Human antibody fc variant-containing bispecific antibody and use thereof

Abstract

The present invention provides a bispecific antibody and a use thereof, the bispecific antibody comprising an Fc domain variant, a first antigen-binding portion, and a second antigen-binding portion. The bispecific antibody of the present invention has the advantages of improved stability with effectiveness at least maintained.

Description

Bispecific antibodies comprising human antibody FC variants and uses thereof

[0001] The present invention relates to a bispecific antibody comprising a human antibody Fc variant and its use.

[0002]

[0003] Protein therapeutics exhibit high specificity for disease targets, resulting in low side effects and toxicity. Therefore, they are rapidly replacing nonspecific small-molecule compound therapeutics and are widely used in clinical practice. Among protein therapeutics, antibody therapeutics and Fc-fusion protein therapeutics, which combine antibody Fc domains, are rapidly developing and are currently in clinical use.

[0004] Therapeutic antibodies exhibit significantly higher target specificity than conventional small-molecule drugs, have low biotoxicity and side effects, and boast an excellent blood half-life of approximately three weeks. These characteristics make them one of the most effective cancer treatments. Indeed, major pharmaceutical companies and research institutes around the world are accelerating research and development of therapeutic antibodies that specifically bind to and effectively eliminate cancer cells, including cancer-causing factors.

[0005] Antibodies provide a link between the humoral and cellular immune systems. While the Fab region of an antibody recognizes antigens, the Fc domain binds to receptors for antibodies (immunoglobulins) on cells (Fc receptors or FcRs) that are differentially expressed by all immunocompetent cells, and have different mechanisms depending on the type of FcγR expressed on the surface of the immune cell to which it binds. Binding of an antibody to a cell-surface Fc receptor triggers several important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (antibody-dependent cellular cytotoxicity, or ADCC), release of inflammatory mediators, control of placental transport, and immunoglobulin production.

[0006] The Fc domain plays a crucial role in the recruitment of immune cells and antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In particular, the ADCC and ADCP effects of antibodies depend on their interaction with Fc receptors, which are present on the surface of many cells. Human Fc receptors are classified into five types, and the type of immune cell recruited depends on which Fc receptor an antibody binds to. For example, the Fc domain of an antibody binds to FcγRⅢa to induce ADCC, binds to FcγRI or FcγRⅡa to induce ADCP, and binds to the circulating complement protein C1q to activate complement-dependent cytotoxicity (CDC) mechanisms. Each of these mechanisms then triggers an immune response to the target antigen bound to the Fab domain, which is responsible for the primary therapeutic effect of therapeutic antibodies.

[0007] This mechanism of action by the antibody Fc region induces the death of defective cells (e.g., cancer, infected cells), and is an important mechanism of action of therapeutic antibodies. However, when the target antigen of a therapeutic antibody is expressed on immune cells rather than defective cells (e.g., cancer, infected cells), such as immune checkpoint inhibitor antibodies that bind to immune cells or bispecific immune cell engaging bispecific antibodies, if the Fc mechanism remains, normal immune cells that are beneficial to the immune action bound by the antibody may be destroyed, or the host immune defense may be activated, causing safety issues and unwanted side effects.

[0008] In particular, immune cell-directing bispecific antibodies, which are antibody therapeutics that bind to antigens on the surface of cancer cells on one side and bind to immune cells on the other side, act to guide immune cells to cancer cells so that they can more effectively eliminate cancer cells, can cause side effects if the antibody has an Fc-mediated immune action mechanism because the immune cells are destroyed and cancer cells cannot be effectively eliminated. In addition, agonist antibodies that bind to target cells and induce cell activation or antagonist antibodies that block the interaction between the target antigen and the ligand also have the problem of lowering the original effect of the antibody, such as becoming toxic to the target cells due to the Fc-mediated immune action mechanism.

[0009] Therefore, in order to solve the problem of causing immune cell death side effects or off-target toxicity to normal cells while maintaining the excellent efficacy of antibodies through target antigen-specific binding, it is essential to eliminate the Fc mechanism of action. To this end, when developing antibodies, IgG2 antibodies, which have the lowest binding affinity to FcγR among human IgG subclasses and therefore a very low immune action mechanism, are considered. However, IgG2 antibodies have multiple allotypes due to disulfide bond exchange in the hinge region, and there is a physical problem of aggregation due to decreased stability. Therefore, IgG4 antibodies, which have the next lowest binding affinity, are considered and are currently being used in clinical development. However, IgG4 antibodies also have a disadvantage in that the stability of the antibody hinge region is insufficient, resulting in exchange of the Fab-arm of the IgG antibody molecule. In addition, they still have considerable binding affinity to FcγRs, and in particular, they have a fairly strong binding affinity (equilibrium dissociation constant of several nM) to FcγRI, so there is a problem that various Fc action mechanisms are activated. Therefore, in order to prevent target cells from being destroyed due to the immune action mechanism of the antibody, an Fc with eliminated binding affinity to FcγRs is required. To meet these needs, there is a need to secure variants with the Fc mechanism eliminated, and based on these variants, there is a need for bispecific antibodies based on Fc variants with suppressed Fc mechanism that can specifically bind to the target while reducing undesirable side effects. In this case, when an Fc variant with suppressed Fc mechanism is applied, it is generally expected that although stability will be improved compared to when the wild type is applied, efficacy will be reduced. Therefore, there is a need to develop a technology that maintains at least the same effect as the cancer cell killing effect compared to when the wild type is applied, even when an Fc variant with suppressed mechanism is applied.

[0010] [Prior Art Literature]

[0011] [Patent Document]

[0012] (Patent Document 1) Republic of Korea Patent Publication No. 10-2024-0022613 (February 20, 2024) Specification

[0013] The present invention aims to provide a bispecific antibody that suppresses the Fc mechanism of action while maintaining at least the effectiveness.

[0014] In addition, the present invention aims to provide a novel use of the dual antibody of the present invention.

