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WO2025220804A1 - Nouvel anticorps dirigé contre cd5 et ses utilisations - Google Patents

Nouvel anticorps dirigé contre cd5 et ses utilisations

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Publication number
WO2025220804A1
WO2025220804A1 PCT/KR2024/011252 KR2024011252W WO2025220804A1 WO 2025220804 A1 WO2025220804 A1 WO 2025220804A1 KR 2024011252 W KR2024011252 W KR 2024011252W WO 2025220804 A1 WO2025220804 A1 WO 2025220804A1
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WO
WIPO (PCT)
Prior art keywords
seq
car
cell
cells
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/KR2024/011252
Other languages
English (en)
Korean (ko)
Inventor
이영호
유승록
정정훈
조현정
이형지
이효빈
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Curocell Inc
Original Assignee
Curocell Inc
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Filing date
Publication date
Application filed by Curocell Inc filed Critical Curocell Inc
Priority claimed from KR1020240101587A external-priority patent/KR20250154905A/ko
Publication of WO2025220804A1 publication Critical patent/WO2025220804A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to a novel antibody against CD5 and its use, and more particularly, to an antibody that specifically binds to CD5 or an antigen-binding fragment thereof or a pharmaceutical composition comprising the same for preventing or treating cancer.
  • 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. The prevalence and mortality rates of cancer continue to rise, with one in eight men and one in 11 women dying from it.
  • the global anticancer drug market is undergoing a paradigm shift, starting with first-generation chemotherapy, and moving to second-generation targeted anticancer drugs and third-generation immunotherapy.
  • 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.
  • the CD5 is a type of differentiation cluster expressed in T cells and B cells, is mainly found in bone marrow or lymphoid tissue, and is overexpressed in T cells more than in B cells, so it is mainly used as a marker for T cells.
  • lymphomas that originate in T cells such as peripheral T-cell lymphoma, anaplastic large cell lymphoma, and extranodal NK/T-cell lymphoma, express CD5; abnormal B cells also express CD5; and some lymphomas that originate in B cells, such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma, also express CD5.
  • T cells such as peripheral T-cell lymphoma, anaplastic large cell lymphoma, and extranodal NK/T-cell lymphoma
  • CD5 abnormal B cells also express CD5
  • some lymphomas that originate in B cells such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma, also express CD5.
  • CLL chronic lymphocytic leukemia
  • CD5 is being actively studied as a target molecule for targeted anticancer therapy, and there is a need for the development of excellent antibody therapeutics that can bind to CD5 with higher affinity and specificity and efficiently inhibit its activity.
  • the technical problem to be achieved by the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to CD5.
  • a technical problem to be achieved by the present invention is to provide a nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof.
  • a technical problem to be achieved by the present invention is to provide an expression vector comprising the nucleic acid molecule.
  • a technical problem to be achieved by the present invention is to provide a host cell including the expression vector.
  • the technical problem to be achieved by the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the antibody or an antigen-binding fragment thereof.
  • one embodiment of the present invention comprises a light chain variable region comprising a light chain CDR1 having an amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 28, and SEQ ID NO: 37; a light chain CDR2 having an amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 29, and SEQ ID NO: 38; and a light chain CDR3 having an amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 30, and SEQ ID NO: 39; and a heavy chain CDR1 having an amino acid sequence of any one of SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 31, and SEQ ID NO: 40; and a heavy chain CDR2 having an amino acid sequence of any one of SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 23, SEQ
  • a heavy chain variable region comprising a heavy chain CDR3 having an amino acid sequence of any one of SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 33, and SEQ ID NO: 42;
  • An antibody or antigen-binding fragment thereof that specifically binds to CD5 is provided.
  • the light chain variable region may include an amino acid sequence of any one of SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25, SEQ ID NO: 34, and SEQ ID NO: 43.
  • the heavy chain variable region may include an amino acid sequence of any one of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, and SEQ ID NO: 44.
  • the light chain variable region and the heavy chain variable region are connected by a linker, and the light chain variable region, the heavy chain variable region, and the linker may be arranged in any one of the following order: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region in the N-terminal to C-terminal direction.
