Conjugates in which FL118 drugs are bound to acid-sensitive linkers and immunoconjugates using the same

Abstract

The present invention relates to a conjugate in which the FL118 drug is linked to an acid-sensitive linker and an immunoconjugate using the same. The present invention is characterized in that one or more FL118 drugs of formula 1 are bound to an antibody or a fragment thereof containing the antigen-binding site via an acid-sensitive linker, and after targeting cancer cells via the antigen-binding site that targets the antigen on the cancer cells, the acid-sensitive linker is decomposed in the acidic environment (pH≦7) around the cancer, liberating at least a portion of the FL118 drug of formula 1, which penetrates the cell membrane and moves into the cells, where it inhibits the action of efflux pumps, concentrating the free FL118 drug of formula 1 within the cells.

Description

[Technical field]

[0001] The present invention relates to a conjugate in which a FL118 drug is bound to an acid-sensitive linker, and an immunoconjugate using the same. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are a new drug platform that selectively delivers a payload with strong anticancer effects to cancer tissues using the high tissue selectivity of antibodies. ADCs selectively deliver a powerful payload that can kill cancer cells even at low pM concentrations to cancer tissues, while minimizing systemic drug exposure, ensuring both anticancer effects and safety.

[0003] Existing ADC developments have pM-level IC at the cellular level. 50 However, the types of drugs with such properties are limited to microtubulin inhibitors, DNA damaging agents, and Top-1 inhibitors, and various problems have appeared. Even when these drugs are used as ADCs, a sufficient therapeutic window is often not ensured, but the side effects experienced by these patients are the same as those of the payload itself, so it is difficult to develop an excellent ADC with a payload whose safety has not been ensured. In addition, the use of these drugs causes resistance to cancer through various mechanisms, but there are currently no ADCs using new payloads to overcome resistance when such resistance appears.

[0004] In other words, there is a strong demand for a new ADC development platform that has a mechanism of action that is differentiated from existing anticancer drugs / payloads, has sufficient efficacy and the potential to overcome resistance, is sufficiently safe even when used alone, and ensures a wide therapeutic window.

[0005] Since the development of ADCs since the 1970s, many efforts have been made to discover new antigens / antibodies, develop payloads for ADCs, develop linkers to combine antibody-payload, improve antibodies for uniform quality and develop CMC routes, etc. As a result, most of the current ADCs are developed using second- or third-generation ADC platforms.

[0006] Although there have been many efforts, primarily from companies such as Genetech and Seattle Genetics in the US and Regochem Bio in Korea, to develop ADCs that can be effectively used to treat various cancers, especially solid tumors, there are still significant unmet needs, such as limited therapeutic window and / or resistance development.

[0007] In the initial stage of ADC development, various ADC candidate substances are developed using available payload condition identifiers, identification of linkers with various properties, and simple binding methods. Through this, suitable payloads for ADCs (picomolar IC such as Calicheamicin, MMAE, PBD, etc.) are identified. 50 The first generation of ADC products, including Mylotarg and Kadcyla, were developed.

[0008] Since then, various second-generation ADCs have been developed by improving CMC (Chemical, Manufacturing and Control) through site-specific conjugation and developing stable linkers that release drugs only in cancer cells, but most of these have not been successful due to issues such as insufficient therapeutic window. These failures have established the fact that, rather than simply using a strong payload, a payload that has a strong anticancer effect and is safe enough to be used alone is necessary.

[0009] Subsequently, Immunomedics and Daiichi Sankyo led the development of third-generation ADCs using payloads that ensured a sufficient therapeutic window to be used as single-administered anticancer drugs in humans, and Trodelvy and Enhertu were approved by the FDA as ADCs for solid tumors.

[0010] [Limitations of existing ADCs (second generation ADCs): Powerful toxins] Many of the previously developed payloads have the problem of not being able to ensure a therapeutic window when applied to solid cancers. To overcome this problem, a powerful toxin is used as the payload, and an ultrastable linker system has been introduced in which the chemical bond is selectively broken by cathepsin B, an enzyme present in cells, and the drug is released.

[0011] A representative ultrastable linker system is the valine-citrulline (Val-Cit) linker developed by Seattle Genetics, Inc. Representative ADCs using this system include Adcetris, Polivy, and Padcev (Figure 13).

[0012] Payloads paired with the Val-Cit linker have very high toxicity, such as Vedotin and related drugs. Although highly stable linker systems such as Val-Cit have been introduced to solve the problem of high toxicity, there is still a limit to securing a therapeutic window due to the toxicity of the strong payload.

[0013] [Limits of 3rd generation ADCs] Instead of the powerful toxins that were mainly used in second-generation anticancer drugs, new ADC anticancer drugs have been developed by incorporating targeted anticancer drugs into the ADC payload, which have a high potential for development as therapeutic drugs on their own. Based on the excellent safety of payloads based on these targeted anticancer drugs, they are being successfully developed as therapeutic drugs for solid tumors (Figure 14).

[0014] FIG. 14 shows examples of ADCs (Enhertu (top) and Trodelvy (bottom)) that use a camptothecin-based compound, a topoisomerase I inhibitor, as a payload as a targeted anticancer agent.

[0015] However, these third-generation ADCs have several limitations, such as limited anti-cancer efficacy due to the use of payloads with relatively limited efficacy compared to the payloads used by existing second-generation ADCs, inconvenience due to the need for high doses, and the occurrence of problems such as resistance development in patients with solid tumors.

[0016] In particular, many ADCs currently in use or in development are focused on the use of payloads with only a restricted mechanism of action, resulting in limited treatment options and resistance development. Therefore, it is necessary to develop new ADCs with new mechanisms of action and the potential to overcome resistant cancers. Summary of the Invention [Problem to be solved by the invention]

[0017] These second- and third-generation ADC platforms suffer from a variety of issues, primarily payload-related toxicity, lack of efficacy, and resistance.

[0018] In order to develop new ADCs, there is an urgent need to develop new payloads that have the following characteristics: (1) equivalent or superior anticancer effects compared to existing payloads; (2) a level of safety in the human body that ensures that the payload itself is safe even when used alone as a drug, thereby ensuring a wide therapeutic window for the ADC; and (3) overcoming the resistance mechanisms of existing payloads. [Means for solving the problem]

[0019] The present invention provides a new payload-linker conjugate that has a mechanism of action differentiated from existing anticancer drugs / payloads, has sufficient anticancer effects and the potential to overcome resistance, maintains sufficient safety even when used alone, and ensures a wide therapeutic window, and a new ADC development platform using the same. Effect of the Invention

[0020] The immunoconjugate of the present invention is a hydrophobic small molecule drug, and by using a combination of a cell membrane-permeable FL118 drug and an acid-sensitive linker, it is possible to solve the problem of insufficient drug concentration entering cells due to the inefficient internalization process by antigen-antibody complexes, and the problem of antibodies having difficulty penetrating deep into cancer tissues, and maximize the bystander effect of entering not only target cells but also surrounding cells.

[0021] The immunoconjugate of the present invention can overcome the problems identified in the development of the first to third generation ADCs, including the emergence of resistance to ADCs and the narrow therapeutic window due to the relatively weak efficacy of the payload used in the third generation ADCs. That is, the immunoconjugate of the present invention can balance strong anticancer effects and in vivo safety compared to existing ADC payloads by using a combination of FL118 drug, which has various resistance overcoming mechanisms, and an acid-sensitive linker, and can exhibit an optimized therapeutic window. In particular, the problems of insufficient efficacy and resistance associated with existing SN-38-based ADCs can be overcome through the strong efficacy unique to FL118, and development can be accelerated by using a biomarker that can identify the patient group that is most likely to respond to FL118. [Brief description of the drawings]

[0022] [Figure 1] Camptothecin-based anticancer drugs: SN-38 (left), Topotecan (center), Exatecan (right) [Diagram 2] FIG. 1 is a schematic diagram showing the mechanism of efficient drug release and resistance overcoming in the immunoconjugate of the present invention. [Diagram 3] This shows the results of a comparative evaluation of cell viability in vitro between FL118 and SN-38. [Figure 4] FIG. 1 is a schematic diagram of an example of a Carrier-Drug Conjugate comprising a FL118 drug-acid sensitive linker. [Figure 5a] The results of confirming the production of DAR4ADC (5a) and DAR8ADC (5b) using SEC, HIC, and LC-MS. [Figure 5b] The results of confirming the production of DAR4ADC (5a) and DAR8ADC (5b) using SEC, HIC, and LC-MS. [Figure 6]1 shows the results of evaluation of Trastuzumab-CL2A-FL118 (DAR 8) in Example 5 in the JIMT-1 cell line. [Figure 7] 1 shows the results of evaluation of Trastuzumab-CL2A-FL118 (DAR 4) in Example 6 in the JIMT-1 cell line. [Figure 8] This shows the evaluation results of Trastuzumab-CL2A-FL118 (DAR 8) in the JIMT-1 Xenograft model of Example 5. Trastuzumab-FL118ADC exhibits excellent efficacy in the Kadcyla-resistant JIMT-1 (Her2 over-expression) In Vivo Xenograft model. [Figure 9] This shows the evaluation results of Cetuximab-CL2A-FL118 (DAR 8) in the MDA-MB-468 cell line in Example 8. [Figure 10] This shows the evaluation results of Cetuximab-CL2A-FL118 (DAR 8) in the MDA-MB-468 Xenograft model of Example 8. Cetuximab-FL118 ADC exhibits excellent efficacy in the MDA-MB-468 (EGFR over-expression) In Vivo Xenograft model. [Figure 11] Example 9: Evaluation of sacituzumab-CL2A-FL118 (DAR 8) in MDA-MB-468 cell line. [Figure 12] This shows the results of comparative evaluation of sacituzumab-CL2A-FL118 (DAR 8) with Trodelvy in the MDA-MB-468 xenograft model in Example 9. The sacituzumab-FL118 ADC exhibits superior efficacy compared to the approved TROP2-targeted ADC Trodelvy in the MDA-MB-468 (TROP2 over-expression) in vivo xenograft model. [Figure 13] ADCs developed using the Val-Cit linker: Adcetris (top), Polivy (middle), and Padcev (bottom). [Figure 14] Schematic diagram of ADCs that use camptothecin-based compounds, which are topoisomerase I inhibitors, as payloads as targeted anticancer drugs: Enhertu (top) and rodelvy (bottom) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The first aspect of the present invention is an immunoconjugate or a pharma- ceutically acceptable salt thereof comprising [FL118 drug of formula 1]-[acid-sensitive linker]-[antibody or a fragment containing an antigen-binding site thereof], in which one or more FL118 drugs of formula 1 are bound to an antibody or a fragment containing an antigen-binding site thereof via an acid-sensitive linker, and after targeting a cancer cell by the antigen-binding site that targets an antigen of the cancer cell, the acid-sensitive linker is decomposed in an acidic environment (pH ≦ 7) around the cancer, and at least a part of the FL118 drug of formula 1 is liberated, and the free FL118 drug of formula 1 penetrates the cell membrane and moves into the cell, and the FL118 drug of formula 1 is transported by an efflux pump (efflux pump). The present invention provides an immunoconjugate or a pharma- ceutical acceptable salt thereof, which is characterized in that the FL118 drug of formula 1 is enriched in free form within cells by suppressing the action of a FL118 pump, and the immunoconjugate to which the FL118 drug of formula 1 is optionally bound is internalized into cells, and the FL118 drug of formula 1 is released from lysosomes.

[0024] [ka]

[0025] A second aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the immunoconjugate of the first aspect or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0026] A third aspect of the present invention provides a drug-linker conjugate, or a pharma- ceutically acceptable salt thereof, in which a FL118 drug of formula 1 is linked to an acid-sensitive linker of formula 2 below:

[0027] A fourth aspect of the present invention provides an immunoconjugate or a pharma- ceutically acceptable salt thereof, comprising: (a) the drug-linker conjugate of the third aspect or a pharma- ceutically acceptable salt thereof; and (b) an antibody or a fragment containing an antigen-binding site thereof, wherein one or more FL118 drugs of Formula 1 are linked to the antibody or the fragment containing the antigen-binding site thereof via an acid-sensitive linker of Formula 2.

[0028] A fifth aspect of the present invention provides a method for preparing a carrier-drug conjugate, comprising binding one or more FL118 drugs of formula 1 to a carrier via an acid-sensitive linker of formula 2, using the drug-linker conjugate of the first aspect or a pharma- ceutically acceptable salt thereof.

[0029] The present invention will now be described. The present invention relates to an immunoconjugate comprising: [FL118 drug of formula 1]-[acid-sensitive linker]-[antibody or a fragment thereof containing an antigen-binding site], (i) an antibody or a fragment thereof containing an antigen-binding site is linked to one or more FL118 drugs of formula 1 via an acid-labile linker; (ii) after targeting the cancer cell with the antigen-binding site that targets the antigen of the cancer cell, in the acidic environment (pH ≦ 7) around the cancer, the acid-sensitive linker is decomposed, and the FL118 drug of formula 1 is at least partially liberated, and the free FL118 drug of formula 1 penetrates the cell membrane and moves into the cell; (iii) the FL118 drug of formula 1 inhibits the action of an efflux pump, thereby enriching the free FL118 drug of formula 1 in the cells; (iv) Optionally, the immune complex to which the FL118 drug of formula 1 is bound is characterized by internalization into cells and release of the FL118 drug of formula 1 from lysosomes (Figure 2).

