Antibody-drug conjugate comprising human antibody fc variant and use thereof

Abstract

The present invention provides an antibody-drug conjugate and a use thereof, the antibody-drug conjugate comprising: an antibody comprising an Fc domain variant and a first antigen-binding portion linked to the Fc domain variant; and a drug linked to the antibody. The antibody-drug conjugate of the present invention exhibits improved stability, maintains at least comparable efficacy, and offers the advantage of being stable.

Description

Antibody-drug conjugates comprising human antibody FC variants and uses thereof

[0001] The present invention relates to an antibody-drug conjugate (ADC) comprising a human antibody Fc variant and uses thereof.

[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 they bind. 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 effector functions 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, binding of the Fc domain of an antibody to FcγRⅢa induces ADCC, binding to FcγRI or FcγRⅡa induces ADCP, and binding to the circulating complement protein C1q activates complement-dependent cytotoxicity (CDC) mechanisms. Subsequently, an immune response is elicited against the target bound to the Fab domain according to each mechanism of action, resulting in 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, in the case of antibody-drug conjugates (ADCs), there is a safety issue that arises from destroying normal cells expressing the target antigen rather than defective cells (cancer, infected cells), and the antibody-drug conjugate may induce safety issues and unwanted side effects by entering and destroying normal cells or immune cells expressing FcγR.

[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 mutants with the Fc mechanism of action eliminated, and antibody-drug conjugates (ADCs) based on Fc mutants with suppressed Fc mechanism of action are needed to specifically bind to the target while reducing undesirable side effects. In this case, when an Fc mutant with suppressed Fc mechanism of action 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 the wild type antibody-drug conjugate even when an Fc mutant with suppressed mechanism of action is applied.

[0010]

[0011] The present invention aims to provide an antibody-drug conjugate (ADC) that suppresses the Fc mechanism of action while maintaining at least the efficacy.

[0012] In addition, the present invention aims to provide a novel use of the antibody-drug conjugate of the present invention.

[0013]

[0014] 1. An antibody-drug conjugate (ADC) comprising:

[0015] (a) an antibody comprising an Fc domain variant in which the amino acid at position 234 (numbered according to the Kabat numbering system) 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 linked to the Fc domain variant; and (b) a drug linked to the antibody.

[0016] 2. In the above 1, the antibody-drug conjugate is an antibody-drug conjugate composed of multiple components.

[0017] 3. An antibody-drug conjugate according to the above 1, wherein the drug is linked to at least one amino acid selected from the group consisting of cysteine ​​and lysine of the antibody.

[0018] 4. An antibody-drug conjugate according to the above 1, wherein the antibody further comprises a second antigen binding portion.

[0019] 5. An antibody-drug conjugate according to 1 above, wherein the antibody and the drug are connected by a linker.

[0020] 6. In the above 1, the human antibody is IgG1, an antibody-drug conjugate.

[0021] 7. In the above 1, the antibody-drug conjugate binds to at least one of the following antigens:

[0022] 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.,

[0023] 8. In the above 1, the antibody-drug conjugate binds to at least one of the following antigens:

[0024] HER2, HER3, EGFR (ErbB1, HER1), CD33, CD20, CD38, BCMA, CD138, FGFR4, GD2, PDGFR, TEM1 / CD248, and TROP-2.

[0025] 9. In the above 1, the drug is at least one selected from the group consisting of auristatin, maytansinoid, tubulysin, calicheamicin, duocarmycin, exatecan, and pyrrolobenzodiazepine, an antibody-drug conjugate.

[0026] 10. In the above 5, the linker is at least one selected from the group consisting of Val-Cit-PABA, Gly-Gly-Phe-Gly, Val-Ala-PABA, sulfo-SPDB, SMCC, SPDB, oxime, Ala-Ala-PABA, CL2A, Val-Cit, hydrazone, mc-VC-PABC, dipeptide, and tetrapeptide, an antibody-drug conjugate.

[0027] 11. A pharmaceutical composition for preventing or treating cancer, comprising an antibody-drug conjugate according to any one of 1 to 10 above as an active ingredient, wherein the drug is an anticancer agent.

[0028] 12. In the above 11, the cancer is 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, lymphatic cancer, 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. A pharmaceutical composition for preventing or treating cancer, comprising at least one selected from the group consisting of:

[0029]

[0030] The antibody-drug conjugate of the present invention has an effect of suppressing the Fc mechanism of action while maintaining at least the efficacy.

[0031] In addition, the antibody-drug conjugate of the present invention has the effect of suppressing aggregation and being stable.

[0032]

[0033] Figure 1 shows an amino acid comparison by aligning the goat IgG Fc sequence and the human IgG Fc sequence.

[0034] Figure 2a shows the analysis of the cell killing ability of antibody-drug conjugates (Trastuzumab antibody, MMAE drug) containing wild type or Fc domain variants against HER2-expressing cancer cell line SKBR-3.

[0035] Figure 2b shows the analysis of the cell killing ability of antibody-drug conjugates (Trastuzumab antibody, MMAE drug) containing wild type or Fc domain variants against HER2-expressing cancer cell line BT474.