  • it may include any one of the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 36, and SEQ ID NO: 45.
  • the antibody or antigen-binding fragment thereof may be a scFv, a Fab fragment, a F(ab') fragment, a F(ab') 2 fragment, or a Fv fragment.
  • another embodiment of the present invention provides a nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof.
  • another embodiment of the present invention provides an expression vector comprising the nucleic acid molecule.
  • another embodiment of the present invention provides a host cell comprising the expression vector.
  • another embodiment of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the antibody or an antigen-binding fragment thereof.
  • the present invention relates to a novel antibody against CD5 and its use.
  • the antibody of the present invention exhibits high binding affinity and inhibitory ability to the CD5 protein, and thus can be utilized as an effective treatment for various CD5-mediated cancer diseases.
  • Figure 1 shows the results of measuring whether the scFv targeting CD5 discovered in the present invention binds to the CD5 protein.
  • FIG. 2 is a drawing showing the configuration of a chimeric antigen receptor (CAR) used in the present invention, and shows the configuration of a CAR including H65, a positive control scFv, and A2, C7 scFv discovered in the present invention, respectively.
  • CAR chimeric antigen receptor
  • Figure 3 shows the results of transducing a CD5 CAR into a CD5 negative cell line and measuring the binding affinity to the recombinant CD5 protein.
  • Figure 4 shows a schematic diagram of the structure of a mutant CD5 prepared to confirm the binding site and the results of confirming the binding site for the CD5 protein of the positive control and A2, C7 scFv.
  • Figure 5 shows the results of measuring the CAR expression pattern of CD5 CAR transduced cells.
  • Figure 6 shows the results of measuring cell viability and growth of CD5 CAR transduced cells.
  • Figure 7 shows the results of measuring the secretion amounts of IFN- ⁇ and TNF- ⁇ during the culture process of CD5 CAR transduced cells.
  • Figure 8 shows the results of measuring cell phenotype based on the expression pattern of each surface marker of CD5 CAR transduced cells.
  • Figure 9 shows the results of measuring the in vitro tumor cell killing ability of CD5 CAR transduced cells.
  • Figure 10 shows the results of measuring the in vivo tumor cell killing ability of CD5 CAR transduced cells.
  • Figure 11 shows the results of measuring the binding of scFv targeting the CD5 membrane-proximal domain discovered in the present invention to the CD5 protein.
  • FIG 12 is a diagram showing the configuration of a chimeric antigen receptor (CAR) used in the present invention, showing the configuration of a CAR using an scFv targeting the CD5 membrane-distal domain and an scFv targeting the CD5 membrane-proximal domain, including H65, a positive control scFv, respectively.
  • CAR chimeric antigen receptor
  • Figure 13 shows the results of transducing a CD5 CAR into a CD5 negative cell line and measuring the binding affinity to the recombinant CD5 protein.
  • Figure 14 shows the results of confirming the binding site for the CD5 protein of the positive control and scFv discovered in the present invention.
  • Figure 15 shows the results of measuring the CAR expression pattern of CD5 membrane-proximal domain targeting CAR transduced cells.
  • Figure 16 shows the results of measuring cell viability and growth of CD5 membrane-proximal domain-targeting CAR transduced cells.
  • Figure 17 shows the results of measuring cell phenotypes based on the expression patterns of each surface marker of CD5 membrane-proximal domain-targeting CAR transduced cells.
  • Figure 18 shows the results of measuring the in vitro tumor cell killing ability of CD5 membrane-proximal domain-targeting CAR transduced cells.
  • Figure 19 shows the results of measuring the in vivo tumor cell killing ability of CD5 membrane-proximal domain-targeting CAR transduced cells.
  • the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to CD5.
  • antibody used in the present invention refers to an immunoglobulin molecule that immunologically binds specifically to an epitope of an antigen and exhibits reactivity.
  • the antibody may include a monoclonal antibody, a polyclonal antibody, an antibody having a full-length chain structure (full-length antibody), a functional fragment having at least an antigen-binding function (antigen-binding fragment), and a recombinant antibody.
  • the above monoclonal antibody refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.