[0030] As shown in Figure 2, the immunoconjugate of the present invention uses an acid-sensitive linker to efficiently release the drug not only within cancer cells after antigen binding but also in the vicinity of the cancer tissue, thereby overcoming the resistance mechanisms associated with ADC treatment.

[0031] In addition, the FL118 drug of formula 1 released from the immune complex of the present invention is not excreted outside the cell by the efflux pump ABCG2, but is secreted by various anti-apoptotic proteins. to It is possible to block resistance caused by tic proteins.

[0032] In its free state, the FL118 drug of formula 1 is a triple-targeted anticancer agent that directly targets Topoisomerase I, a well-established anticancer target, and simultaneously inhibits the Bcl family, including Survivin, a resistance protein involved in resistance mechanisms, and inhibits the action of efflux pumps.

[0033] For an ADC to function effectively, it is not enough to simply maximize potent, antigen-selective cytotoxicity against cellular targets in vitro; it must also be able to successfully penetrate cancer tissues and deliver drugs efficiently in vivo or in actual patients.

[0034] The immunoconjugate of the present invention binds to cancer cells via an antigen-binding site that targets an antigen on the cancer cells. After targeting, the tumor is exposed to the acidic environment (pH ≦ 7) around the tumor. SensationThe hydrophilic linker is degraded, at least partially liberating the FL118 drug of formula 1, and since the free FL118 drug of formula 1 is a hydrophobic small molecule, it can penetrate deep into the cancer tissue and move into the cell through the cell membrane.

[0035] Therefore, the immunoconjugate of the present invention can rapidly release a drug in the tumor microenvironment around the cancer by using an acid-sensitive linker that is decomposed in the acidic environment (pH≦7) around the cancer. The free FL118 drug has a low molecular weight and, unlike antibodies, has a high penetration ability into cancer tissue, solving the problem of existing ADCs in that antibodies have difficulty penetrating deep into cancer tissue.

[0036] Thus, the cells into which the free form of the FL118 drug of formula 1 is transported may be the targeted cancer cells and / or surrounding cells.

[0037] In addition, the FL118 drug of formula 1 is (a) a hydrophobic small molecule that can permeate cell membranes, and (b) is not excreted outside the cells by efflux pumps. Therefore, the FL118 drug released from the immunoconjugate of the present invention by degradation of the extracellular linker around the cancer can rapidly accumulate in the cancer tissue and maintain a high concentration for a long time. After penetrating the cell membrane and exerting cytotoxicity within the cells, it can cause apoptosis and then be released, and can continuously penetrate the cell membranes of surrounding cells to move into the cells and act there.

[0038] That is, the drug-linker conjugate of the present invention is (i) the use of an acid-sensitive linker such as the CL2A linker of formula 2 in order to introduce a large amount of free hydrophobic small molecule drugs into cells and / or penetrate deep into tissues after liberating hydrophobic small molecule drugs that can penetrate cell membranes in the tumor microenvironment around cancer, which is an acidic environment (pH≦7) and play a role within the cells; and (ii) In the tumor microenvironment around the cancer, which is an acidic environment (pH ≦ 7), a large amount of free drug released by decomposition of the acid-sensitive linker penetrates the cell membrane and moves into the cell, where it is highly enriched within the cell. This is characterized by the use of FL118 drug of formula 1, which is a hydrophobic small molecule drug that can penetrate the cell membrane and inhibit the action of efflux pumps at the same time; Another advantage is that the use of these combinations exploits the organic mechanism of action of the FL118 drug-acid-labile linker.

[0039] The FL118 drug of formula 1 and the acid-sensitive linker are preferably linked by a carbonate or ester bond so that they are decomposed in an acidic environment (pH≦7) and the free FL118 drug of formula 1 is released upon decomposition of the acid-sensitive linker (Figure 4).

[0040] In the present invention, the bond between the [acid-sensitive linker] and the [antibody or a fragment containing an antigen-binding site thereof] may be such that a thiol group contained in the antibody or antigen-binding fragment thereof is bonded to a maleimide group or maleic hydrazide group of the acid-sensitive linker via a "click" reaction as shown in Reaction Scheme 1.

[0041] [ka]

[0042] The present invention also provides drug-linker conjugates in which the FL118 drug is linked to various acid-sensitive linkers, and uses the same to provide carrier-drug conjugates in which the FL118 drug is linked to various carriers via various acid-sensitive linkers.

[0043] Furthermore, the present invention also provides a method for preparing a carrier-drug conjugate, which comprises using the above-mentioned drug-linker conjugate of the present invention or a pharma- ceutically acceptable salt thereof to bind one or more FL118 drugs of formula 1 to a carrier via an acid-sensitive linker of formula 2.

[0044] The structure of a carrier-drug conjugate bound to a carrier using a drug-linker conjugate according to the present invention is illustrated in FIG.

[0045] [Competitive advantages of FL118 drugs linked via acid-sensitive linkers] The immune complex of the present invention is targeted to cancer cells by the antigen-binding site that targets the antigen of the cancer cells, and is then decomposed mainly in the acidic environment (pH ≦ 7) around the cancer and / or partially in the blood due to its acid-sensitivity. Therefore, in terms of the anti-cancer effect and stability issues due to the mechanism of action of the free FL118 drug of formula 1, which begins outside the cells, the competitive advantages of the free FL118 drug of formula 1 are considered as follows.

[0046] The FL118 drug of formula 1 is a potent Topoisomerase I inhibitor that has the same camptothecin core structure as existing commercial Top1 inhibitors such as SN-38, topotecan, and exatecan (Figure 1). Even when administered alone, it has a differentiated anticancer effect and excellent stability compared to SN-38 in various cancer cell models and animal models, and has a wide therapeutic window. It is a drug.

[0047] Specifically, the FL118 drug has a similar or higher level of Top1 inhibitory effect as SN-38 in cancer cells, and is 5 to 20 times more potent than SN-38 in various cancer cell lines, i.e., has a low IC 50The compound showed cytotoxicity with IC values ​​of <100 nM against most cancer cells in 140 cell lines derived from various cancers (Figure 3). 50 It has shown extremely strong anti-cancer effects.

[0048] FL118 drug can overcome various resistance mechanisms of SN-38 / Exatecan. Camptothecin-based anticancer drugs such as SN-38 have resistance in the form of drugs being excreted outside the cell due to overexpression of ABCG2 transporter, but FL118 drug is not affected by ABCG2 transporter, so it is possible to overcome this resistance.

[0049] On the other hand, camptothecin-based anticancer drugs initially show excellent anticancer responses when administered to patients, but due to epigenetic silencing of the Top1 gene and Top2 dependence in cancer cells, strong resistance to these drugs appears. On the other hand, the drug FL118 showed strong efficacy in xenograft models of cancer cell lines in which Top1 is not expressed due to epigenetic silencing or knock-out.

[0050] The FL118 drug inhibits anti-apoptotic proteins (APs), another major cause of cancer drug resistance. to The development of resistance can be blocked by potently inhibiting the expression of certain inhibitors of inflammatory cytokines (e.g., Survivin, cIAP2, and XIAP) at low concentrations.

[0051] The FL118 drug has an optimal PK / safety profile for the application of targeted drug delivery (e.g., carrier-drug conjugates). When the FL118 drug is administered alone, it is rapidly metabolized / excreted in the blood and exhibits only low concentrations, but it is rapidly accumulated in cancer tissues immediately after administration and maintains high concentrations for a long time. For example, when applied as an ADC, it ensures maximum selectivity between tumor tissues and normal tissues.

[0052] The FL118 drug has been shown to have excellent safety profiles through GLP toxicity studies in rats and beagle dogs, making it suitable as a payload for ADCs with a secured therapeutic window.

[0053] FL118 drug showed superior efficacy compared to SN-38 in various cancer cell line xenograft models.

[0054] The FL118 drug showed potent tumor regression efficacy against SN-38 in colon cancer, head and neck cancer, pancreatic cancer, etc., when administered in vivo at a similar amount to SN-38.

[0055] FL118 maintained a strong anticancer effect even when FL118 was administered after inducing SN-38 resistance in tumor xenografts. The potential of FL118 to overcome various resistance mechanisms to SN-38 / Exatecan was confirmed in vivo.

[0056] That is, in the conjugate of [FL118 drug of formula 1]-[acid-sensitive linker], the FL118 drug has a competitive advantage as a payload in the following respects: (i) Producing potent anti-cancer effects by selectively inhibiting / degrading additional anti-cancer targets DDX5, UBE2T and USP2a in addition to the well-validated anti-cancer drug target Top1; (ii) It exhibits 5-10 times stronger cellular cytotoxicity against SN-38 and exhibits equal or stronger cellular cytotoxicity against exatecan, ensuring a strong pharmacological effect sufficient for ADC and / or other drug conjugates; (iii) Unlike SN-38 and Exatecan, it is not an ABCG2 or P-GP substrate, thus circumventing resistance mechanisms due to overexpression of these transporter proteins; (iv) demonstrated potent efficacy in animal models of cancer in which epigenetic silencing of Top1 / over-expression of Top2 occurs, a major mechanism of resistance to Top1 inhibitors; (v) Anti-apoptotic proteins, a common mechanism of resistance in most cancers to It is possible to circumvent resistance by fundamentally blocking overexpression of the tic protein; (vi) Biomarkers (DDX5, K-ras, p53) that predict anti-cancer responses in patients and companion diagnostic technologies have already been established. By obtaining customized biomarkers, personalized treatment through companion diagnostics is possible, and this has shown particularly strong efficacy in p53 / K-ras mutant cancer cells with poor prognosis.

[0057] In particular, the FL118 drug of formula 1 can maximize the efficacy of modulation of the main drug target through an additional mechanism of action that (i) simultaneously inhibits / inhibits the Bcl family, including the resistance protein Survivin, and (ii) inhibits the action of efflux pumps.

[0058] In other words, the FL118 drug bound to the carrier of the present invention by an acid-sensitive linker has been selected based on a well-designed polypharmacology approach that breaks away from the conventional drug development method of targeting one drug target with one drug, which has limited therapeutic effects, and instead targets various drug targets that need to be controlled simultaneously to obtain excellent therapeutic effects. This makes it possible to treat intractable diseases for which no clear therapeutic effects have been shown with existing therapeutic agents.

[0059] In addition, in the case of recurrent / resistant cancers, there has been a problem in that sufficient effects cannot be obtained by simply regulating the activity of one drug target, and safety cannot be guaranteed when targeting an unspecified number of drug targets at the same time. However, the FL118 drug bound to the carrier of the present invention by an acid-sensitive linker utilizes two or more appropriately selected drug targets, thereby exhibiting a synergistic effect in terms of efficacy while at the same time ensuring sufficient safety.

[0060] [Competitive advantage of immunoconjugates in which FL118 drugs are linked via acid-sensitive linkers] The immunoconjugate of the present invention uses the FL118 drug, which inhibits the activity of efflux pumps, linked via an acid-sensitive linker, thereby reflecting the competitive advantage of the FL118 drug described above compared to the camptothecin-based anticancer drugs with a similar structure used in Enhertu and Trodelvy, and can overcome resistance and ensure a wider therapeutic window, which is differentiated from currently developed third-generation ADCs (Enhertu and Trodelvy).

[0061] In particular, the immunoconjugate according to the present invention can show a strong resistance-overcoming effect by further inhibiting cancer resistance-inducing proteins DDX5, UBE2T, and USP2a in addition to inhibiting Topoisomerase I, a tumor target verified through the FL118 drug. Therefore, the immunoconjugate according to the present invention can be used as a resistance-overcoming ADC in the field where ADCs based on existing SN-38, such as Trop-2 / CEACAM-5, have been successfully developed.

[0062] In other words, in the case of Trop-2-SN-38 ADC, it has been successfully developed for many cancers such as TNBC, bladder, and gastric cancer, but there are still problems with SN-38 resistance (overexpression of drug efflux transporter, epigenetic silencing of Top1, and increased levels of anti-apoptotic proteins). to The FL118 drug of formula 1 according to the present invention - acid-sensitive An immunoassay comprising an antibody or a fragment thereof containing an antigen-binding site thereof and a linker (indirect) By utilizing a composite, the above problems can be overcome.

[0063] The immune complex of the present invention can identify optimal responding patients from among patients expressing antigens such as Trop-2 and CEACAM-5 by utilizing relevant biomarkers such as DDX5, K-ras, and p53, thereby ensuring optimal therapeutic responsiveness.

[0064] [Payloads for various Carrier-Drug Conjugates] The FL118 drug satisfies all of the following characteristics (1) to (5), and the present invention is characterized in that the FL118 drug is selected as a payload for various carrier-drug conjugates: (1) Among the drugs in this series, Exatecan, SN-38, etc. have been developed as payloads for ADCs; (2) It utilizes topoisomerase I inhibitors (Irinotecan, Topotecan, etc.), an anti-cancer mechanism whose efficacy and safety have been verified in clinical trials. Topoisomerase I inhibitors have been shown to have excellent anti-cancer effects in various refractory solid cancers, including colorectal cancer, lung cancer, breast cancer, and ovarian cancer; (3) Overcome many of the resistance mechanisms of payload targets among existing camptothecin-based drugs, ensuring efficacy in resistant / recurrent cancers; (4) It is a drug that has a wide therapeutic window that ensures both anticancer efficacy and safety even when administered alone; and (5) PK that remains in cancer tissue for a long time but is rapidly eliminated when released into the bloodstream Since it has a specific profile, it can ensure a wider therapeutic window when used as a payload for ADC, and it is stable even when released from ADC in the vicinity of cancer cells.