[0015]

[0016] 1. A dual antibody comprising:

[0017] (a) An Fc domain variant in which the amino acid at position 234 (numbered according to the Kabat numbering system) in a wild type human antibody Fc domain is substituted with alanine (A), the amino acid at position 235 is substituted with proline (P), and the amino acid at position 329 is substituted with leucine (L);

[0018] (b) a first antigen binding portion linked to the Fc domain variant; and

[0019] (c) A second antigen binding portion linked to the above Fc domain variant.

[0020] 2. In the above 1, the double antibody is a double antibody composed of multiple molecules.

[0021] 3. A dual antibody characterized in that in the above 1, the first antigen binding portion or the second antigen binding portion is connected by a linker.

[0022] 4. A bispecific antibody characterized in that the human antibody in the above 1 is IgG1.

[0023] 5. In the above 1, the first antigen binding portion or the second antigen binding portion is a dual antibody characterized in that it binds to at least one of the following antigens:

[0024] Cancer target antigens, antigen-presenting cell-associated proteins, proteins expressed on activated lymphocytes, and receptor or receptor complex proteins.

[0025] 6. In the above 1, the first antigen binding portion or the second antigen binding portion is a dual antibody characterized in that it binds to at least one of the following antigens:

[0026] 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-ab1, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, Claudin 3, Claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto 1 growth factor, CS1, CTLA-4, CXCR2, CXORF61, Cyclin Bl, CYP1B1, Cadherin-3, Cyderin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, Ephrin A4, Ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-associated antigen 1,Fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gp100, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-1 1Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrin (α4, αvβ3, αvβ5, αvβ6, α4β1, α4β7, α5β1, α6β4, αⅡbβ3, including integrins), integrin alpha V, intestinal carboxyl esterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, Legumain, Lewis Y, LFA 1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, Melan A / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, Nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NYBR- 1, NY-ESO-1, o-Icetyl-GD2, OR51E2, OY-TES1, p53, p53 mutation, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA- 1 / Galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostase, prostate cancer cells, prostein, Pseudomonas aeruginosa, rabies antigen, survivin and telomerase, PRSS21, PSCA,PSMA, PTK7, RAGE-1, RANKL, Ras mutation, respiratory syncytial virus, rhesus factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoint, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1, and XAGE1.,

[0027] 7. In the above 1, the first antigen binding portion or the second antigen binding portion is a dual antibody characterized in that it binds to at least one of the following antigens:

[0028] HER2, HER3, EGFR (ErbB1, HER1), CD33, CD20, CD38, BCMA, CD138, FGFR4, GD2, PDGFR, TEM1 / CD248, and TROP-2.

[0029] 8. In the above 1, the first antigen binding portion or the second antigen binding portion is a dual antibody characterized in that it binds to at least one of the following antigens:

[0030] CD40, OX40L, Endoglin, DEC-205, 4-1BBL, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC5A, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD1A, HVEM, CD32B, PD-Ll, BDCA-2, XCR-1, and CCR2.

[0031] 9. In the above 1, the first antigen binding portion or the second antigen binding portion is a dual antibody characterized in that it binds to at least one of the following antigens:

[0032] Immunoglobulin gene superfamily members, TNF receptor superfamily members, integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins, and complement control proteins.

[0033] 10. In the above 1, the first antigen binding portion or the second antigen binding portion is a dual antibody characterized in that it binds to at least one of the following antigens:

[0034] CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152 / CTLA-4, PD-1, and ICOS.

[0035] 11. In the above 1, the first antigen binding portion or the second antigen binding portion is a dual antibody characterized in that it binds to at least one of the following antigens:

[0036] CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3.

[0037] 12. In the above 1, the first antigen binding portion or the second antigen binding portion is a dual antibody characterized in that it binds to at least one of the following antigens:

[0038] CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103 and CD104.

[0039] 13. In the above 1, the first antigen binding portion or the second antigen binding portion is a dual antibody characterized in that it binds to at least one of the following antigens:

[0040] C, S, and I type lectins.

[0041] 14. A pharmaceutical composition for preventing or treating cancer, comprising a dual antibody according to any one of 1 to 13 above as an active ingredient.

[0042] 15. In the above 14,

[0043] The cancer is characterized in that at least one selected from the group consisting of brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. Pharmaceutical composition.

[0044] 16. An antibody-drug conjugate (ADC) comprising a dual antibody according to any one of the above 1 to 13 and a drug.

[0045] 17. A pharmaceutical composition for preventing or treating cancer, comprising an antibody-drug conjugate according to the above 16 as an active ingredient, wherein the drug is an anticancer agent.

[0046]

[0047] The dual antibody of the present invention has the effect of suppressing the mechanism of action of Fc, thereby improving stability and maintaining at least the effectiveness.

[0048]

[0049] Figure 1 shows an amino acid comparison by aligning the goat IgG Fc sequence and the human IgG Fc sequence.

[0050] Figure 2a shows the analysis of the binding affinity of single or double antibodies containing wild type or Fc domain variants to the antigen CD40.

[0051] Figure 2b shows the analysis of the binding affinity of single or double antibodies containing wild type or Fc domain variants to the antigen CD47.

[0052] Figure 3a shows the analysis of the binding affinity of single or double antibodies containing wild type or Fc domain variants to FcγRⅠ.

[0053] Figure 3b shows the binding affinity of single or double antibodies containing wild type or Fc domain variants to FcγRⅡa-131H.

[0054] Figure 3c shows the binding affinity of single or double antibodies containing wild type or Fc domain variants to FcγRⅢa-158V.

[0055] Figure 4 shows the analysis of the binding affinity of single or double antibodies containing wild type or Fc domain variants to C1q.

[0056] Figures 5a and 5b show the analysis of T cell activation of single or double antibodies containing wild type or Fc domain variants.

[0057] Figure 6a shows the analysis of red blood cell counts in a preliminary toxicity test of a CD40-CD47 targeting bispecific antibody.

[0058] Figure 6b shows the analysis of platelet counts in a preliminary toxicity test of a CD40-CD47 targeting dual antibody.

[0059] Figure 6c shows the analysis of lymphocyte counts in a preliminary toxicity test of a CD40-CD47 targeting bispecific antibody.

[0060] Figure 6d shows the analysis of neutrophil counts in a preliminary toxicity test of a CD40-CD47 targeting bispecific antibody.