  • the full-length antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain being capable of being linked to a heavy chain by a disulfide bond.
  • the above antibody comprises a heavy chain (HC) and a light chain (LC) polypeptide, and the heavy chain and light chain may include a variable region and a constant region.
  • the constant region is a site that mediates binding of the antibody to various types of cells of the immune system (such as T cells) and host tissues containing components of the complement system.
  • the constant region has the same function regardless of the type of antigen as long as it is an antibody of the same type derived from the same species, and the amino acid sequence constituting the constant region is also the same or has a high degree of similarity between antibodies.
  • the constant region can be divided into a heavy chain constant region (which may be abbreviated as CH) and a light chain constant region (which may be abbreviated as CL).
  • variable region is an antibody portion having specificity for an antigen, and can be divided into a heavy chain variable region (which may be abbreviated as VH) and a light chain variable region (which may be abbreviated as VL).
  • VH heavy chain variable region
  • VL light chain variable region
  • the variable region may include three CDRs (complementary-determining regions) and four FRs (framework regions).
  • the CDRs may be ring-shaped portions involved in antigen recognition, and specificity for an antigen may be determined according to the amino acid sequence of the CDRs.
  • the CDRs may be referred to as CDR1, CDR2, and CDR3 according to their order, and depending on which polypeptide among the heavy and light chains they are CDRs of, the heavy chain variable region may be referred to as CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region may be referred to as CDR-L1, CDR-L2, and CDR-L3.
  • FR can be referred to as FR-H1, FR-H2, FR-H3, FR-H4 for the heavy chain variable region, and FR-L1, FR-L2, FR-L3, FR-L4 for the light chain variable region.
  • the antigen-binding fragment of the present invention refers to any fragment of the antibody of the present invention that retains the antigen-binding function of the antibody.
  • the antigen-binding fragment may be referred to interchangeably with terms such as "fragment”, "antibody fragment”, etc., and the antigen-binding fragment may be, for example, an scFv, a Fab fragment, an F(ab') fragment, an F(ab') 2 fragment, or an Fv fragment.
  • the above scFv is known to have superior antigen binding affinity than VH or VL alone, as VH and VL are linked by a linker to form a continuous protein chain. Any linker commonly used in the art may be used as the linker connecting VH and VL.
  • the linker may be a peptide link and may have a length of about 10 to 25 amino acids.
  • the linker may include a hydrophilic amino acid such as glycine (G) and/or serine (S). More specifically, the linker may be GGSSRSSSSGGGGSGGGG.
  • the antibody of the present invention or an antigen-binding fragment thereof comprises a light chain variable region comprising a light chain CDR1 having an amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 28, and SEQ ID NO: 37; a light chain CDR2 having an amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 29, and SEQ ID NO: 38; and a light chain CDR3 having an amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 30, and SEQ ID NO: 39; and a heavy chain CDR1 having an amino acid sequence of any one of SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 31, and SEQ ID NO: 40; a heavy chain CDR2 having an amino acid sequence of any one of SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 23, SEQ
  • the light chain variable region may include, for example, any one of the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25, SEQ ID NO: 34, and SEQ ID NO: 43.
  • the heavy chain variable region may include, for example, any one of the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, and SEQ ID NO: 44.
  • the antibody or antigen-binding fragment thereof may comprise an amino acid sequence of any one of SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 36, and SEQ ID NO: 45.
  • the antibody or antigen-binding fragment thereof of the present invention may further include, for example, a heavy chain constant region and/or a light chain constant region of an antibody derived from a human, and, as long as the antibody or antigen-binding fragment thereof does not inhibit the property of specifically binding to CD5, the heavy chain constant region and/or the light chain constant region of the antibody derived from a human may be used without limitation in type or amino acid sequence.
  • the scope of the antibody or antigen-binding fragment of the present invention includes variants having conservative amino acid substitutions in the CDR region, and may include variants for the amino acid sequences listed above within the range capable of specifically recognizing CD5.
  • additional changes may be made to the amino acid sequence of the antibody to further improve its half-life, biocompatibility, and other biological properties.
  • 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 described sequences.
