WO2018030806A1 - Cytokine fusionnée à un hétérodimère fc d'immunoglobuline et composition pharmaceutique la contenant - Google Patents

Cytokine fusionnée à un hétérodimère fc d'immunoglobuline et composition pharmaceutique la contenant Download PDF

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WO2018030806A1
WO2018030806A1 PCT/KR2017/008676 KR2017008676W WO2018030806A1 WO 2018030806 A1 WO2018030806 A1 WO 2018030806A1 KR 2017008676 W KR2017008676 W KR 2017008676W WO 2018030806 A1 WO2018030806 A1 WO 2018030806A1
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region
cells
protein
domain
mil
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PCT/KR2017/008676
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English (en)
Korean (ko)
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김용성
정근옥
하지희
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아주대학교산학협력단
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Priority to EP17839824.4A priority Critical patent/EP3511340A4/fr
Priority to US16/323,839 priority patent/US10696722B2/en
Application filed by 아주대학교산학협력단 filed Critical 아주대학교산학협력단
Priority to BR112019002394-1A priority patent/BR112019002394B1/pt
Priority to JP2019506697A priority patent/JP6993403B2/ja
Priority to CN201780062851.0A priority patent/CN110267977A/zh
Priority to SG11201901071TA priority patent/SG11201901071TA/en
Priority claimed from KR1020170101594A external-priority patent/KR102050463B1/ko
Priority to CA3033475A priority patent/CA3033475A1/fr
Priority to MX2019001651A priority patent/MX2019001651A/es
Priority to AU2017310163A priority patent/AU2017310163B2/en
Publication of WO2018030806A1 publication Critical patent/WO2018030806A1/fr
Priority to ZA2019/00772A priority patent/ZA201900772B/en
Priority to US16/886,177 priority patent/US11078249B2/en
Priority to US16/886,184 priority patent/US11692019B2/en
Priority to AU2021273642A priority patent/AU2021273642A1/en
Priority to US18/323,124 priority patent/US20230416325A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/24—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]

Definitions

  • the present invention includes a first Fc region and a second Fc region of an antibody (immunoglobulin) heavy chain constant region (Fc) pair,
  • Heterodimeric Fc-fused protein having a subunit of a bioactive protein bound to at least one of the N-terminus or C-terminus of the first Fc region and / or the second Fc region.
  • the first Fc region and the second Fc region are heterodimeric Fc-fused proteins, characterized in that the CH3 domain is mutated to promote the formation of heterodimers and
  • It relates to a pharmaceutical composition comprising the heterodimer-fusion protein.
  • Heterodimeric Fc-fused protein according to the present invention is a form and structure in which two or more subunits form a protein complex so that a protein constituting one bioactive protein exists in nature. It can be fused to the antibody heavy chain constant region (Fc), there is an advantage that can maintain the activity as it exists in nature.
  • Fc antibody heavy chain constant region
  • the half-life of the bioactive protein contained in the heterodimer-fusion protein is significantly increased, and various biological activities in the body are maintained for a long time. There is an advantage that can be sustained.
  • heterodimer-fusion protein in which the subunit of the bioactive protein is fused to the N-terminus or C-terminus of the antibody heavy chain constant region heterodimer Fc according to the present invention is expressed after purification, compared with the wild type Fc-based fusion protein. This is easy.
  • Immunoglobulin G IgG
  • IgM immunoglobulin M
  • IgD immunoglobulin D
  • IgE immunoglobulin A
  • homodimerization between two identical heavy chains is performed by the final domain of the constant region of the antibody (CH3 domain for IgG, IgD, IgA, CH4 domain for IgM, CH2 and CH4 domain for IgE). It is induced by non-covalent interactions between and disulfide bonds between hinge regions.
  • Antibody-derived heterodimeric heavy chain constant region (Hterodimeric Fc) technology allows specific non-covalent linkages between the last domain of the constant region (CH3 domain), which contributes significantly to the homologous duplication of earlier spontaneous antibodies (IgG, IgM, IgA, IgD, IgE). Heterogeneous redundancy is preferred, and it is a technique to create heterologous heavy chain invariant regions by engineering them to have a bond that is not preferred or rejected. More specifically, genetic modifications induce mutations in the CH3 domains of two different antibody heavy chains, forming two heterozygotes with very similar structures to naturally occurring antibodies and minimal variation in sequence. (US Patent No. 7,695,936; Korean Patent No. 1,522,954).
  • the heterodimeric heavy chain constant region technology is a basic technology for making double antibodies.
  • CH3 domain mutants known to induce heterodimers are known to be mostly asymmetric by structure-based rational design of the antibody on the surface of CH3 domain interaction. It was prepared by introducing a red pair of mutations (Spreter Von Kreudenstein et al., 2014).
  • the A107 mutant used in the present invention is a high yield heterodimeric heavy chain constant region selected from a human antibody heteroduplex heavy chain constant region library prepared using a yeast cell surface expression system, and a hydrophobic core of the CH3 domain interaction surface. Induces mutations in the conserved and charged amino acids to form complementary hydrophobic bonds (K409W CH3A -D399V / F405T CH3B ) and to form hydrogen bonds (K370E CH3A -E357N CH3B ) to form heterodimers (Choi et al. 2016; Korean Patent Application No. 2015-0142181).
  • the native protein complex structure existing in nature is formed as it is, and the activity of the original protein is not only properly expressed. There is no such form of development that can be sustained for a long time.
  • heterodimer variants comprising Fc regions from IgG1 as well as other previously reported isotype antibodies such as IgG2, IgG3, and IgG4, Using this, two or more different subunits are formed, and two or more subunits form one protein complex to bind one or more subunits of a protein showing physiological activity to the ends of the Fc region.
  • the present invention was completed by developing a novel therapeutic fusion protein in the form of a heterodimeric Fc-fused protein.
  • a protein consisting of two or more different subunits in the present invention, wherein the two or more subunits form a protein complex and exhibit physiological activity is preferably Interleukin-12, IL-12) can be used.
  • Interleukin-12 increases the activity of immune cells, such as cytotoxic T Lymphocytes (CTL) and natural killer cells (NK), among other immune cells, to directly kill tumors or interferon Inhibits tumorigenesis by activating the immune response in the tumor microenvironment where the immune response is suppressed through the secretion of pro-inflammatory cytokine such as gamma (IFN- ⁇ )
  • CTL cytotoxic T Lymphocytes
  • NK natural killer cells
  • an optional tag for further purification only at the C terminus of one Fc region as shown in FIGS.
  • the method of constructing a fusion protein has been used by fusion or by purifying the Fc region and protein separately in high purity.
  • this form is not only costly in producing large amounts of protein, but also requires research to further optimize the purification process.
  • the problem to be solved in the present invention is a heterozygote-fusion heterozygous form of a new type of antibody heavy chain constant region in which two or more subunits form a protein complex so that the activity of the protein showing physiological activity can be sufficiently maintained for a long time. It is to provide a protein (heterodimeric Fc-fused protein).
  • the antibody heavy chain constant region heterodimeric Fc-fused protein according to the present invention has two or more subunits that form a protein complex to form a protein complex that exhibits physiological activity as it exists in nature. By simulating as much as possible, it is a form which can maintain the activity as it exists in nature.
  • the half-life of the fusion protein is significantly increased by fusion of the antibody heavy chain constant region, so that various physiological activities in the body can be maintained for a long time.
  • the present invention also provides a pharmaceutical composition comprising the antibody heavy chain constant region heterodimeric Fc-fused protein, and a composition and treatment method for the treatment of diseases, in particular cancer, using the same. do.
  • the present invention includes a first Fc region and a second Fc region of an antibody (immunoglobulin) heavy chain constant region (Fc) pair,
  • heterodimeric Fc-fused protein in which a bioactive protein is bound to at least one of the N-terminus or C-terminus of the first Fc region and / or the second Fc region,
  • the physiologically active protein is composed of two or more different subunits (subunit), two or more different subunits (subunit) is characterized in that to form a protein complex (protein complex) to show the physiological activity,
  • the first Fc region and the second Fc region provide a heterodimeric Fc-fused protein, characterized in that the CH3 domain is mutated to promote formation of a heterodimeric Fc.
  • the present invention also provides a pharmaceutical composition comprising the heterologous-fusion protein and a composition and a method for the treatment of diseases, in particular cancer, using the same.
  • 1 (A) to 1 (C) is a diagram showing a strategy for obtaining a fusion protein in the form of monomers and heterodimers using existing wild-type human antibody heavy chain constant region.
  • FIG. 1 (D) shows an antibody-cytokine (Immunocytokine) construct by fusing monomeric cytokines to an IgG-type antibody containing a KnoH-in-hole heterodimeric heavy chain constant region (KiH heterodimeric Fc) variant in the existing literature. It is a figure which shows an example.
  • Figure 2 (A) and 2 (B) is a diagram showing the monomer and heterodimer fusion protein forms that can be constructed using heterodimeric heavy chain constant region.
  • Figure 2 (C) is a diagram showing a heterodimeric fusion protein form in a human antibody of the IgG form containing a heterodimeric heavy chain constant region.
  • Figure 3 is a view showing the results of selecting and comparing the sequence of the sequence of each human antibody immunoglobulin G isotype CH3 domain for the production of CH3 domain variants for heterodimer formation by human antibody isotype.
  • FIG. 4 is a structural modeling of heterologous heavy double chain constant region variants of the heterologous type having the mutation-induced sequence at the position selected in FIG. 3, and the resulting modeling structure is compared with the wild type IgG1 based A107 mutant and It is a figure which shows the result of an analysis.
  • FIG. 5 is a schematic diagram of a vector for expressing heterologous heavy chain constant regions of homologous type constructed by sequence and structural analysis in animal cells. Heterozygous heavy chain constant region variants of each homotype, including the mutated hinge region, were cloned into the vector using the restriction enzyme NotI / HindIII.
  • FIG. 6 is a schematic diagram schematically illustrating a scFv-Fc CH3A / Fc CH3B co-expression system for evaluating the degree of heterodimer formation of heteroduplex heavy chain constant region variants due to the duplex size difference of the expressed protein. to be.
  • FIG. 7 shows a single-chain antibody fragment (scFv) fused with a single chain antibody fragment (scFv) constructed to evaluate the formation yield of heteroduplex of the antibody heavy chain constant region by the CH3 mutation pair as shown in FIG. 6. .1 Schematic diagram for cloning into vectors.