[0065] [Linker technology enabling efficient drug delivery] Commonly used linkers in ADCs include acid-labile hydrazones, protease-labile peptides, and reducing agent-sensitive disulfides, as well as the so-called "non-cleavable" thioether linkers used in ADCs.

[0066] Existing supertoxins such as MMAE, Calicheamicin, and PBD In the case of ADCs that utilize er toxin, a stable linker system (a feature of second-generation ADCs) was used, which aims to minimize the separation of the drug from the blood before it reaches the cancer tissue.

[0067] The principle by which most second-generation ADCs function in cancer cells is that in the first step, the antibody moiety that constitutes the ADC binds to an antigen that is overexpressed in the cancer cell; in the second step, the ADC that has bound to the antigen on the surface of the cancer cell through an antigen-antibody reaction is transported inside the cancer cell via endosomes and lysosomes; in the third step, the antigen-antibody moiety is degraded in the lysosome, releasing an enzyme (cathepsin B) drug; and in the final step, the cancer cell is killed by the drug released inside the cancer cell.

[0068] In order to develop a new ADC that can be selectively used for various solid cancers, it is essential to utilize a linker system that surpasses the drug delivery efficiency of existing ADCs. In order to develop an ADC targeting a new antigen beyond the already established drug targets Trop-2, Her2, and Folate Receptor, ADCs should be developed for slow internalizing antigens such as CEACAM-5 and / or cancer-specific antigens such as NY-ESO-1 / HLA complex. However, it is difficult to deliver a sufficient amount of drug with the limited drug delivery efficiency of the existing Val-Cit or MAC-glucuronide Linker systems.

[0069] In particular, for efficient drug delivery to antigens such as CEACAM-5, the antibody-drug bond must remain stable in the blood and / or surrounding environment of normal tissues, but must be capable of rapidly releasing the drug in the environment surrounding cancer cells, such as the tumor microenvironment.

[0070] If the drug can be released quickly even in the tumor microenvironment surrounding the cancer, it can also be advantageously used to target some antigens that have limited ADC uptake rate (representatively, CEACAM-5, various cancer specific antigen-HLA complexes, etc.).

[0071] Furthermore, since the FL118 drug has an excellent therapeutic window even when used alone, immunoconjugates using the FL118 drug are required to have a linker with a release profile that delivers the drug quickly and efficiently after first reaching the cancer tissue, rather than stability that minimizes the separation of the FL118 drug in the blood.

[0072] Therefore, the acid-sensitive linker used in the present invention is one that is decomposed in the acidic environment (pH ≦ 7) around the cancer, and was selected taking advantage of the fact that the free FL118 drug of formula 1 is a hydrophobic small molecule and can penetrate deep into the cancer tissue, penetrate the cell membrane, and move into the cell.

[0073] According to the present invention, the antibody to which the FL118 drug of formula 1 is bound via an acid-sensitive linker binds to an antigen overexpressed on the surface of cancer cells, similar to the first step in which second-generation ADCs work in cancer cells, but some of them undergo an intracellular processing step like second-generation ADCs, and the rest of them can release the drug due to the low pH around the cancer cells. The drug released from the cancer tissue then moves to the inside of the cancer cells by diffusion, and not only does it not pass through endosomes or lysosomes, but also acts on the cancer cells immediately without enzyme (cathepsin B) reaction to induce apoptosis. Therefore, compared to second-generation ADCs that can only release drugs through enzyme reactions, the antigen selectivity of cancer cells is the same, but the main feature of the immunoconjugate of the present invention is that it is possible to maximize the drug release and delivery efficiency into cancer cells by using a pH-sensitive linker.

[0074] In the [FL118 drug of formula 1]-[acid-sensitive linker] of the present invention, it is preferable that the FL118 drug of formula 1 and the acid-sensitive linker are linked by a carbonate or ester bond so that the free FL118 drug of formula 1 is released when the acid-sensitive linker is decomposed.

[0075] Generally, carbamate bonds provide superior stability of drug linkers in terms of hydrolysis compared to ester and carbonate bonds. However, the present invention is characterized in that labile ester or carbonate bonds are used instead of carbamate bonds to design the FL118 drug to be detachable from the drug linker both extracellularly and intracellularly around the cancer cells in the acidic environment (pH) of the cancer cells.

[0076] The pH of blood is kept constant at 7.3 to 7.4. Therefore, the FL118 drug is not cleaved from the acid-sensitive linker in blood, and even if it is cleaved, the release rate of the FL118 drug from the ADC at the neutral pH of serum is further reduced compared to that in tumor tissues with an acidic environment.

[0077] Unlike humans, mouse plasma contains high levels of esterase activity and readily cleaves ester and carbonate functional groups, which could be problematic in the mouse model experiments of Example 12. However, it was confirmed that the FL118 drug-containing ADC did not cause side effects such as weight loss due to the attack of normal cells when FL118 was released from the blood (Example 12).

[0078] CL2A, the linker used in Trodelvy, an existing FDA-approved ADC, is a linker that meets all of the following characteristics: (i) stability during storage after production, (ii) stability in the blood upon administration (almost no free payload exposed in plasma), and (iii) rapid release of the payload in cancer tissue.

[0079] The acid-sensitive linker used in the present invention may utilize the CL2A linker, which has already been proven to be successfully applied to the development of ADCs of camptothecin-based compounds through the successful development of Trodelvy and IMMU-130, and may be designed as shown in the following formula 2 to selectively and efficiently deliver the FL118 drug to cancer tissues. That is, in the present invention, the acid-sensitive linker may be derived from a compound of the following formula 2: [ka] wherein X1 and X2 are each independently -H or -halogen; Y is -NH-, -NR A - or null; Z is -C1-C4 alkyl-, -C3-C6 cycloalkyl-, -(C1-C2 alkyl)-(C3-C6 cycloalkyl)-, -(C3-C6 cycloalkyl)-(C1-C2 alkyl)-, or -(C1-C2 alkyl)-(C3-C6 cycloalkyl)-(C1-C2 alkyl)-; W is -R B -, -M- -R B -M-, -MR B -or-R B -M.R. C - and; R A ~R C are each independently C1-C4 alkyl; M is [ka] and n is an integer from 5 to 9.

[0080] In the present invention, the length of the linker, i.e., n in the above formula 2, may be an integer of 5 to 9, specifically, n may be an integer of 6 to 8, and more specifically, n may be 7, but is not limited thereto. Even if it is outside the above range, if there is no significant difference in effect due to the change in the linker length, it is naturally all included in the equivalent scope of the present invention.

[0081] Since the solubility of a drug can be improved by placing a polyethylene glycol (PEG) spacer between the drug and the carrier, the linker in formula 2 contains a low-molecular-weight PEG moiety that contains a limited number (n = 5-9) of PEG monomers.

[0082] The acid-sensitive linker of formula 2 is a customized linker that can be optimized depending on the properties of the targeting target, payload, and carrier.

[0083] The FL118 drug of formula 1 has a site that is easy to attach to various linkers to prepare carrier-drug conjugates. For example, the alcohol group site of the drug of formula 1 can be used as an attachment site to a linker.

[0084] Therefore, in the present invention, [FL118 drug of formula 1]-[acid-sensitive linker] may be one in which the alcohol group site of the FL118 drug of formula 1 is linked to the alcohol group site of the acid-sensitive linker of formula 2.

[0085] Experiments confirmed that various conjugates of FL118 drug-acid-sensitive linker were stable at pH 7.4 and / or plasma conditions, but readily released the drug under tumor lysosome conditions (pH 4.5 and proteolytic enzyme), and demonstrated that FL118 can be attached to various antibodies using the CL2A linker, a linker system currently used in FDA-approved ADCs.

[0086] In addition, a method was established that allows easy preparation of FL118ADC, which is DAR2, through site-specific conjugation. The A of DAR4 and 8 was prepared by partial reduction of the interchain SS bond of the antibody and conjugation. A method for producing DCs was established (Figure 5).

[0087] The stability of the obtained ADC was evaluated, and it was confirmed that it can be stably stored under low temperature conditions when stored in a lyophilized powder form or in solution, which allows for reconstitution.

[0088] Each of the resulting ADCs with DARs of 2, 4, and 8 was confirmed to retain a similar level of antigen-binding affinity to the original antibody.

[0089] In the present invention, the [FL118 drug of formula 1]-[acid-sensitive linker] conjugate can be any one selected from the group consisting of compounds represented by the following formulas 3 to 5.

[0090] [ka] [ka] [ka] (wherein each n is independently an integer from 5 to 9).

[0091] In order to improve the efficiency of drug delivery, the acid-sensitive linker of the present invention develops and applies a multivalent linker system that can overcome the problem of low DAR, which is considered to be a drawback of site-specific conjugation, and ensures a method for preparing high DAR ADCs of DAR4-12 even when using site-specific conjugation by attaching 2 to 3 payloads to one attachment site.

[0092] For this purpose, in the present invention, a Bridgeable Linker system as exemplified in formulas 4 and 5 can be used as a linker.

[0093] This Bridgeable Linker system can increase the efficiency of CMC and simplify the process so that DAR4 ADC can be easily produced while maintaining the fast release property of the payload in cancer tissue, which is the greatest advantage of the CL2A linker system. It can produce site-specific antibody-drug conjugates of DAR4 without separate antibody engineering. When disulfide-SS- (disulfide-SS-) present in the antibody is reduced, two thiols (-SH) are formed, and the thiols thus generated form a 2:1 conjugate with the new bridgeable linker-FL118 compound, so that the antibody-drug conjugate of DAR4 can be easily produced as a single product by reacting all four disulfides in a general antibody.

[0094] [Immune complex] In the present invention, the term "immunoconjugate" refers to a complex in which a cytotoxic drug-linker conjugate is bound to an antibody or an antigen-binding fragment thereof.

[0095] An antibody-drug conjugate (ADC) is an example of an immune conjugate, and therefore, in the present invention, the description of an ADC and the description of an immune conjugate can be used interchangeably.

[0096] In the present invention, the term "drug-linker conjugate" refers to an immunoconjugate or a carrier-drug conjugate. The term "carrier-drug conjugate" refers to a material for preparing an immune complex or a carrier-drug conjugate, and means that an antibody, a fragment containing the antigen-binding site of the antibody, or a carrier is not linked to the antibody, and can be used as an immune complex or a carrier-drug conjugate by binding to any antibody, a fragment containing the antigen-binding site of the antibody, or a carrier depending on the purpose.

[0097] When the immune complex is administered in vivo, one of its components, an antibody or a fragment thereof containing the antigen-binding site, binds to a target antigen and then releases a drug, allowing the drug to act on the target cell and / or surrounding cells. As a targeted drug, the immune complex is expected to have excellent efficacy and reduced side effects.

[0098] Factors that have a significant impact on the efficacy of immunoconjugates include (1) drug potency, (2) drug linker stability, and (3) efficient on-target drug release. Because the efficacy is influenced by a complex combination of various factors, it is very difficult to predict the efficacy of an immunoconjugate, which is a combination of these factors, based only on the facts known about each factor.

[0099] Immunoconjugates designed to release drugs after internalization have the problem that if the internalization process is inefficient, a sufficient concentration of active drug cannot be delivered to the inside of the cell. Even if a hydrophobic drug is used as a cytotoxic drug, it is difficult to expect a bystander cell-killing phenomenon against surrounding cells.

[0100] In order to solve such problems, the immune complex of the present invention is (i) Using an acid-sensitive linker to liberate a hydrophobic drug that can penetrate the cell membrane in the tumor microenvironment (pH ≦ 7) around the cancer and play a role inside the cell, and then introduce a large amount of the free hydrophobic drug into the cell; (ii) In the acidic environment (pH ≦ 7) surrounding cancer cells, the acid-sensitive linker is decomposed to release the drug, and a large amount of the free drug penetrates the cell membrane and is highly enriched in the cells. This is characterized by the use of FL118 drug of formula 1, which is a hydrophobic drug that can penetrate the cell membrane and inhibit the action of efflux pumps at the same time (Figure 2).

[0101] The FL118 drug is a topoisomerase I inhibitor and can exhibit cytotoxicity against cancer cells, but there have been no reports of its use in the preparation of immune complexes.

[0102] The FL118 drug of formula 1 is (a) a hydrophobic small molecule that can permeate cell membranes, and (b) is not effluxed out of cells via efflux pumps, so it can accumulate quickly in cancer tissues and maintain high concentrations for a long time. It is concentrated and exerts cytotoxicity inside cells, and after causing apoptosis, it is excreted and can continuously penetrate the cell membranes of surrounding cells to move into the cells and act there.

[0103] The immunoconjugate of the present invention is targeted to cancer cells by the antigen-binding site that targets the antigen of the cancer cells, and then some of the acid-sensitive linkers are decomposed mainly in the acidic environment (pH≦7) around the cancer and / or in the blood, and the mechanism of action of the free FL118 drug of formula 1 mainly begins outside the cells. Therefore, it is a surprising result that the excellent anticancer effect and stability described above could not be predicted until the results were confirmed through experiments.

[0104] The FL118 drug of formula 1 is also named (4S)-4-ethyl-4-hydroxy-8,9-methylenedioxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione (PubChem CID 72403).

[0105] Although not limited thereto, the compound of formula 1 may have C 1-3 It is self-evident that simple functional groups such as alkyl, hydroxy, halogen, and amine groups can be added, or existing hydroxy, ethyl, and oxo functional groups can be replaced with other functional groups or removed, as long as they exhibit the same efficacy as the immunoconjugates of the present invention.