  • 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.
  • the present invention relates to a nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof.
  • nucleic acid molecule as used in the present invention 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 the 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.
  • the present invention relates to an expression vector comprising the nucleic acid molecule.
  • the above expression vector may comprise an expression control sequence operably linked to the above nucleic acid.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • vectors include plasmids, i.e., circular double-stranded DNA fragments into which additional DNA segments can be ligated.
  • viral vectors i.e., vectors into which additional DNA segments can be ligated within the viral genome.
  • Such vectors are capable of autonomous replication within a host cell into which they are introduced. Examples include bacterial vectors having a bacterial origin of replication and episomal mammalian vectors.
  • vectors e.g., non-episomal mammalian vectors
  • any vector can express a gene operably linked to the vector.
  • expression control sequence refers to a polynucleotide sequence necessary for ligating a coding sequence to express and process the coding sequence.
  • Expression control sequences include appropriate transcription initiation, termination, promoter, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that increase translation efficiency (i.e., Kozak consensus sequences), and sequences that enhance protein stability, and, if desired, sequences that promote protein secretion.
  • the nature of such expression control sequences varies depending on the host organism.
  • such expression control sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence
  • such expression control sequences include a promoter and a transcription termination sequence.
  • the expression control sequence includes at least all components whose presence is essential for the expression and processing process, and may include additional components whose presence is advantageous, such as a leader sequence and a fusion partner sequence.
  • the present invention relates to a host cell comprising the above expression vector.
  • a "host cell” as used herein is a cell that can be used to express a nucleic acid, for example, a nucleic acid of the present invention.
  • the host cell may be a prokaryotic organism, for example, Escherichia coli, or a eukaryotic organism, for example, a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell, or insect cell), or a hybridoma.
  • a prokaryotic organism for example, Escherichia coli
  • a eukaryotic organism for example, a unicellular eukaryote (e.g., yeast or other fungi)
  • a plant cell e.g., tobacco or tomato plant cell
  • an animal cell e.g., human cell, monkey cell, hamster cell
  • host cells examples include the COS-7 cell line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or derivatives thereof such as Veggie CHO and related cell lines grown in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31) or the DHFR-deficient CHO line DX-B11 (see Urlaub et al., 1980, Proc. Natl. Acad. Sci.
  • COS-7 cell line of monkey kidney cells ATCC CRL 1651
  • L cells C127 cells
  • 3T3 cells ATCC CCL 163
  • CHO Chinese hamster ovary
  • the host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, after which the nucleic acid can be expressed in the host cell.
  • the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising the antibody or an antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof is as described above.
  • cancer refers to or means a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.
  • the cancer may include all cancers or carcinomas that can be treated with the pharmaceutical composition of the present invention, for example, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, follicular T-cell lymphoma, anaplastic large-cell lymphoma, adult T-cell leukemia/lymphoma, hepatosplenic T-cell lymphoma, mycosis fungoides, Sezary syndrome, extranodal NK/T-cell lymphoma, primary cutaneous gamma/delta T-cell lymphoma, cutaneous T-cell lymphoma, It may be mantle cell lymphoma, T-cell acute lymphobalstic lymphoma, chronic lymphoblastic leukemia, thymic carcinoma, non-Hodgkin lymphoma, diffuse large cell lymphoma, small lymphocytic lymphoma, or T-cell neoplasm.
  • peripheral T-cell lymphoma for example, peripheral
  • Treatment as used in the present invention means an activity that improves or favorably changes symptoms caused by cancer.
  • prevention means preventing the onset, recurrence, or transmission of a disease or disorder, or one or more symptoms caused by the disease/disorder, and may include prophylactic treatment for potential candidates.
  • the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier, which is commonly used in formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
  • a pharmaceutically acceptable carrier which is commonly used in formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stea
  • the pharmaceutical composition of the present invention may further include, in addition to the above components, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.
  • a lubricant e.g., a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.