  • Figure 8 is a transient expression of co-transformation in HEK293F cells for evaluating the formation ability of the heteroduplex described in Figure 6 animal cell expression vector introduced with a CH3 mutation pair constructed in accordance with the expression system described in Figures 5 and 7 And, after purification, 5 ⁇ g of protein was isolated on SDS-PAGE under non-reducing conditions for evaluation of heterodimer antibody formation ability, and analyzed by size and combination through Coomasie Blue staining. At this time, wild type Fc using wild type CH3 was used as a negative control.
  • FIG. 9 is a diagram showing the result of Western blot using the anti-human IgG-AP conjugated antibody with AP enzyme after protein separation by SDS-PAGE in the same manner as in FIG. 8. .
  • Figure 10 (A) is a schematic diagram showing the form of the endogenous interleukin 12 cytokine unfused to the heavy chain constant region, which is a control of the present invention.
  • FIG. 10 (B) is a schematic diagram showing the form of a bi-IL-12-Fc fusion protein in which an interleukin 12 cytokine linked with an amino acid linker is fused to a wild-type IgG4 Fc as a comparative example of the present invention.
  • FIG. 10 (C) is a schematic diagram showing the form of a mono-IL-12-Fc fusion protein in which the interleukin 12 cytokine is fused with a ⁇ 4-A107 variant made of IgG4 in a heterologous heavy double chain constant region variant of the present invention. to be.
  • FIG. 10C are schematic diagrams of vectors for expressing and purifying the fusion protein of the embodiment of the present invention (FIG. 10C) in animal cells.
  • Figure 12 is a schematic diagram of a vector for the expression and purification of the fusion protein of the comparative example (Fig. 10 (B) in the animal cell of the present invention.
  • FIG. 13 transiently expresses and purifies the animal cell expression vectors of FIGS. 11 (A) and (B) constructed with human and mouse interleukin genes through cotransformation into HEK293F cells, and then amplifies 5 ⁇ g of protein. It is a diagram showing the results of the analysis on the size and combination form by separating on the SDS-PAGE of the condition, Coomasie Blue staining.
  • FIG. 14 shows the results of analyzing the fusion proteins of FIG. 13 using size exclusion chromatography.
  • FIG. 15 shows peripheral blood immune cells (PHA-activated PBMCs) that induced receptors for IL-12 by treating normal peripheral blood immune cells (normal PBMCs) having no receptor for IL-12 and phytohaemagglutinin (PHA). It is a figure which shows the result of having confirmed the binding ability of the mono-hIL-12-Fc and the wild type bi-hIL-12-Fc which were constructed about with the flow cytometer (FACS).
  • PHA-activated PBMCs peripheral blood immune cells
  • FACS flow cytometer
  • FIG. 16 shows Fc (A107), recombinant human IL-12 (rhIL-12), and bi-hIL-12 in peripheral blood immune cells (PHA-activated PBMCs) inducing receptors for IL-12 by treating PHA, a mitogen.
  • Figure showing the results of measuring the cell proliferation according to the concentration-specific treatment of -Fc and mono-hIL-12-Fc through the WST-1 cell proliferation assay.
  • FIG. 17 is a view showing the results of measuring the concentration of IFN- ⁇ in the culture supernatant cultured in Figure 16 by ELISA
  • FIG. 18 shows normal peripheral blood binding ability of mono-mIL-12-Fc and bi-mIL-12-Fc constructed using the property that mouse IL-12 binds to human IL-12 receptor as well as mouse IL-12 receptor.
  • PHA peripheral blood immune cells
  • FIG. 19 shows Fc (A107), recombinant mouse IL-12 (rmIL-12), bi-mIL-12-Fc in peripheral blood immune cells (PHA-activated PBMCs) inducing receptors for IL-12 by PHA treatment.
  • cell proliferation according to the concentration-specific treatment of mono-mIL-12-Fc is a diagram showing the results measured by the WST-1 cell proliferation assay.
  • Figure 20 (A) shows Fc (A107), rmIL-12, bi-mIL-12-Fc and mono-mIL-12-Fc when the tumor size is 100 mm3 in Balb / c mice transplanted with CT26 HER2 / Neu cancer cells It is a diagram showing the results of confirming the change in tumor volume measured during intraperitoneal administration and the size of tumor after lethality of mice at the end of administration.
  • FIG. 20 (B) is a graph showing the weight change of the mouse periodically measured during the experiment of FIG. 20 (A).
  • FIG. 21 (A) shows bi-mIL-12-Fc and mono-mIL-12-Fc intraperitoneally administered twice a week at the tumor size of 300 mm 3 in CT26 HER2 / Neu transplanted Balb / c mice. It is a figure which measured the change of the tumor volume.
  • Figure 21 (B) is a graph showing the change in the volume of the tumor of the individual mice periodically measured in the experimental procedure of Figure 21 (A).
  • Figure 21 (C) is a diagram showing the result of confirming the tumor size by killing the mouse 3 days after the last administration of Figure 21 (A).
  • Figure 21 (D) is a graph showing the weight change of the mouse periodically measured in the experimental procedure of Figure 21 (A).
  • FIG. 21 (E) is a graph of alanine aminotransferase (ALT) which is an indicator of liver toxicity by collecting blood from the facial vein of the mouse on day 1 after the last administration of FIG. 21 (A).
  • ALT alanine aminotransferase
  • Figure 22 (A) is a graph measuring the increase in the number of CD4 + T cells, CD8 + T cells and NK cells in the spleen after 3 days of the last administration of Figure 21 (A).
  • Figure 22 (B) is a view showing the total number of immune cells, CD4 + T cells, CD8 + T cells infiltrated into the tumor after 3 days of the third administration of Figure 21 (A).
  • Figure 23 (A) is the result of measuring the IFN-g concentration in the serum isolated by collecting blood from the facial vein of the mouse 24 hours after the last administration of Figure 21 (A) by ELISA.
  • FIG. 23 (B) shows bi-mIL-12-Fc and mono-mIL-12 at a molar concentration of 1 ⁇ g rmIL-12 when the tumor size was 300 mm 3 in Balb / c mice transplanted with CT26 HER2 / Neu cancer cells.
  • Figure 23 (C) is a graph measuring the cytotoxic effect on CT26 HER2 / Neu cancer cells of cytotoxic T cells isolated from the spleen by killing the mouse 3 days after the last administration of Figure 21 (A).
  • Figure 23 (D) shows the cytotoxic effect of cytotoxic T cells isolated from the spleen by killing mice 3 days after the third administration of Figure 21 (A) does not express CT26 HER2 / Neu cancer cells expressing tumor antigens It is a figure analyzed by the flow cytometer using 4T1 cells.
  • Figure 23 (E) is a graph measuring the cytotoxic effect on CT26 HER2 / Neu cancer cells of natural killer cells isolated from the spleen by killing the mouse 3 days after the third administration of Figure 21 (A).
  • Figure 24 (A) is a graph measuring the number of CD8 + effect T cells isolated from the spleen by lethal mice three days after the last administration of Figure 21 (A).
  • Figure 24 (B) is a graph measuring the number of CD8 + effect memory T cells isolated from the spleen by lethal mice three days after the last administration of Figure 21 (A).
  • Figure 24 (C) is a graph measuring the number of CD8 + central memory T cells isolated from the spleen by killing the mouse three days after the last administration of Figure 21 (A).
  • FIG. 24 (D) shows CT26 HER2 / Neu in Balb / c mice of the same age as those surviving 120 days after administration of 1 ⁇ g mono-IL-12-Fc in FIG. 21 (A). Cancer cells are transplanted to measure the change in the tumor volume of the mouse.
  • FIG. 24 (E) shows memory precursor effect cells (KLRG1 - IL-7R + ) and short-lived effect cells (KLRG1) among CD8 + T cells present in the spleen after 3 days of the third administration of FIG. 21 (A).
  • + IL-7R - is a diagram of analyzing the percentage of by flow cytometry (flow cytometry).
  • FIG. 25 (A) shows the ratio of CD8 + T cells with high expression of T-bet, a transcription factor that inhibits the differentiation of memory cells by killing mice after 3 days of the third administration of FIG. 21 (A). It is a graph analyzed by measuring with a flow cytometer.
  • FIG. 25 (B) shows CD8 + T with high expression of Eomes and low expression of T-bet that promote the differentiation of memory cells by killing mice after 3 days of the third administration of FIG. 21 (A). It is a graph analyzed by measuring the percentage of cells by flow cytometry.
  • Figure 25 (C) shows CT26 HER2 / Neu Balb / c mice transplanted with cancer cells were intraperitoneally administered with bi-mIL-12-Fc and mono-mIL-12-Fc at a molar concentration of 1 ⁇ g rmIL-12 when the tumor size was 300 mm 3. After time, the expression level of phosphorylated STAT4 in CD8 + T cells isolated from inguinal lymph nodes was measured by flow cytometry.
  • Figure 25 (D) is a graph measuring the ratio of CD8 + T cells expressing T-bet inhibiting the differentiation of memory cells in the inguinal lymph nodes 72 hours after one intraperitoneal administration of Figure 25 (C) by flow cytometry.
  • FIG. 25 (F) shows that CD8 + T cells isolated from the spleen and inguinal lymph nodes of normal Balb / c mice were stimulated with mono-mIL-12-Fc and bi-mIL-12-Fc cross-linked with an antibody that recognizes Fc.
  • the ratio of CD8 + T cells expressing T-bet is a graph measured by flow cytometry.
  • FIG. 26 is a schematic diagram showing the differentiation-induced mechanism of memory precursor effector and memory cells induced by mono-mIL-12-Fc and the differentiation-induced mechanism of short-lived effect cells induced by bi-mIL-12-Fc.
  • the present invention includes a first Fc region and a second Fc region of an antibody (immunoglobulin) heavy chain constant region (Fc) pair,
  • heterodimeric Fc-fused protein in which a bioactive protein is bound to at least one of the N-terminus or C-terminus of the first Fc region and / or the second Fc region,
  • the bioactive protein is composed of two or more different subunits (subunit), the two or more different subunits (subunit) is characterized in that the protein complex (protein complex) to form a physiological activity,
  • the first Fc region and the second Fc region are for a heterodimeric Fc-fused protein, characterized in that the CH3 domain is mutated to promote formation of a heterodimer.
  • Fc region or “heavy chain constant region” means a region including a CH2 domain, a CH3 domain, and a hinge domain derived from an antibody. However, in the case of IgE, it means a region including a CH2 domain, a CH3 domain, a CH4 domain, and a hinge domain.