[0106] As described above, the immune complex of the present invention has a technical feature of using the FL118 drug and an acid-sensitive linker, so that any antibody or a fragment containing the antigen-binding site thereof can be bound to the immune complex according to the desired purpose, and all of these are included in the scope of the present invention. In one specific example of the present invention, trastuzumab, cetuximab, and sacituzumab were bound to the FL118-linker complex of the present invention to prepare immune complexes, and their excellent anticancer effects were confirmed.

[0107] In the present invention, the immune complex may have an average drug-to-antibody ratio (DAR) of 2-12, preferably a DAR of 4-12.

[0108] [ADC platform design] The ADC platform according to the present invention can be designed based on the following basic design concepts. (1) Targeting an antigen that is more than 10 times more prevalent in cancer cells than in normal cells; (2) an acid-sensitive linker that allows efficient drug release in cancer cells; (3) FL118 is a drug that has demonstrated high tumor regression effects and is safe even when used alone.

[0109] When applying the ADC platform according to the present invention as described above, various antibodies can be utilized, and resistance and resistivity against existing third-generation ADCs, Trodelvy and Enhertu, can be overcome.

[0110] [Preparation Process of FL118 Drug-CL2A Linker-Antibody Platform] <Synthesis of FL118 Drug-CL2A Linker> The FL118 drug-CL2A linker complex can be synthesized through two main processes. In Process 1, the intermediate N3-PEG8-Lys(MMT)-PABOH is synthesized, and in Process 2, it is combined with FL118 to synthesize the final substance, FL118-CL2A.

[0111] More specifically, in Process 1, Fmoc-Lys(MMT)-OH and 4-Aminobenzyl alcohol are synthesized by amide coupling using EEDQ. After removing the protecting group Fmoc, it is reacted with N3-PEG8-COOH using EEDQ, and then separated and purified to synthesize the intermediate N3-PEG8-Lys(MMT)-PABOH.

[0112] In Process 2, the previously synthesized N3-PEG8-Lys(MMT)-PABOH and FL118 are used to synthesize N3-PEG8-Lys(MMT)-PABC-FL118 using Triphosgene and DMAP. MCC-alkyne, which is a part necessary for binding to the antibody, is synthesized by click reaction of the azide of N3-PEG8-Lys(MMT)-PABOH using CuBr, PPh3, and DIPEA. Then, the MMT, which is the amine protecting group of lysine, is removed to synthesize the final substance, the FL118 drug-CL2A linker complex.

[0113] [Antibody and FL118-CL2A Conjugation] To bind an antibody (e.g., trastuzumab, a monoclonal antibody targeting Her2, cetuximab, a monoclonal antibody targeting EGFR, or sacituzumab, an antibody targeting Trop-2) to the FL118 drug-CL2A linker conjugate, conjugation is performed via a bond between a cysteine ​​residue generated by reducing the disulfide bond of the antibody and a maleimide functional group of the FL118 drug-CL2A linker conjugate.

[0114] At this time, to prepare ADCs of DAR4 or 8, the disulfide bonds of the antibody are reduced using 2.8 or 7.5 times (DAR4 and 8 ADCs, respectively) of TCEP (tris(2-carboxyethyl)phosphine hydrochloride) compared to the antibody in a reaction buffer (20 mM histidine, 150 mM sodium chloride, pH 6.0), and the antibody is reacted using a desalting column, after which the residual TCEP is removed.

[0115] The reduced antibody is reacted with 10 or 12 times (DAR4, 8ADC, respectively) FL118-CL2A in the reaction buffer compared to the antibody. After reaction with a desalting column, the remaining FL118-CL2A is removed and the buffer is exchanged with 18 mM MES (pH 6.5).

[0116] The prepared ADC was subjected to sterile filtration using a 0.2 μm PVDF membrane filter, and 0.07 mM polysorbate 80 (PS80) and 20 mM trehalose dehydrate were added, lyophilized, and then stored.

[0117] [Target antigen_drug target] When designing the antibody-drug conjugates (ADCs) or immunoconjugates of the invention, targeting can be expanded beyond cancer cells to include infectious disease organisms and / or cells involved in autoimmune diseases.

[0118] Thus, the cells targeted by the antibody or fragment containing the antigen-binding site thereof may be cancer cells, cells associated with infectious disease organisms and / or autoimmune diseases.

[0119] Non-limiting examples of target antigens include antigens that are preferentially distributed on the surface of cancer cells, such as Her2, FolR, and PSMA, and antigens expressed by cancer cells that are also distributed in low abundance in normal tissues, such as Trop2.

[0120] Cancer cell specific antigens include, for example, 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, AFP, Aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, ALCAM, ALK, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOCl, AR, aromatase, ASPH, ATX, AX1, AXL, AZGP1 (zinc-a-glycoprotein), B4 GALNT1, B7, B7.1, B7.2, B7-H1, B7-H3, B7-H4, B7-H6, BAD, BAFF, BAG1, BAI1, BCR, BCL2, BCL6, BCMA, BDNF, BLNK, BLR1(MDR15), BIyS, BMP1, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMP10, BMPR1A, BMPR1B, BMPR2, BPAG1(プレクチン), BRCA1, C19orflO(IL27w), C3, C4A, C5, C5R1, CA6, CA9, CANT1, CAPRIN-1, CAS P1, CASP4, CAV1, CCBP2(D6 / JAB61), CCL1(1-309), CCLI1(エオタキシン), CCL13(MCP-4), CCL15(MIP-Id), CCL16(HCC-4), CCL17(TARC), CCL18(PARC), CCL1 9(MIP-3b), CCL2(MCP-1), MCAF, CCL20(MIP-3a), CCL21(MEP-2), SLC, exodus-2, CCL22(MDC / STC-I), CCL23(MPIF-I), CCL24(MPIF-2 / エオタキシン-2), CCL2 5(TECK), CCL26(エオタキシン-3), CCL27(CTACK / ILC), CCL28, CCL3(MIP-Ia), CCL4(MIPIb), CCL5(RANTES), CCL7(MCP-3), CCL8(mcp-2), CCNA1, CCNA2, CCN D1, CCNE1, CCNE2, CCR1(CKR1 / HM145), CCR2(mcp-IRB / RA), CCR3(CKR3 / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6),CCR7(CKR7 / EBI1), CCR8, CDw198(CMKBR8 / TERI / CKR-L1), CCR9(GPR-9-6). )、CCRL1(VSHK1),CCRL2(L-CCR),CD13,CD164,CD19,CDH6,CDIC,CD2,CD20 CD21, CD200, CD22, CD23, CD24, CD27, CD28, CD29, CD3, CD33, CD35, CD37C D38, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD45RB, CD47, CD52, CD56, CD 69 CD70, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD97, CD9 9, CD117, CD125, CD137, CD147, CD179b, CD223, CD279, CD152, CD274, CDH1( E-カドヘリ) CDH1O, CDH12, CDH13, CDH18, CDH19, CDH2O, CDH3, CDH5, CDH7, CD H8, CDH9, CDH17, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKN1A(p21Wap1 / Cip1) 、CDKN1B(p27Kip1) 、CDKN1C 、CDKN2A(p16INK4a) 、CDKN2B(CDKN2C)C DKN3, CEA, CEACAM5, CEACAM6, CEBPB, CERI, CFC1B, CHGA, CHGB (Chit inase) CHST1O, CIK, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLF SF7, CKLFSF8, CLDN3, CLDN6, CLDN7(Cold-7), CLDN18, CLEC5A, CLEC6A, CL EC11A, CLEC14A, CLN3, CLU(CLU), CMKLR1, CMKOR1(RDC1), CNR1, C-MET. COL18A1, COLIA1, COL4A3, COL6A1, CR2, Crypto, CRP, CSF1(M-CSF), CSF2(GM). -CSF), CSF3(GCSF), CTAG1B(NY-ESO-1), CTLA4, CTL8, CTNNB1(b-テニ), CT SB(テシB), CX3CL1(SCYD1), CX3CR1(V28), CXCL1(GRO1), CXCL1O(IP-IO).CXCLI1(1-TAC / IP-9)、CXCL12(SDF1)、CXCL13、CXCL14、CXCL16、CXCL2(GRO2)、CXCL3(GRO3)、CXCL5(ENA-78 / LIX)、CXCL6(GCP-2)、CXCL9(MIG)、CXCR3(GPR9 / CKR-L2)、CXCR4、CXCR6(TYMSTR / STRL33 / Bonzo)、CYB5、CYC1、CYSLTR1、DAB2IP、DES、DKFZp451J0118、DLK1、DNCL1、DPP4、E2F1、Engel、Edge、Fennel、EFNA3、EFNB2、EGF、EGFR、ELAC2、ENG、Enola、ENO2、ENO3、EpCAM、EPHA1、EPHA2、EPHA3、EPHA4、EPHA5、EPHA6、EPHA7、EPHA8、EPHA9、EPHA10、 EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, EPHRIN-A1, EPHRIN-A2, EPHRINA3, EPHRIN-A4, EPHRIN-A5, EPHRIN-A6, EPHRIN-B1, EPHRIN-B2, EPHRIN-B3, EPHB4, EPG, ERBB2 (HER-2), ERBB3, ERBB4, EREG, ERK8, estrogen receptor, Earl, ESR2, F3 (TF), FADD, FAP, farnesyltransferase, FasL, FASNf, FCER1A, FCER2, FCGR3A, FGF, FGF1 (aFGF), FGF10, FGF1 1, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2(bFGF), FGF20, FGF21, FGF22, FGF23, FGF3 (int-2), FGF4(HST), FGF5, FGF6(HST-2), FGF7(KGF), FGF8, FGF9, FGFR1, FGFR2, FGFR3, FGFR4, FIGF(VEGFD) , FIL1 (EPSILON), FBL1 (ZETA), FLJ12584, FLJ25530, FLRT1 (fibronectin), FLT1, FLT-3, FOLR1, FOS, FOSL1 (FRA-1), FR-alpha, FY (DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GD2, GD3, GDF5, GFI1, GFRA1 , GGT1, GM-CSF, GNAS1, GNRH1, GPC1, GPC3, GPNB, GPR2(CCR10), GPR31, GPR44, GPR81(FKSG80), GRCC1O(C1O), GRP, GSN(Gelsolin), GSTP1, GUCY2C, HAVCR1, HAVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, Hedgehog, HER3, H GF, HIF1A, HIP1, histamine and histamine receptor, HLA-A, HLA-DR, HLA-DRA, HLA-E, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-a, IFNA1, IFNA2, IFNA4, IFNA5, EFNA6, BFNA7, IFNB1, IFN gamma, IFNW1, IGBP1, IGF1, IGFIR,IGF2, IGFBP2, IGFBP3, IGFBP6, DL-1, ILIO, ILIORA, ILIORB, IL-1, IL1R1(CD121a), IL1R2(CD121b), IL-IRA, IL-2, IL2RA(CD25), IL2RB(CD122), IL2RG(CD132), IL-4, IL-4R(CD123), IL-5, IL5RA(CD125), IL3RB(CD131), IL-6, IL6RA, (CD126), IR6RB(CD130), IL-7, IL7RA(CD127), IL-8, CXCR1(IL8RA), CXCR2, (IL8RB / CD128), IL-9, IL9R(CD129), IL-10, IL10RA(CD210), IL10RB(CDW210B), IL-11, IL11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, ILIA, ILIB, ILIF10, ILIF5, IL1F6, ILIF7, IL1F8, DL1F9, ILIHYI, ILIR1, IL1R2, ILIRAP, ILIRAPLI, ILIRAPL2, ILIRL1, IL1RL2, ILIRN, IL2, IL20, IL20RA, IL21R, IL22, IL22R, IL22RA2, IL23, DL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA, IL4, 1L4, IL6ST (glycoprotein 130), ILK, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (α6 integrin), ITGAV, ITGB3, ITGB4 (β4 integrin), JAG1, JAK1, JAK3, JTB, JUN, K6HF, KAI1, KDR, KIT, KITLG, KLF5 (GC Box BP), KLF6, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, KRT1, KRT19 (keratin 19), KRT2AKRTHB6(hair(hair)-complex II with scale), L1CAM, LAG3, LAMA5, LAMP1, LEP(hair), Lewis Y (LeY), LILRB1, Lingo-p75, Lingo-Troy, LGALS3BP, LRRC15, LPS LTA(TNF-b), LTB, LTB4R(GPR16), LTB4R2, LTBR, ​​LY75, LYPD3, MACMAR CKS, MAG, OMgp, MAGEA3, MAGEA6, MAP2K7(c-Jun), MCP-1, MDK, MIB1 midkine, MIF, EGYPT, MJP-2, MLSN, MK, MKI67(Ki-67), MMP2, MMP9, MS4 A1, MSMB, MT3(mucin-UI), mTOR, MTSS1, MUC1(mucin), MUC16, MYC MYD88, NCK2, NCR3LG1, Neurocan, NFKBI, NFKB2, NGFB (NGF) NGFR, NgR-Lingo, NgRNogo66, (Nogo), NgR-p75, NgR-Troy, NMEI(NM2). 3A) NOTCH, NOTCH1, NOTCH3, NOX5, NPPB, NROB1, NROB2, NRID1, NR1D2, N.S R1H2, NR1H3, NR1H4, NR112, NR113, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1 NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6 A1, NRP1, NRP2, NT5E, NTN4, NY-ESO1, ODZI, OPRDI, P2RX7, PAP, PART1 PATE, PAWR, P-linkage, PCA3, PCD1, PD-L1, PCDGF, PCNA, PDGFA, PDGFB, PD GFRA, PDGFRB, PECAMI, L1-CAM, peg-linkage, PF4(CXCL4), PGF, PGR. Liquids (phosphacan), PIAS2, PI3, PIK3CG, PLAU(uPA), PLG, PLXDC I, PKC, PKC-link, PPBP(CXCL7), PPID, PR1, PRAME, PRKCQ, PRKD1, PRL ROC, PROK2, PSAP, PSCA, PSMA, PTAFR, PTEN, PTHR2, PTGS2(COX-2), PTN.PVRIG, RAC2 (P21Rac2), RANK, RANK ligand, RARB, RGS1, RGS13, RGS3, RNFI1O (ZNF144), Ron, ROBO2, ROR1, RXR, S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYE1 (endothelial monocyte-activating cytokine), SDF2, SERPENA1, SERPINA3, SERPINB5 (maspin), SERPINEI (PAI-I), SERPINFI, SHIP-1, SHIP-2, SHB1, SHB2, SHBG, SfcA Z, SLAMF7, SLC2A2, SLC33A1, SLC43A1, SLC44A4, SLC34A2, SLIT2, SPP1, SPRR1B(Spr1), ST6GAL1, ST8SIA1, STAB1, STATE, STEAP, STEAP2, TB4R2, TBX21, TCP1O, TDG F1, TEK, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, THIL, THBS1 (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor (tissue factor), TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TNF, TNF-a, TNFAIP2(B94), TNFAIP3, TNFRSFI1A, TNFRSF1A , TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6(Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSF1O(TRAIL), TNFRSF10A, TNFRSF10B, TNFRSF12A, TNFRS F17, TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFRSF14 (HVEM), TNFSF15 (VEGI), TNFSF18, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-like receptor,TOP2A (Topoisomerase Iia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM2, TROP2, TRPC6, TSLP, TWEAK, Tyrosinase, uPAR, VEGF, VEGFB, VEGFC, versican, VHL C5, VLA-4, WT1, Wnt-1, XCL1 (Lymphotactin), XCL2 (SCM-Ib), XCRI (GPR5 / CCXCR1), YY1, ZFPM2, <h2 style=";text-align:left;direction:ltr">CLEC4C(BDCA-2、DLEC、CD303、CDH6、CLECSF7)、CLEC4D(MCL、CLECSF8)、CLE C4E(Mincle)、CLEC6A(デクチン-2)、CLEC5A(MDL-1、CLECSF5)、CLEC1B(CLEC-2) )、CLEC9A(DNGR-1)、CLEC7A(デクチン-1)、CLEC11A、PDGFRa、SLAMF7、GP6(GPVI) )、LILRA1(CD85I)、LILRA2(CD85H、ILT1)、LILRA4(CD85G、ILT7)、LILRA5(CD) 85F、ILT11)、LILRA6(CD85b、ILT8)、LILRB1、NCR1(CD335、LY94、NKp46)、NC R3(CD335、LY94、NKp46)、NCR3(CD337、NKp30)、OSCAR、TARM1、CD30、CD300C 、CD300E、CD300LB(CD300B)、CD300LD(CD300D)、KIR2DL4(CD158D)、KIR2DS 、KLRC2(CD159C、NKG2C)、KLRK1(CD314、NKG2D)、NCR2(CD336、NKp44)、PILRB SIR PA、SIRPB1(CD172B)、TREM1(CD354)、TREM2、KLRF1(NKp80)、17-1A、SLAM7、 MSLN、CTAG1B / NY-ESO-1、MAGEA3 / A6、ATP5I(Q06185)、OAT(P29758)、AIFM1( Q9Z0X1)、AOFA(Q64133)、MTDC(P18155)、CMC1(Q8BH59)、PREP(Q8K411)、YM EL1(O88967)、LPPRC(Q6PB66)、LONM(Q8CGK3)、ACON(Q99KI0)、ODO1(Q6059) 7)、IDHP(P54071)、ALDH2(P47738)、ATPB(P56480)、AATM(P05202)、TMM93( Q9CQW0)、ERGI3(Q9CQE7)、RTN4(Q99P72)、CL041(Q8BQR4)、ERLN2(Q8BFZ9)、TERA(Q01853), DAD(P61804), CALX(P35564), CALU(O35887), VAPA(Q9WV55), MOGS(Q8 0UM7), GANAB (Q8BHN3), ERO1A (Q8R180), UGGG1 (Q6P5E4), P4HA1 (Q60715), HYEP (Q9D3 79), CALR(P14211), AT2A2(O55143), PDIA4(P08003), PDIA1(P09103), PDIA3(P27773 ), PDIA6(Q922R8), CLH(Q68FD5), PPIB(P24369), TCPG(P80318), MOT4(P57787), NICA( P57716), BASI (P18572), VAPA (Q9WV55), ENV2 (P11370), VAT1 (Q62465), 4F2 (P10852), ENOA (P17182), ILK (O55222), GPNMB (Q99P91), ENV1 (P10404), ERO1A (Q8R180), CLH (Q68FD5), DSG1A (Q61495), AT1A1 (Q8VDN2), HYOU1 (Q9JKR6), TRAP1 (Q9CQN1), GRP75 (P38647), ENPL (P08113), CH60 (P63038), or CH10 (Q64433), but is not limited thereto.