  • Suitable pharmaceutically acceptable carriers and preparations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
  • the appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, body weight, sex, degree of disease symptoms, food, administration time, administration route, excretion rate, and reaction sensitivity, and a generally skilled physician can easily determine and prescribe an effective dosage for the desired treatment. Meanwhile, the dosage of the pharmaceutical composition of the present invention is not limited there
  • the pharmaceutical composition of the present invention can be administered orally or parenterally.
  • parenterally it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. It is preferable that the route of administration of the pharmaceutical composition of the present invention be determined depending on the type of disease to which it is applied.
  • the pharmaceutical composition of the present invention can be manufactured in a unit dosage form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains, and the method can be performed.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.
  • CD5 is a target cell surface protein used in CAR-T therapy to treat T-cell tumors.
  • CAR-T therapy to treat T-cell tumors.
  • two new CD5-targeting scFvs for the antigen-binding region of the CAR structure were discovered through phage display.
  • the scFv proteins were conjugated to the CD5-positive Jurkat cell line and the CD5-negative NLAM-6 cell line, which constitutively express CD5 on their cell surfaces, and binding was confirmed by flow cytometry (Fig. 1). Comparison with the negative control NALM-6 cell line and the isotype control confirmed binding of the two discovered scFvs to the Jurkat cell line.
  • the structure of the CD5 CAR comprised in the CD5 CAR-T therapeutic agent for T cell treatment of the present invention is shown in Fig. 2.
  • the CD5 CAR has a structure in which a CD8 signal sequence domain; an anti-CD5 scFv sequence (VL-linker-VH domain); a c-myc-derived myc-tag domain; a CD8-derived hinge domain; a CD8-derived transmembrane domain; a 4-1BB-derived intracellular signaling domain; a CD3z-derived intracellular signaling domain and a stop codon, TAA, are sequentially linked.
  • Three types of CAR structures are shown in Fig. 2 according to the type of anti-CD5 scFv sequence, among which H65 scFv is a positive control scFv.
  • each CAR molecule composed of the developed scFvs was transduced into the CD5-negative K562 cell line.
  • CAR molecule expression was confirmed in K562 cell lines expressing each CAR molecule using a myc-tag (Fig. 3a).
  • Recombinant human CD5 protein was bound to the three manufactured cell lines and the untransduced K562 cell line, and the degree of binding according to the amount of recombinant human CD5 protein treated was analyzed by flow cytometry (Fig. 3b). It was confirmed that the CD5 binding affinity of the CAR molecules of each structure was no different.
  • a mutant CD5 (DX23) was prepared by deleting the domain 1 (D1), which is the most distant from the membrane among the three extracellular domains of the CD5 protein
  • a mutant CD5 (DXX3) was prepared by deleting the extracellular domain 1 and the extracellular middle domain 2, and wild-type CD5.
  • Each CD5 sequence was sequentially followed by a P2A self-cleaving peptide sequence linked to glycine (G)-serine (S)-glycine (G), an eGFP sequence, and a stop codon TAA (Fig. 4a).
  • the three mutant CD5 sequences were transduced into K562 cells, a CD5-negative cell line, and the transduction efficiency was analyzed by flow cytometry (Fig. 4b).
  • the CD5 CAR molecule shown in Fig. 2 was transduced into T cells.
  • the measured raw values were normalized to the GFP expression level of the cell lines used in each co-culture (Fig. 4c).
  • CD4-positive or CD8-positive T cells were isolated and purified from peripheral blood mononuclear cells (PBMCs) of healthy donors.
  • the purified T cells were designated “TCs.”
  • the frozen TCs were thawed, and T cell activation was induced for 48 hours using TransAct coated with CD3 and CD28 antibodies.
  • the activated T cells were designated “ATs.”
  • the ATs were treated with a lentiviral vector to transduce the CD5 CAR gene and cultured for 24 hours to transduce the CD5 CAR gene.
  • the transduced T cells were harvested and cultured in CAR-T culture medium for an additional 10 days.
  • the cultured T cells were designated “CTs.”
  • H65CAR5 transduced with H65 CAR, A2CAR5 transduced with A2 CAR, and C7CAR5 transduced with C7 CAR were harvested, and CAR molecule expression was confirmed using myc-tag (Fig. 5).