  • the expression “the first Fc region and the second Fc region are mutated to promote the formation of heterodimers” indicates that antibodies present in nature are homodimers in which two Fc regions have the same sequence. (homodimer) form, which causes mutations in some sequences of these Fc regions, thereby promoting the formation of heterodimers through specific non-covalent linkages between the first and second Fc regions and homodimers. It means that the formation of is reduced or preferably mutated so that it hardly occurs.
  • the mutations to facilitate the formation of a heterodimer of the first Fc region and the second Fc region according to the present invention are each of the CH3 domains included in the antibody-derived first and second Fc regions. May include mutations that facilitate the formation of this heterodimer.
  • heterodimeric Fc or Fc heterodimer includes a first Fc region and a second Fc region, and the first Fc region and the second Fc region are heterodimers. It means a heterodimer, characterized in that the CH3 domain is modified to promote the formation of).
  • the first Fc region and the second Fc region in the present invention may be derived from an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, and IgE, respectively, preferably Wherein the first Fc region and the second Fc region are derived from IgG1, IgG2, IgG3, or IgG4, respectively,
  • first Fc region and the second Fc region may be characterized in that it is derived from an isotype antibody.
  • the mutation of the CH3 domain may be characterized in that it comprises one or more mutations selected from the following group. All mutation positions in the present invention are according to the EU index.
  • substitution of the amino acid residue at the K370 position of the CH3 domain of the first Fc region is K370E, K370R, K370M, K370D or K370H,
  • substitution of the amino acid residue at the E357 position of the CH3 domain of the second Fc region may be E357N, E357D, E357A, E357I, E357G or E357M, and the substitution of the amino acid residue at the S364 position may be S364T or S364W. .
  • substitution of the amino acid residue at the K409 position in the CH3 domain of the first Fc region is K409W
  • the substitution of the amino acid residue at the F405 position of the CH3 domain of the second Fc region is F405T
  • amino acid residue at the D399 position is The substitution may be characterized as being D399V.
  • amino acid residue change such as K370E means that the K at position 370 is changed to E, and all amino acid residue changes in the present invention are used as the same meaning.
  • the mutation of the CH3 domain of the first Fc region or the second Fc region may include one or more mutations selected from the following group. (However, the mutation position is according to the EU index)
  • the CH3 domains of the first Fc region and the second Fc region as described above may further include the following bonds.
  • the mutation of the CH3 domain may be characterized in that it comprises one or more mutations selected from the following group.
  • the substitution of the amino acid residue at the K360 position of the CH3 domain of the first Fc region is K360E
  • the substitution of the amino acid residue at the E347 position of the CH3 domain of the second Fc region may be E347R.
  • Substitution of the amino acid residue at the K409 position of the CH3 domain of the first Fc region is K409W
  • Substitution of the amino acid residue at the F405 position of the CH3 domain of the second Fc region is F405T
  • Substitution of the amino acid at the D399 position is It may be characterized by the D399V.
  • the mutation of the CH3 domain of the first Fc region or the second Fc region may include one or more mutations selected from the following group. (However, the mutation position is according to the EU index)
  • the CH3 domains of the first Fc region and the second Fc region as described above may further include the following bonds.
  • the CH3 domains included in the antibody-derived first and second Fc regions of the present invention are each
  • It may be characterized by having a sequence selected from the group consisting of the amino acid sequence represented by the sequence number of.
  • the antibody-derived first Fc region and the second Fc region preferably have a sequence of the CH3 domain described in Table 1 derived from IgG4.
  • the subunit of the bioactive protein may be bound only to one of the N-terminus or the C-terminus of the first Fc region or the second Fc region,
  • One or more different subunits of one (one) bioactive protein may be respectively bound to the N-terminus or C-terminus of the first Fc region and the second Fc region (FIG. 2 ( B) and FIG. 2 (C)).
  • the "subunit of the bioactive protein is bonded only to one terminal of either the N-terminus or the C-terminus of the first Fc region or the second Fc region", the first Fc region or the second Fc One of the subunit (s) of the bioactive protein is bound to either end of the N-terminal or C-terminal end of the region, and the remaining subunit (s) of the bioactive protein are linker-mediated.
  • the linker is preferably an amino acid linker, but is not limited thereto.
  • the one or more different subunits of one (one) bioactive protein is respectively bonded to each of the N-terminal or C-terminal of the first Fc region and the second Fc region.
  • One or more different subunit (s) of the bioactive protein are bound to both the first Fc region and the second Fc region N-terminus, respectively, or the bioactive protein is bound to both the C-terminus of the first Fc region and the second Fc region.
  • one or more different subunits of the bioactive protein are respectively bound to both the N-terminus and the C-terminus of the first Fc region and the second Fc region.
  • the binding of the terminal of the first Fc region and / or the second Fc region and the subunit of the bioactive protein is genetic fusion. It can be characterized by fused by
  • first Fc region and the second Fc region and a subunit of the bioactive protein may be combined in a linker-mediated form.
  • the linker is preferably an amino acid linker, but is not limited thereto.
  • heterodimeric-fusion protein heterodimeric Fc-fused protein
  • the bioactive protein is composed of two or more different subunits, and the two or more different subunits are characterized by forming a (one) protein complex to represent the biological activity.
  • the bioactive protein is selected from the group consisting of interleukin 12 (IL-12), interleukin 23 (IL-23), interleukin 27 (IL-27), interleukin 35 (IL-35), and follicle stimulating hormone (FSH).
  • IL-12 interleukin 12
  • IL-23 interleukin 23
  • IL-27 interleukin 27
  • IL-35 interleukin 35
  • FSH follicle stimulating hormone
  • the most preferred bioactive protein according to the present invention is interleukin 12 (IL-12).
  • IL-12 Interleukin-12
  • IL-12 consists of two subunits of p35 (IL-12A) and p40 (IL-12B), and its bioactive form is p70, a heterodimer of p35 and p40. . In nature, IL-12 must be present in the form of p70, a heterodimer of p35 and p40 in order to have activity.
  • the antibody heavy chain constant region heterologous double-fusion protein according to the present invention was implemented.
  • At least one subunit of a bioactive protein comprising a first Fc region and a second Fc region according to the present invention, at one or more of the ends of the first and second Fc regions Heterodimeric Fc-fused protein in the antibody heavy chain constant region to which (subunit) is bound,
  • At least one subunit (s) constituting one bioactive protein is bound only at either end of the N-terminus or C-terminus of the first Fc region and the second Fc region, and the rest
  • the subunit (s) may be linked by a linker
  • one or more different subunit (s) of one bioactive protein may be bound to each of the N-terminus and / or C-terminus of the first Fc region and the second Fc region, respectively. have.
  • the p35 or p40 subunit is attached to only one terminal of either the N-terminus or the C-terminus of the first Fc region or the second Fc region, and the remaining subunits are the first Fc region or the second Fc region.
  • the linker may be linked to a subunit of p35 or p40 that is bound to either the N-terminus or C-terminus of the Fc region to form a heterodimer-fusion protein (FIGS. 2B and 2C). ) Reference).
  • any one subunit selected from p35 and p40 is coupled to the N-terminus or C-terminus of the first Fc region, and the other subunit is N-terminus or the C-terminus of the second Fc region.
  • Units can form heterodimer-fusion proteins having a combined form (see FIGS. 2 (B) and 2C).
  • the physiologically active protein is interleukin 12 (IL-12),
  • the p35 or p40 subunit of interleukin 12 is bound to only one of the N-terminus or C-terminus of the first Fc region or the second Fc region, and the remaining subunits are linker-mediated. Form a subunit bound to either the N-terminus or C-terminus of the 1 Fc region or the second Fc region, or
  • P35 and p40 subunits of interleukin 12 are respectively bound to the N-terminus or C-terminus of the first Fc region and the second Fc region.
  • heterodimeric-fusion protein heterodimeric Fc-fused protein
  • the hinge region included in the N-terminus in the first Fc region and the second Fc region may be characterized by mutation of a cysteine residue included in the hinge region.
  • the mutation of the cysteine residue in the hinge region is a cysteine residue of the upper hinge region except for the cysteine residue in the core hinge region for heterodimer formation.
  • the first Fc region and the second Fc region are included in a whole antibody (whole antibody) form consisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE. can do.
  • the "whole antibody form” means the CH2 domain, CH3 domain and hinge region (including CH4 domain in IgE) in the Fc region in the case of IgG, IgA and IgD, in addition to the CH1 domain, VH.
  • intact form is meant an antibody comprising a domain, a CL domain and a VL domain.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody heavy chain constant region heterodimer-fusion protein according to the present invention.
  • the use of the pharmaceutical composition according to the invention is characterized in that it depends on the use of the bioactive protein contained in the antibody heavy chain constant heterodimer-fusion protein.
  • the bioactive protein contained in the antibody heavy chain constant region heterodimer-fusion protein according to the present invention is IL-12 or one or more subunits thereof, and thus the present invention includes IL-12 as a bioactive protein. It provides a pharmaceutical composition for the treatment of cancer comprising an antibody heavy chain constant region heterodimer-fusion protein.
  • Cancers treatable with a pharmaceutical composition for treating cancer comprising an antibody heavy chain constant region heterodimer-fusion protein comprising IL-12 or one or more subunits thereof as the bioactive protein include colorectal cancer, melanoma, breast cancer, Pancreatic cancer, kidney cancer, prostate cancer, ovarian cancer, small intestine cancer, esophageal cancer, cervical cancer, lung cancer, lymphoma and hematologic cancer may be selected from the group consisting of, but not limited to.
  • compositions according to the invention may additionally include a pharmaceutically acceptable carrier.
  • a “pharmaceutically acceptable carrier” is a substance that can be added to the active ingredient to help formulate or stabilize a formulation and does not cause significant deleterious toxic effects on the patient.
  • the carrier refers to a carrier or diluent that does not irritate the patient and does not inhibit the biological activity and properties of the heterodimer-fusion protein according to the present invention.
  • Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary.
  • diluents may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • Other carriers are described, for example, in Remington's Pharmaceutical Sciences (E. W. Martin).
  • compositions include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions for immediate administration.
  • the use of such media and agents for pharmaceutically active substances is known in the art.
  • the composition is preferably formulated for parenteral injection.
  • the compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.
  • the carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (eg glycerol, propylene glycol and liquid polyethylene glycols, etc.) and suitable mixtures thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition.
  • Sterile injectable solutions can be prepared by incorporating the required amount of the heterodimer-fusion protein in an appropriate solvent with one or a combination of ingredients described above as required, followed by sterile microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those described above.