[0036] Figure 2c shows the analysis of the cell killing ability of antibody-drug conjugates (Trastuzumab antibody, Tesirine drug) containing wild type or Fc domain variants against HER2-expressing cancer cell line SKBR-3.

[0037] Figure 2d shows the analysis of the cell killing ability of antibody-drug conjugates (Trastuzumab antibody, Tesirine drug) containing wild type or Fc domain variants against HER2-expressing cancer cell line BT474.

[0038] Figure 2e shows the analysis of the cell killing ability of antibody-drug conjugates (Pertuzumab antibody, MMAE drug) containing wild type or Fc domain variants against HER2-expressing cancer cell line BT474.

[0039] Figure 3a shows the analysis of the cell killing ability of antibody-drug conjugates (Cetuximab antibody, MMAE drug) containing wild type or Fc domain variants against EGFR expressing cancer cell line MDA-MB-468.

[0040] Figure 3b shows the analysis of the cell killing ability of antibody-drug conjugates (Nimotuzumab antibody, MMAE drug) containing wild type or Fc domain variants against EGFR expressing cancer cell line MDA-MB-468.

[0041] Figure 3c shows the analysis of the cell killing ability of antibody-drug conjugates (Necitumumab antibody, MMAE drug) containing wild type or Fc domain variants against EGFR expressing cancer cell line MDA-MB-468.

[0042] Figure 4 shows the analysis of the cell killing ability of antibody-drug conjugates containing wild type or Fc domain variants against EGFR expressing cancer cell line (MDA-MB-468).

[0043] Figure 5a shows the analysis of the cell killing ability of antibody-drug conjugates (Gemtuzumab antibody, MMAE drug) containing wild type or Fc domain variants against CD33-expressing cancer cell line THP-1.

[0044] Figure 5b shows the analysis of the cell killing ability of antibody-drug conjugates (Gemtuzumab antibody, MMAE drug) containing wild type or Fc domain variants against CD33-expressing cancer cell line U937.

[0045] Figure 5c shows the analysis of the cell killing ability of antibody-drug conjugates (Gemtuzumab antibody, MMAE drug) containing wild type or Fc domain variants against CD33 expressing cancer cell line MV-4-11.

[0046] Figure 5d shows the analysis of the cell killing ability of antibody-drug conjugates (Gemtuzumab antibody, MMAE drug) containing wild type or Fc domain variants against CD33 expressing cancer cell line HL-60.

[0047] Figure 6a shows the analysis of the cell killing ability of antibody-drug conjugates (Atezolizumab antibody, MMAE drug) containing wild type or Fc domain variants against the PD-L1 expressing cancer cell line Calu-1.

[0048] Figure 6b shows the analysis of the cell killing ability of antibody-drug conjugates (Atezolizumab antibody, MMAE drug) containing wild type or Fc domain variants against PD-L1 expressing cancer cell line KAPRAS 299.

[0049] Figure 7a shows the analysis of the aggregation effect of an antibody-drug conjugate containing a human antibody Fc variant through SEC-HPLC.

[0050] Figure 7b shows the analysis of the aggregation effect of an antibody-drug conjugate containing a human antibody Fc variant through HIC DAR analysis.

[0051] Figure 7c shows an analysis of the aggregation effect of an antibody-drug conjugate containing a human antibody Fc variant through Concentration Measurement.

[0052] Figure 8a shows the analysis of the cell killing ability of antibody-drug conjugates (Nimotuzumab antibody, MMAE drug) containing wild type or Fc domain variants against EGFR expressing cancer cell line A431.

[0053] Figure 8b shows the analysis of the cell killing ability of antibody-drug conjugates (Cetuximab antibody, MMAE drug) containing wild type or Fc domain variants against EGFR expressing cancer cell line A431.

[0054] Figures 9a and 9b show in vivo tests analyzing the tumor suppression effect according to the administered dose of an antibody-drug conjugate containing a wild type or Fc domain variant.

[0055] Figure 10a shows the analysis of the tumor suppression effect over time of an antibody-drug conjugate (Nimotuzumab antibody, MMAE drug) containing a wild type or Fc domain variant.

[0056] Figure 10b shows the analysis of the tumor suppression effect over time of antibody-drug conjugates (Cetuximab antibody, MMAE drug) containing wild type or Fc domain variants.

[0057] The present invention provides an antibody-drug conjugate comprising a human antibody Fc variant and uses thereof.

[0058] 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.

[0059] 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.

[0060] Throughout the specification, the term "and / or" includes each and every combination of one or more of the mentioned components.

[0061] 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.

[0062] In the present invention, an "antibody-drug conjugate" is formed by linking an antibody and a drug, and the antibody refers to an antibody containing at least one antigen-binding portion. In this case, "antigen" encompasses any conjugate capable of binding to the antigen-binding portion, regardless of its name.

[0063] An antibody-drug conjugate according to one embodiment of the present invention comprises an Fc domain variant in which the amino acid at position 234 (numbered according to the Kabat numbering system) is substituted with alanine (A), the amino acid at position 235 with proline (P), and the amino acid at position 329 with leucine (L) in a wild-type human antibody Fc domain, and an antibody comprising a first antigen-binding portion linked to the Fc domain variant, and a drug linked to the antibody. Such an antibody-drug conjugate maintains efficacy while inhibiting the Fc mechanism of action. In addition, the antibody-drug conjugate according to one embodiment of the present invention may be formed in multiple units. In one embodiment of the present invention, even when formed in multiple units, since mutual aggregation is suppressed contrary to expectations, stability can be ensured.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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).