  • H65CAR5 and A2CAR5 showed similar CAR molecule positivity rates, while C7CAR5 showed a statistically significant lower CAR molecule positivity rate (*p ⁇ 0.05, **p ⁇ 0.01, *** ⁇ 0.001, ns: not significant).
  • T-cell-targeting CARs can lead to fratricide, a phenomenon in which CAR-T cells kill each other during culture. This phenomenon results in negative outcomes, including low cell viability, poor growth, cytokine release during culture, and cell exhaustion due to continuous stimulation.
  • Fig. 6a H65CAR5 and A2CAR5 showed similar cell viability, while C7CAR5 showed a statistically significant higher cell viability (*p ⁇ 0.05, **p ⁇ 0.01, *** ⁇ 0.001, ns: not significant).
  • the growth of CD5 CAR-transduced T cells was measured. The number of CD5 CAR-transduced T cells after 10 days of additional culture was measured compared to the number of CD5 CAR-transduced T cells after 3 days of transduction and additional culture, and the growth rate of CD5 CAR-transduced T cells when cultured for 7 days was confirmed (Fig. 6b).
  • H65CAR5 and A2CAR5 showed similar cell growth rates, while C7CAR5 showed a statistically significant higher cell growth rate (*p ⁇ 0.05, **p ⁇ 0.01, *** ⁇ 0.001, ns: not significant).
  • C7CAR5 showed a statistically significant higher cell growth rate (*p ⁇ 0.05, **p ⁇ 0.01, *** ⁇ 0.001, ns: not significant).
  • cytokines released into the culture medium during the culture process was measured (Fig. 7). A portion of the culture supernatant was recovered and stored in a -80°C ultra-low temperature freezer. After the culture procedure was completed, the stored culture supernatant was thawed, and the concentrations of INF- ⁇ and TNF- ⁇ cytokines were measured using a cytometric bead array. It was confirmed that both cytokines were released at a statistically significantly lower level in C7CAR5 after 6 days of additional culture (*p ⁇ 0.05, **p ⁇ 0.01, *** ⁇ 0.001, ns: not significant). This confirmed that C7CAR5 among CD5 CAR-transduced T cells exhibited a lower level of autophagy.
  • CD5 CAR transduced T cells were harvested and centrifuged at 600 x g for 3 minutes at room temperature. The supernatant was removed, suspended in assay medium, and the cell number was measured. The suspension was diluted to 3 x 10 3 cells/100 ⁇ L using assay medium to prepare CAR-positive cells. GFP fluorescent target cell lines were added to a 96-well plate at 1 x 10 4 cells/well (1 x 10 5 x cells/mL, 100 ⁇ L). 100 ⁇ L of CD5 CAR transduced T cells prepared so that the ratio of E (CD5 CAR transduced T cells): T (CD5 positive (Jurkat) or negative (NALM-6) target cell line) was 0.3:1 was added to each well.
  • a Target Only group was also prepared by adding 100 ⁇ L of assay medium instead of CD5 CAR transduced T cells.
  • the amount of GFP fluorescence emitted from each well was calculated as the amount of target cell line by taking advantage of the fact that the killed target cell line loses GFP fluorescence.
  • GFP fluorescence was measured using an IncuCyte real-time cell analysis system and normalized using the following formula: (total GFP fluorescence per well at each measurement time point / total GFP fluorescence per well at the first measurement time point) * 100 (Fig. 9).
  • the tumor killing ability of CD5 CAR-transduced T cells against the Jurkat cell line, a CD5-positive cell line was observed.
  • NOG mice specific pathogen-free mice (SPF) NOD.Cg-Prkdc scid IL2 ⁇ g tm1Sug /JicKoat mice (manufactured by Coretech Co., Ltd.) were used.
  • Jurkat cells expressing firefly luciferase to be used for mouse tumor transplantation were prepared by thawing the frozen cell line and subculturing once every 3 to 4 days. All cancer cells were harvested and centrifuged on the day of tumor transplantation into the mouse. The supernatant was removed, suspended using cell line culture medium, and the cell number was measured.
  • a cell suspension was prepared at a concentration of 5 x 10 6 cells/mL using D-PBS.