[0121] The target antigen may be an antigen that is 10 times more abundant in cancer cells than in normal cells.

[0122] In particular, the present invention provides a novel payload, FL of Formula 1, that can overcome the shortcomings of existing ADCs. By utilizing a next-generation ADC platform focused on -118 drugs, we will be able to provide a best-in-class ADC targeting Trop-2, an antigen selectively overexpressed on the surface of solid tumors, and a first-in-class ADC targeting EGFR, a known cancer-related antigen for which no related ADC has yet been developed due to safety / efficacy issues.

[0123] [Antibody or a fragment containing the antigen-binding site thereof] According to the present invention, an immune complex comprising [FL118 drug of formula 1]-[acid-sensitive linker]-[antibody or a fragment containing an antigen-binding site thereof] can exert an organic mechanism of action according to the use of the combination of [FL118 drug of formula 1]-[acid-sensitive linker], regardless of the type, as long as the antibody or a fragment containing an antigen-binding site thereof targets an antigen on a cancer cell. In particular, the free FL118 drug is 5 to 20 times more potent than SN-38 in various cancer cell lines, i.e., has a low level of IC 50 Cytotoxicity is expressed numerically, and the evaluation results for 140 cell lines derived from various cancers showed IC<100nM for the majority of cancer cells. 50 In addition, FL118 showed superior efficacy to SN-38 in various cancer cell line xenograft models, Rat This is because FL118 shares the advantage of being able to secure a therapeutic window for immunoconjugates that use this as a payload, as it has excellent safety through GLP toxicity testing in beagle dogs. In addition, the FL118 drug utilizes a topoisomerase I inhibitor, an anti-cancer mechanism whose efficacy and safety have been verified in clinical trials. Topoisomerase I inhibitor (Irinotecan This is because the anticancer drugs used in clinical trials (Irinotecan, Topotecan, etc.) have been shown to have excellent anticancer effects against various refractory solid cancers, including colorectal cancer, lung cancer, breast cancer, and ovarian cancer.

[0124] Furthermore, in Examples 11 and 12, the anticancer effect of the antibody-FL118 conjugate was demonstrated using antibodies targeting Her2, EGFR, and TROP2, respectively.

[0125] As used herein, the term "antibody" refers to a protein molecule that acts as a ligand to specifically recognize an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, and whole antibodies. The term also includes chimeric antibodies and bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. The term further includes single-chain antibodies, chain antibodies, derivatives of antibody constant regions, and artificial antibodies based on scaffold proteins that retain the binding function to FcRn. A whole antibody has a structure with two full-length light chains and two full-length heavy chains, and each light chain is bonded to a heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG has subtypes including IgG1, IgG2, IgG3, and IgG4.

[0126] As used herein, the term "fragment containing an antigen-binding site" may refer to any fragment of an antibody that retains the antigen-binding activity of the antibody. Examples of antibody fragments include, but are not limited to, single-chain antibodies, Fd, Fab, Fab', F(ab')2, dsFv, or scFv. The Fd refers to the heavy chain portion contained in the Fab fragment. The Fab has a structure that includes a light chain variable region, a heavy chain constant region, and a first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is produced by forming disulfide bonds between cysteine ​​residues in the hinge region of Fab'. Fv (variable fragment) refers to the minimum antibody fragment that has only a heavy chain variable region and a light chain variable region. In double disulfide Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by a disulfide bond, and in single chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally linked by a covalent bond via a peptide linker. Such antibody fragments can be obtained using protease (for example, Fab fragments can be obtained by restricting and cleaving the whole antibody with papain, and F(ab')2 fragments can be obtained by cleaving with pepsin), or can be preferably produced through genetic recombination techniques.

[0127] [Pharmaceutically acceptable salts] In this specification, pharma- ceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, including, for example, salts of inorganic ions including sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc., and salts of inorganic acids such as hydrochloric acid, phosphoric acid, and sulfuric acid, as well as salts of organic acids such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotic acid, and acetylsalicylic acid, and salts of amino acids such as lysine, arginine, and guanidine. In addition, salts of organic ions such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium, and benzethonium that can be used in pharmaceutical reactions, purification processes, and isolation processes. However, the types of salts referred to in the present invention are not limited to these listed salts.

[0128] [Various Carrier-Drug Conjugates] The [FL118 drug of formula 1]-[acid-sensitive linker] conjugate of the present invention can be applied to various types of drug carriers in addition to antibodies, and unlike antibodies, it can fully penetrate deep into cancer tissues. By using a carrier having the property of easy CMC, it can be effectively used in various applications.

[0129] Therefore, the method for producing a carrier-drug conjugate of the present invention includes the steps of: The present invention is characterized in that the [FL118 drug of formula 1]-[acid-sensitive linker of formula 2] conjugate or a pharma- ceutically acceptable salt thereof is used to link one or more drugs of formula 1 to a carrier via a linker of formula 2.

[0130] In this case, the carrier may be an antibody, a lipibody, and / or an aptamer.

[0131] A description of antibodies and non-limiting examples thereof are provided above.

[0132] Aptamers are biopolymers based on DNA / RNA, and have antigen-binding affinity at the level of several nM, similar to antibodies, and can therefore be used as tumor tissue-selective drug carriers.

[0133] Lipibodies are a new class of immune proteins found in fish, which can recognize antigens in the same way as antibodies and nanobodies (single chain antibodies derived from mammals such as llamas), have an antigen binding affinity of several nM, and can be used as a selective carrier of drugs for cancer tissues. In particular, they are non-immunogenic, so they can be used as drug carriers, and at the same time, their low molecular weight can increase their penetration into cancer cells, making it easier to continuously produce lipibodies of uniform quality and suitable for the production of drug conjugates with increased penetration into cancer tissues.

[0134] [In the case of ADC, its role as an antibody therapeutic agent] The ADCs of the present invention may also serve as fundamental therapeutic antibodies, which have played an important role in the treatment of diseases such as cancer and autoimmunity over the past decade.

[0135] Antibodies have the ability to distinguish between antigens and cells in the body, and therefore have an excellent screening function that selectively acts on antigens. Antibody therapeutic drugs utilize this antigen-selective binding property of antibodies as a treatment for diseases. Antibodies are Y-shaped, with antigen binding sites on each arm, and complementarity determining regions (CDRs) allow the antibody to selectively recognize the target antigen. There are five main types of antibodies (IgM, IgD, IgG, IgA, and IgE / Immunoglobulin (Ig)), and IgG antibody therapeutic drugs are mainly used.

[0136] Antibody therapeutics can selectively react to antigens and remove them. To achieve this, various mechanisms are utilized, but the main features of the mechanisms used for antibody therapeutics are as follows:

[0137] The first mechanism is blocking function. Antibodies selectively bind to target receptors on cells and block the physiological functions of the target cells. Antibodies bind to ligands or receptors expressed on the cell surface and block the target signaling pathway, causing loss of cell activity, growth inhibition, apoptosis, etc. due to reduced signaling.

[0138] When an antibody binds to a receptor, it changes the structure of the receptor or prevents factors that should bind to the receptor from binding, inhibiting intracellular signal transduction, thereby regulating cell growth and differentiation. When an antibody binds to a receptor, the receptor enters the cell via an intracellular influx mechanism, reducing the number of receptors on the cell surface, thereby regulating the physiological mechanism of the cell. A representative antibody therapeutic agent that shows excellent effects through this mechanism is trastuzumab (Herceptin®), which targets HER2-positive breast cancer, one of the human epidermal growth factor receptors (HER / EGFR / ERBB).

[0139] The second mechanism is antibody-dependent cell-mediated cytotoxicity (ADCC). Cancer cells have specific receptors or overexpressed receptors that are different from those of normal cells, and the immune system recognizes these receptors and produces antibodies that bind to the cell surface. When NK cells recognize Fc receptor (FcγRIII) and are activated, apoptosis is induced.

[0140] The third mechanism is complement-dependent cytotoxicity (CDC). It binds to the surface of cancer cells, activates complement protein (C1q), triggers the classical pathway, attacks the cell membrane, and creates pores that cause lysis of the target cell. Other mechanisms include opsonization, neutralization, and inflammatory responses.