[0061] Figure 6e shows the analysis of monocyte counts in a preliminary toxicity test of a CD40-CD47 targeting bispecific antibody.

[0062] Figure 6f shows the analysis of spleen weight in a preliminary toxicity test of a CD40-CD47 targeting dual antibody.

[0063] Figure 6g shows an analysis of mouse body weight changes in a preliminary toxicity test of a CD40-CD47 targeting dual antibody.

[0064] Figure 7a shows an analysis of the efficacy of a single administration of an mCD40-mCD47 targeting dual antibody in a mouse model.

[0065] Figure 7b shows an analysis of the deviation of the efficacy test through a single administration of the mCD40-mCD47 targeting bispecific antibody in a mouse model.

[0066] The present invention provides a bispecific antibody comprising a human antibody Fc variant and uses thereof.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0068] Hereinafter, the advantages and features of the present invention, and the methods for achieving them, will be clarified by referring to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided only to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0069] Throughout the specification, the term "and / or" includes each and every combination of one or more of the mentioned components.

[0070] The terminology used herein is for the purpose of describing embodiments and is not intended to limit the present invention. In this specification, the singular also includes the plural unless specifically stated otherwise. As used herein, the terms "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components and / or steps.

[0071] In the present invention, the term "dual antibody" includes a "dual-specific antibody" and refers to an antibody comprising at least two antigen-binding sites. In this case, "antigen" encompasses a target antigen capable of binding to the antigen-binding site, regardless of its name.

[0072] One embodiment of the present invention comprises a bispecific antibody in which the amino acid at position 234 (numbered according to the Kabat numbering system) in the wild-type human antibody Fc domain is substituted with alanine (A), the amino acid at position 235 is substituted with proline (P), and the amino acid at position 329 is substituted with leucine (L). Such a bispecific antibody maintains efficacy while inhibiting the Fc mechanism of action.

[0073] In the present invention, not only the conventional one-letter and three-letter codes for natural amino acids are used, but also the generally accepted three-letter codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. are used.

[0074] The amino acids referred to by abbreviations in the present invention are described as follows according to the IUPAC-IUB nomenclature: Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S, Threonine: T, Tryptophan: W, Tyrosine: Y and Valine: V.

[0075] In the present invention, the position of a specific amino acid in the amino acid sequence means a position numbered according to the Kabat numbering system.

[0076] The term “amino acid modification / variation” as used herein refers to the substitution, insertion, and / or deletion, preferably substitution, of an amino acid in a polypeptide sequence. The term “amino acid substitution” or “substitution” as used herein refers to the replacement of an amino acid at a specific position in the polypeptide sequence of a wild-type human antibody Fc domain with another amino acid. For example, a human antibody Fc domain variant comprising an L235P substitution refers to a human antibody Fc domain variant in which leucine (L), the 235th amino acid residue in the amino acid sequence of a wild-type human antibody Fc domain, is replaced with proline (P).

[0077] A bispecific antibody according to one embodiment of the present invention is an antibody comprising an Fc domain variant in which the amino acid at position 234 is substituted with alanine (A), the amino acid at position 235 is substituted with proline (P), and the amino acid at position 329 is substituted with leucine (L) in a wild-type human antibody Fc domain, and a first antigen-binding portion and a second antigen-binding portion connected thereto.

[0078] In one embodiment, the bispecific antibody of the present invention may comprise any one amino acid substitution selected from the group consisting of D265L, D265M, D265N, H268Q, Y296F, Y300F, and E333V in addition to L234A, L235P, and P329L of a wild-type human antibody Fc domain.

[0079] In one embodiment, the bispecific antibody of the present invention may comprise singular or plural antibodies. In one embodiment of the present invention, even when composed of multiple antibodies, aggregation is suppressed, thereby ensuring stability.

[0080] In one embodiment, the bispecific antibody of the present invention may have a first antigen-binding portion and / or a second antigen-binding portion directly and / or indirectly linked to an Fc domain variant.

[0081] In one embodiment, the bispecific antibody of the present invention may comprise an Fc domain variant and a first antigen-binding portion linked by a linker.

[0082] In one embodiment, the bispecific antibody of the present invention may comprise an Fc domain variant and a second antigen-binding portion linked by a linker.

[0083] In one embodiment of the present invention, the linker of the bispecific antibody of the present invention may include at least one of the linker sequences of [Table 1], but is not limited thereto. The linker may directly or indirectly connect the first antigen-binding portion and / or the second antigen-binding portion to the Fc domain variant. For example, additional structures may be included between the linker and the Fc domain variant, or between the linker and the first antigen-binding portion and / or the second antigen-binding portion, and indirect connection may be achieved by such additional structures.

[0084] NameStructureMolecular weightCharacteristicFlexible linker(G)LinkerGly-Gly-Ser(75.07)nFlexible Peptide linker(GGGGS)n Linker(Gly-Gly-Gly-Gly-Ser)n(333.3)nFlexible Peptide linkerRigid linker(EAAAK)n Linker(Glu-Ala-Ala-Ala-Lys)n(488.54)nAlpha helix-forming linker(XP)n Linker(X-Pro)n(X+115.13)nProline-rich linkerDisulfide linkerDisulfide linkerCys-Cys224.3Reversible redox in response to oxidant

[0085] In one embodiment, the antibody (immunoglobulin) can be a human antibody IgA, IgM, IgE, IgD, or IgG, or a variant thereof. The human antibody can be IgG1, IgG2, IgG3, or IgG4, with IgG1 being preferred.

[0086] In one embodiment, the antibody (immunoglobulin) may be a human antibody IgG1 or a variant thereof, wherein the Fc domain of the wild-type IgG1 may comprise the amino acid sequence of SEQ ID NO: 7.

[0087] In one embodiment, the bispecific antibody comprising the Fc variant of the present invention may have reduced effector function compared to a wild-type human antibody Fc domain.