  • sterile powders for the preparation of sterile injectable solutions some methods of preparation include vacuum drying and freeze-drying (freeze-drying), which produce a powder of the active ingredient and any further desired ingredients from its presterilized-filtered solution. )to be.
  • compositions according to the invention may be administered orally or parenterally at dosages and frequencies that may vary depending on the severity of the suffering patient.
  • the composition may be administered to the patient as a bolus or by continuous infusion as needed.
  • the pharmaceutical compositions according to the invention may be administered rectally, intravenously, subcutaneously, intrauterinely or intratracerebrovascularly, but are not limited thereto.
  • the pharmaceutical composition for treating cancer comprising the antibody heavy chain constant region heterodimer-fusion protein comprising IL-12 may be used for combination therapy with other anticancer agents, and the other anticancer agents may be cytotoxic T.
  • Cells and / or Natural Killer (NK) cells are preferred, but are not limited to any of the other anticancer agents that can be used in the art and can be used for combination therapy.
  • a pharmaceutical composition for treating cancer comprising an antibody heavy chain constant region heterodimer-fusion protein comprising interleukin 12 is used for combination treatment with cytotoxic T cells and / or Natural Killer (NK) cells. Occation,
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CTL cytotoxic T lymphocytes
  • CTLs cytotoxic T lymphocytes
  • the present invention provides a method of treating or preventing a disease comprising administering to a patient in need thereof a pharmaceutical composition comprising an antibody heavy chain constant region heterodimer-fusion protein according to the present invention.
  • the treatable or preventable disease depends on the use of the bioactive protein contained in the antibody heavy chain constant heterodimer-fusion protein,
  • the present invention provides a method of treating or preventing a patient suffering from cancer selected from the group consisting of breast cancer, pancreatic cancer, kidney cancer, prostate cancer, ovarian cancer, small intestine cancer, esophageal cancer, cervical cancer, lung cancer, lymphoma and hematologic cancer.
  • Example 1 Design of antibody heavy chain constant region CH3 domain variants for heterodimer formation by human antibody isotype (sequence analysis)
  • Figure 3 is a comparison of the sequence of each human antibody immunoglobulin G (IgG) isotype CH3 domains listed.
  • IgG human antibody immunoglobulin G
  • Each amino acid sequence was identified in the International ImMunoGeneTics information system (IMGT; URL: http://www.imgt.org/).
  • IMGT International ImMunoGeneTics information system
  • the sequence of G3m (s, t) which has been reported to maintain serum half-life similar to other IgG isotypes among various Gm allotypes, was used (Stapleton NM et al., 2011). ).
  • the 409th amino acid sequence unlike IgG1, IgG2, and IgG3, had a sequence conserved in all homotypes at the positions of the other mutations except that it had a different sequence from IgG4 to arginine. It was.
  • the position having the same amino acid sequence number was selected as a position for transplanting the A107 mutant pair into an isoform other than IgG1. All amino acid position notations in the present invention follow the EU index (numbering).
  • Example 2 Design of antibody heavy chain constant region CH3 domain variants for heterodimer formation by human antibody isotype (structural modeling)
  • each mutant shown in FIG. 3 shows whether the mutation can be stably introduced to form a heterodimer.
  • Structural modeling uses an online modeling server (URL: https://swissmodel.expasy.org/; Biasini M et al., 2014) as a template of the structure of an already known Immunoglobulin Fc heterodimer variant (PDB ID: 4X98). Predicted through.
  • the heterologous mutant A107 heavy chain constant region of each isotype designed through the sequence analysis of Example 1 and the structural analysis of Example 2 is a site-directed mutation performed by a person skilled in the art using synthetic oligonucleotides (Macrogen, Korea) Site-directed mutagenesis) was performed using the NotI / HindIII restriction enzyme in-frame to sequence the signal sequence-hinge-CH2-CH3 to the animal cell expression vector pcDNA3.1 (+) (Invitrogen, USA). Cloning was carried out (see FIG. 5).
  • the hinge region used here is a cysteine residue of the upper hinge region except for the cysteine residue in the core hinge region for duplex formation. Substituted with serine residues to prevent formation.
  • serine residues to prevent formation.
  • only 15 amino acids in the C-terminal region of the central hinge region among the 47 amino acid sequences of the G3m (s, t) allotypes maintain high antibody-specific effect functions (ADCC, CDC) of IgG3. This was confirmed through the literature (Dall'Acqua WF et al., 2006), whereby only 15 amino acids of the C terminus of the sequence shown in FIG. 5 were used.
  • Table 2 shows amino acid sequence information of the CH3 region in the wild type and A107 heavy chain constant heterodimeric variant pairs of the present invention.
  • Purified antibodies in the scFv-Fc CH3A / Fc CH3B co-expression system were scFv-Fc CH3A homodimer (103 kDa), scFv-Fc CH3A / Fc CH3B heterodimer (78 kDa), Fc CH3B homodimer (53 kDa) Since the molecular weights of) are different, it is possible to compare the degree of heterodimer formation on SDS-PAGE.
  • Fc CH3B vector a vector constructed in Example 3 was used, and additionally, scFv was introduced only to the N terminus of Fc CH3A , that is, of pcDNA3.1 (+)-scFv-hinge-CH2-CH3A (scFv-Fc CH3A ). Vectors expressed in the format were cloned.
  • Figure 7 shows a schematic diagram of the animal cell expression vector pcDNA3.1 (+)-scFv-hinge-CH2-CH3A (scFv-Fc CH3A ) vector used in the scFv-Fc CH3A / Fc CH3B co-expression system.
  • the scFv antibody used is an antibody connecting the VH and VL regions of hAY4a, which is an enhanced version of the humanized antibody hAY4 that specifically binds DR4 (Lee, Park et al. 2010). Cloning was performed using NotI restriction enzyme and BsiWI restriction enzyme located immediately before the hinge region. Wild type Fc was constructed in the same format (scFv-Fc / Fc) as a control for the variants.
  • HEK293-F cells Upon 200 mL transfection in a shake flask, HEK293-F cells were seeded in 100 ml of medium at a density of 2.0 ⁇ 10 6 cells / ml and incubated at 150 rpm, 8% CO 2 .
  • the resulting heavy and light chain plasmids were diluted in 10 ml FreeStyle 293 expression medium (Invitrogen) with 125 ⁇ g of heavy chain and 125 ⁇ g of light chain in total, 250 ⁇ g (2.5 ⁇ g / ml), with 10 ml of dilute 750 ⁇ g of PEI. The mixture was mixed with the medium (7.5 ⁇ g / ml) and reacted for 10 minutes at room temperature.
  • the reacted mixed medium was put in the cells seeded with 100 ml in advance, incubated at 150 rpm and 8% CO 2 for 4 hours, and then the remaining 100 ml of FreeStyle 293 expression medium was added.
  • the proteins produced by the cells ie antibodies containing heavy chain constant region variants, are secreted out of the cells by the cells and accumulated in the medium. Therefore, the protein was purified using a protein A Sepharose column (GE healthcare) from the cell culture supernatant collected by centrifugation at 2500 rpm for 20 minutes after cell culture.
  • the purification method referred to the standard protocol provided by the Protein A column company, and the purified protein was measured by absorbance at a wavelength of 562 nm using a solution in a BCA protein assay kit (Thermo), and the amount was determined according to the drawn standard curve. was quantified.
  • Example 5 5 ⁇ g of the antibody including the heterologous heterologous heterologous heterologous heterologous variant A107 heavy chain in Example 5 was analyzed on SDS-PAGE under 12% non-reducing conditions (FIG. 8).
  • the homodimer of the CH3A variant was 103 kD
  • the homodimer of the CH3B variant was 53 kD
  • the monomer of the CH3B variant was 25 kD
  • the heterodimer of the CH3A and CH3B variants was 78 kD.
  • Western blots were performed together to determine the extent of more sophisticated homodimers.
  • Western blot was performed by separating anti-human IgG-AP conjugated antibody (Sigma) using a method performed by those skilled in the art after separating 0.1 ⁇ g of protein, which is less than SDS-PAGE analysis, in 12% non-reducing conditions ( 9).
  • the IgG1 heterodimer into which the wild-type CH3 domain was introduced as a control group showed all homodimers of CH3A / CH3B and CH3A: CH3B heterodimers on SDS-PAGE, whereas IgG2, IgG3, Human antibody isotype A107 heavy chain constant region heterodimer variants incorporating A107 heterodimerization mutations into IgG4 were found to form heterodimers with similar or higher yields than previously reported IgG1-based A107 mutants. .
  • the Fc monomer (Half Fc) containing CH3A or CH3B was also observed, which is one of the characteristics of naturally occurring IgG4, and the hinge region (particularly, before the Fab-arm exchange in the blood) occurs. , 228 th serine in the central hinge region). This is due to the characteristic of forming half Fc (Liu H et al., 2012).
  • Example 7 Construction of human / mouse IL-12 fusion protein
  • the IgG4-based variant ( ⁇ 4-A107) was used.
  • Persistent Interleukin 12 fusion protein was constructed.
  • Interleukin 12 in nature consists of two different units of p35 monomer (p35; IL-12A) and p40 monomer (p40; IL-12B), which are active by interacting with each other to form heterodimers.
  • p35; IL-12A p35 monomer
  • p40 monomer p40 monomer
  • Has Formation of such heterodimers is achieved by binding more stably with one disulfide bond present between two units. Therefore, we tried to maintain the heterodimer form of cytokines in nature by connecting each monomer to different heterodimeric Fc variants (CH3A or CH3B).
  • the heavy chain constant region heterodimeric variant for the construction of the fusion protein was used ⁇ 4-A107 to introduce a heterodimer by introducing an A107 mutation based on IgG4.
  • intrinsic functions such as ADCC / CDC of IgG1 in the construction of immunocytokine, which is a fusion form of antibody and cytokine, promote the clearance in vivo.
  • the fusion protein was constructed using an IgG4 isotype that shows little function of ADCC / CDC compared to IgG1 (Gillies SD et al., 1999).
  • (C) is a fusion protein to which the CH3 mutant pair produced in the present invention is introduced.
  • Human interleukin 12 (hIL-12, Uniprot entry name P29460, P29459; SEQ ID NO: 17-18) and mouse interleukin 12 (mIL-12, Uniprot entry name P43432, P43431; SEQ ID NO: 19-20) are both mature except signal sequence Only the DNA sequence encoding the form was amplified and cloned using an NotI / BsiWI restriction enzyme in an in-frame as shown in FIGS. 11 (A) and (B) in an animal cell expression vector containing a ⁇ 4-A107 variant. They were named mono-hIL-12-Fc and mono-mIL-12-Fc, respectively.