[0068] An antibody-drug conjugate, which is one embodiment of the present invention, is an antibody comprising an Fc domain variant in which the amino acid at position 234 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), a first antigen-binding portion linked thereto, and a drug linked thereto.

[0069] In one embodiment, the antibody-drug conjugate of the present invention may further comprise, in addition to the above substitutions, any one amino acid substitution selected from the group consisting of substitution of the amino acid at position 265 with leucine (L), methionine (M), or asparagine (N), substitution of the amino acid at position 268 with glutamine (Q), substitution of the amino acid at position 296 with phenylalanine (F), substitution of the amino acid at position 300 with phenylalanine (F), and substitution of the amino acid at position 333 with valine (V).

[0070] In one embodiment, the antibody-drug conjugate of the present invention may comprise singular or plural conjugates.

[0071] In one embodiment, the drug in the antibody-drug conjugate of the present invention may comprise a drug linked to at least one amino acid selected from the group consisting of cysteine ​​and lysine of the antibody.

[0072] In one embodiment of the present invention, the antibody-drug conjugate may include at least one of the drugs in [Table 1] below, but is not limited thereto.

[0073] ClassificationDrugsDrug structureDrug mechanismTubulin inhibitorAuristatins Maytansinoids induce apoptosis by disrupting microtubule growth by promoting tubulin polymerization. Tubulysins induce apoptosis by blocking the polymerization of tubulin dimers and inhibiting the formation of mature microtubules. DNA damaging agents that induce apoptosis by inhibiting tubulin polymerization. Calicheamicins Duocarmycins bind to the groove of the DNA double strand, causing DNA strand breaks and inducing cell death. Exatecans bind to the minor groove of DNA and induce apoptosis by alkylating the nucleobase adenine at the N3 position. Pyrrolobenzodiazepines bind to the topoisomerase I and DNA complex, prevent DNA religation, and induce DNA damage, thereby inducing apoptosis. Induces apoptosis by creating cross-links between DNA strands

[0074]

[0075] In one embodiment, the antibody in the antibody-drug conjugate of the present invention may further comprise a second antigen binding portion, and the second antigen binding portion may be linked to an Fc domain variant.

[0076] In one embodiment, the antibody of the present invention may be directly and / or indirectly linked to the Fc domain variant through the first antigen-binding portion and / or the second antigen-binding portion. The first antigen-binding portion and / or the second antigen-binding portion may be directly or indirectly linked to the Fc domain variant through a linker. 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 the linker may be indirectly linked through such additional structures. The linker may be comprised of a known amino acid sequence that links the wild-type Fc domain and the antigen-binding portion present in a wild-type antibody.

[0077] In one embodiment, the antibody-drug conjugate of the present invention may comprise an antibody and a drug linked by a linker.

[0078] In one embodiment of the present invention, the linker is preferably, but not necessarily, chemically stable to extracellular conditions, and may be designed to be cleaved, sacrificed, and / or otherwise specifically degraded within the cell (cleavable linker). Alternatively, a linker that is not designed to be specifically cleaved or degraded within the cell (non-cleavable linker) may be used. In addition, a wide variety of linkers useful for linking drugs to antibodies in the context of ADCs are known in the art, such as site-specific linkers, PEG linkers, and the like. The linker may be connected by a linker comprising at least one of these linkers, but is not limited thereto.

[0079] In one embodiment, the linker connecting the antibody and the drug of the antibody-drug conjugate of the present invention may be at least one selected from the group consisting of Val-Cit-PABA, Gly-Gly-Phe-Gly, Val-Ala-PABA, sulfo-SPDB, SMCC, SPDB, oxime, Ala-Ala-PABA, CL2A, Val-Cit, hydrazone, mc-VC-PABC, dipeptide, and tetrapeptide, but is not limited thereto.

[0080] 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.

[0081] 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: 22.

[0082] In one embodiment, an antibody-drug conjugate comprising an Fc variant of the present invention may have reduced effector function compared to a wild-type human antibody Fc domain.

[0083] 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.

[0084] "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.

[0085] "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.

[0086] “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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] The antibody-drug conjugate comprising the Fc variant 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.

[0091] The nucleic acid encoding the Fc domain variant portion of the antibody-drug conjugate 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 of 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 a nucleic acid encoding the Fc domain variant of 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 of the present invention can be produced using Escherichia coli, which has low production costs and high industrial utility, as a host cell.

[0092] In one embodiment, an antibody-drug conjugate comprising an Fc variant of the present invention can be prepared by: culturing a host cell into which a nucleic acid encoding an Fc domain variant has been introduced under conditions suitable for protein expression; purifying or isolating the expressed Fc domain variant from the host cell; and linking the Fc domain variant and a linker-drug.

[0093] An antibody-drug conjugate, which is an embodiment of the present invention, can be obtained, for example, through the following process.

[0094] 1) Add TCEP working solution to the antibody and incubate at 25°C to 37°C for 2 hours to reduce the antibody.