[0141] Therapeutic antibodies are generally engineered from multiple perspectives for a specific therapeutic purpose. By performing a multi-step process of targeting, the antibody is produced in an optimized form. Furthermore, antibody engineering techniques that have been applied to the development of such therapeutic monoclonal antibodies are being extended and applied to the development of optimized ADCs. For example, currently most therapeutic antibodies and ADCs in clinical development are IgG1, with only a few using IgG2 and IgG4. The selection of antibody isotype is equally important for therapeutic antibodies and ADCs. This is because the antibody isotype affects the functions of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). In addition, IgG4 becomes functionally monovalent through in vivo Fab arm exchange. In addition, the efficacy of ADCs depending on the isotype and the Fc-domain engineering of antibodies can be applied to improve the in vivo efficacy of ADCs.

[0142] [Pharmaceutical composition for preventing or treating cancer] The present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the above-mentioned immunoconjugate according to the present invention or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0143] The immune complex of the present invention specifically binds to an antigen on a cancer cell, releases a drug inside or outside the cancer cell, and exhibits cytotoxicity, and therefore can be useful for treating or preventing cancer. The anticancer activity of the immune complex of the present invention is as described above.

[0144] In the present invention, the cancer includes all cancers treatable by inhibition of topoisomerase I and / or inhibition of one or more cancer-associated survival genes selected from the group consisting of survivin, Mcl-1, XIAP, and cIAP2, and may be solid cancer or blood cancer, such as pseudomyxoma, intrahepatic biliary tract cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphocytic leukemia, basal cell carcinoma, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder, bile duct cancer, colorectal cancer, and chronic myeloid leukemia. , chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, paranasal sinus, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain tumors, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue tumors, malignant bone cancer, malignant The cancer may be at least one selected from the group consisting of, but is not limited to, primary cancer, primary lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, carcinoid tumor, gastrointestinal stromal cancer, Wilms' tumor, breast cancer, sarcoma cancer, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain tumor, anterior mediastinal tumor, rectal cancer, rectal carcinoid tumor, vaginal cancer, spinal cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer, and thyroid cancer. In addition, the cancer includes not only primary cancer but also metastatic cancer.

[0145] According to one embodiment of the present invention, there is provided a method for treating or preventing cancer, comprising administering a therapeutically effective amount of the immunoconjugate to a subject in need thereof. The subject may be a mammal, including a human.

[0146] The term "therapeutically effective amount" as used herein refers to an amount of said immunoconjugate effective for the treatment or prevention of cancer. Specifically, a "therapeutically effective amount" refers to an amount effective for medical treatment. It means an amount sufficient to treat a disease with a reasonable applicable benefit / risk ratio, and an effective dose level can be determined by factors including the type and severity of the individual, age, sex, type of disease, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, drugs used simultaneously, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with commercially available therapeutic agents. It can also be administered in a single dose or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that provides maximum effect with a minimum amount without side effects, and since the immune complex of the present invention shows a dose-dependent effect, the dosage can be easily determined by those skilled in the art depending on various factors such as the patient's condition, age, sex, and complications. The active ingredient of the pharmaceutical composition of the present invention is excellent in safety, so it can be used even at a dosage higher than the determined dosage.

[0147] According to one embodiment of the present invention, the present invention provides a use of the immunoconjugate for the preparation of a medicament for the treatment or prevention of cancer. The immunoconjugate for preparing the medicament may be mixed with an acceptable adjuvant, diluent, carrier, etc., and may be prepared as a composite preparation together with other active ingredients to have a synergistic effect of the active ingredients. All references to uses, compositions, and methods of treatment of the present invention apply equally unless they contradict each other. EXAMPLES

[0148] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in more detail with reference to the following examples, which are merely intended to clearly illustrate the technical features of the present invention and are not intended to limit the scope of the present invention.

[0149] Example 1: Preparation of FL118-Linker1 Conjugate [ka]

[0150] Step 1: (9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxy) Synthesis of (methyl)phenyl)amino)-6-(((4-methoxyphenyl)diphenylmethyl)amino)-1-oxohexan-2-yl)carbamate [ka]

[0151] To a suspension of 4-aminobenzyl alcohol (0.202 g, 1.639 mmol) and Fmoc-Lys(Mmt)-OH (1.00 g, 1.561 mmol) in dichloromethane (10 mL) was added EEDQ (0.405 g, 1.623 mmol). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with dichloromethane (10 mL) and washed with water (2×5 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a yellowish foam, which was purified by flash column chromatography (24 g Si, heptane / ethyl acetate 0-100%, dichloromethane loading). The product fractions were combined and concentrated under reduced pressure to give the title compound (928 mg, 80%) as a white foam. LCMS(sc_basic):522[M-Fmoc-H] -

[0152] Step 2: Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide [ka]

[0153] (9H-Fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)phenyl)amino)-6-(((4-methoxyphenyl)diphenylmethyl)amino)-1-oxohexan-2-yl)carbamate (928 mg, 1.244 mmol) was dissolved in diethylamine (10 mL, 97 mmol) and stirred at room temperature. After 2 hours, the solvent was concentrated under reduced pressure and the sticky residue was washed twice with heptane. The residue was then dissolved in dichloromethane and precipitated with heptane. The solvent was poured off and the procedure was repeated. Evaporation gave the product as a white foam (Yd: 677 mg, purity <90%).

[0154] The product was dissolved in 5 mL of dichloromethane and precipitated with 20 mL of heptane, then the solvent was poured off and evaporated to dryness to give 627 mg, 96% of the title compound as a white foam. LCMS(sc_acid):522[MH] -

[0155] Step 3: Synthesis of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide [ka]

[0156] To a pale yellow solution of O-(2-azidoethyl)-O'-(N-diglycolyl-2-aminoethyl)-heptaethylene glycol (662 mg, 1.193 mmol) and (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (625 mg, 1.193 mmol) in dichloromethane (10 mL) was added EEDQ (300 mg, 1.203 mmol). The reaction mixture was stirred at room temperature for 3 h. The reaction was concentrated under reduced pressure. The crude product (1.68 g) was purified by column chromatography (silica gel, ethyl acetate / methanol 0-10%) to afford the title compound as a thick colorless oil.

[0157] Yd: 1.02g LCMS:purity>95%. m / z1082 M+Na, 1058 MH 1 H NMR (d6-DMSO): Consistent with structure. Contains residual ethyl acetate. 1 H NMR (400 MHz, DMSO) δ 10.06(s, 1H), 8.15(d, J=8.0 Hz, 1H), 8.08(t, J=5.8 Hz, 1H), 7.58-7.49(m, 2H), 7.37(m, 4H), 7.25(m, 8H), 7.15(m, 2H), 6.86-6.76(m, 2H), 5.11(t, J=5.7 Hz, 1H), 4.43(m, 3H), 4.00(s, 2H), 3.96(d, J=4.4 Hz, 2H), 3.70(s, 3H), 3.59(m, 2H), 3.57-3.44(m, 30H), 3.40(m, 5H), 3.25(q, J=6.1 Hz, 2H), 2.43(m, 1H), 1.93(m, 2H), 1.64(m, 2H), 1.54-1.22(m, 4H)

[0158] Further drying under vacuum gave 0.968 g of a colorless oil. Yield: 76%. Used without further analysis.

[0159] Step 4: Synthesis of 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl)cyclohexane-1-carboxamide [ka]

[0160] Succinimidyl 4-(N-maleimidomethyl)cyclohexanecarboxylate (1 The crude product (white solid, 950 mg) was purified by column chromatography (silica gel, 24 g, dichloromethane / methanol 0-2%). The product fractions were combined and concentrated in vacuo to give a white solid, followed by trituration with cold dichloromethane. Filtration afforded the title compound as a white solid. Yield: 577 mg, 73%.

[0161] LCMS(sc_acid):275[M+H] + 1H NMR (400 MHz, DMSO) δ 8.47(s, 1H), 8.16(d, J=7.9 Hz, 1H), 8.09(t, J=5.8 Hz, 1H), 7.58(d, J=8.4 Hz, 2H), 7.52(d, J=2.9 Hz, 2H), 7.41-7.20(m, 12H), 7.14(t, J=7.3 Hz, 2H), 6.94(s, 1H), 6.81(d, J=8.9 Hz, 2H), 6.28(d, J=2.0 Hz, 2H), 5.50(s, 2H), 5.24(s, 2H), 5.17-5.01(m, 2H), 4.49-4.40(m, 1H), 4.0 1(s, 2H), 3.99-3.91(m, 2H), 3.69(s, 3H), 3.62-3.45(m, 30H), 3.45-3.37(m, 4H), 3.28-3.21(m, 2H), 2.46-2.40(m, 1H), 2.25-2.02(m, 2H), 2.01-1.81(m, 2H), 1.74-1.54(m, 2H), 1.53-1.41(m, 2H), 1.40-1.19(m, 2H), 0.89(t, J=7.4 Hz, 3H).

[0162] Step 5: Synthesis of 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate [ka]

[0163] FL118 (50 mg, 0.127 mmol) and 4-dimethylaminopyridine (DAMP) (58.4 mg, 0.478 mmol) were dissolved in 8 mL of dichloromethane, and triphosgene (11.4 mg, 0.038 mmol) was dissolved in 2 mL of dichloromethane and added rapidly. The yellow reaction mixture was stirred for 30 min. After 1 h, (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-zadotriacontanamido-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (81 mg, 0.076 mmol) dissolved in 2 mL of dichloromethane was added and stirred for 30 min. The reaction mixture was then concentrated in vacuo. The crude product was taken up in DMSO and purified by basic preparative HPLC. The product fractions were combined and lyophilized to give 64 mg, 57% of the title compound as a yellow solid.

[0164] LCMS(sc_base):1206[M-MMT+H] + 1 H NMR (400 MHz, DMSO) δ 8.47(s, 1H), 8.16(d, J=7.9 Hz, 1H), 8.09(t, J=5.8 Hz, 1H), 7.58(d, J=8.4 Hz, 2H), 7.52(d, J=2.9 Hz, 2H), 7.41-7.20(m, 12H), 7.14(t, J=7.3 Hz, 2H), 6.94(s, 1H), 6.81(d, J=8.9 Hz, 2H), 6.28(d, J=2.0 Hz, 2H), 5.50(s, 2H), 5.24(s, 2H), 5.17-5.01(m, 2H), 4.49-4.40(m, 1H), 4.0 1(s, 2H), 3.99-3.91(m, 2H), 3.69(s, 3H), 3.62-3.45(m, 30H), 3.45-3.37(m, 4H), 3.28-3.21(m, 2H), 2.46-2.40(m, 1H), 2.25-2.02(m, 2H), 2.01-1.81(m, 2H), 1.74-1.54(m, 2H), 1.53-1.41(m, 2H), 1.40-1.19(m, 2H), 0.89(t, J=7.4 Hz, 3H).

[0165] Step 6: Synthesis of 4-((S)-35-(4-((4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate [ka]

[0166] 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl)cyclohexane-1-carboxamide (25 mg, 91 μmol) and 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide)benzyl((S)-7-ethyl-8,1 1-Dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate (65 mg, 44.0 μmol), copper(I) bromide (2.5 mg, 17.43 μmol) and triphenylphosphine (2.3 mg, 8.77 μmol) were dissolved in dichloromethane (2.0 mL) and N,N-diisopropylmethylamine (0.023 mL, 132 μmol) was added.

[0167] The reaction mixture was observed to quickly turn into a pale yellow suspension and was stirred for 28 hours. The reaction mixture was then concentrated in vacuo. The crude product was taken up in DMSO and purified by basic preparative HPLC. The product fractions were combined and lyophilized to give 62 mg, 80% of the title compound as a white solid. LCMS(sc_base):1480[M-MMT+H] +

[0168] Step 7: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate [ka]

[0169] 4-((S)-35-(4-((4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33 -Nonaoxa-3,9-diazapentatriacontanamide)benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate (65 mg, 0.037 mmol) was dissolved in 0.7 mL of dichloromethane. Anisole (15 μl, 0.137 mmol) and dichloroacetic acid (32 μl, 0.387 mmol) were added dropwise. The reaction mixture was stirred for 30 min. MTBE (5 mL) was added and the mixture was stirred vigorously. The solid was allowed to settle and the solvent was poured off. The residue was washed twice with 5% dichloromethane in MTBE and once with MTBE. The solid was then dried under reduced pressure at room temperature. The title compound (FL118-linker compound) was obtained as a pale yellow solid and stored at -20 °C.

[0170] Yd: 53mg, 97% LCMS(an_acid):1480[M+H] + 1H NMR (400 MHz, DMSO) δ 10.18(s, 1H), 8.48(s, 1H), 8.24-8.17(m, 2H), 8.09(t, J=5.7 Hz, 1H), 7.81(s, 1H), 7.68(s, 3H), 7.62-7.57(m, 2H), 7.53(s, 2H), 7.31(d, J=8.6 Hz, 2H), 7.01(s, 2H), 6.94(s, 1H), 6.29(s, 2H), 6.01(s, 1H), 5.50(s, 2H), 5.25(s, 2H), 5.17-5.01(m, 2H), 4.47(m, 3H), 4.25(d, J=5.6 Hz, 2H), 4.08-3.95(m, 4H), 3.77(t, J=5.2 Hz, 2H), 3.54-3.41(m, 34H), 3.28-3.20(m, 4H), 2.78(s, 2H), 2.18-1.99(m, 3H) ), 1.83-1.67(m, 4H), 1.65-1.45(m, 6H), 1.42-1.19(m, 5H), 0.94-0.84(m, 5H).

