-65; and Pearson et al. Meth. Mol. Biol.(1994) 24:307-31.

[0025] As used herein, the term "bispecific antibody" refers to an antibody comprising two different antigen-binding regions defined by different amino acid sequences. Typically, the two antigen-binding regions included in a bispecific antibody are each specific for a different antigen or epitope.

[0026] According to a specific embodiment of the present invention, the antigen-binding fragment of the anti-CD55 antibody included in the bispecific antibody is in the form of scFv.

[0027] According to a specific embodiment of the present invention, the antigen-binding fragment of the antibody that specifically binds to CD55 comprises scFv.

[0028] According to a specific embodiment of the present invention, the antigen-binding fragment of the antibody that specifically binds to CD55 is in the form of VL-linker-VH-CH2-CH3.

[0029] According to a specific embodiment of the present invention, the antigen-binding fragment of the anti-CD20 antibody included in the bispecific antibody is in the Fab form.

[0030] According to a specific embodiment of the present invention, the antigen-binding fragment of the antibody that specifically binds to CD20 comprises Fab.

[0031] According to a specific embodiment of the present invention, the antigen-binding fragment of the antibody that specifically binds to CD20 is in the form of VL-CK-linker-VH-CH1-CH2-CH3.

[0032] According to a specific embodiment of the present invention, the antibody that specifically binds to CD20 is Rituximab.

[0033] The bispecific antibody of the present invention may comprise an Fc region comprised of first and second subunits capable of stably assembling. The term "Fc region" as used herein refers to the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region of a full-length antibody, and includes both wild-type sequence Fc regions and variant Fc regions. The Fc region of an IgG comprises IgG CH2 and IgG CH3 domains. The CH3 region of the bispecific antibody of the present invention may be a wild-type or variant CH3 domain (e.g., a CH3 domain having a "knob" introduced into one chain thereof and a corresponding "cavity" introduced into the other chain thereof).

[0034] According to a specific embodiment of the present invention, the bispecific antibody of the present invention can use a knob-into-hole method to prevent unwanted pairing between the heavy chains of CD20 X CD55. The "knob-into-hole" method [US 5,731,168; US7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); Carter, J Immunol Meth 248, 7-15 (2001)] introduces a protrusion ("knob") at the interface of the first polypeptide and a corresponding cavity ("hole") at the interface of the second polypeptide so that the protrusion can be positioned in the cavity to promote heterodimer formation and inhibit homodimer formation. The protrusion is formed by replacing a small amino acid side chain with a larger side chain (e.g., tyrosine or tryptophan) at the interface of the first polypeptide. By replacing the large amino acid side chain with a smaller side chain (e.g., alanine or threonine), a complementary cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide.

[0035] According to a specific embodiment of the present invention, the bispecific antibody of the present invention may be in a form in which an antigen-binding fragment of an anti-CD55 antibody in the form of VL-linker-VH-CH2-CH3 and an antigen-binding fragment of an anti-CD20 antibody in the form of VL-CK-linker-VH-CH1-CH2-CH3 are linked.

[0036] According to a specific embodiment of the present invention, the bispecific antibody of the present invention may use a linker composed of 10 to 100, more specifically 15 to 60, Gly and Ser to prevent unwanted pairing between the heavy chains of CD20 X CD55.

[0037] Specifically, the bispecific antibody of the present invention may be in a form in which CD55-scFv-knob (VL-linker20-VH-CH2-CH3) and CD20-Fab-hole (VL-CK-linker40-VH-CH1-CH2-CH3) are combined.

[0038] linker20 contains 20 of the above Gly or Ser, examples of which may include, but are not limited to, Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Ser.

[0039] linker40 contains 20 of the above Gly or Ser, examples of which may include, but are not limited to, Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Ser.

[0040] The term “affinity” in this specification means the strength of binding between an antibody or an antigen-binding fragment thereof and an antigen, and may also be expressed as “binding force.”

[0041] The present invention can provide a nucleic acid molecule encoding the antibody of the present invention or an antigen-binding fragment thereof described above.