[0095] 2) Prepare the Linker-payload by dissolving it in a DMSO solution of a specific concentration. At this time, the volume of the Linker-payload solution required for each conjugation reaction is calculated based on the molar concentration.

[0096] 3) The antibody and Linker-payload solution prepared in a 10% DMSO environment are mixed at the target molar ratio and reacted at 10°C for 1 hour.

[0097] 4) The antibody-drug conjugate generated after the reaction is purified using Amicon Ultra-15 Centrifugal Filter Ultracel-30K and finally filtered using a 0.22 μm filter.

[0098] 5) The obtained conjugate undergoes QC analysis by SEC-HPLC, LC-MS, and HPLC-HIC.

[0099] In one embodiment, the antibody of the antibody-drug conjugate of the present invention binds to 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, 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 mutations, 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 It may bind to at least one antigen selected from the group consisting of CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1 and XAGE1, and preferably may 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, and preferably may 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] “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.

[0101] "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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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).

[0106] 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.

[0107] 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.

[0108] In one aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody-drug conjugate of the present invention as an active ingredient, wherein the drug is an anticancer agent.

[0109] 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 a non-human animal) an antibody-drug conjugate of the present invention.

[0110] The above animal may be a mammal.

[0111] The above animal may be an animal requiring administration of the antibody-drug conjugate of the present invention.

[0112] The above animal may be an animal that has developed or is likely to develop cancer.

[0113] Additionally, the antibody-drug conjugate of the present invention administered above may be an effective amount of the antibody-drug conjugate.

[0114] The present invention also provides a use of the antibody-drug conjugate of the present invention for the manufacture of a preparation for treating or preventing cancer.

[0115] The drug of the antibody-drug conjugate of the present invention may be an anticancer agent.

[0116] 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.

[0117]

[0118] Hereinafter, the present invention will be described in more detail with examples.

[0119] Example 1. Production of antibodies with Fc domain variants

[0120] (1) Fc variant design

[0121] 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).

[0122] (2) Production of antibodies containing wild type or Fc variants

[0123] Among the previously designed Fc variants, the Fc variants containing L234A / L235P / P329L substitutions or the wild-type Fc were cloned into the model antibody heavy chain gene to construct an expression vector. The heavy chain gene and the 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 the expression vector gene were mixed in a 4:1 ratio and incubated at room temperature for 20 minutes. 2 × 10 6After transfection into Expi293F animal cells cultured at a density of 10 cells / ml, the cells were cultured for 7 days under the conditions of 37°C, 125 rpm, and 8% CO2, and then centrifuged to collect only the supernatant. 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 Fc variant or wild-type Fc, stirred at 4°C for 16 hours, spun down, and the resin was recovered and washed with 2 ml of 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 SDS-PAGE analysis was performed. As a result, antibodies containing high-purity glycosylated Fc variants or wild-type Fc as shown in [Table 2] below could be obtained. [Table 2] shows the amino acid sequences constituting the heavy and light chains of each model antibody containing an Fc variant or a model antibody containing a wild-type Fc, and the types of antibodies.

[0124] Classification Amino acid sequence type Heavy chain Light chain Trastuzumab Fc-WT SEQ ID NO: 1 SEQ ID NO: 3 IgG1 Trastuzumab Fc-SB SEQ ID NO: 2 Cetuximab Fc-WT SEQ ID NO: 4 SEQ ID NO: 6 Cetuximab Fc-SB SEQ ID NO: 5 Gemtuzumab Fc-WT SEQ ID NO: 7 SEQ ID NO: 9 IgG4 Gemtuzumab Fc-SB SEQ ID NO: 8 Nimotuzumab Fc-WT SEQ ID NO: 10 SEQ ID NO: 12 IgG1 Nimotuzumab Fc-SB SEQ ID NO: 11 Necitumumab Fc-WT SEQ ID NO: 13 SEQ ID NO: 15 Necitumumab Fc-SB SEQ ID NO: 14 Atezolizumab Fc-WT SEQ ID NO: 16 SEQ ID NO: 18 Atezolizumab Fc-SB SEQ ID NO: 17 Pertuzumab Fc-WT SEQ ID NO: 19 SEQ ID NO: 21 Pertuzumab Fc-SB sequence number 20

[0125] In addition, if sequence numbers 1 to 22 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.

[0126] 서열번호아미노산 서열명칭1EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKTrastuzumab Heavy chain Fc-WT2EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALLAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKTrastuzumab Heavy chainFc-SB3DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECTrastuzumab Light chain4QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKCetuximab Heavy chainFc-WT5QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALLAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKCetuximab Heavy chain Fc-SB6DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECCetuximab Lightchain7EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGemtuzumab Heavy chain Fc-WT8EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALLAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGemtuzumab Heavy chainFc-SB9DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGemtuzumab Light chain10QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKNimotuzumab Heavy chainFc-WT11QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALLAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKNimotuzumab Heavy chain Fc-SB12DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECNimotuzumab Lightchain13QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSSASTKGPSVLPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKNecitumumab Heavy chain Fc-WT14QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSSASTKGPSVLPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALLAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKNecitumumab Heavy chainFc-SB15EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQYGSTPLTFGGGTKAEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECNecitumumab Light chain16EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAtezolizumab Heavy chainFc-WT17EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALLAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAtezolizumab Heavy chain Fc-SB18DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAtezolizumab Lightchain19EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPertuzumab Heavy chain Fc-WT20EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALLAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPertuzumab Heavy chainFc-SB21DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECPertuzumab Light chain22DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKWild-type human antibody IgG1 Fc domain