[0171] Example 2: Preparation of FL118-Linker2 Conjugate Step 1: Synthesis of tert-butyl ((4-(prop-2-yn-1-ylcarbamoyl)cyclohexyl)methyl)carbamate [ka]

[0172] To a solution of Boc-tranexamic acid (trans) (1.00 g, 3.89 mmol) in N,N-dimethylformamide (10 mL) was added HATU (1.77 g, 4.66 mmol) and stirred for 10 min. Propargylamine (0.25 mL, 3.90 mmol) was added followed by diisopropylamine (0.71 mL, 4.07 mmol). The mixture, which turned into a yellow solution, was stirred at room temperature overnight and most of the DMF was evaporated under reduced pressure. The mixture was then diluted with ethyl acetate and water. The aqueous layer was separated and the organic layer was washed with water and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated to give a beige solid (1.4 g).

[0173] The solid was triturated with dichloromethane and then filtered to give the title compound (493 mg, 43%) as an off-white solid. The filtrate was purified by column chromatography (silica gel, DCM / methanol 0-5%) to give another batch of the title compound (315 mg, 27%).

[0174] SC_ACID:239[M-tBu+H] + 1 H NMR (400 MHz, CDCl3)δ 5.57(s, 1H), 4.55(s, 1H), 4.04(dd, J=5.2, 2.6 Hz, 2H), 2.98(t, J=6.5 Hz, 2H), 2.23(t, J=5.1 Hz, 1H), 2.09-1.99(m, 1H), 1.98-1.89(m, 2H), 1.89-1.80(m, 2H), 1.52-1.39(m, 12H), 1.02-0.90(m, 2H).

[0175] Step 2: Synthesis of 4-(aminomethyl)-N-ethynylcyclohexane-1-carboxamide [ka]

[0176] tert-Butyl ((4-(prop-2-yn-1-ylcarbamoyl)cyclohexyl)methyl)carbamate (800 mg, 2.72 mmol) was suspended in 5 mL of dioxane and HCl (4 N in dioxane) (10 mL, 40.0 mmol) was added to obtain a clear solution. The reaction mixture was stirred at room temperature for 30 min, and it was observed that a solid precipitated quickly. The reaction mixture was evaporated to dryness and co-evaporated twice with DCM. The crude product was dissolved in 1 mL of methanol and loaded onto SCX-2 (2×5 g). After flushing the column with methanol until neutral, the product was eluted with NH3 in methanol (7 N). The product fractions were dried and evaporated to give the title compound (520 mg, 98%) as a white solid.

[0177] SC_ACID:195[M+H] + 1 H NMR (400 MHz, D2O) (crude, HCl salt) δ 3.86 (d, J=2.5 Hz, 2H), 2.79 (d, J=7.1 Hz, 2H), 2.51 (t, J=2.5 Hz, 1H), 2.22-2.11(m, 1H), 1.87-1.74(m, 4H), 1.65-1.54(m, 1H), 1.42-1.28(m, 2H), 1.07-0.94(m, 2H).

[0178] Step 3: Synthesis of 4-((3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl)cyclohexane-1-carboxamide [ka]

[0179] A solution of 2,3-dibromomaleic acid (200 mg, 0.730 mmol) in acetic acid (2.5 mL) was stirred at reflux for 45 min, followed by the addition of 4-(aminomethyl)-N-ethynylcyclohexane-1-carboxamide (135 mg, 0.749 mmol) and stirring at reflux for 2 h. The reaction mixture was evaporated to dryness, triturated with dichloromethane, and filtered to give some product. The filtrate was purified by column chromatography (silica gel, heptane / ethyl acetate 0-100%) to give some additional product. The two batches were combined to give the title compound (153 mg, 48%) as a yellow solid.

[0180] SC_ACID:433[M+H] + , double Br pattern 1H NMR (400 MHz, DMSO) δ 8.14(t, J=5.5 Hz, 1H), 3.81(dd, J=5.5, 2.5 Hz, 2H), 3.29(s, 2H), 3.05(t, J=2.5 Hz, 1H), 2.08-1.99(m, 1H), 1.69(d, J=12.3 Hz, 4H), 1.52(s, 1H), 1.32-1.20(m, 2H), 0.96-0.83(m, 2H).

[0181] Step 4: Synthesis of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide [ka]

[0182] To a pale yellow solution of O-(2-azidoethyl)-O′-(N-diglycolyl-2-aminoethyl)-heptaethylene glycol (1.06 g, 1.910 mmol) and (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (1.0 g, 1.910 mmol) in dichloromethane (10 mL) was added EEDQ (0.476 g, 1.910 mmol). The reaction mixture was stirred at room temperature for 7.5 h and concentrated under reduced pressure. The crude product (1.68 g) was purified by column chromatography (silica gel, ethyl acetate / methanol 0-10%) to obtain the dark colorless title compound (1.53 g, 76% yield). SC_ACID_M1200:1082[M+Na] + , 1058[MH] -

[0183] Step 5: Synthesis of 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate [ka]

[0184] FL118 (60 mg, 0.153 mmol) and DMAP (70.1 mg, 0.573 mmol) were mixed in dry dichloromethane (8 mL). Triphosgene (20.4 mg, 0.069 mmol) dissolved in dry dichloromethane (1.75 mL) was added in one portion, and the brownish suspension was stirred at room temperature for 1 h. (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (162 mg, 0.153 mmol) in dry dichloromethane (1.75 mL) was added in one portion. The reaction mixture was evaporated in vacuo and the residue was dissolved in DMSO, filtered and purified by basic preparative MPLC. The product fractions were combined and lyophilized to give the title compound as a white fluffy solid (81 mg, 35%). SC_BASE_M700-1900:1206[M-MMT+H] +

[0185] Step 6: 4-((S)-35-(4-((4-((3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)-methyl)-1H-1,2,3-triazol-1-yl)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6 Synthesis of 12,15,18,21,24,27,30,33-Nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbamate [ka]

[0186] 4-((3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl)cyclohexane-1-carboxamide (105 mg, 0.243 mmol), 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide)benzyl((S)-7-ethyl-8,11-dioxa So-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate (120 mg, 0.081 mmol), copper(I) bromide (4.7 mg, 0.032 mmol) and triphenylphosphine (4.3 mg, 0.016 mmol) were dissolved in 1.5 mL of dichloromethane, and diisopropylethylamine (0.043 mL, 0.243 mmol) was added to the mixture, and a yellow suspension was quickly formed. The reaction mixture was stirred at room temperature for 6 hours and purified by column chromatography (silica gel 12 g, dichloromethane / methanol 0-10%) to obtain the title compound (94 mg, 60%) as a yellow solid.

[0187] SC_ACID:820[M+2H-MMT] 2+ / 2、doubly charged ion 1 H NMR (400 MHz, DMSO) δ 10.12 (s, 1H), 8.47 (s, 1H), 8.22-8.01 (m, 4H), 7.81 (s, 1H), 7.57 (d, J=8.4 Hz, 2H), 7.52 (d, J=2.5 Hz, 2H), 7.37 (d, J=7.9 Hz, 4H), 7.32-7.21 (m, 9H), 7.19-7.10 (m, 2H), 6.94 (s, 1H), 6.81 (d, J=8.7 Hz, 2H), 6.28 (s, 2H), 5.49 (s, 2H), 5.24 (s, 2H), 5.16-5.01 (m, 2H), 4.47 (t, J=5.2 Hz, 3H), 4.25 (d, J=5.8 Hz, 2H), 4.07-3.94 (m, 7H), 3.77 (t, J=5.3 Hz, 2H), 3.69 (s, 3H), 3.62 (s, 3H), 3.52-3.44 (m, 30H), 3.42 (t, J=6.0 Hz, 3H), 3.26-3.22(m, 3H), 3.17-3.11(m, 2H), 2.18-2.04(m, 3H), 1.96-1.88(m, 2H), 1.73-1.64(m, 5H), 1.51-1.44(m, 2H), 0.90(t, J=7.3 Hz, 3H).

[0188] Project 7: Synthesis of FL118-Rincon 2 complex

change

[0189] 4-((S)-35-(4-((4-((3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)-methyl)-1H-1,2,3-triazol-1-yl)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide)benzene To ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate (94 mg, 0.049 mmol) was added anisole (0.064 mL, 0.590 mmol), a solution of dichloroacetic acid (0.049 mL, 0.590 mmol) in dichloromethane (2.5 mL), and the yellow solution was stirred at room temperature for 45 min. MTBE (8 mL) was then added, and the mixture was stirred until a light brown solid precipitated. Most of the solvent was removed, and the solid was triturated five times with 8 mL of MTBE, then dried under reduced pressure to give the title compound (73 mg, 91%) as a light yellow solid.

[0190] UPLC_AN_ACID:820[M+2H] 2+ / 2, doubly charged ion SC_ACID_M700-2000:1640[M+H] + , double Br pattern 1H NMR (400 MHz, DMSO) δ 10.17(s, 1H), 8.48(s, 1H), 8.25-8.16(m, 2H), 8.09(t, J=5.9 Hz, 1H), 7.82(s, 1H), 7.67-7.56(m, 5H), 7.53(s, 2H), 7.32(d, J=8.3 Hz, 2H), 6.94(s, 1H), 6.29(s, 2H), 5.50(s, 2H), 5.25(s, 2H), 5.17-5.02(m, 2H), 4.51-4.44(m, 3H), 4.25(d, J=5.6) Hz, 2H), 4.08-3.95(m, 4H), 3.78(t, J=5.2 Hz, 2H), 3.54-3.40(m, 30H), 3.28(t, J=6.0 Hz, 4H), 2.77(d, J=6.0 Hz, 2H), 2.21-2.01(m, 3H), 1.81-1.63(m, 6H), 1.60-1.46(m, 3H), 1.41-1.21(m, 6H), 0.89(t, J=7.3 Hz, 3H).

[0191] Example 3: Preparation of FL118-Linker3 Conjugate Step 1: Synthesis of (9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)phenyl)amino)-6-(((4-methoxyphenyl)diphenylmethyl)amino)-1-oxohexan-2-yl)carbamate [ka]

[0192] To a solution of 4-aminobenzyl alcohol (0.404 g, 3.28 mmol) and Fmoc-Lys(Mmt)-OH (2 g, 3.12 mmol) in dichloromethane (20 mL) was added EEDQ (0.809 g, 3.25 mmol) and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with dichloromethane (20 mL) and washed with water (20 mL). The organic layer was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to give a yellowish foam. Purification by flash column chromatography (40 g Si, heptane / ethyl acetate 0-100%) gave the title compound (1.77 g, 76%) as a white foam.

[0193] The results of the analysis were confirmed to be consistent with literature data. SC_ACID:768[M+Na] +

[0194] Step 2: Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide [ka]

[0195] (9H-Fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)phenyl)amino)-6-(((4-methoxyphenyl)diphenylmethyl)amino)-1-oxohexan-2-yl)carbamate (1.77 g, 2.373 mmol) was dissolved in diethylamine (19.5 mL, 189 mmol) and stirred at room temperature for 1 h, after which the solvent was evaporated under reduced pressure. The residue was dissolved in DCM (5 mL) and precipitated with heptane (25 mL) followed by solvent pouring. The process was repeated and then evaporated to give the title compound as a white foam (1.3 g).

[0196] SC_ACID:522[MH] - The results of the analysis were confirmed to be consistent with literature data.

[0197] Step 3: Synthesis of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide [ka]

[0198] To a pale yellow solution of O-(2-azidoethyl)-O'-(N-diglycolyl-2-aminoethyl)-heptaethylene glycol (1.059 g, 1.910 mmol) and (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (1.0 g, 1.910 mmol) in dichloromethane (10 mL) was added EEDQ (0.476 g, 1.910 mmol), and the reaction mixture was stirred at room temperature for 7.5 h and then concentrated under reduced pressure. The crude product was then purified by column chromatography (silica gel, ethyl acetate / methanol 0-10%) to afford the title compound as a thick colorless oil (1.53 g, 76%). SC_ACID_M1200:1082[M+Na] + , 1058[MH] -

[0199] Step 4: Synthesis of 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate [ka]

[0200] FL118 (70 mg, 0.178 mmol) and DMAP (82 mg, 0.669 mmol) were mixed in dry dichloromethane (10 mL), and then triphosgene dissolved in dry dichloromethane (2 mL) was added in one portion. The brownish suspension was stirred for 1 h. LCMS (sample in MeOH) confirmed nearly complete conversion to the methyl carbonate corresponding to FL118. Then, (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (189 mg, 0.178 mmol) in dry dichloromethane (2 mL) was added in one portion. The reaction mixture was stirred for 30 min and then evaporated in vacuo The residue was taken up in DMSO, filtered and purified by basic preparative MPLC to give the title compound (178 mg, 67%) as a white solid. SC_BASE_M1800:1500[M+Na] + , 1206[M-MTT+H] +

[0201] Step 5: Synthesis of 4-((S)-35-amino-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate [ka]

[0202] 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide)benzyl((S)-7-ethyl-8,11-dioxo-7,8,11 To a solution of ,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate (177 mg, 0.120 mmol) was added water (2.25 mL), followed by a solution of trimethylphosphine (1.0 M) in THF (0.239 mL, 0.239 mmol). The mixture was stirred at room temperature for 1 h to give a yellow-pink solution. The mixture was dried, concentrated, and used directly in the next step. SC_BASE_M700-1900:1452[M+H] +

[0203] Step 6: Synthesis of 3-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)propanoic acid [ka]

[0204] 2,3-Dibromomaleic acid (500 mg, 1.826 mmol) was dissolved in acetic acid (15 mL) and heated at reflux for 30 min. To the solution was added a solution of di-tert-butyl 1-(3-(tert-butoxy)-3-oxopropyl)-2-methylhydrazine-1,2-dicarboxylate (615 mg, 1.643 mmol) in acetic acid (2 mL) and the mixture was further stirred at reflux for 3 h. The mixture was then cooled at room temperature and concentrated and co-evaporated with toluene (2x) and dichloromethane (2x) to give an orange oil. It was then purified by flash column chromatography (24 g; 50-100% ethyl acetate in n-heptane + 1% acetic acid) to give the title compound (408 mg) as a pale yellow oil.