[0042] As used herein, the term "nucleic acid molecule" has a comprehensive meaning including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base moieties are modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90:543-584). The sequence of a nucleic acid molecule encoding the heavy and light chain variable regions of the present invention may be modified. The modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0043] The nucleic acid molecule of the present invention is also interpreted to include a nucleotide sequence that exhibits substantial identity with the nucleotide sequence described above. The substantial identity refers to a nucleotide sequence that exhibits at least 80% homology, in one specific example at least 90% homology, and in another specific example at least 95% homology, when the nucleotide sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0044] As used herein, the term "chimeric antigen receptor (CAR)" refers to a receptor protein engineered to confer a novel ability to target a specific protein on a T cell. The receptor is chimeric because it combines both antigen-binding and T-cell activating functions into a single receptor. A CAR is a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain (a transmembrane domain), and at least one intracellular domain (an intracellular signaling domain). A "chimeric antigen receptor (CAR)" is sometimes referred to as a "chimeric receptor," a "T-body," or a "chimeric immune receptor (CIR)." An "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding a specific antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transduces a signal that causes the activation or inhibition of a biological process in a cell.

[0045] As used herein, the term “domain” means a region of a polypeptide that folds into a specific structure independently of other regions.

[0046] The term "transmembrane domain" as used herein may be derived from a natural polypeptide or may be artificially designed. A transmembrane domain derived from a natural polypeptide may be obtained from any membrane-bound or transmembrane protein. For example, a transmembrane domain of the T cell receptor α or β chain, CD3 zeta chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR may be used. An artificially designed transmembrane domain is a polypeptide that primarily comprises hydrophobic residues, such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is found at each terminus of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or polypeptide linker, for example, a linker having a length of 2 to 10 amino acids, may be arranged between the transmembrane domain and the intracellular domain. In one embodiment, a linker sequence having a continuous sequence of glycine-serine may be used.

[0047] The nucleic acid encoding the CAR of the present invention can be inserted into a vector, and the vector can be introduced into a cell. For example, a viral vector, such as a retroviral vector (including an oncoretroviral vector, a lentiviral vector, and a pseudotyped vector), an adenoviral vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector, a Sendai virus vector, an Epstein-Barr virus (EBV) vector, and a HSV vector can be used. It is preferable to use a viral vector that lacks replication ability so as not to self-replicate in infected cells.

[0048] For example, when using a retroviral vector, a suitable packaging cell can be selected based on the packaging signal sequence and LTR sequence possessed by the vector to produce retroviral particles using the packaging cell. Examples of packaging cells include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-12, and Psi-Crip. Retroviral particles can also be produced using 293 cells or 293T cells, which have high transfection efficiency. Many types of retroviral vectors produced based on retroviruses and packaging cells that can be used for packaging retroviral vectors are widely commercially available from many companies.

[0049] CAR-T cells bind to specific antigens via the CAR, thereby transmitting signals to the cells, resulting in cell activation. Activation of CAR-expressing cells varies depending on the type of host cell and the intracellular domain of the CAR, and can be identified based on indicators such as cytokine release, enhanced cell proliferation rate, and changes in cell surface molecules. For example, the release of cytotoxic cytokines (e.g., tumor necrosis factor, lymphotoxin) from activated cells causes the destruction of target cells expressing the antigen. In addition, the release of cytokines or changes in cell surface molecules stimulate other immune cells, such as B cells, dendritic cells, NK cells, and macrophages.

[0050] Cells expressing CARs can be used as therapeutic agents for diseases. The therapeutic agent comprises cells expressing CARs as the active ingredient and may further comprise suitable excipients.

[0051] The term "prevention" as used herein means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to developing the disease or condition.