[0127] Example 2. Production of antibody-drug conjugates having Fc domain variants or wild-type Fc

[0128] (1) Production of antibody-drug conjugates with wild-type Fc

[0129] To the model antibody in a polypropylene tube, 10 mM TCEP (Tris(2-carboxyethyl) phosphine hydrochloride, SIGMA-ALDRICH) aqueous solution was added at a ratio of 2.00-3.00 times per antibody molecule. Additionally, PBS buffer was added to adjust the final antibody concentration to 5 mg / mL (4.5 mg / mL when Gemtuzumab model antibody was used), and the mixture was incubated at 37°C for 2 hours to reduce the disulfide bond between the light and heavy chains of the antibody. The reduced antibody solution was cooled down at room temperature for approximately 10 minutes, and then linker-payload (10 mM, 8.0 times per antibody molecule) dissolved in DMSO was added and incubated at 25°C for 1 hour (final DMSO concentration, 10% v / v). Subsequently, the above reaction solution was purified using an Amicon® ultrafiltration column (Merck, 30K MWCO), and the final ADC conjugate was stored at 30 mM His-Hac, pH 6.0. The concentration of the purified ADC was measured using UV, the monomer ratio (SEC monomer%) was analyzed by SEC-HPLC, and the DAR was analyzed by HIC-HPLC.

[0130] (2) Production of antibody-drug conjugates with Fc domain variants

[0131] To the model antibodies containing Fc domain variants contained in polypropylene tubes, 10 mM TCEP (Tris(2-carboxyethyl) phosphine hydrochloride, SIGMA-ALDRICH) aqueous solution was added at a ratio of 2.00-3.00 times per antibody molecule. Additionally, PBS buffer was added to adjust the final antibody concentration to 5 mg / mL (4.5 mg / mL when Gemtuzumab model antibody was used), and the mixture was incubated at 37°C for 2 hours to reduce the disulfide bond between the light and heavy chains of the antibody. The reduced antibody solution was cooled down at room temperature for approximately 10 minutes, and then linker-payload (10 mM, 8.0 times per antibody molecule) dissolved in DMSO was added and incubated at 25°C for 1 hour (final DMSO concentration, 10% v / v). Subsequently, the above reaction solution was purified using an Amicon® ultrafiltration column (Merck, 30K MWCO), and the final ADC conjugate was stored at 30 mM His-HAc, pH 6.0. The concentration of the purified ADC was measured using UV, and the monomer ratio (SEC monomer%) was analyzed by SEC-HPLC, and the DAR was analyzed by HIC-HPLC.

[0132] As a result, 18 different antibody-drug conjugates were produced according to the type of model antibody used and the type of linker-drug as shown in [Table 4] below. Each item according to the classification in [Table 4] indicates the type of antibody, whether the Fc domain is mutated (WT, wild type; SB, stealth body, which is an Fc domain mutant), type of linker, and type of drug. For example, Trastuzumab Fc-WT-VC-MMAE refers to an antibody-drug conjugate in which the Fc portion of the model antibody Trastuzumab is wild type and VC is used as a linker to link it to the drug MMAE (Monomethyl auristatin E).

[0133] ClassificationFab regionFc region1. Trastuzumab Fc-WT-VC-MMAEAnti-HER2wild type2. Trastuzumab Fc-SB-VC-MMAEAnti-HER2mutant3. Trastuzumab Fc-WT-VC-TesirineAnti-HER2wild type4. Trastuzumab Fc-SB-VC-TesirineAnti-HER2mutant5. Pertuzumab Fc-WT-VC-MMAEAnti-HER2wild type6. Pertuzumab Fc-SB-VC-MMAEAnti-HER2mutant7. Cetuximab Fc-WT-VC-MMAEAnti-EGFRwild type8. Cetuximab Fc-SB-VC-MMAEAnti-EGFRmutant9. Necitumumab Fc-WT-VC-MMAEAnti-EGFRwild type10. Necitumumab Fc-SB-VC-MMAEAnti-EGFR mutant 11. Nimotuzumab Fc-WT-VC-MMAEAnti-EGFR wild type 12. Nimotuzumab Fc-SB-VC-MMAEAnti-EGFR mutant 13. Gemtuzumab Fc-WT-VC-MMAEAnti-CD33 wild type 14. Gemtuzumab Fc-SB-VC-MMAEAnti-CD33 mutant 15. Atezolizumab Fc-WT-VC-MMAEAnti-PD-L1 wild type 16. Atezolizumab Fc-SB-VC-MMAEAnti-PD-L1 mutant 17. Nimotuzumab Fc-SB-VC-TesirineAnti-EGFR mutant 18. Nimotuzumab Fc-SB-GGFG-DeruxtecanAnti-EGFR mutant

[0134]

[0135] Example 3. Confirmation of the ability of antibody-drug conjugates having human antibody Fc domain variants or wild-type Fc to kill cells that do not express HER2 target antigen.