  • the human / mouse p35 monomer was assigned 15 flexible peptide linkers (G 4 S) 3 Linker between the p35 monomer and the hinge region to allow sufficient interaction with the p40 monomer.
  • G 4 S flexible peptide linkers
  • bi-hIL-12-Fc and bi-mIL-12- fused with human interleukin 12 (hIL-12) and mouse interleukin 12 (mIL-12) were fused with wild-type IgG4 Fc (wt IgG4).
  • Fc was constructed.
  • IL-12 which is active only by heteroduplexing
  • two units are connected to the 15 peptide linkers, and then the animal cell expression vector containing the ⁇ 4-A107 variant is shown in FIG. 12. It was cloned in-frame using NotI / BsiWI restriction enzyme.
  • the comparative example is a fusion protein of the type used in previous studies to make IL-12 fusion proteins (Lisan S. Peng et al., 1999).
  • Table 3 shows the amino acid sequences for the mature forms of the monomers of human and mouse interleukin 12 used to construct the fusion protein.
  • Example 8 IL-12 fusion protein expression / purification
  • the mono-IL-12-Fc fusion protein of FIG. 10C shows 1: 1 ratio of expression vectors of human / mouse IL-12.p40- ⁇ 4-A107A and human / mouse IL-12.p35- ⁇ 4-A107B. It was expressed / purified in the same manner as in Example 5.
  • the bi-IL-12-Fc fusion protein of FIG. 10 (B) was expressed / purified via a single transfection of the human / mouse scIL-12-IgG4 Fc (wt) expression vector. All fusion proteins were expressed / purified at a similar level of 12-13 mg per 100 ml HEK293F cell culture.
  • SEC 14 is a result of size-exclusion chromatography (SEC) of the fusion proteins. Some polymers were observed in the Mono-hIL-12-Fc fusion protein.
  • Example 8 The binding ability of the mono-hIL-12-Fc expressed and purified in Example 8 to the IL-12 receptor was compared with bi-hIL-12-Fc.
  • FIG. 15 shows the result of confirming that the mono-hIL-12-Fc constructed as compared with bi-hIL-12-Fc showed binding ability to the IL-12 receptor by FACS Calibur (BD Biosciences).
  • PBMC immune cells
  • PHA Sigma-Aldrich
  • T and NK cells were treated for 72 h to stimulate T and NK cells.
  • PHA treatment has been reported to express IL-12 receptors on T cells and NK cells as the immune cells divide.
  • 1 ⁇ 10 6 cells / ml of PBMC was added to RPMI1640 medium containing 10% FBS, and PHA was added at a concentration of 10 ⁇ g / ml as a mitogen, followed by incubation for 72 hours at 37 degrees and 5% CO 2 incubator.
  • Normal PBMC and PHA activated PBMC were washed with cold PBS (pH 7.4) and 5 ⁇ 10 5 cells were prepared for each sample.
  • Fc (A107), bi-hIL-12-Fc and mono-hIL-12-Fc were added at a concentration of 1 ⁇ M, reacted at 4 ° C. for 30 minutes, and washed with cold PBS (pH 7.4).
  • FITC fluorescence-linked secondary antibody Sigma-Aldrich
  • FACS Calibur BD Bioscience
  • bi-hIL-12-Fc and mono-hIL-12-Fc did not bind to normal PBMCs that did not express IL-12 receptors, but only to PBMCs that were activated by PHA and expressed IL-12 receptors. Therefore, the binding capacity of mono-hIL-12-Fc to IL-12 receptor was confirmed to be the same as bi-hIL-12-Fc.
  • IL-12 recombinant human IL-12
  • FIG. 16 shows WST-1 cell proliferation test results confirming cell proliferation ability by Fc (A107), rhIL-12, bi-hIL-12-Fc and mono-hIL-12-Fc in PHA-activated PBMCs.
  • PBMC PBMC (2 ⁇ 10 4, 50 ⁇ l) activated with PHA was added to 96-well plate (SPL, Korea) and serially diluted with RPMI1640 medium containing 10% FBS as in Example 9.
  • Example 11 Evaluation of the ability to induce IFN- ⁇ secretion of mono-hIL-12-Fc fusion protein against PBMC
  • FIG. 17 shows ELISA results of measuring the amount of IFN- ⁇ secretion by Fc (A107), rhIL-12, bi-hIL-12-Fc and mono-hIL-12-Fc in PHA activated PBMC.
  • Example 10 in order to measure IFN- ⁇ concentration in cell culture cultured for 72 hours in Example 10, a human IFN- ⁇ capture antibody (Thermo Fisher Scientific) was placed in a 96-well plate (Thermo Fisher Scientific, Korea) for ELISA. After coating for 12 hours, washed with PBST (PBS with 0.1% Tween-20), 1% BSA (PBS with 1% bovine serum albumin) was added and blocked for 1 hour at room temperature. After washing with PBST (PBS with 0.1% Tween-20), the culture cultured in Example 2 was diluted 5 times with 1% BSA, added 100 ⁇ l, and then reacted at room temperature for 2 hours.
  • PBST PBS with 0.1% Tween-20
  • BSA PBS with 1% bovine serum albumin
  • mono-hIL-12-Fc showed that IFN- ⁇ secretion ability to PBMC is similar or higher than rhIL-12.
  • Example 8 The binding ability of the mono-mIL-12-Fc expressed and purified in Example 8 to the IL-12 receptor was compared with bi-mIL-12-Fc.
  • FIG. 18 is a flow cytometry result confirming that mono-mIL-12-Fc constructed as compared with bi-mIL-12-Fc shows binding ability to IL-12 receptor.
  • mouse IL-12 was reported to bind not only mouse IL-12 receptor but also human IL-12 receptor, and analyzed in the same manner as in Example 9.
  • bi-mIL-12-Fc and mono-mIL-12-Fc did not bind to normal PBMCs that did not express IL-12 receptors, but only to PBMCs that were activated by PHA and expressed IL-12 receptors. Therefore, the binding capacity of mono-mIL-12-Fc to IL-12 receptor was confirmed to be the same as bi-mIL-12-Fc.
  • WST-1 confirmed the cell proliferation ability by Fc (A107), recombinant mouse IL-12 (rmIL-12), bi-mIL-12-Fc and mono-mIL-12-Fc in PHA activated PBMC Cell proliferation assay results.
  • Example 13 cell proliferation in PBMCs activated with PHA of mono-mIL-12-Fc was confirmed. It was confirmed whether the effect of mono-mIL-12-Fc is the same in vivo.
  • 20 (A) and 20 (B) show the results of measuring tumor growth inhibitory activity of mono-mIL-12-Fc in 100 mm 3 tumors.
  • mice hair of 4 week old female Balb / c mice (NARA Biotech, Korea) was removed with a razor and CT26 HER2 / Neu colorectal cancer cells (1 ⁇ 10 6 cells / mouse) were diluted in 150 ⁇ L PBS and transplanted into the mouse subcutaneous. It was.
  • Figure 21 (A), 21 (B) and 21 (C) is the result of measuring the tumor growth inhibitory activity in mice in vivo of mono-mIL-12-Fc administered at different concentrations in mice with 300 mm 3 tumor size to be.
  • mice hair of 4 week old female Balb / c mice (NARA Biotech, Korea) was removed with a razor and CT26 HER2 / Neu colorectal cancer cells (1 ⁇ 10 6 cells / mouse) were diluted in 150 ⁇ L PBS and transplanted into the mouse subcutaneous. It was.
  • bi-IL-12-Fc was mono-mIL-12-Fc at a molar concentration of 1 ⁇ g IL-12 or less even for large tumors. Compared with that, the effect of inhibiting tumor growth was significantly higher.
  • the same molar concentration of mono-mIL-12-Fc was effective in removing tumors in 40% of mice.
  • 73% of the tumors were cleared by only 5 doses of mono-mIL-12-Fc at a concentration of 0.5 ⁇ g IL-12 where bi-mIL-12-Fc did not remove tumors.
  • Figure 21 (D) is the result of measuring the weight change in vivo toxicity of mono-mIL-12-Fc administered by concentration.
  • the weight of the mice administered as shown in Figure 21 (A) was measured twice a week to observe the weight loss.
  • the weight of the tumor was increased as the tumor size increased, but it was confirmed that there was no weight loss in the mice administered with all concentrations of bi-mIL-12-Fc and mono-mIL-12-Fc. Therefore, mono-mIL-12-Fc was not considered to be in vivo toxic to induce weight loss.
  • Figure 21 (D) is the result of measuring alanine aminotransferase (ALT) which is an indicator of liver toxicity.
  • blood was collected from the facial vein at 24 hours of the last administration in the mouse of FIG. 21 (A). Blood was left at room temperature for 2 hours to induce coagulation, and then centrifuged at 8000 rpm for 10 minutes to recover serum from the upper layer.
  • blood was collected from the facial vein of the mouse 24 hours after the last IL-12-Fc fusion protein administration. The blood was left at room temperature for 2 hours to induce coagulation, and then centrifuged at 8000 rpm for 10 minutes to recover serum from the upper layer.
  • ALT measurement substrate solution (alanine and ⁇ -ketoglutarate mixed solution) was taken in a 15 ml test tube and incubated in a 37 ° C constant temperature water bath for 5 minutes.
  • the mice transplanted with the tumor were treated with bi-mIL-12-Fc and mono-mIL-12-Fc, and blood samples were collected by diluting the serum 10-fold and adding 200 ⁇ l and shaking. Incubate for 30 minutes in a water bath. 1 ml of color solution (2,4-dinitrophenyl-1-hydrazone) was added to the test tube taken out of the constant temperature water bath, followed by mixing at room temperature for 20 minutes.
  • Example 16 Evaluation of immune cell proliferation induction capacity of mono-mIL-12-Fc in vivo
  • Figure 22 (A) is the result of confirming the increase in the number of CD4 + T cells, CD8 + T cells and NK cells in the spleen after 3 days of the last administration of Figure 21 (A).
  • the mouse spleen was extracted at 34 days after tumor transplantation, pulverized with a wire mesh in Petri dishes and washed with 10 ml of medium containing 2% FBS. Thereafter, 1 ml of red blood cell lysis buffer was added to erythrocytes, and red blood cells were lysed and washed with PBS to prepare splenocyte turbidity, and the number of cells was counted with a hemocytometer. Spleen lymphocytes were stained at 4 ° C. for 30 minutes with antibodies recognizing CD45, CD3, CD4, CD8 and CD49b bound to APC, FITC, PE or PE-cy5 and washed with cold PBS (pH 7.4).