[0088] In one embodiment, the effector function can be an Fc-mediated effector function selected from C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), Fc-receptor binding including Fc-gamma receptor binding, protein A-binding, protein G-binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, Fc-containing polypeptide internalization, target downmodulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

[0089] "Antibody" refers to a substance produced within the immune system in response to antigen stimulation. Its type is not particularly limited and can be obtained naturally or non-naturally (e.g., synthetically or recombinantly). Antibodies are highly stable both in vitro and in vivo, with long half-lives, making them advantageous for mass expression and production. Furthermore, antibodies inherently possess a dimeric structure, resulting in extremely high avidity. A complete antibody consists of two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond. The constant region of antibodies is divided into the heavy chain constant region and the light chain constant region. The heavy chain constant region has the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and the subclasses are gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has the kappa (κ) and lambda (λ) types.

[0090] "Antigen" refers to a substance that specifically binds to an antibody or T-cell receptor (TCR). In the present invention, it is used to encompass any target substance that binds to an antibody. Typically, when an antigen enters the body, it induces immune activity, triggering a corresponding antigen-specific immune response.

[0091] “Heavy chain” is interpreted to mean a full-length heavy chain and fragments thereof, comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and three constant region domains CH1, CH2 and CH3, and a hinge. In addition, “light chain” is interpreted to mean a full-length light chain and fragments thereof, comprising a variable region domain VL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a constant region domain CL.

[0092] The "Fc domain", "Fc fragment" or "Fc region" forms an antibody together with the Fab domain / fragment, wherein the Fab domain / fragment is composed of the variable region (VL) of the light chain and the variable region (VH) of the heavy chain, the constant region (CL) of the light chain and the first constant region (CH1) of the heavy chain, and the Fc domain / fragment is composed of the second constant region (CH2) and the third constant region (CH3) of the heavy chain.

[0093] Antibodies can be isolated or purified by a variety of methods known in the art. Standard purification methods include chromatography, electrophoresis, immunoassays, precipitation, dialysis, filtration, concentration, and chromatofocusing. As is known in the art, various natural proteins, such as bacterial proteins A, G, and L, bind to antibodies and can be used for purification. Often, purification using specific fusion partners may be possible.

[0094] Antibodies include not only whole antibody forms but also functional fragments of antibody molecules. Whole antibodies have a structure with two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond. A functional fragment of an antibody molecule is a fragment that retains the antigen-binding function. Examples of antibody fragments include (i) a Fab fragment, which consists of the variable region (VL) of the light chain and the variable region (VH) of the heavy chain, and the constant region (CL) of the light chain and the first constant region (CH1) of the heavy chain; (ii) a Fd fragment, which consists of the VH and CH1 domains; (iii) a Fv fragment, which consists of the VL and VH domains of a single antibody; (iv) a dAb fragment, which consists of a VH domain; (v) separate CDR regions; (vi) a F(ab')2 fragment, which is a bivalent fragment containing two linked Fab fragments; (vii) single-chain Fv molecules (scFv) joined by a peptide linker that joins the VH domain and the VL domain to form an antigen-binding site; (viii) bispecific single-chain Fv dimers (PCT / US92 / 09965); and (ix) diabodies (WO94 / 13804), which are multivalent or multispecific fragments produced by gene fusion.

[0095] The Fc variant-containing bispecific antibody of the present invention can be prepared by any method known in the art. Various methods for this purpose are described in Molecular Cloning - A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons.

[0096] The nucleic acid encoding the Fc domain variant portion according to the bispecific antibody comprising the Fc variant of the present invention can be inserted into an expression vector for protein expression. The expression vector typically includes the protein operably linked, i.e., in a functional relationship, with regulatory sequences, a selectable marker, an optional fusion partner, and / or additional elements. The Fc variant according to the present invention can be produced by a method of inducing protein expression by culturing a host cell transformed with the nucleic acid under an appropriate condition, preferably an expression vector containing the nucleic acid encoding the Fc domain variant according to the present invention. Various suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and may vary depending on the host cell used. Preferably, the Fc domain variant according to the present invention can be produced using Escherichia coli, which has a low production cost and high industrial utility, as a host cell.

[0097] In one embodiment, the bispecific antibody comprising the Fc variant of the present invention can be produced by a step of culturing a host cell into which a nucleic acid encoding an Fc domain variant and an antigen-binding portion has been introduced under conditions suitable for protein expression; and a step of purifying or isolating the Fc domain variant expressed from the host cell.

[0098] In one embodiment, the antigen binding portion (first antigen binding portion, second antigen binding portion) of the bispecific antibody of the present invention may bind to at least one of a cancer target antigen, an antigen-presenting cell-associated protein, a protein expressed on activated lymphocytes, and a receptor or receptor complex protein.

[0099] In one embodiment, the antigen binding portion (first antigen binding portion, second antigen binding portion) of the bispecific antibody of the present invention is 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-ab1, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, Claudin 3, Claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto 1 growth factor, CS1, CTLA-4, CXCR2, CXORF61, Cyclin Bl, CYP1B1, Cadherin-3, Cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, Ephrin A4, Ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3,Folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, Fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gp100, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-1 1Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, Integrins (including α4, αvβ3, αvβ5, αvβ6, α4β1, α4β7, α5β1, α6β4, αⅡbβ3, integrins), integrin alpha V, intestinal carboxyl esterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, Legumain, Lewis Y, LFA 1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, Melan A / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, Nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NYBR- 1, NY-ESO-1, o-Icetyl-GD2, OR51E2, OY-TES1, p53, p53 mutation, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA- 1 / Galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostase, prostate cancer cells, prostein,Pseudomonas aeruginosa, rabies antigen, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutation, respiratory syncytial virus, rhesus factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoint, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, It may bind to at least one selected from the group consisting of TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1 and XAGE1, and preferably bind to at least one antigen selected from the group consisting of HER2, HER3, EGFR (ErbB1, HER1), CD33, CD20, CD38, BCMA, CD138, FGFR4, GD2, PDGFR, TEM1 / CD248, and TROP-2.

[0100] In one embodiment, the antigen binding portion (first antigen binding portion, second antigen binding portion) of the bispecific antibody of the present invention may bind to at least one of OX40L, Endoglin, DEC-205, 4-1BBL, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC5A, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD1A, HVEM, CD32B, BDCA-2, XCR-1, and CCR2.