[0052] As used herein, the term "treatment" means (a) inhibiting the development of a disease, condition, or symptom; (b) alleviating the disease, condition, or symptom; or (c) eliminating the disease, condition, or symptom. When the composition of the present invention is administered to a subject, it inhibits the activity of the CD55 receptor, thereby restoring the complement immune mechanism and inhibiting the proliferation of cancer cells, thereby inhibiting the progression of cancer or eliminating or alleviating the symptoms caused by it. Therefore, the composition of the present invention may be a composition for treating cancer on its own, or may be used as a therapeutic adjuvant to enhance the therapeutic response by being administered together with another pharmacological agent, for example, a therapeutic agent whose mechanism of action is complement-dependent cytotoxicity (CDC). Accordingly, the terms "treatment" or "therapeutic agent" as used herein include the meaning of "therapeutic adjuvant" or "therapeutic adjuvant."

[0053] As used herein, the term "administration" or "administering" refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject, thereby forming an identical amount in the subject's body. The term "therapeutically effective amount" as used herein refers to the content of the composition in which the pharmacological ingredient in the composition is contained in an amount sufficient to provide a therapeutic or preventive effect to the subject to whom the pharmaceutical composition of the present invention is to be administered, and thus includes a "prophylactically effective amount."

[0054] The term "subject" as used herein includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus macaque. Specifically, the subject of the present invention is a human.

[0055] In the present invention, the first domain may be characterized by including a complementarity determining region (CDR) composed of LCDR1 represented by SEQ ID NO: 1, LCDR2 represented by SEQ ID NO: 2, LCDR3 represented by SEQ ID NO: 3, HCDR1 represented by SEQ ID NO: 4, HCDR2 represented by SEQ ID NO: 5, and HCDR3 represented by SEQ ID NO: 6.

[0056] In the present invention, the second domain may be characterized by including a complementarity determining region (CDR) consisting of LCDR1 represented by SEQ ID NO: 7, LCDR2 represented by SEQ ID NO: 8, LCDR3 represented by SEQ ID NO: 9, HCDR1 represented by SEQ ID NO: 10, HCDR2 represented by SEQ ID NO: 11, and HCDR3 represented by SEQ ID NO: 12 or 13.

[0057] In the present invention, the first domain may be characterized by including one selected from the group consisting of a light chain variable region represented by SEQ ID NO: 14 and a heavy chain variable region represented by SEQ ID NO: 15; a light chain variable region represented by SEQ ID NO: 16 and a heavy chain variable region represented by SEQ ID NO: 17; and a light chain variable region represented by SEQ ID NO: 16 and a heavy chain variable region represented by SEQ ID NO: 18.

[0058] In the present invention, the second domain may be characterized by including one selected from the group consisting of a light chain variable region represented by SEQ ID NO: 19 and a heavy chain variable region represented by SEQ ID NO: 20; a light chain variable region represented by SEQ ID NO: 21 and a heavy chain variable region represented by SEQ ID NO: 22; and a light chain variable region represented by SEQ ID NO: 21 and a heavy chain variable region represented by SEQ ID NO: 23.

[0059] In the present invention, it may be characterized by a first domain represented by sequence number 24 and a second domain represented by sequence number 25.

[0060] In the present invention, it may be characterized in that one of the Fc regions of the first domain and the second domain has a knob structure and the other has a hole structure.

[0061] In the present invention, the CAR may be characterized by further comprising a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

[0062] In the present invention, the CD19 antigen binding domain may be characterized as represented by SEQ ID NO: 32 or SEQ ID NO: 33.

[0063] In the present invention, the immune cell may be characterized as being a T cell, NK cell, NKT cell, or macrophage.

[0064] In the present invention, the cancer may be selected from the group consisting of breast cancer, colon cancer, colorectal cancer, liver cancer, pancreatic cancer, lung cancer, stomach cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, melanoma, rectal cancer, anal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphoma, hepatocellular carcinoma, glioblastoma, bladder cancer, liver tumor, salivary gland cancer, kidney cancer, thyroid cancer, head and neck cancer, and brain cancer, but is not limited thereto.

[0065] In addition, the present invention provides (i) an anti-CD20 / anti-CD55 bispecific antibody comprising a first domain that specifically binds to CD20 and a second domain that specifically binds to CD55; and

[0066] (ii) a method for preventing or treating cancer, comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising an immune cell expressing a chimeric antigen receptor (CAR) comprising a CD19 antigen binding domain.