[0136] HER2 target antigen is not expressed, and the HER2 target antibody-drug conjugates containing the Fc domain variants or wild-type Fc produced through Examples 1 and 2 were treated with OCI-AML-2, MV-4-11, U937, and EOL-1 cancer cells that express FcγR, to confirm the cell killing ability. As a result, in the case of the antibody-drug conjugates using the human antibody Fc domain variant, the cell killing ability was significantly suppressed, confirming that the mechanism of action of Fc was greatly suppressed (Table 5).

[0137] Trastuzumab Fc-WT-VC-MMAETrastruzumab Fc-SB-VC-MMAECellsIC50(nM)IC50(nM)OCI-AML-21.854No effectMV-4-115.293No effectU9376.362No effectEOL-120.630No effectJurkat(No FcγR)No effectNo effectHEK293(No FcγR)No effectNo effect

[0138] Example 4. Confirmation of the ability of antibody-drug conjugates having human antibody Fc domain variants or wild-type Fc to kill cells that do not express EGFR target antigens.

[0139] EGFR target antigen is not expressed, and the cell killing ability was confirmed by treating THP-1, MV-4-11, HL-60, and EOL-1 cancer cells that express FcγR with the Fc domain variants produced through Examples 1 and 2 or EGFR target antibody-drug conjugates containing wild-type Fc (Cetuximab Fc-WT-VC-MMAE or Cetuximab Fc-SB-VC-MMAE). As a result, in the case of the antibody-drug conjugates using the human antibody Fc domain variant, the cell killing ability was significantly suppressed, confirming that the mechanism of action of Fc was suppressed (Table 6).

[0140] Cetuximab Fc-WT-VC-MMAECetuximab Fc-SB-VC-MMAECellsIC50(nM)IC50(nM)THP-16.355No effectMV-4-1164.090No effectHL60517.000No effectEOL-1566.600No effectJurkat(No FcγR)No effectNo effectHEK293(No FcγR)No effectNo effect

[0141] Example 5. Confirmation of the cell killing ability of an antibody-drug conjugate having a human antibody Fc domain variant or wild-type Fc expressing a target antigen (HER2)

[0142] This time, the HER2-targeting antibody-drug conjugates produced in Examples 1 and 2 were treated on cancer cell lines (SKBR-3, BT474) expressing the HER2 target antigen to confirm their cell killing ability. As a result, it was confirmed that both antibody-drug conjugates using Fc domain variants or wild-type Fc had similar cell killing ability against cancer cell lines expressing the target antigen, regardless of which drug, MMAE or Tesirine, was combined. Therefore, it was confirmed that the cancer cell killing efficacy was maintained even when the Fc domain variant was introduced (Figs. 2a to 2e).

[0143] Example 6. Confirmation of the cell killing ability of antibody-drug conjugates having human antibody Fc domain variants or wild-type Fc expressing target antigen (EGFR)

[0144] The EGFR target antibody-drug conjugates prepared in Examples 1 and 2 were treated to a cancer cell line (MDA-MB-468) expressing the EGFR target antigen to confirm their cell killing ability. As a result, it was confirmed that all antibody-drug conjugates containing Fc domain variants or wild-type Fc had similar cell killing ability in cancer cell lines expressing the target antigen, regardless of which antibody among Cetuzimab, Nimotuzumab, or Necitumumab was included. Therefore, it was confirmed that the cancer cell killing efficacy was maintained even when the Fc domain variant was introduced (Figures 3a to 3c).

[0145] Example 7. Cytotoxicity test of antibody-drug conjugates having human antibody Fc domain variants against target antigen (EGFR)

[0146] The cytotoxicity of the EGFR target antibody-drug conjugates (Nimotuzumab Fc-SB-GGFG-Deruxtecan (Dxd), Nimotuzumab Fc-SB-VC-Tesirine (PBD)) produced in Examples 1 and 2 against a cancer cell line (MDA-MB-468) expressing the EGFR target antigen was tested.

[0147] Specifically, cells expressing EGFR were cultured, diluted in medium, and adjusted to the required density. 80 μL was dispensed per well into a 96-well culture plate and cultured overnight in a CO₂ incubator at 5% CO₂ and 37℃. The payload solution was serially diluted in a 1:5 ratio to prepare a 9-point concentration response curve. Each well was treated with these diluted solutions (20 μL diluted payload + 80 μL medium) to obtain final payload concentrations of 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, and 0.0026 nM. Control wells were treated with only cells and medium. After culturing for 144 hours under 5% CO₂ and 37℃ conditions, the cells were measured using CelltiterGlo. As a result, it was confirmed that the survival rate of EGFR expressing cells was lower in the antibody-drug conjugate containing the Fc domain variant than in the antibody-drug conjugate containing the wild-type Fc domain. In other words, the effectiveness of the antibody-drug conjugate containing the Fc domain variant was confirmed (Figure 4). In Figure 4, Nimotuzumab Variant6-Dxd represents the result for Nimotuzumab Fc-SB-GGFG-Deruxtecan, Nimotuzumab Variant6-PBD represents the result for Nimotuzumab Fc-SB-VC-Tesirine, and huIgG-Vc-MMAE represents the result for the antibody-drug conjugate in which the wild-type human antibody IgG is linked to the VC-MMAE linker-payload.