[0101] In one embodiment, the antigen binding portion (first antigen binding portion, second antigen binding portion) of the bispecific antibody of the present invention may bind to at least one of an immunoglobulin gene superfamily member, a TNF receptor superfamily member, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, and a complement control protein.

[0102] In one embodiment, the antigen binding portion (first antigen binding portion, second antigen binding portion) of the bispecific antibody of the present invention may bind to at least one of CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152 / CTLA-4, PD-1, and ICOS.

[0103] In one embodiment, the antigen binding portion (first antigen binding portion, second antigen binding portion) of the bispecific antibody of the present invention may bind to at least one of CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3.

[0104] In one embodiment, the antigen binding portion (first antigen binding portion, second antigen binding portion) of the bispecific antibody of the present invention may bind to at least one of CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, and CD104.

[0105] In one embodiment, the antigen binding portion (first antigen binding portion, second antigen binding portion) of the bispecific antibody of the present invention may bind to at least one of C-type, S-type, and I-type lectins.

[0106] “Prevention” means any act of inhibiting or delaying the occurrence, spread and recurrence of cancer by administering a pharmaceutical composition according to the present invention.

[0107] "Treatment" refers to any action that kills cancer cells or improves or beneficially alters the symptoms of cancer through administration of the composition of the present invention. Those skilled in the art to which the present invention pertains will be able to accurately determine the criteria for diseases for which the composition of the present invention is effective, and determine the degree of improvement, enhancement, and cure, by referring to materials provided by the Korean Medical Association and other sources.

[0108] The term "therapeutically effective amount" used in combination with the active ingredient in the present invention means the amount of a pharmaceutically acceptable salt of the composition effective in preventing or treating the target disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the administration method, the target site, the condition of the patient, etc. Therefore, the dosage for use in humans should be determined as an appropriate amount by taking both safety and efficacy into consideration. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal testing. Such considerations in determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.

[0109] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dosage level may be determined based on factors including the patient's health condition, cancer type and severity, drug activity and sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, combination or concurrent use of drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0110] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0111] The composition of the present invention may also include a carrier, diluent, excipient, or a combination of two or more thereof commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and examples thereof include compounds described in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other common additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into a main-use dosage form such as an aqueous solution, suspension, or emulsion, or into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the field or the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0112] The composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally in the form of an injection) or orally, depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and it is more preferable to administer it once or several times a day.

[0113] Liquid preparations for oral administration of the composition of the present invention include suspensions, solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0114] In one aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the dual antibody of the present invention as an active ingredient.

[0115] In one embodiment, the present invention also provides a method of treating, preventing, ameliorating or inhibiting cancer, comprising administering to an animal (including a human or an animal other than a human) a bispecific antibody of the present invention.

[0116] The above animal may be a mammal.

[0117] The above animal may be an animal requiring administration of the dual antibody of the present invention.

[0118] The above animal may be an animal that has developed or is likely to develop cancer.

[0119] In addition, the administered dual antibody of the present invention may be an effective amount of the dual antibody.

[0120] In addition, the present invention provides a use of the dual antibody of the present invention for the manufacture of a preparation for treating or preventing cancer.

[0121] In one embodiment, the cancer can be at least one selected from the group consisting of brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

[0122] In one embodiment, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising an antibody-drug conjugate (ADC) comprising a dual antibody of the present invention and a drug as an active ingredient, wherein the drug is an anticancer agent.

[0123]

[0124] Hereinafter, the present invention will be described in more detail with examples.

[0125] Example 1. Production of antibodies with Fc domain variants

[0126] (1) Fc variant design

[0127] 다양한 동물유래의 IgG 항체들 중 고트(goat)의 IgG가 인간 FcγRs와 교차반응성(cross reactivity)이 매우 낮으므로(S. T. Jung et al. (2010) "Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells", Proceedings of the National Academy of Sciences USA (PNAS), 107:2, 604-609; S. T. Jung, et al. (2013) "Effective phagocytosis of low Her2 tumor cell lines with engineered, aglycosylated IgG displaying high FcγRⅡa affinity and selectivity", ACS Chemical Biology, 8:2, 368-375; M. Jo et al. (2018) "Engineered aglycosylated full-length IgG Fc variants exhibiting improved FcγRⅢa binding and tumor cell clearance", mAbs, 2:10, 278-289; H. W. Yoon et al.(2019) "Optimal combination of beneficial mutations for improved ADCC effector function of aglycosylated antibodies", Molecular Immunology, 114, 62-71), by comparing the sequences of goat IgG Fc and human IgG Fc, we designed human glycosylated Fc variants derived from goat IgG Fc sequence by introducing mutations centered on four regions of IgG antibody Fc (Lower hinge, B / C loop, C' / E loop, and F / G loop) predicted to interact with human FcγRs (Fig. 1).

[0128] (2) Production of bispecific antibodies containing wild type or Fc variants

[0129] An expression vector was constructed by cloning together the Fc variant containing the L234A / L235P / P329L substitutions among the previously designed glycosylated Fc variants or the wild-type Fc and the human Sirpα domain gene (binding to CD47) and mouse CD40L (binding to CD40) domain genes into the human antibody IgG1 heavy chain gene. The heavy chain gene and light chain gene were first mixed in a 1:1 ratio in Freestyle 293 expression medium (Gibco, 12338-018), and then PEI (polyethylenimine, Polyscience, 23966) and expression vector gene were mixed in a 4:1 ratio and incubated at room temperature for 20 minutes. Then, the mixture was transfected into Expi293F animal cells cultured at a density of 2 × 106 cells / ml, and cultured for 7 days under the conditions of 37°C, 125 rpm, and 8% CO2. After centrifugation, only the supernatant was recovered. The supernatant was equilibrated with 25×PBS and filtered through a 0.2 μm syringe filter. Protein A resin was added to the filtered culture medium containing the purified human Sirpα-mouse CD40L, human Sirpα, and wild-type Fc or mutants having the mouse CD40L domain, stirred at 4°C for 16 hours, spun down, and recovered the resin, which was washed with 2 ml 1× PBS. 300 μl of 100 mM glycine (pH 2.7) was added to the resin for elution, and 100 μl of 1 M Tris-HCl (pH 8.0) was used for neutralization. Amicon Ultra-4 centrifugal filter units 30K (Merck Millipore, UFC503096) were used to change the buffer, and highly pure human Sirpα-mouse CD40L, human Sirpα, and wild-type Fc or mutant Fc antibodies having the mouse CD40L domain were produced through SDS-PAGE analysis.