  • CD45 + CD3 + CD4 + cell populations CD45 + CD3 + CD8 + cell populations
  • CD45 + CD3 - CD49b + cell populations CD4 + T cells, CD8 + T cells, and NK cells.
  • the ratio of total splenocytes was calculated and the number of CD4 + T cells, CD8 + T cells and NK cells increased after mono-mIL-12-Fc administration by multiplying the number of cells counted with hemocytometer. .
  • mono-mIL-12-Fc infiltrated tumors based on the report that the increase in the number of CD4 + T cells and CD8 + T cells, which are adaptive immune cells infiltrated into tumors, is important (Schreiber et al., 2011). It was analyzed whether the number of adaptive immune cells increased. When 6 mono-mIL-12-Fc were administered to a large number of mice without tumors, the number of immune cells infiltrated into the tumors of mice after 3 administrations was analyzed.
  • Figure 22 (B) is the result showing the total number of immune cells, CD4 + T cells and CD8 + T cells infiltrated into the tumor by 3 days after the third administration of the 21 (A).
  • the tumor of the mouse was extracted and weighed on the 24th day of tumor transplantation, and then pulverized using a wire mesh and collagenase (100 ⁇ g / ml) in Petri dish, followed by 2% FBS. 10 ml of the medium containing the was added and centrifuged at 50 g for 5 minutes to remove the parenchyma. Thereafter, 1 ml of red blood cell lysis buffer was added to erythrocytes, and red blood cells were lysed, washed with PBS to prepare a cell suspension, and the number of cells was counted with a hemocytometer. Cells isolated from tumors were stained at 4 ° C.
  • CD45 + T cells and tumor infiltrating CD8 + T cells were defined.
  • bi-mIL-12-Fc and mono-mIL-12-Fc showed the number of total immune cells, CD4 + T cells, and CD8 + T cells infiltrating tumors in tumor-transplanted mice. It can be confirmed that increased.
  • Mono-mIL-12-Fc increased the number of total immune cells, CD4 + T cells, and CD8 + T cells infiltrated tumors more significantly than bi-mIL-12-Fc. Therefore, mono-mIL-12-Fc significantly increased the number of CD4 + T cells and CD8 + T cells infiltrating tumors than bi-mIL-12-Fc.
  • Example 17 Evaluation of cytokine secretion and cytotoxicity function of mono-mIL-12-Fc in vivo immune cells
  • IL-12 is known to inhibit the growth of cancer cells by increasing IFN-g secretion of T cells and NK cells (Trinchieri, 2003). IL-12 also has an anticancer effect by enhancing direct cytotoxic effects on cancer cells of cytotoxic T cells and natural killer cells. Therefore, we analyzed whether the anti-cancer effect of Mono-IL-12-Fc was due to the increase of serum IFN-g concentration and the direct cytotoxic effect of cytotoxic T cells and natural killer cells on cancer cells.
  • Figure 23 (A) is the result of measuring the IFN-g concentration in the serum isolated by collecting blood from the facial vein of the mouse 24 hours after the last administration of Figure 21 (A) by ELISA.
  • FIG. 20 blood was collected from the facial vein of the mouse 24 hours after the last mIL-12-Fc fusion protein administration in FIG. 20 (A).
  • the blood was left at room temperature for 2 hours to induce coagulation, and then centrifuged at 8000 rpm for 10 minutes to recover serum from the upper layer.
  • a mouse IFN- ⁇ capture antibody was coated in a 96-well plate for ELISA (Thermo Fisher Scientific) for 12 hours, followed by washing with PBST (PBS with 0.1% Tween-20).
  • 1% BSA PBS with 1% bovine serum albumin
  • bi-mIL-12-Fc was not capable of inducing IFN- ⁇ secretion by NK cells and T cells, and thus, bi-mIL-12-Fc was administered in FIG. 23 (A).
  • mono-mIL-12-Fc and bi-mIL-12-Fc were administered only once, and then blood IFN- ⁇ levels were measured over time.
  • FIG. 23 (B) shows IFN- ⁇ in serum by time after intraperitoneal administration of bi-mIL-12-Fc and mono-mIL-12-Fc to Balb / c mice transplanted with CT26 HER2 / Ne colorectal cancer cells. Concentration was measured by ELISA.
  • a mouse IFN- ⁇ capture antibody was coated in a 96-well plate for ELISA (Thermo Fisher Scientific) for 12 hours, followed by washing with PBST (PBS with 0.1% Tween-20).
  • PBST PBS with 0.1% Tween-20
  • 1% BSA PBS with 1% bovine serum albumin
  • the serum was diluted 10-fold with 1% BSA and reacted at room temperature for 2 hours.
  • biotin-coupled mouse IFN- ⁇ detection antibody was bound for 1 hour at room temperature.
  • the bi-mIL-12-Fc-administered group showed a similar concentration of IFN- ⁇ in the blood to the mono-mIL-12-Fc-treated group until day 5 in the tumor-grafted mice. There was no inherent deficiency in the ability of -Fc to induce the secretion of IFN- ⁇ in effector cells.
  • Figure 23 (C) is a graph measuring the cytotoxic effect on CT26 HER2 / Neu cancer cells of cytotoxic T cells isolated from the spleen by killing the mouse 3 days after the last administration of Figure 21 (A).
  • mice 72 hours after the last cytokine administration in FIG. 21 (A), mice were killed and spleens were removed and spleens and PBS were put into a 60 mm dish containing 70 micron mesh and ground.
  • the cells obtained by centrifugation were lysed with red blood cell hemolysis buffer, and then washed with PBS and washed with PBS, an APC conjugated antibody that recognizes CD3 (Thermo Fisher Scientific), and a PE conjugated antibody that recognizes CD8 (Thermo Fisher Scientific).
  • the reaction was carried out for 30 minutes at 4 °C. After washing with PBS, cytotoxic T cells (CD3 + CD8 + ) were isolated by FACS Aria III (BD biosciences, Korea).
  • CT26 HER2 / Neu cancer cells were stained with calcein AM (Thermo Fisher Scientific Inc., 10 ⁇ M). After CT26 HER2 / Neu cancer cells (2 ⁇ 10 6 ) were suspended in 2 ml of DPBS, 2 ⁇ l of calcein AM (10 mM) was added and mixed, followed by reaction for 45 minutes at 5% CO 2 and 37 ° C.
  • a RPMI1640 with 10% FBS was added 10 ml into three times, washed, and then put into a 2 ⁇ 10 4 cells per well in a 96-well plate cytotoxic T cells (1 ⁇ 10 5/100 ⁇ l / well) into each 5 % CO 2 , incubated for 4 hours at 37 °C incubator.
  • Live CT26 HER2 / Neu cancer cells that fluoresce green and dead CT26 HER2 / Neu cancer cells that do not fluoresce green were analyzed by flow cytometry to express the cytotoxic effect of cytotoxic T cells in percentage.
  • Cytotoxic T cells isolated from tumor-grafted mice treated with Mono-mIL-12-Fc were cytotoxic T cells isolated from tumor-grafted mice administered bi-mIL-12-Fc or cytotoxic T cells isolated from the control group.
  • the cytotoxic effect on CT26 HER2 / Neu cancer cells, which are more target cells, was higher, and the tumor suppression effect of mono-mIL-12-Fc was a direct cytotoxic effect of cancer cells by some cytotoxic T cells.
  • Figure 23 (D) is the third dose of Figure 21 (A) to determine whether the cytotoxic effect of cytotoxic T cells enhanced by administration of mono-IL-12-Fc in tumor-grafted mice is cancer antigen specific. Three days later, the cytotoxic effect of cytotoxic T cells isolated from the spleen by lethal mice was measured using CT26 HER2 / Neu cancer cells expressing tumor antigen and 4T1 cells not expressing tumor antigen.
  • mice 72 hours after the three-time administration of mono-IL-12-Fc in FIG. 20 (A), mice were killed and spleens were removed, and spleen and PBS were placed in a 60 mm dish containing 70 micron mesh. Pulverized. Cytotoxic T target cells in CT26 HER2 / Neu tumor cells and cytotoxic T Target cells 4T1 cytotoxic effect in the same way as FIG. 21 (C) to measure CT26 HER2 / Neu cancer with 4T1 tumor cells to a non-cellular in the cell Were stained with calcein AM (Thermo Fisher Scientific Inc., 10 ⁇ ).
  • calcein AM Thermo Fisher Scientific Inc., 10 ⁇
  • the RPMI1640 with 10% FBS was added 10 ml into three times, washed, and then put into a 2 ⁇ 10 4 cells per well in a 96-well plate cytotoxic T cells (1 ⁇ 10 5/100 ⁇ l / well) into each 5 % CO 2 , incubated for 4 hours at 37 °C incubator.
  • Green Fluorescent Live CT26 HER2 / Neu Dead CT26 HER2 / Neu with no green fluorescence with cancer cells or 4T1 cancer cells Cancer cells or 4T1 cancer cells were analyzed by flow cytometry to express the cytotoxic effects of cytotoxic T cells in percentage. As a result, it was confirmed that the cytotoxic effect of the cytotoxic T cells enhanced by the administration of mono-mIL-12-Fc was target cell specific.
  • Figure 23 (E) is a result of measuring the cytotoxic effect on CT26 HER2 / Neu cancer cells of natural killer cells isolated from the spleen by killing the mouse 3 days after the third administration of Figure 21 (A).
  • mice were killed and spleens were removed, and spleens and PBS were put in a 60 mm dish containing 70 micron mesh and ground.
  • the cells obtained by centrifugation were lysed with red blood cell hemolysis buffer, washed with PBS, and washed with PBS, and APC-bound antibody (Thermo Fisher Scientific) that recognizes CD3 and PE-bound antibody (Thermo Fisher Scientific) that recognizes CD49b.
  • the reaction was carried out for 30 minutes at 4 °C. After washing with PBS, natural killer cells (CD3 - CD49b + ) were separated by FACS Aria III (BD biosciences, Korea).
  • CT26 HER2 / Neu Cancer cells were stained with calcein AM (Thermo Fisher Scientific Inc., 10 ⁇ M) to measure the cytotoxicity of the target cells in CT26 HER2 / Neu cancer cells of NK cells.