[0148] Example 8. Confirmation of the cell killing ability of antibody-drug conjugates having human antibody Fc domain variants or wild-type Fc expressing target antigen (CD33)

[0149] The CD33 targeting antibody-drug conjugates (Gemtuzumab Fc-WT-VC-MMAE or Gemtuzumab-Fc-SB-VC-MMAE) produced in Examples 1 and 2 were treated to cancer cell lines (THP-1, U937, MV-4-11, HL-60) expressing the CD33 target antigen to confirm their cell killing ability. As a result, it was confirmed that both antibody-drug conjugates containing Fc domain variants or wild-type Fc had similar cell killing abilities in cancer cell lines expressing the target antigen. Therefore, it was confirmed that the cancer cell killing efficacy was maintained even when the Fc domain variant was introduced (Figures 5a to 5d).

[0150] Example 9. Confirmation of the cell killing ability of antibody-drug conjugates having human antibody Fc domain variants or wild-type Fc expressing target antigen (PD-L1)

[0151] The cell killing ability was confirmed by treating a cancer cell line (Calu-1, KARPAS 299) expressing the PD-L1 target antigen with the PD-L1 target antibody-drug conjugate (Atezolizumab Fc-WT-VC-MMAE or Atezolizumab Fc-SB-VC-MMAE) produced in Examples 1 and 2. As a result, it was confirmed that both antibody-drug conjugates containing Fc domain variants or wild-type Fc had similar cell killing abilities in cancer cell lines expressing the target antigen. Therefore, it was confirmed that the cancer cell killing efficacy was maintained even when the Fc domain variant was introduced (Figures 6a and 6b).

[0152] Example 10. Evaluation of the aggregation effect of antibody-drug conjugates having human antibody Fc domain variants or wild-type Fc.

[0153] Among the antibody-drug conjugates containing the wild-type Fc domain or the Fc domain variants produced in Examples 1 and 2, the aggregation effect of the antibody-drug conjugates using Cetuximab or Gemtuzumab as the antibody was tested. Specifically, (1) a freeze / thaw test in which the test sample was subjected to three cycles of freezing (-80°C) - thawing (room temperature), and then the initial sample and the experimental sample were compared by QC analysis. (2) a 4°C stability test in which the test sample was stored at 4°C for 7 days, and then the initial sample and the sample after 7 days were compared by QC analysis. (3) the test sample was sealed and stored at 40°C for 7 days, and then samples were collected on days 0, 1, 3, and 7, respectively, and stored frozen at -80°C. Thereafter, a 40°C stability test in which all samples were subjected to QC analysis and compared (Figs. 7a to 7c). In Figures 7a to 7c, Cetuximab-variant06-VcMMAE represents the results for Cetuximab Fc-SB-VC-MMAE, and Gemtuzumab-variant06-VcMMAE represents the results for Gemtuzumab Fc-SB-VC-MMAE. Figure 7a shows the results analyzed by SEC-HPLC technique, and the y-axis of the bar graph represents the monomer ratio. Figure 7b shows the results of HIC-DAR analysis, and the y-axis represents the DAR (Drug-to-Antibody Ratio) value. Figure 7c shows the results of measuring concentration, and the y-axis represents the concentration (mg / ml).

[0154] The results of the stability impact evaluation of this example are shown in [Table 7]. In the case of the antibody-drug conjugate using the Cetuximab model antibody, when the Fc domain variant of the present invention was applied, the aggregation was reduced by about 40-50% on the 1st and 3rd days under 40℃ conditions compared to when the wild type was applied. In the case of the antibody-drug conjugate using the Gemtuzumab model antibody, the Fc domain variant of the present invention showed aggregation reduced by about 80% on the 3rd day under 40℃ conditions compared to the wild type, and aggregation was also reduced by about 50% on the 7th day. That is, it was confirmed that the stability of the antibody-drug conjugate was improved by the introduction of the Fc domain variant.

[0155] TestCetuximab-WT_Vc-MMAECetuximab-SB-Vc-MMAEGemtuzumab-WT-Vc-MMAEGemtuzumab-SB-Vc-MMAE4℃ 1 week StableStableStableStableFreeze-thaw 3 timesStableStableStable40℃ 1 day 7.2% increase in aggregation, no change in concentration4.5% increase in aggregation, no change in concentrationApparent HIC-DAR 13.0% increase in aggregation3.0% increase in aggregation, no change in concentration40℃ 3 days 10.4% increase in aggregation, no change in concentration5.6% increase in aggregation, no change in concentrationApparent HIC-DAR 22.9% increase in aggregation, 32.4% increase in aggregation5.7% increase in aggregation, no change in concentration40℃ 7 days 16.2% increase in aggregation, no change in concentration14.2% increase in aggregation, no change in concentrationApparent HIC-DAR increased by 23.9%, coagulation increased by 57.4%, and concentration decreased by 23.2%. Coagulation increased by 11.2%, and concentration did not change.