[0130] As a result, antibodies CPP-001-01 to CPP-001-06 containing amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 6 were obtained as shown in [Table 2] below. Each of the tables represents six types of single antibodies or double antibodies having an antigen-binding portion of anti-CD47 and / or anti-CD40, where WT (Wild Type) means an antibody containing a wild type Fc domain, and SB (Stealth Body) means an antibody containing a variant Fc domain.

[0131] CategoryFab regionFc regionDomain 1Domain 2CPP-001(Anti-CD47 / CD40 surrogate)CPP-001-1_WTAnti-mouse CD47Anti-mouse CD40Wild typeCPP-001-2_SBAnti-mouse CD47Anti-mouse CD40MutantCPP-001-3_WTAnti-mouse CD47-Wild typeCPP-001-4_SBAnti-mouse CD47-MutantCPP-001-5_WT-Anti-mouse CD40Wild typeCPP-001-6_SB-Anti-mouse CD40Mutant

[0132] In addition, if sequence numbers 1 to 7 are organized, they are as shown in [Table 3] below. [Table 3] shows the amino acid sequence of each sequence number (amino acids at positions 234, 235, and 329 numbered according to the Kabat numbering system in the amino acid sequence are highlighted and underlined) and which antibody contains the amino acid sequence of each sequence number.

[0133]

[0134] Example 2. Confirmation of binding affinity of dual antibodies to antigens, FcγR, and C1q.

[0135] (1) Confirmation of binding affinity to antigen

[0136] To confirm the CD40 and CD47 binding affinity of purified antibodies containing wild-type Fc or Fc domain variants produced in Example 1, ELISA analysis was performed. Specifically, 50 μl each of CD40-GST and CD47-GST, diluted to 4 μg / ml in 0.05 M Na2CO3 (pH 9.6), were immobilized in a flat-bottom polystyrene high-bind 96-well microplate (Costar, 3590) at 4°C for 16 hours, washed, and 100 μl of 4% skim milk (GenomicBase, SKI400) was added and blocked at room temperature for 1 hour. Subsequently, washing was performed four times with 180 μl of 0.05% PBST. After washing four times, 50 μl of antibodies containing wild-type Fc or Fc domain variants serially diluted with 1% skim milk were dispensed into each well and reacted for 1 hour at room temperature. After washing, antibody reaction was performed for 1 hour at room temperature using 50 μl of HRP-Protein L (GenScript, M00098) and washed again. 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added at 50 μl each to develop color, and 2 M H2SO4 was added at 50 μl each to terminate the reaction. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek). For reference, commercially available antibodies (Commercial anti-mCD40, Commercial anti-mCD47) were also analyzed in the same manner. As a result, it was confirmed that the expressed antibodies (CPP-001-1_WT, CPP-001-2_SB, CPP-001-3_WT, CPP-001-4_SB, CPP-001-5_WT, CPP-001-6_SB) had binding affinity for each antigen (Fig. 2a and Fig. 2b).

[0137] (2) Confirmation of binding affinity to FcγR

[0138] Similarly, ELISA analysis was performed to determine the binding affinity of purified antibodies containing wild-type Fc or Fc domain variants to FcγRs.

[0139] Specifically, 50 μl of each FcγRs-GST (FcγRIGST, FcγRⅡa-131H-GST, or FcγRⅢa-158V-GST) diluted to 4 μg / ml in 0.05 M Na2CO3 (pH 9.6) was immobilized in a flat-bottom polystyrene high-bind 96-well microplate (Costar, 3590) at 4°C for 16 h, washed, and 100 μl of 4% skim milk (GenomicBase, SKI400) was added, followed by blocking for 1 h at room temperature. Washing was performed four times with 180 μl of 0.05% PBST. After washing four times, 50 μl of purified wild-type Fc or Fc domain variant antibody serially diluted with 1% skim milk was dispensed into each well and reacted for 1 hour at room temperature. After washing, antibody reaction was performed for 1 hour at room temperature using 50 μl of HRP-Protein L (GenScript, M00098) and washed again. 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added at 50 μl each to develop color, and 2 M H2SO4 was added at 50 μl each to terminate the reaction. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek). For reference, commercially available antibodies (Commercial anti-mCD40, Commercial anti-mCD47) were also analyzed in the same manner. As a result, it was confirmed that the CPP-001-2_SB, CPP-001-4_SB, and CPP-001-6_SB antibodies containing human antibody Fc domain variants all had their binding affinity eliminated, and the CPP-001-1_WT, CPP-001-3_WT, and CPP-001-5_WT antibodies having wild-type Fc all had their binding affinity maintained (Figures 3a to 3c).That is, the effect of eliminating binding affinity to FcγR by introducing the Fc domain variant was confirmed.

[0140] (3) Confirmation of binding affinity to C1q

[0141] ELISA analysis was performed to determine the C1q binding affinity of purified antibodies containing wild-type Fc or Fc domain variants.

[0142] Specifically, 50 μl each of human Sirpα-mouse CD40L, human Sirpα, and antibodies containing wild-type Fc and Fc domain variants having mouse CD40L domains, diluted to 4 μg / ml in 0.05 M Na2CO3 (pH 9.6), were immobilized in a flat-bottom polystyrene high-bind 96-well microplate (Costar, 3590) at 4°C for 16 h, and then blocked with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 h. After washing four times with 180 μl of 0.05% PBST, 50 μl of C1q (Quidel, A400) protein serially diluted with 1% skim milk was dispensed into each well and reacted for 1 h at room temperature. After washing the plate, 50 μl of anti-C1q-HRP (Invitrogen, PA1-84324) was added to each well, incubated at room temperature for 1 hour, and washed again. 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added at 50 μl each to develop color, and the reaction was terminated by adding 50 μl each of 2 M H2SO4. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek). For reference, commercially available antibodies (Commercial anti-mCD40, Commercial anti-mCD47) were also analyzed in the same manner. As a result, it was confirmed that the CPP-001-2_SB, CPP-001-4_SB, and CPP-001-6_SB antibodies containing human antibody Fc domain variants all had their binding affinity eliminated, and the CPP-001-1_WT, CPP-001-3_WT, and CPP-001-5_WT antibodies having wild-type Fc all had their binding affinity maintained (Fig. 4).That is, the introduction of the Fc domain variant confirmed the effect of eliminating binding affinity to C1q.