  • CT26 HER2 / Neu cancer cells (2 ⁇ 10 6 ) were suspended in 2 ml of DPBS, 2 ⁇ l of calcein AM (10 mM) were mixed and allowed to react for 45 minutes at 5% CO 2 , 37 ° C.
  • a RPMI1640 with 10% FBS was added 10 ml into three by three times, and then each well in a 96-well plate into a 2 ⁇ 10 4 cells into natural killer cells (1 ⁇ 10 5/100 ⁇ l / well) , respectively 5% Incubated for 4 hours in a CO 2 , 37 °C incubator.
  • Live CT26 HER2 / Neu cancer cells that fluoresce green and dead CT26 HER2 / Neu cancer cells that do not fluoresce green were analyzed by flow cytometry to express the cytotoxic effect of natural killer cells in percentage.
  • Natural killer cells isolated from tumor-grafted mice treated with Mono-mIL-12-Fc were more targeted than natural killer cells isolated from bi-mIL-12-Fc-treated mice or with cytotoxic T cells isolated from the control group.
  • the cytotoxic effect on CT26 HER2 / Neu cancer cells, which are the cells, was high, and the tumor suppression effect of mono-mIL-12-Fc was a direct cytotoxic effect of cancer cells by some natural killer cells.
  • the generation of adaptive immunity in tumor-grafted mice is assessed by effector memory CD8 + T cells and memory CD8 + T cell formation.
  • Tumor elimination effect by Mono-mIL-12-Fc was determined by the effect memory CD8 + T cells and memory CD8 + T cell formation.
  • 24 (A), 24 (B) and 24 (C) show effect CD8 + T cells, effect memory CD8 + T cells and memory CD8 generated when mono-mIL-12-Fc was administered to mice with tumors, respectively. + The result of measuring the number of T cells.
  • the mouse spleen was extracted at 34 days after tumor transplantation, pulverized using a wire mesh in Petri dishes, washed with 10 ml of medium containing 2% FBS, and then erythrocyte hemolysis buffer 1 ml of red blood cell lysis buffer was added to erythrocytes, and then washed with PBS to prepare a splenocyte suspension, and the number of cells was counted with a hemocytometer.
  • Splenocytes were stained at 4 ° C. for 30 minutes with antibodies recognizing CD3, CD8, CD62L and IL-7 receptor (IL-7R) bound to PE-cy5, PE, FITC or APC, and cold stained with PBS (pH 7.4).
  • CD3 + CD8 + CD62L low IL-7R low Cell groups CD3 + CD8 + CD62L low IL-7R hi cell populations and CD3 + CD8 + CD62L hi IL-7R hi cell populations were defined as effect CD8 + T cells, effect memory CD8 + T cells and memory CD8 + T cells, respectively.
  • the number of effect CD8 + T cells, effect memory CD8 + T cells, and memory CD8 + T cells after mono-mIL-12-Fc administration was analyzed by multiplying the number of cells counted by hemocytometer. .
  • mono-mIL-12-Fc increased the number of effect memory CD8 + T cells and memory CD8 + T cells in a tumor-grafted mouse compared with the control group.
  • bi-mIL-12-Fc increased the number of effect memory CD8 + T cells and memory CD8 + T cells only in the group administered with a molar concentration such as 0.5 ⁇ g IL-12, and the molar like 1 ⁇ g IL-12.
  • the concentration group did not increase the number of effect memory CD8 + T cells and memory CD8 + T cells. Therefore, mono-mIL-12-Fc significantly increased the number of effect memory CD8 + T cells and memory CD8 + T cells than bi-mIL-12-Fc.
  • Figure 24 (D) is in the mice survived 120 days after the administration in 21 (A) to FIG 1 ⁇ g mono-IL-12-Fc CT26 HER2 / Neu Cancer cells are transplanted to measure the change in the tumor volume of the mouse.
  • FIG. 21 (A) the last 1 ⁇ g mono-IL-12- was applied to female Balb / c mice (NARA Biotech, Korea) that matched the age of the surviving mice after administration of 1 ⁇ g mono-IL-12-Fc.
  • the hair of the mouse was removed with a razor and CT26 HER2 / Neu cells (1 ⁇ 10 6 cells / mouse) were transplanted into the mouse subcutaneously diluted in 150 ⁇ L PBS.
  • additional 1 ⁇ g mono-IL-12-Fc of the measurement without the administration of the tumor twice a week, and the volume (V) of the tumor was calculated as V L x W 2/2.
  • bi-mIL-12-Fc was compared with mono-mIL-12-Fc in the number of spleen CD8 + T cells, the number of effect memory CD8 + T cells, and central memory CD8 in mice transplanted with tumors.
  • the effect of increasing the number of + T cells was observed to be low.
  • the effect CD8 + T cells are partially differentiated into memory CD8 + T cells through memory precursor effector cells (MPEC) and are mostly short-lived. It has been reported to die as short-lived effector cells (SLEC).
  • MPEC memory precursor effector cells
  • SLEC short-lived effector cells
  • FIG. 24 (E) shows memory precursor effect cells (KLRG1 - IL-7R + ) and short-lived effect cells (KLRG1) among CD8 + T cells present in the spleen after 3 days of the third administration of FIG. 21 (A). + IL-7R - an analysis of the rate of).
  • the mouse spleen was extracted on the 24th day of tumor transplantation, and then pulverized using a wire mesh in Petri dishes and washed with 10 ml of medium containing 2% FBS. Thereafter, 1 ml of red blood cell lysis buffer was added to lyse red blood cells, and then washed with PBS to prepare a cell suspension. Splenocytes were stained at 4 ° C. for 30 minutes with antibodies recognizing CD3, CD8, KLRG1 and IL-7 receptor (IL-7R) bound to PE-cy5, PE, FITC or APC, and cold stained with PBS (pH 7.4).
  • IL-7R IL-7 receptor
  • bi-mIL-12-Fc did not increase the proportion of memory precursor cells compared to the control group, but rather increased the number of short-lived cells. Therefore, mono-mIL-12-Fc promoted the production of memory precursor cells more than bi-mIL-12-Fc and significantly increased the number of effect memory CD8 + T cells and memory CD8 + T cells, resulting in a higher tumor removal effect. Confirmed.
  • Example 20 Evaluation of the Effect of Mono-mIL-12-Fc on the Expression of Transcription Factors Related to Memory Cell Differentiation Induction
  • T-bet a transcription factor that differentiates CD8 + T cells into short-lived effect cells
  • Eomes eomesodermin
  • 25 (A) and 25 (B) show the percentage of CD8 + T cells with high expression of T-bet that inhibits the differentiation of memory cells in the spleen by killing mice 3 days after the third dose of FIG. 21 (A). This is the result of measuring the ratio of CD8 + T cells with high expression of Eomes and low expression of T-bet that promotes the differentiation of memory cells by flow cytometry.
  • the mouse spleen was extracted on the 24th day of tumor transplantation, and then pulverized using a wire mesh in Petri dishes and washed with 10 ml of medium containing 2% FBS. Thereafter, 1 ml of red blood cell lysis buffer was added to lyse red blood cells, and then washed with PBS to prepare a cell suspension. Splenocytes were stained with PE-cy5 or FITC-coupled CD3 and CD8 antibodies for 30 minutes at 4 ° C, washed with cold PBS (pH 7.4), and stained with transcription factor Foxp3 / Transcription Factor.
  • the cells were fixed and permeabilized using Staining Buffer Set (Thermo Fisher Scientific), and then stained for 30 minutes at 4 ° C. with an antibody recognizing T-bet or Eomes bound to PE or efluor 660.
  • Permeabilization buffer was added and analyzed by flow cytometry FACS Calibur (BD Bioscience) and Flow jo (Thermo Fisher Scientific). Each sample was analyzed by dot plot to analyze the ratio of CD3 + CD8 + T-bet high cell population and CD3 + CD8 + Eomes + T-bet low cell population.
  • mono-mIL-12-Fc reduced the proportion of CD3 + CD8 + T-bet high cell populations in a tumor-dependent mouse compared to the control group, and decreased the concentration of CD3 + CD8 + Eomes + T-bet low cells. It was confirmed that the ratio was increased.
  • bi-mIL-12-Fc decreased the proportion of CD3 + CD8 + T-bet high cell population only in the group administered with 0.5 ⁇ g IL-12, and CD3 + CD8 + Eomes + T-bet low
  • the percentage of cell population was increased, and in the group administered with a molar concentration such as 1 ⁇ g IL-12, the percentage of CD3 + CD8 + T-bet high cell population was decreased or the percentage of CD3 + CD8 + Eomes + T-bet low cell population was decreased. There was no increasing effect.
  • mono-mIL-12-Fc reduces the proportion of CD3 + CD8 + T-bet high cell population and increases the ratio of CD3 + CD8 + Eomes + T-bet low cell population than bi-mIL-12-Fc.
  • the number of CD8 + T cells and memory CD8 + T cells was significantly increased to confirm that the tumor removal effect was high.
  • CD8 + T cells when stimulated with inflammatory cytokines such as IL-12 in the presence of T cell receptor and co-stimulatory signals, increase phosphorylation of STAT4, and phosphorylated STAT4 (pSTAT4) migrates inside the nucleus to the T-bet enhancer. It is known to bind to increase expression of T-bet. Therefore, when 8 ⁇ g IL-12 at a molar concentration of bi-mIL-12-Fc is administered, CD8 + T cells are differentiated into short-lived cells.
  • Fc administration increased the expression of pSTAT4 and T-bet when CD8 + T cells were activated in the groin lymph nodes, the tumor draining lymph nodes of mice transplanted with tumors than mono-mIL-12-Fc. Cognitive was measured.
  • Figure 25 (C) shows the molar concentrations of bi-mIL-12-Fc and mono-mIL-12-Fc at the same molar concentration of 1 ⁇ g rmIL-12 when the tumor size was 300 mm 3 in CT26 HER2 / Ne transplanted Balb / c mice.
  • the expression level of phosphorylated STAT4 in CD8 + T cells isolated from inguinal lymph nodes 24 hours after one intraperitoneal administration was measured by flow cytometry.
  • Inguinal lymph node cells were stained with PE-cy5 or FITC-coupled CD3 and CD8 antibodies for 30 minutes at 4 ° C, washed with cold PBS (pH 7.4), and fixed with cold methanol. The inguinal lymph node cells were then washed with cold PBS (pH 7.4), and then stained with cold PBS (pH 7.4) for 30 minutes at 4 ° C with an antibody recognizing pSTAT4 bound to APC, followed by FACS Calibur (flow cytometry). BD Bioscience) and Flow jo (Thermo Fisher Scientific). Each sample was analyzed by dot plot to compare the amount of pSTAT4 expressed in CD3 + CD8 + T cells. As a result, compared with mono-mIL-12-Fc, bi-mIL-12-Fc increased the expression of pSTAT4 when CD8 + T cells were activated in the groin lymph nodes of tumor-grafted mice.