[0156] Example 11. Validation of Antibody-Drug Conjugates Having Human Antibody Fc Domain Variants or Wild-Type Fc in a Mouse Tumor Transplantation Model

[0157] (1) Confirmation of effectiveness according to administered dose

[0158] In Examples 1 and 2, antibody-drug conjugates prepared using Nimotuzumab or Cetuximab as model antibodies were treated with EGFR-expressing cancer cell lines (A431) to confirm their cell killing ability (Figs. 8a and 8b). Then, the efficacy according to the administered dose was confirmed in a syngeneic mouse model in which EGFR-expressing cancer cell lines were transplanted. Specifically, 6-week-old female Balb / c mice were acclimatized for 2 weeks, and at 8 weeks of age, EGFR-expressing cancer cell lines subcultured in DMEM supplemented with 10% FBS and 1X antibiotics were detached using tripsin-EDTA, and 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. 3After group separation upon reaching the target, body weight was measured and then the test substance was administered. The test substance was diluted in Corning® PBS (Phosphate-Buffered Saline) and administered as a single dose of 200 uL per animal through the caudal vein at concentrations of 2 mg / kg, 5 mg / kg, and 10 mg / kg. As a result, in the case of the antibody-drug conjugate containing nimotuzumab, the antibody-drug conjugate containing the wild type and the Fc domain variant completely suppressed tumors at both 5 mg / kg and 10 mg / kg, but in the 2 mg / kg dose group, a weak tumor suppression effect was confirmed only in the antibody-drug conjugate containing the Fc domain variant from day 14. In the case of antibody-drug conjugates containing cetuximab, both the wild type and the antibody-drug conjugates containing the Fc domain variant were confirmed to completely suppress tumors at 5 mg / kg and 10 mg / kg, and in the 2 mg / kg dose group, only the antibody-drug conjugate containing the Fc domain variant showed tumor suppression efficacy of about 50% or more (Figs. 9a and 9b).

[0159] (2) Validation of efficacy over time in a mouse tumor transplantation model

[0160] For antibody-drug conjugates containing nimotuzumab, both antibody-drug conjugates containing human antibody Fc domain wild-type and mutants were confirmed to strongly suppress tumors up to 49 days at 5 mg / kg. In the case of antibody-drug conjugates containing Fc domain mutants, it was confirmed that tumor suppression activity was strong from the beginning, whereas in the case of antibody-drug conjugates containing wild-type, tumor suppression activity was not observed until day 4, and tumor suppression activity was confirmed to be observed after day 4.

[0161] In the case of antibody-drug conjugates containing cetuximab, both antibody-drug conjugates containing Fc domain variants and wild-type Fc showed similarly strong tumor suppression activity until day 11 at 5 mg / kg, but the antibody-drug conjugate containing wild-type Fc began to show weakened tumor suppression activity from day 14, and the antibody-drug conjugate containing Fc domain variants began to show weakened tumor suppression activity from day 32 (Figs. 10a and 10b).

[0162] From the above results, it can be seen that the antibody-drug conjugate of the present invention has the effect of suppressing the Fc mechanism of action, thereby enhancing safety, maintaining at least efficacy, and suppressing aggregation, thereby providing stability. Furthermore, it can be seen that the antibody-drug conjugate of the present invention can also be used for anticancer purposes.

Claims

1. An antibody-drug conjugate (ADC) comprising: (a) an antibody comprising 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), and a first antigen-binding portion linked to the Fc domain variant; and (b) A drug linked to the above antibody.

2. In paragraph 1, The above antibody-drug conjugate is an antibody-drug conjugate composed of multiple molecules.

3. In paragraph 1, An antibody-drug conjugate, wherein the drug is linked to at least one amino acid selected from the group consisting of cysteine ​​and lysine of the antibody.

4. In paragraph 1, An antibody-drug conjugate characterized in that the antibody further comprises a second antigen binding portion.

5. In paragraph 1, An antibody-drug conjugate characterized in that the antibody and the drug are linked by a linker.

6. In paragraph 1, An antibody-drug conjugate characterized in that the human antibody is IgG1.

7. In paragraph 1, An antibody-drug conjugate characterized in that the antibody 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., 8. In paragraph 1, An antibody-drug conjugate characterized in that the antibody 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.

9. In paragraph 1, An antibody-drug conjugate, characterized in that the drug is at least one selected from the group consisting of auristatin, maytansinoid, tubulysin, calicheamicin, duocarmycin, exatecan, and pyrrolobenzodiazepine.

10. In paragraph 5, An antibody-drug conjugate, characterized in that the linker is at least one selected from the group consisting of Val-Cit-PABA, Gly-Gly-Phe-Gly, Val-Ala-PABA, sulfo-SPDB, SMCC, SPDB, oxime, Ala-Ala-PABA, CL2A, Val-Cit, hydrazone, mc-VC-PABC, dipeptide, and tetrapeptide.

11. A pharmaceutical composition for preventing or treating cancer, comprising an antibody-drug conjugate according to any one of claims 1 to 10 as an active ingredient, wherein the drug is an anticancer agent.

12. In paragraph 11, 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, lymphatic cancer, 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.

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