[0067] In addition, the present invention provides (i) an anti-CD20 / anti-CD55 bispecific antibody comprising a first domain that specifically binds to CD20 and a second domain that specifically binds to CD55; and

[0068] (ii) Provides a composition comprising an immune cell expressing a chimeric antigen receptor (CAR) comprising a CD19 antigen binding domain as an active ingredient for use in the prevention or treatment of cancer.

[0069] In addition, the present invention provides (i) an anti-CD20 / anti-CD55 bispecific antibody comprising a first domain specifically binding to CD20 and a second domain specifically binding to CD55 for the manufacture of a drug for preventing or treating cancer; and

[0070] (ii) provides a use of an immune cell expressing a chimeric antigen receptor (CAR) comprising a CD19 antigen binding domain.

[0071]

[0072] By using a pharmaceutical composition for combination administration comprising a bispecific antibody and CAR-T according to the present invention, anticancer activity can be significantly increased by maximizing cancer cell attack through multiple different targets, and the problem of decreased anticancer activity of CAR-T due to decrease or loss of CD19 target factor in tumor cells can be overcome, and thus, it can be usefully utilized in various industrial fields including the pharmaceutical industry as an effective cancer prevention and / or treatment agent.

[0073]

[0074] Figures 1a to 4d illustrate CAR-T Expansion according to one embodiment of the present invention.

[0075] Figures 5a and 5b show the results of cell line phenotype analysis according to one embodiment of the present invention.

[0076] Figure 6 shows the results of evaluating the cell killing efficacy of rituximab on six types of lymphoma cell lines according to one embodiment of the present invention.

[0077] Figure 7a shows the results of evaluating the cell killing efficacy of a dual-specific antibody and CAR-T combination therapy on Raji cells according to one embodiment of the present invention.

[0078] Figure 7b shows the results of evaluating the cell killing efficacy of a combination therapy of a bispecific antibody and CAR-T according to one embodiment of the present invention on Mino cells.

[0079] Figure 7c is a schematic diagram of a combination therapy of dual antibodies and CAR-T according to one embodiment of the present invention.

[0080] Figure 7d shows the main results of the combination therapy of dual antibodies and CAR-T according to one embodiment of the present invention.

[0081] Figure 8a shows the results of confirming CD55 expression of anti-CD19 CAR-T cells according to one embodiment of the present invention.

[0082] Figure 8b is a diagram illustrating the possibility that a bispecific antibody according to one embodiment of the present invention may exhibit toxicity to anti-CD19 CAR-T.

[0083] Figures 8c and 8d show the results of toxicity evaluation of a bispecific antibody against CAR-T cells according to one embodiment of the present invention (Donor #1).

[0084] Figures 8e and 8f show the results of toxicity evaluation of a bispecific antibody against CAR-T cells according to one embodiment of the present invention (Donor #2).

[0085] Figure 9a is a schematic diagram illustrating CAR-T resistance due to antigen loss according to one embodiment of the present invention.

[0086] Figure 9b illustrates a method for producing CD19-Raji cells for an in vitro antigen loss model according to one embodiment of the present invention.

[0087] Figure 9c shows the results of verifying CD19 expression of CD19-Raji cells according to one embodiment of the present invention.

[0088] Figure 10a shows the results of co-culturing CD19- Raji cells and CD19+ Raji cells according to one embodiment of the present invention.

[0089] Figure 10b shows the results of a luminescence-based evaluation of the possibility of overcoming CAR-T resistance due to antigen loss in a dual antibody and CAR-T combination therapy according to one embodiment of the present invention.

[0090] Figure 10c shows the results of a flow-based evaluation of the possibility of overcoming CAR-T resistance due to antigen loss in a dual antibody and CAR-T combination therapy according to one embodiment of the present invention.

[0091] Figure 10d is a schematic diagram of the effect of combination therapy of a dual antibody and CAR-T according to one embodiment of the present invention.