  • Figure 25 (D) is a result of measuring the ratio of CD8 + T cells expressing T-bet inhibiting the differentiation of memory cells in the groin lymph nodes 72 hours after one intraperitoneal administration of Figure 25 (C) by flow cytometry.
  • Inguinal lymph node cells were stained with PE-cy5 or FITC-bound CD3 and CD8 antibody for 30 minutes at 4 ° C, washed with cold PBS (pH 7.4), and stained with transcription factor Foxp3 / Transcription.
  • Cells were fixed and permeabilized using a Factor Staining Buffer Set (Thermo Fisher Scientific). Afterwards, the antibody recognizes T-bet conjugated with PE or APC, and then stained at 4 ° C. for 30 minutes, and then added a permeabilization buffer to FACS Calibur (BD Bioscience) and Flow jo (Thermo Fisher Scientific). ). Each sample was analyzed by dot plot to compare the proportion of CD3 + CD8 + T cells expressing T-bet.
  • bi-mIL-12-Fc increased the expression of T-bet when CD8 + T cells were activated in the groin lymph nodes of mice transplanted with tumors. Therefore, the differentiation of CD8 + T cells into monolithic cells when bi-mIL-12-Fc at the same molar concentration as 1 ⁇ g IL-12 was observed in the peritumor lymph nodes of mice transplanted with tumors than mono-mIL-12-Fc. It was confirmed that the expression of pSTAT4 and T-bet was increased when CD8 + T cells were activated in the inguinal lymph nodes (tumor draining lymph nodes).
  • spleen and groin lymph nodes were extracted from normal Balb / c mice, pulverized using a wire mesh in Petri dishes, and washed with 10 ml of medium containing 2% FBS. Thereafter, 1 ml of red blood cell lysis buffer was added to lyse red blood cells, and then washed with PBS to prepare a cell suspension. Lymphocytes were stained for 30 minutes at 4 ° C with antibodies bound to PE-binding CD8, washed with cold PBS (pH 7.4), and then combined with anti-PE microbeads (Miltenyi Biotec) for 15 minutes. Miltenyi Biotec) was used to isolate CD8 + T cells.
  • pSTAT4 and T-bet were stained by the method of FIGS. 25 (C) and 25 (D), respectively, and analyzed by flow cytometry, FACS Calibur (BD Bioscience) and Flow jo (Thermo Fisher Scientific). Each sample was analyzed by dot plot to compare the amount of pSTAT4 or T-bet expressed in CD8 + T cells. As a result, when mono-mIL-12-Fc was cross-linked with an antibody that recognizes Fc, the CD8 + T cells were stimulated by two molecules of IL-12, similar to those treated with bi-mIL-12-Fc. As a result, the expression of pSTAT4 and T-bet was increased.
  • mono-mIL-12-Fc induces less expression of pSTAT4 and T-bet in CD8 + T cells than bi-mIL-12-Fc so that CD8 + T cells have a memory effect.
  • Differentiation into cells leads to differentiation into effect memory cells and central memory cells, so even at low concentrations (molar concentrations such as 0.5 ⁇ g IL-12), the tumors of mice transplanted with tumors can be removed to extend the lifespan of the mice. .
  • bi-mIL-12-Fc induces the expression of pSTAT4 and T-bet in CD8 + T cells to differentiate into short-lived effect cells and to prevent differentiation into memory cells, thus molar concentrations such as mono-mIL-12-Fc
  • molar concentrations such as mono-mIL-12-Fc
  • the tumor was not completely removed from the transplanted mouse, and cytotoxic CD8 + T in the effector phase was directly administered at a higher concentration (molar concentration such as 2 ⁇ g IL-12).
  • the cells must be expanded to remove the tumor.
  • Heterodimeric Fc-fused protein according to the antibody heavy chain constant region according to the present invention by forming a protein complex of two or more subunits to mimic the physiologically active protein forms in nature, It can maintain the activity as it exists in nature, and also has the advantage that the half-life in the body of the bioactive protein can be significantly extended.
  • heterodimeric-fusion protein heterodimeric Fc-fused protein
  • the heterodimeric-fusion protein form according to the present invention has the advantage that it is easy to prepare a monovalent heterodimer-fusion protein without further purification process optimization.

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Abstract

La présente invention concerne une protéine fusionnée Fc hétérodimère et une composition pharmaceutique comprenant la protéine fusionnée Fc hétérodimère, dans la protéine fusionnée Fc hétérodimère comprenant une première région Fc et une seconde région Fc d'une paire de régions Fc d'une immunoglobuline tandis que une sous-unité d'une protéine biologiquement active est liée à au moins l'un des N-terminaux ou C-terminaux de la première région Fc et/ou de la seconde région Fc, la première région Fc et la seconde région Fc sont des domaines CH3 modifiés pour favoriser la formation d'un hétérodimère. Dans la protéine fusionnée Fc hétérodimère selon la présente invention, deux sous-unités ou plus forment un complexe protéique, de telle sorte qu'une protéine constituant une protéine biologiquement active peut être fusionnée à Fc dans une forme et une structure d'origine lorsque la protéine existe dans la nature et ainsi la protéine peut maintenir une activité d'origine lorsque la protéine existe dans la nature. L'utilisation de la protéine fusionnée Fc hétérodimère selon la présente invention accroît d'une manière remarquable la demi vie du in vivo d'une protéine biologiquement active contenu dans la protéine fusionnée Fc hétérodimère et ainsi divers types d'activités biologiques peuvent être maintenues pour longtemps dans le corps.
PCT/KR2017/008676 2016-08-10 2017-08-10 Cytokine fusionnée à un hétérodimère fc d'immunoglobuline et composition pharmaceutique la contenant WO2018030806A1 (fr)

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MX2019001651A MX2019001651A (es) 2016-08-10 2017-08-10 Citocina fusionada a fc heterodimerico, y composicion farmaceutica que comprende la misma.
CA3033475A CA3033475A1 (fr) 2016-08-10 2017-08-10 Cytokine a fusion fc heterodimerique et composition pharmaceutique la comprenant
BR112019002394-1A BR112019002394B1 (pt) 2016-08-10 2017-08-10 Proteína heterodimérica fundida com fc e seu uso
US16/323,839 US10696722B2 (en) 2016-08-10 2017-08-10 Heterodimeric Fc-fused cytokine and pharmaceutical composition comprising the same
CN201780062851.0A CN110267977A (zh) 2016-08-10 2017-08-10 细胞因子免疫球蛋白Fc融合异二聚体和包含其的药物组合物
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AU2017310163A AU2017310163B2 (en) 2016-08-10 2017-08-10 Heterodimeric Fc-fused cytokine and pharmaceutical composition comprising the same
EP17839824.4A EP3511340A4 (fr) 2016-08-10 2017-08-10 Cytokine fusionnée à un hétérodimère fc d'immunoglobuline et composition pharmaceutique la contenant
JP2019506697A JP6993403B2 (ja) 2016-08-10 2017-08-10 ヘテロダイマーＦｃ融合サイトカインおよびそれを含む医薬組成物
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US16/886,177 US11078249B2 (en) 2016-08-10 2020-05-28 Heterodimeric Fc-fused cytokine and pharmaceutical composition comprising the same
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AU2021273642A AU2021273642A1 (en) 2016-08-10 2021-11-26 Heterodimeric Fc-fused cytokine and pharmaceutical composition comprising the same
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CN110396133A (zh) * 2018-04-25 2019-11-01 中国科学院生物物理研究所 一种以白介素12为活性成分的融合蛋白型药物前体
WO2020086758A1 (fr) * 2018-10-23 2020-04-30 Dragonfly Therapeutics, Inc. Protéines hétérodimères fusionnées à un fragment fc
WO2020072821A3 (fr) * 2018-10-03 2020-06-18 Xencor, Inc. Protéines de fusion fc hétérodimères d'il -12
US11078249B2 (en) 2016-08-10 2021-08-03 Ajou University Industry-Academic Cooperation Foundation Heterodimeric Fc-fused cytokine and pharmaceutical composition comprising the same
WO2021246720A1 (fr) * 2020-06-01 2021-12-09 머스트바이오 주식회사 Anticorps bispécifique ou fragment de liaison à l'antigène de celui-ci, et procédé de préparation associé
US11471490B2 (en) 2017-07-03 2022-10-18 Torque Therapeutics, Inc. T cells surface-loaded with immunostimulatory fusion molecules and uses thereof
US11851466B2 (en) 2019-10-03 2023-12-26 Xencor, Inc. Targeted IL-12 heterodimeric Fc-fusion proteins
WO2024086739A1 (fr) 2022-10-20 2024-04-25 Synthekine, Inc. Procédés et compositions de mutéines il12 et de mutéines il2
RU2819097C2 (ru) * 2018-10-03 2024-05-14 Ксенкор, Инк. Il-12 гетеродимерные слитые белки fc
JP7500619B2 (ja) 2019-05-30 2024-06-17 アムジエン・インコーポレーテツド 抗体の二量体化を促進するためのヒンジ領域の操作

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RU2819097C2 (ru) * 2018-10-03 2024-05-14 Ксенкор, Инк. Il-12 гетеродимерные слитые белки fc
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JP2022505871A (ja) * 2018-10-23 2022-01-14 ドラゴンフライ セラピューティクス， インコーポレイテッド ヘテロ二量体ｆｃ融合タンパク質
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US11787864B2 (en) 2018-10-23 2023-10-17 Dragonfly Therapeutics, Inc. Heterodimeric Fc-fused proteins
JP7500619B2 (ja) 2019-05-30 2024-06-17 アムジエン・インコーポレーテツド 抗体の二量体化を促進するためのヒンジ領域の操作
US11851466B2 (en) 2019-10-03 2023-12-26 Xencor, Inc. Targeted IL-12 heterodimeric Fc-fusion proteins
WO2021246720A1 (fr) * 2020-06-01 2021-12-09 머스트바이오 주식회사 Anticorps bispécifique ou fragment de liaison à l'antigène de celui-ci, et procédé de préparation associé
WO2024086739A1 (fr) 2022-10-20 2024-04-25 Synthekine, Inc. Procédés et compositions de mutéines il12 et de mutéines il2

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