[0143] Example 3. T cell activation analysis based on CD40-CD47 targeting bispecific antibodies.

[0144] FACS analysis was performed to confirm the activation effect of CD4+ and CD8+ immune cells of purified antibodies containing wild-type Fc or Fc domain variants produced in Example 1. Specifically, BMDCs collected from mouse bone marrow were cultured at 3 x 10 in cRPMI containing 20 ng / mL GM-CSF. 6 / mL cultured and differentiated, immature BMDCs were obtained on the 7th day. EL4 cells, which are APC cells cultured in the BMDCs obtained in this way, were treated with test substances, and co-cultured for 24 hours. On the 8th day, pan T cells were obtained from splenocytes obtained from mouse spleen using a Pan CD3 T cell isolation kit (#480031, Biolegend) and stained with eFluor670 (#65-0840-85, Thermo scientific). After removing media and EL4 cells from the co-cultured BMDCs, 5 X 10 pan T cells were cultured together with 100 U / mL IL-2. 5 The dogs were treated and co-cultured for 72 hours. On the 11th day, all cells were obtained and stained with antibodies such as anti-CD44 and anti-CD25, and then analyzed using FACS.

[0145] As a result, when treated with a mouse CD40 and mouse CD47 targeting bispecific antibody (CPP-001-1_WT) having a wild-type Fc or a mouse CD47 targeting monospecific antibody (CPP-001-4_SB) containing an Fc domain variant, the activation of CD4+ and CD8+ T cells was not promoted but rather suppressed, whereas when treated with a mouse CD40 and mouse CD47 targeting bispecific antibody (CPP-001-2_SB) containing an Fc domain variant, the activation of both CD4+ and CD8+ T cells was promoted. This promotion of T cell activation and T cell proliferation was confirmed by the increase in the proportion of cells expressing CD25+ among the CD8+CD44+ population (Fig. 5a and Fig. 5b). These results demonstrate that mouse CD40 and mouse CD47-targeting bispecific antibodies containing Fc domain variants can promote T cell activation, and in particular, significantly induce CD8+ T cell activation through antigen cross-presentation by dendritic cells.

[0146] Example 4. Preliminary in vivo toxicity analysis of CD40-CD47 targeting bispecific antibodies in mice

[0147] In order to confirm the hematological toxicity of the purified antibody containing the wild-type Fc or Fc domain variant produced in Example 1, a single-dose toxicity test was performed on Balb / c mice. Specifically, 6-week-old female Balb / c mice were acclimatized for 1 week, and at 7 weeks of age, purified antibodies containing the wild-type Fc or Fc domain variants diluted in Chung-Oe NS Injection (CHD21022, Chung-Oe Pharmaceutical) were administered at a concentration of 10 mg / kg per mouse at a dose of 5 mL / kg. The mice were randomly separated into groups of 5 and their body weights were measured on days 0, 1, 3, 5, and 12 after administration. Five mice per group were anesthetized with respiratory anesthetic (isoflurane) on each day and blood samples were collected from the posterior vena cava and abdominal aorta. EDTA-2K was used as an anticoagulant during this process. The collected blood was measured using a blood analyzer (XN-10, Sysmex) and hematological analysis was performed. The red blood cell count, platelet count, lymphocyte count, neutrophil count, and monocyte count were measured. In addition, the spleen was removed at autopsy and its weight was measured. For reference, the same analysis was performed on the positive control group (CD47 positive control group, CD40 positive control group).

[0148] As a result, in the case of the CD47 control antibody used as a positive control antibody when administering antibodies to the mouse model, a decrease in RBCs, platelets, and lymphocytes was confirmed from day 1 to day 5 of administration, and the dual antibody administration group containing the wild-type Fc domain (CPP-001-1_WT) also showed a similar decrease. However, the dual antibody administration group containing the Fc domain variant (CPP-001-2_SB) did not show such a decrease in blood cells, and it was confirmed that they were maintained within the normal range (Figs. 6a to 6c). Neutrophils and monocytes, which are immune cells that appear when an inflammatory response is induced, rapidly increased in both the CD47 control antibody and the dual antibody administration group containing the wild-type Fc domain on day 1 and 3, but it was confirmed that the immune cells were maintained within the normal range when the dual antibody containing the Fc domain variant was administered (Figs. 6d and 6e). Furthermore, we confirmed that the spleen, a representative immune organ, became enlarged when administered with a dual antibody containing a control antibody and a wild-type Fc domain, and body weight also decreased on days 1 and 3 of administration, which was then confirmed to recover. In contrast, splenomegaly and body weight loss were not observed in the dual antibody group containing the Fc domain variant (Figures 6f and 6g). These preliminary toxicity results confirm the safety of antibody technology containing Fc domain variants against indicators of side effects common to conventional antibody-based cancer immunotherapies.

[0149] Example 5. Validation of efficacy through a single administration of a mCD40-mCD47 targeting bispecific antibody in a mouse model.

[0150] In order to confirm the efficacy of the purified antibody containing the wild-type Fc or Fc domain variant produced in Example 1 in a mouse model, a single-dose efficacy test was performed in a syngeneic mouse model transplanted with the CT26 mouse colon cancer cell line. Specifically, 6-week-old female Balb / c mice were acclimatized for 2 weeks, and then at 8 weeks of age, the CT26 cell line was subcultured in DMEM supplemented with 10% FBS and 1X antibiotics. The cell number was measured, and then mixed with Matrigel (356231, discovery labware inc) and 1x10 5 After making it into 0.1 mL, it was implanted subcutaneously into the right flank of a mouse anesthetized with 200 mg / kg tribromoethanol (Averin) using a 1 mL syringe. The tumor grew to 50-100 mm. 3 After group separation upon reaching the target, weights were measured and the test substance was administered. The test substance was diluted in Corning® PBS (Phosphate-Buffered Saline) and administered as a single dose of 10 mg / kg, 200 uL per animal via the caudal vein. Tumor volumes were measured using a Vernier caliper on days 0, 4, 7, 11, 14, and 18 after administration.