[0092]

[0093] Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended solely to illustrate the present invention and should not be construed as limiting the scope of the present invention. The following examples are provided to more fully explain the present invention to those of average skill in the art.

[0094]

[0095] [Example]

[0096] Example 1: Anti-CD20 / anti-CD55 bispecific antibody

[0097] The sequences of the anti-CD20 / anti-CD55 bispecific antibodies produced according to the present invention are as shown in Tables 1 to 3 below.

[0098]

[0099]

[0100]

[0101] The sequences of the anti-CD20 antibody Rituximab and the anti-CD55 antibody 4-1H used in the present invention are as shown in Tables 4 and 5 below.

[0102]

[0103]

[0104]

[0105] Example 2: Anti-CD19 CAR-T

[0106] 2-1: PBMC / T cell isolation

[0107] PBMCs were isolated from the blood of healthy donors using Ficoll, and T cells were finally isolated from PBMCs using the EasySep Human T cell Enrichment Kit (STEMCELL).

[0108]

[0109] 2-2: Production of lentivirus encoding anti-CD19 CAR-T gene

[0110] HEK 293T, a lentivirus production cell line, was cultured at 37°C in a CO25% incubator using GIBCO®RPMI Media-1640 containing 10% Fetal Bovine Serum, 1% penicillin streptomycin, 1% Glutamax, and 1% HEPES. When HEK 293T cells reached 60-70% confluence, they were transfected with plasmids for virus production using 116 μl of Lipofectamine 2000 (plasmids used: 15 μg of expression plasmid with CAR gene inserted, 18 μg of Gag / pol, 18 μg of REV, and 7 μg of VSV-G). After 24 and 48 hours, the culture medium containing the virus was harvested, centrifuged at 15,000 g overnight to concentrate the virus, and stored at -80°C.

[0111]

[0112] 2-3: CAR-T Expansion

[0113] Primary T cells from healthy donors were activated using anti-CD3 / anti-CD28 beads at a ratio of 3 beads: 1 cell. 24 hours after T cell activation, lentiviruses containing the anti-CD19 CAR gene were treated, and 5 days later, anti-CD3 / anti-CD28 beads were removed, and CAR expression was confirmed using flow cytometry after staining using anti-CD19 CAR antibody PE. Subculture was performed every 48 hours at 0.8Х10 cells / ml and 0.5Х10 cells / ㎠, and when the anti-CD19 CAR-T cell size reached 350-400 fL, the cells were frozen and stored in a nitrogen tank (Figs. 1a to 4d).

[0114] The sequence of the anti-CD19 CAR-T used in the present invention is as shown in Table 6 below.

[0115]

[0116]

[0117]

[0118] Example 3: Evaluation of antitumor activity of bispecific antibody and CAR-T combination therapy.

[0119] 3-1: Cell line phenotypic analysis

[0120] To evaluate the antitumor activity of the combination therapy of anti-CD19 CAR-T and antibody produced in Example 2, lymphoma cell lines known to be CD19, CD20, and CD55 positive, Daudi, SU-DHL-4, SU-DHL-5, Raji, Mino, and Jiyoye, were identified.

[0121]

[0122] After staining cells with antibodies, the expression of CD19, CD20, and CD55 was confirmed by flow cytometry. As a result of the expression check, the expression of CD55 was low in SU-DHL-5, and the expression of CD20 was very low in Jiyoye. All cell lines were cultured in GIBCO®RPMI Media 1640 containing 10% Fetal Bovine Serum, 1% penicillin streptomycin, 1% Glutamax, and 1% HEPES at 25% CO2 and 37°C (Figures 5a and 5b).

[0123]

[0124] 3-2: Selection of cell lines to be used for evaluating the combined effects of dual antibodies and CAR-T through rituximab reactivity analysis.