  • the prepared cell suspension was injected into the tail vein of one mouse for each experimental group at 0.2 mL, and cancer cells were transplanted at 1 x 10 6 cells/head.
  • 200 ⁇ L of D-luciferin at a concentration of 15 mg/mL was intraperitoneally administered per mouse (20-25 g body weight), and then the mice were anesthetized with 2% isoflurane for inhalation.
  • Small animal bioimaging Perkin Elmer, IVIS Spectrum Series was performed 10-15 minutes after D-luciferin administration to confirm cancer cell transplantation.
  • CD5 CAR-transduced T cells Eight days after cancer cell transplantation, cultured CD5 CAR-transduced T cells were harvested and centrifuged at 600 xg for 3 minutes at room temperature. The supernatant was removed, suspended in assay medium, and the cell number was measured. Centrifugation was performed again, the supernatant was removed, and a cell suspension was prepared using D-PBS at a concentration of 5 x 10 6 cells/mL based on CD5 CAR-positive T cells. The prepared cell suspension was injected into the tail vein of one mouse for each experimental group at 0.2 mL, and CD5 CAR-transduced T cells were transplanted at 1 x 106 cells/head.
  • small animal bio-imaging was performed on all animals once or twice a week during the experimental period to monitor the amount of cancer cells. In addition, all animals were observed for death to evaluate the survival rate (Fig. 10).
  • the small animal bio-imaging results showed that C7CAR5 exhibited more sustained anti-cancer efficacy than the other groups (Fig. 10a).
  • the graph of the small animal bio-imaging results for each individual mouse also showed that C7CAR5 exhibited more sustained anti-cancer efficacy than the other groups (Fig. 10b).
  • the survival rate results showed that C7CAR5 survived longer than the other groups (Fig. 10c). This confirmed that C7CAR5 among CD5 CAR-transduced T cells had a higher in vivo tumor cell killing ability.
  • C7CAR5 has a lower degree of self-killing and superior in vivo tumor cell killing ability than H65CAR5 and A2CAR5, which utilize the existing positive control H65 scFv.
  • the target binding site of C7CAR5 is the membrane-proximal domain, unlike the other CAR5s. Therefore, in order to invent a superior CAR structure in the composition of CAR-T therapeutics for treating T cell tumors, the present inventors discovered a CD5 membrane-proximal domain-targeting scFv to be newly applied to the antigen-binding site in the CAR structure through the phage display method.
  • the scFv protein was bound to a CD5-negative K562 cell line, a K562 cell line transduced with wild-type CD5, and a K562 cell line transduced with a mutant CD5 (DXX3) that retains only the membrane-proximal domain, and the binding was confirmed through flow cytometry (Fig. 11).
  • a number of scFvs binding to the K562-CD5 (DXX3) cell line were identified, and among them, three scFvs that also bind to K562-CD5 (WT) were finally selected.
  • the structure of the CD5 CAR comprised in the CD5 CAR-T therapeutic agent for T cell treatment of the present invention is shown in Fig. 12.
  • the CD5 CAR has a structure in which a CD8 signal sequence domain; an anti-CD5 scFv sequence (VL-linker-VH domain); a c-myc-derived myc-tag domain; a CD8-derived hinge domain; a CD8-derived transmembrane domain; a 4-1BB-derived intracellular signaling domain; a CD3z-derived intracellular signaling domain and a stop codon, TAA, are sequentially linked.
  • Six types of CAR structures are shown in Fig. 12 according to the type of anti-CD5 scFv sequence. Among the six types of CAR structures, two types of CARs including the positive control H65 scFv target the CD5 membrane-distal domain, and four types of CAR structures target the CD5 membrane-proximal domain.
  • each CAR molecule composed of the developed scFvs was transduced into the CD5-negative K562 cell line.
  • the expression of the CAR molecules in the K562 cell lines expressing each CAR molecule was confirmed using the myc-tag (Fig. 13a).
  • Recombinant human CD5 protein was bound to the six manufactured cell lines and the untransduced K562 cell line, and the degree of binding was analyzed by flow cytometry (Fig. 13b).