[0151] As a result, when comparing the test groups (G5, G6) administered with mCD47 or mCD40-targeting single antibodies and the test group (G4) administered with mCD47 and mCD40-targeting single antibodies, the test group administered with the combination showed a tumor volume reduction effect similar to the test group administered with mCD47-targeting single antibodies (G6). In the case of dual antibodies, the test group administered with dual antibodies containing wild-type Fc (G2) showed an effect similar to the test group administered with mCD47 and mCD40-targeting single antibodies (G4), whereas the test group administered with dual antibodies containing Fc domain variants (G3) showed the highest tumor volume reduction effect. In addition, it was confirmed that there was a significant difference in efficacy compared to the test group administered with vehicle (G1) (Fig. 7a).

[0152] Furthermore, since efficacy was confirmed with a single administration, most test groups showed large variations, but in contrast, the bispecific antibody containing the Fc domain variant exhibited the strongest tumor suppression effect with almost no inter-individual variation (Fig. 7b). This demonstrates that the bispecific antibody containing the Fc domain variant has the effect of activating and suppressing the anti-cancer immune function of CD8+ T cells by inducing the activation of dendritic cells and macrophages.

[0153] From the above results, it can be seen that the bispecific antibody of the present invention suppresses the Fc mechanism of action, thereby enhancing stability and maintaining at least some efficacy. Furthermore, it can be seen that the bispecific antibody of the present invention can also be used for anticancer purposes.

Claims

1. A dual antibody comprising: (a) An Fc domain variant in which the amino acid at position 234 (numbered according to the Kabat numbering system) in a wild type human antibody Fc domain is substituted with alanine (A), the amino acid at position 235 is substituted with proline (P), and the amino acid at position 329 is substituted with leucine (L); (b) a first antigen binding portion linked to the Fc domain variant; and (c) A second antigen binding portion linked to the above Fc domain variant.

2. In paragraph 1, The above double antibody is a double antibody composed of multiple molecules.

3. In paragraph 1, A bispecific antibody characterized in that the first antigen binding portion or the second antigen binding portion is connected to the Fc domain variant by a linker.

4. In paragraph 1, A bispecific antibody characterized in that the above human antibody is IgG1.

5. In paragraph 1, A bispecific antibody characterized in that the first antigen binding portion or the second antigen binding portion binds to at least one of the following antigens: Cancer target antigens, antigen-presenting cell-associated proteins, proteins expressed on activated lymphocytes, and receptor or receptor complex proteins.

6. In paragraph 1, A bispecific antibody characterized in that the first antigen binding portion or the second antigen binding portion binds to at least one of the following antigens: 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-ab1, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, Claudin 3, Claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto 1 growth factor, CS1, CTLA-4, CXCR2, CXORF61, Cyclin Bl, CYP1B1, Cadherin-3, Cyderin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, Ephrin A4, Ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-associated antigen 1,Fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gp100, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-1 1Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrin (α4, αvβ3, αvβ5, αvβ6, α4β1, α4β7, α5β1, α6β4, αⅡbβ3, including integrins), integrin alpha V, intestinal carboxyl esterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, Legumain, Lewis Y, LFA 1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, Melan A / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, Nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NYBR- 1, NY-ESO-1, o-Icetyl-GD2, OR51E2, OY-TES1, p53, p53 mutation, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA- 1 / Galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostase, prostate cancer cells, prostein, Pseudomonas aeruginosa, rabies antigen, survivin and telomerase, PRSS21, PSCA,PSMA, PTK7, RAGE-1, RANKL, Ras mutation, respiratory syncytial virus, rhesus factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoint, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1, and XAGE1., 7. In paragraph 1, A bispecific antibody characterized in that the first antigen binding portion or the second antigen binding portion binds to at least one of the following antigens: HER2, HER3, EGFR (ErbB1, HER1), CD33, CD20, CD38, BCMA, CD138, FGFR4, GD2, PDGFR, TEM1 / CD248, and TROP-2.

8. In paragraph 1, A bispecific antibody characterized in that the first antigen binding portion or the second antigen binding portion binds to at least one of the following antigens: CD40, CD47, OX40L, Endoglin, DEC-205, 4-1BBL, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC5A, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD1A, HVEM, CD32B, PD-Ll, BDCA-2, XCR-1, and CCR2.

9. In paragraph 1, A bispecific antibody characterized in that the first antigen binding portion or the second antigen binding portion binds to at least one of the following antigens: Immunoglobulin gene superfamily members, TNF receptor superfamily members, integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins, and complement control proteins.

10. In paragraph 1, A bispecific antibody characterized in that the first antigen binding portion or the second antigen binding portion binds to at least one of the following antigens: CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152 / CTLA-4, PD-1, and ICOS.

11. In paragraph 1, A bispecific antibody characterized in that the first antigen binding portion or the second antigen binding portion binds to at least one of the following antigens: CD27, CD40, CD47. CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3.

12. In paragraph 1, A bispecific antibody characterized in that the first antigen binding portion or the second antigen binding portion binds to at least one of the following antigens: CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103 and CD104.

13. In paragraph 1, A bispecific antibody characterized in that the first antigen binding portion or the second antigen binding portion binds to at least one of the following antigens: C-, S-, and I-type lectins.

14. A pharmaceutical composition for preventing or treating cancer, comprising a dual antibody according to any one of claims 1 to 13 as an active ingredient.

15. In paragraph 14, The cancer is characterized in that at least one selected from the group consisting of brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. Pharmaceutical composition.

16. An antibody-drug conjugate (ADC) comprising a dual antibody according to any one of claims 1 to 13 and a drug.

17. A pharmaceutical composition for preventing or treating cancer, comprising an antibody-drug conjugate according to Article 16 as an active ingredient, wherein the drug is an anticancer agent.

Images