[0125] Rituximab is a representative treatment for lymphoma and is included in the first-line treatment of lymphoma. If dual antibody and CAR-T combination therapy is used clinically for lymphoma, resistance to rituximab is likely. Therefore, evaluating the efficacy of dual antibody and CAR-T combination therapy is desirable in situations where rituximab's therapeutic effect is limited. To select an appropriate cell line for evaluating the efficacy of dual antibody and CAR-T combination therapy, the cytotoxic effect of rituximab was analyzed using the cell line described in Example 3-1.

[0126] Lymphoma cell lines were engineered to express luciferase, and cytotoxicity was assessed using bioluminescence quantification. 5x10 4 After seeding cancer cells, rituximab was added at a 1 / 2 dilution starting from 10 μg / ml or at a 1 / 10 dilution starting from 100 μg / ml. Human complement serum was then added at a concentration of 5% and cultured. After 48 hours of culture, D-luciferin was added to the cultured cells, and luminescence was measured after incubation for 10 minutes at 37°C. A control group of target cells cultured alone without rituximab was used, and the degree of cytotoxicity was expressed as a percentage by calculating as follows.

[0127] (Cancer only - Sample) / Cancer only x 100 = Killing %

[0128] As a result, Daudi, SU-DHL-4, and SU-DHL-5 were mostly killed by rituximab at concentrations higher than 1 μg / ml, while Raji, Mino, and Jiyoye showed resistance to rituximab (Fig. 6). Based on the expression results of CD19, CD20, and CD55, Raji and Mino were selected as cell lines to be used for evaluating the combined effect of dual antibodies and CAR-T.

[0129]

[0130] 3-3: Evaluation of the efficacy of dual antibody and CAR-T combination in Raji and Mino cell lines.

[0131] The combined effect of dual antibodies and CAR-T was evaluated using the Raji and Mino cell lines selected in Example 3-2. 5x10 4 After seeding luciferase-expressing Raji or Mino cells, rituximab, SBU-C, or SBU2081 were added to each cell at a predetermined concentration. Next, human complement serum was added at a concentration of 5%, and finally, anti-CD19 CAR-T was added at an Effector:Tumor (E:T) ratio of 1.25:1 for Raji and 0.625:1 for Mino, and co-cultured. Subsequent processes were carried out in the same manner as in Example 3-2.

[0132] As a result, the combination therapy demonstrated a higher cell killing effect compared to either dual antibodies or CAR-T alone in Raji and Mino cell lines (Figures 7a to 7d). These results suggest that the combination of targeted therapies targeting different antigens can exhibit additive effects without interfering with each other, resulting in superior efficacy compared to either alone.

[0133] Furthermore, in Raji, SBU2081 demonstrated a combination effect with CAR-T at lower concentrations than rituximab (Fig. 7a). In Mino, SBU-C and SBU2081 demonstrated superior combination effect with CAR-T at lower concentrations than rituximab (Fig. 7b). These results suggest that bispecific antibodies with added CD55 control may exhibit superior efficacy in combination with CAR-T compared to anti-CD20 monoclonal antibodies.

[0134]

[0135] 3-4: Toxicity evaluation of dual antibodies against CAR-T

[0136] Since CD55 can be expressed on immune cells including T cells, the expression of CD55 in the produced anti-CD19 CAR-T was confirmed using the method of Example 3-1, and as a result, the anti-CD19 CAR-T expressed CD55 (Fig. 8a). Since the dual antibody can bind to CD55 and induce CAR-T cell death (Fig. 8b), the toxicity of the dual antibody on CAR-T was evaluated.

[0137] 5x10 4 After seeding anti-CD19 CAR-T cells, antibodies were diluted by half from 20 μg / ml. Human complement serum was then added to a concentration of 5% and cultured. After 120 hours of culture, the cells were stained with dye-labeled antibodies for flow cytometry. Antibodies used included anti-CD3 and anti-CD19 CAR antibodies to label T cells, and counting beads were used to measure cell numbers.

[0138] A control group of monocultured anti-CD19 CAR-T cells without antibody was used, and the degree of cytotoxicity was expressed as a percentage by calculating as follows.