  • the crude results were normalized by dividing the binding affinity to the recombinant human CD5 protein by the expression level of the CAR molecule (Fig. 13c). It was confirmed that the binding affinity of the CAR molecules of each structure to CD5 did not differ (*p ⁇ 0.05, **p ⁇ 0.01, *** ⁇ 0.001, ns: not significant).
  • the CD5 CAR molecule shown in Fig. 12 was transduced into T cells.
  • the measured raw values were normalized to the GFP expression level of the cell line used in each co-culture (Fig. 14).
  • all three identified scFvs, C11CAR5, F8CAR5, and D9CAR5 released INF- ⁇ even when only the CD5 membrane-proximal domain was present. Therefore, it was confirmed that the C11, F8, and D9 scFvs bind to the extracellular membrane-proximal domain.
  • CD5 CAR-transduced T cells were prepared in the same manner as above. NTD without CAR gene transduction, H65CAR5 transduced with H65 CAR, A2CAR5 transduced with A2 CAR, C7CAR5 transduced with C7 CAR, C11CAR5 transduced with C11 CAR, F8CAR5 transduced with F8 CAR, and D9CAR5 transduced with D9 CAR were harvested, and the expression of CAR molecules was confirmed at each time point using myc-tag (Fig. 15).
  • cell viability was measured at the end of culture (Fig. 16a). H65CAR5 and A2CAR5 showed similar cell viability, and CAR5 targeting the CD5 membrane-proximal domain showed a statistically significant higher cell viability (*p ⁇ 0.05, **p ⁇ 0.01, *** ⁇ 0.001, ns: not significant).
  • the growth potential of the entire cell was measured (Fig. 16b). The total number of cells after the end of culture was measured relative to the number of cells used for transduction to determine the growth potential of the entire cell during the entire culture period.
  • H65CAR5 and A2CAR5 showed similar total cell growth rates, and CAR5 targeting the CD5 membrane-proximal domain showed a statistically significant higher total cell growth rate (*p ⁇ 0.05, **p ⁇ 0.01, *** ⁇ 0.001, ns: not significant).
  • the growth potential of CD5 CAR-transduced T was measured. The number of CD5 CAR-transduced T cells after 10 days of additional culture was measured compared to the number of CD5 CAR-transduced T cells after 3 days of transduction and additional culture, and the growth rate of CD5 CAR-transduced T cells when cultured for 7 days was confirmed (Fig. 16c).
  • H65CAR5 and A2CAR5 showed similar cell growth rates, and D9CAR5 showed a statistically significant higher CD5 CAR-transduced T cell growth rate (*p ⁇ 0.05, **p ⁇ 0.01, *** ⁇ 0.001, ns: not significant). This confirmed that among CD5 CAR-transduced T cells, CAR5 targeting the CD5 membrane-proximal domain had a low level of self-killing.
  • the in vivo tumor cell killing ability was analyzed in the same manner as above (Fig. 19).
  • Small animal bioimaging results showed that the CD5 membrane-proximal domain-targeting CAR5 group exhibited stronger tumor cell killing ability than the CD5 membrane-distal domain-targeting CAR5 group. This confirmed that among CD5 CAR-transduced T cells, the CD5 membrane-proximal domain-targeting CAR5 group had a higher tumor cell killing ability in vivo.

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Abstract

La présente invention concerne un nouvel anticorps dirigé contre CD5 et ses utilisations. L'anticorps selon la présente invention présente une affinité de liaison élevée et une grande activité inhibitrice contre la protéine CD5, et peut ainsi être utilisé en tant qu'agent thérapeutique efficace pour diverses maladies cancéreuses à médiation par CD5.
PCT/KR2024/011252 2024-04-18 2024-07-31 Nouvel anticorps dirigé contre cd5 et ses utilisations Pending WO2025220804A1 (fr)

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KR20240051967 2024-04-18
KR10-2024-0051967 2024-04-18
KR1020240101587A KR20250154905A (ko) 2024-04-18 2024-07-31 Cd5에 대한 신규 항체 및 이의 용도
KR10-2024-0101587 2024-07-31

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