[0139] (CAR-T only - Sample) / CAR-T only x 100 = Killing %

[0140] As a result, no dose-dependent decrease in anti-CD19 CAR-T cell counts was observed in either Donor #1 or #2 by SBU-C, SBU2041, or SBU2081 (Figures 8c to 8f). These results indicate that the dual antibody does not induce CAR-T cell death and, more broadly, that there is no CAR-T toxicity caused by the dual antibody.

[0141]

[0142] 3-5: Evaluation of the possibility of overcoming CAR-T resistance induced by antigen loss through dual antibody and CAR-T combination

[0143] Reports that CD19 antigen loss induces resistance to anti-CD19 CAR-T [Lownik et al., Clin Cancer Res 30(14), 2895-2904 (2024); Zhang et al., J Immunother Cancer 8(2), e001150 (2020)] have revealed that antigen loss is a major cause of CAR-T treatment failure (Fig. 9a). Therefore, strategies to circumvent CAR-T resistance due to antigen loss are essential for CAR-T therapy. The combination of dual antibodies and CAR-T targets multiple antigens simultaneously, and thus has the potential to overcome resistance due to the loss of a single antigen. Therefore, we established an in vitro model that simulates the clinical environment of antigen loss and evaluated the possibility of overcoming CAR-T resistance induced by antigen loss of dual antibodies and CAR-T in that model.

[0144] CD19 loss was induced in Raji cells using the CRISPR / CAS9 method (Fig. 9b). CD19 expression in Raji cells (CD19- Raji) in which CD19 loss was induced was confirmed using the method of Example 3-1, confirming that CD19 was knocked out (Fig. 9c).

[0145] The efficacy of dual antibodies and CAR-T was confirmed in an in vitro model simulating the clinical environment of antigen loss in which CD19+ Raji and CD19- Raji were co-cultured in a 1:1 ratio (Fig. 10a). The efficacy was confirmed using the luminescence-based method of Example 3-2 and the flow-based method of Example 3-4. In the flow-based method, anti-CD22 antibody was used to label Raji cells. The concentration of antibodies used in combination was rituximab 100 μg / ml or SBU-C, SBU2041, SBU2081 20 μg / ml, and anti-CD19 CAR-T was used at an Effector:Tumor (E:T) ratio of 1.25:1.

[0146] As a result, in the anti-CD19 CAR-T alone treatment group and the rituximab combination group, the apoptosis of CD19+ Raji cells was induced and the growth was controlled, but the growth of CD19- Raji cells was not controlled (Figs. 10b and 10c). In contrast, in the anti-CD19 CAR-T and SBU-C, SBU2041, or SBU2081 combination group, the apoptosis of both CD19+ Raji cells and CD19- Raji cells was induced and the growth was controlled (Figs. 10b and 10c). These results imply that the combination therapy of dual antibodies and CAR-T can be applied as a strategy to overcome the resistance of CAR-T caused by antigen loss (Fig. 10d).

[0147]

[0148] The foregoing description of the present invention is provided for illustrative purposes only, and it will be apparent to those skilled in the art that various modifications and variations are possible without departing from the technical spirit of the present invention. The scope of the present invention is indicated by the claims that follow rather than the detailed description set forth above, and all changes or modifications derived from the claims and their equivalents should be construed as being included within the scope of the present invention.

[0149]

[0150] The pharmaceutical composition for combination administration comprising a bispecific antibody and CAR-T according to the present invention can be usefully utilized in various industrial fields including the pharmaceutical industry as an effective cancer prevention and / or treatment agent, and thus has industrial applicability.

Claims

1. (i) an anti-CD20 / anti-CD55 bispecific antibody comprising a first domain that specifically binds to CD20 and a second domain that specifically binds to CD55; and (ii) A pharmaceutical composition for combination administration for the prevention or treatment of cancer, comprising an immune cell expressing a chimeric antigen receptor (CAR) comprising a CD19 antigen binding domain as an active ingredient.

2. A pharmaceutical composition according to claim 1, characterized in that the first domain comprises a complementarity determining region (CDR) consisting of LCDR1 represented by SEQ ID NO: 1, LCDR2 represented by SEQ ID NO: 2, LCDR3 represented by SEQ ID NO: 3, HCDR1 represented by SEQ ID NO: 4, HCDR2 represented by SEQ ID NO: 5, and HCDR3 represented by SEQ ID NO:

6.

3. A pharmaceutical composition according to claim 1, characterized in that the second domain comprises a complementarity determining region (CDR) consisting of LCDR1 represented by SEQ ID NO: 7, LCDR2 represented by SEQ ID NO: 8, LCDR3 represented by SEQ ID NO: 9, HCDR1 represented by SEQ ID NO: 10, HCDR2 represented by SEQ ID NO: 11, and HCDR3 represented by SEQ ID NO: 12 or 13.

4. A pharmaceutical composition according to claim 1, wherein the first domain comprises one selected from the group consisting of a light chain variable region represented by SEQ ID NO: 14 and a heavy chain variable region represented by SEQ ID NO: 15; a light chain variable region represented by SEQ ID NO: 16 and a heavy chain variable region represented by SEQ ID NO: 17; and a light chain variable region represented by SEQ ID NO: 16 and a heavy chain variable region represented by SEQ ID NO:

18.

5. A pharmaceutical composition according to claim 1, wherein the second domain comprises one selected from the group consisting of a light chain variable region represented by SEQ ID NO: 19 and a heavy chain variable region represented by SEQ ID NO: 20; a light chain variable region represented by SEQ ID NO: 21 and a heavy chain variable region represented by SEQ ID NO: 22; and a light chain variable region represented by SEQ ID NO: 21 and a heavy chain variable region represented by SEQ ID NO:

23.

6. A pharmaceutical composition according to claim 1, wherein the anti-CD20 / anti-CD55 bispecific antibody comprises a first domain represented by SEQ ID NO: 24 and a second domain represented by SEQ ID NO:

25.

7. A pharmaceutical composition characterized in that, in the first paragraph, one of the Fc regions of the first domain and the second domain has a knob structure and the other has a hole structure.

8. A pharmaceutical composition according to claim 1, characterized in that the CAR further comprises a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

9. A pharmaceutical composition according to claim 1, wherein the CD19 antigen binding domain is represented by SEQ ID NO: 32 or SEQ ID NO:

33.

10. A pharmaceutical composition according to claim 1, characterized in that the immune cell is a T cell, NK cell, NKT cell or macrophage.

11. A pharmaceutical composition according to claim 1, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, colorectal cancer, liver cancer, pancreatic cancer, lung cancer, stomach cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, melanoma, rectal cancer, anal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphoma, hepatocellular carcinoma, glioblastoma, bladder cancer, liver tumor, salivary gland cancer, kidney cancer, thyroid cancer, head and neck cancer, and brain cancer.

12. In the first paragraph, the pharmaceutical composition is characterized in that the anti-CD20 / anti-CD55 bispecific antibody and the immune cells expressing the CAR are mixed, or the anti-CD20 / anti-CD55 bispecific antibody and the immune cells expressing the CAR are formulated separately and administered simultaneously or sequentially. 13.(i) an anti-CD20 / anti-CD55 bispecific antibody comprising a first domain that specifically binds to CD20 and a second domain that specifically binds to CD55; and (ii) A method for preventing or treating cancer, comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising an immune cell expressing a chimeric antigen receptor (CAR) comprising a CD19 antigen binding domain. 14.(i) an anti-CD20 / anti-CD55 bispecific antibody comprising a first domain that specifically binds to CD20 and a second domain that specifically binds to CD55; and (ii) Use of a composition comprising an immune cell expressing a chimeric antigen receptor (CAR) comprising a CD19 antigen binding domain as an active ingredient for the prevention or treatment of cancer.

15. (i) an anti-CD20 / anti-CD55 bispecific antibody comprising a first domain specifically binding to CD20 and a second domain specifically binding to CD55 for the manufacture of a drug for the prevention or treatment of cancer; and (ii) Use of an immune cell expressing a chimeric antigen receptor (CAR) comprising a CD19 antigen binding domain.

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