[0205] SC_ACID:357[M+H] + , 2Br isotope pattern 1 H NMR (400 MHz, DMSO) δ 12.48 (s, 1H), 4.27 (d, J=7.4 Hz, 3H), 3.56 (s, 5H), 2.62 (t, J=7.4 Hz, 3H).

[0206] Step 7: Synthesis of 4-((S)-39-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazanonatriacontanamido)benzyl((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate [ka]

[0207] To a solution of 3-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)propanoic acid (128 mg, 0.360 mmol) in peptide grade N,N-dimethylformamide (4 mL) was added HATU (137 mg, 0.360 mmol), and the solution was mixed with 4-((S)-35-amino-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6 After adding 174 mg, 0.120 mmol) of benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate [crude], the mixture was stirred for 15 min. Then, DIPEA (0.063 mL, 0.360 mmol) was added and the mixture was stirred at room temperature for 1 h.

[0208] The reaction mixture was directly purified by acidic preparative MPLC, and the product fractions were combined and lyophilized to obtain the product (120 mg) as a white solid, which was analyzed to be a 4 / 1 mixture of the title compound and the MMT-deprotected compound. SC_ACID_M1000-1800:1790[M+H] + , 2Br-isotope pattern

[0209] Step 8: Synthesis of FL118-Linker3 Conjugate [ka]

[0210] 4-((S)-39-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazanonatriacontanamide)benzyl(( To a solution of S-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate (115 mg, 0.064 mmol), anisole (0.028 mL, 0.257 mmol) was added, followed by dichloroacetic acid (0.064 mL, 0.771 mmol). The reaction mixture was confirmed to turn bright orange and stirred at room temperature for 45 minutes. After adding MTBE (1.5 mL) to the reaction mixture, the suspension was vigorously stirred for 2 to 3 minutes. After stopping the stirring, a precipitate was confirmed to form at the bottom of the vial. After removing the solvent with a pipette, MTBE (10 mL) was added, and the trituration process was repeated six times. The remaining suspension was dried under reduced pressure at 40° C. to give the title compound (90 mg, 92%) as a white solid.

[0211] UPLC_AN_ACID_M1600:1518[M+H] + 1H NMR (400 MHz, DMSO) δ 10.18(s, 1H), 8.48(s, 1H), 8.21(d, J=8.0 Hz, 1H), 8.10(dd, J=10.0, 5.0 Hz, 2H), 7.81-7.56(m, 5H), 7.53(s, 2H), 7.32(d, J=8.4 Hz, 2H), 6.94(s, 1H), 6.29(s, 2H), 5.82(s, 1H), 5.50(s, 2H), 5.25(s, 2H), 5.19-5.01(m, 2H), 4.47(q, J=7.9 Hz, 1H), 4.26(t, J=7.0 Hz, 2H), 4.11-3.91(m, 4H), 3.54(s, 3H), 3.51-3.40(m, 30H), 3.30-3.24(m, 2H), 3.15(q, J=5.8 Hz, 2H), 2.83-2.72(m, 2H), 2.44(t, J=7.0 Hz, 2H), 2.22-2.08(m, 2H), 1.83-1.62(m, 2H), 1.61-1.48(m, 2H), 1.44-1.20(m, 3H), 0.89(t, J=7.4 Hz, 3H).

[0212] Example 4: Preparation of FL118-Linker 4 Conjugate Step 1: Synthesis of (9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)phenyl)amino)-6-(((4-methoxyphenyl)diphenylmethyl)amino)-1-oxohexan-2-yl)carbamate [ka]

[0213] To a solution of 4-aminobenzyl alcohol (0.404 g, 3.28 mmol) and Fmoc-Lys(Mmt)-OH (2 g, 3.12 mmol) in dichloromethane (20 mL) was added EEDQ (0.809 g, 3.25 mmol) and stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane (40 mL) and washed with water (40 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to give a yellowish foam. Purification by flash column chromatography (40 g silica, Hept / ethyl acetate 0-100%) gave the title compound (1.54 g, 66%) as a white foam. SC_ACID:273[MMT] + , 522[M-Fmoc-H] -

[0214] Step 2: Synthesis of (9H-fluoren-9-yl)methyl ((S)-1-((4-((((((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)oxy)carbonyl)oxy)methyl)phenyl)amino)-6-(((4-methoxyphenyl)diphenylmethyl)amino)-1-oxohexan-2-yl)carbamate [ka]

[0215] FL118 (50 mg, 0.127 mmol) and DMAP (58.4 mg, 0.478 mmol) were mixed in dry dichloromethane (7 mL), triphosgene (17.02 mg, 0.057 mmol) dissolved in dry dichloromethane (1.5 mL) was added in one portion, and the brown suspension was stirred for 30 minutes. Then, (9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)phenyl)amino)-6-(((4-methoxyphenyl)diphenylmethyl)amino)-1-oxohexan-2-yl)carbamate (95 mg, 0.127 mmol) in dry dichloromethane (1.5 mL) was added in one portion, slowly obtaining a brown solution. The reaction mixture was evaporated to dryness to give a brown / grey foam which was then purified by column chromatography (12 g; 0-5% methanol in dichloromethane) to give the title compound (107 mg, 72%) as an orange / brown foam.

[0216] SC_ACID:892[M-MMT+H] + 1 H NMR (400 MHz, CDCl3)δ 8.07(s, 1H), 7.86(s, 1H), 7.71(d, J=7.6 Hz, 2H), 7.56-7.27(m, 16H), 7.25-7.11(m, 10H), 7.09(s, 1H), 6.78(d, J=8.8 Hz, 2H), 6.12(s, 2H), 5.68(d, J=17.1 Hz, 1H), 5.37(d, J=17.0 Hz, 1H), 5.28-5.04(m, 5H), 4.46(d, J=6.6 Hz, 2H), 4.15(s, 2H), 3.75(s, 3H), 3.48-3.38(m, 1H), 2.32-2.20(m, 1H), 2.19-2.08(m, 3H), 1.50(s, 3H), 1.40-1.28(m, 2H), 0.98(t, J=7.5 Hz, 3H).

[0217] Step 3: Synthesis of 4-((S)-2-amino-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamido)benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate [ka]

[0218] (9H-fluoren-9-yl)methyl ((S)-1-((4-((((((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)oxy)carbonyl)oxy)methyl)phenyl)amino)-6-(((4-methoxyphenyl)diphenylmethyl)amino)-1-oxohexan-2-yl)carbamate (162 mg, 0.139 mmol) was dissolved in dry dichloromethane (2.5 mL), diethylamine (1.3 mL, 12.44 mmol) was added, and the mixture was stirred at room temperature for 2 hours to confirm the formation of a suspension. The reaction mixture was evaporated to dryness to yield the title compound (154mg) as a yellow / beige solid which was used in the next step. SC_ACID:964 {M+Na] + , 670[M-MMT+H] +

[0219] Step 4: Synthesis of DBCO-PEG-acid [ka]

[0220] A solution of DBCO-PEG6-amine TFA salt (196 mg, 0.270 mmol) and diisopropylethylamine (0.708 mL, 4.05 mmol) in dichloromethane (2.5 mL) was stirred at room temperature for 10 minutes. Diglycolic anhydride (62.7 mg, 0.540 mmol) was then added and the reaction mixture was stirred at room temperature overnight. Water (0.125 mL) was then added and stirred at room temperature for 4 hours. The mixture was diluted with dichloromethane (2 mL) to obtain a clear solution, which was dried over sodium sulfate, filtered and concentrated to obtain a yellow turbid oil (340 mg) containing the title compound and residual DIPEA, which was used in the next step. SC_ACID:728[M+H] +

[0221] Step 5: Synthesis of dibenzocyclooctyne-PEG-Lys(MMT)-PABC-FL118 [ka]

[0222] To DBCO-PEG-acid (296 mg, 0.244 mmol) in dichloromethane (10 mL) was added HATU (111 mg, 0.292 mmol) and the mixture was stirred at room temperature for 20 min. 4-((S)-2-amino-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamido)benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl)carbonate (270 mg, 0.244 mmol) was added and the mixture was stirred at room temperature. The solution was purified by flash column chromatography (12 g silica; 0-10% methanol in dichloromethane), and fractions 11-13 were combined and concentrated to give an off-white solid (139 mg), which was purified by basic preparative MPLC to give the title compound (86 mg, 21%) as a white solid.

[0223] SC_BASE_M700-2000:1652[M+H] + 1 H NMR (400 MHz, DMSO) δ 10.15 (s, 1H), 8.47 (s, 1H), 8.15 (d, J=8.0 Hz, 1H), 8.08 (t, J=5.8 Hz, 1H), 7.76 (t, J=5.6 Hz, 1H), 7.70-7.65 (m, 1H), 7.64-7.49 (m, 6H), 7.49-7.40 (m, 3H), 7.39-7.10 (m, 20H), 6.93 (s, 1H), 6.81 (d, J=8.9 Hz, 2H), 6.28 (d, J=2.1 Hz, 2H), 5.50 (s, 2H), 5.23 (s, 2H), 5.15-4.98 (m, 3H), 4.48-4.40 (m, 1H), 4.03 -3.93(m, 4H), 3.69(s, 3H), 3.50-3.37(m, 23H), 3.30-3.20(m, 4H), 3.12-3.03 (m, 2H), 2.62-2.54 (m, 1H), 2.45-2.40 (m, 1H), 2.27-2.09 (m, 3H), 2.02-1.89 ( m, 3H), 1.79-1.57 (m, 3H), 1.51-1.44 (m, 2H), 1.37-1.22 (m, 3H), 0.89 (t, J=7.4 Hz, 3H).

[0224] Project 6: Synthesis of FEG-FEG-Lys-PABC-FL118 (FL118-Lys-PABC-4 complex)

change

[0225] The compound obtained in step 5 was dissolved in dichloromethane (0.5 mL) and dried over sodium sulfate overnight. The solution was then transferred to a reaction tube and evaporated to dryness. Anisole (0.052 mL, 0.472 mmol) and dichloroacetic acid (0.039 mL, 0.472 mmol) dissolved in dichloromethane (4 mL) were added, and the yellow reaction mixture was stirred at room temperature for 30 minutes. tert-Butyl methyl ether (15 mL) was added, and a suspension was formed, but the product was not sufficiently precipitated, so the solvent was evaporated at room temperature under reduced pressure. tert-Butyl methyl ether (5 mL) was added to obtain a suspension, and the mixture was stirred at 0°C and the solvent was removed with a pipette. The mixture was triturated four times with MTBE (5 mL) to obtain the title compound (56 mg) as a light yellow solid.

[0226] AN_BASE_M700-2000:1379[M+H] + 1 H NMR (400 MHz, DMSO) δ 10.19(s, 1H), 8.48(s, 1H), 8.21(d, J=8.1 Hz, 1H), 8.10(t, J=5.8 Hz, 1H), 7.80-7.64(m, 4H), 7.64-7.56(m, 3H), 7.56-7.16(m, 11H), 6.94(s, 1H), 6.29(s, 2H) , 6.14(s, 1H), 5.50(s, 2H), 5.24(s, 2H), 5.19-4.98(m, 3H), 4.53-4.43(m, 1H), 4.10-3.93(m, 4H), 3.53-3.40(m, 23H), 3.30-3.27(m, 4H), 2.77(s, 2H), 2.63-2.54(m, 1H), 2.28-2.08(m, 3H) ), 2.04-1.93(m, 1H), 1.80-1.66(m, 2H), 1.61-1.49(m, 2H), 1.44-1.27(m, 2H), 0.89(t, J=7.4 Hz, 3H).

[0227] Example 5: Preparation of anti-HER2 antibody-linker1-FL118 immune complex (DAR 8) Anti-HER2 antibody (trastuzumab) was reduced with 5 mM TCEP in the original buffer (20 mM histidine, 150 mM sodium chloride, pH 6.0) and the reaction vial was placed in an incubator-shaker at 22° C. with a rotation speed of 60 rpm for 6 hours. After reduction, TCEP was removed via a spin desalting column (40K, 2 mL).

[0228] DMSO and FL118-Linker 1 compound solution (10 mg / mL stock in DMSO, 12 eq. relative to antibody) were added to the reduced antibody solution for a final DMSO concentration of 10%. The reaction mixture was mixed appropriately and the reaction vial was left at 4° C. for 2 hours.

[0229] After conjugation, the reaction mixture was exchanged with 18 mM MES (pH 6.5) through a spin desalting column (40K, 5 mL). The product was then sterile filtered through a 0.2 μm PVDF single-use filter. The resulting immunoconjugate was characterized and lyophilized after the addition of 0.07 mM MPS80 and 20 mM trehalose dehydrate.

[0230] The DAR of the immune complex of Example 5 was determined using HIC and MS, and the average MS-DAR The value was confirmed by 7.14 and HIC-DAR value of 8.00 (Table 1 and Figure 5b).

[0231] [Table 1]

[0232] Example 6: Preparation of anti-HER2 antibody-linker1-FL118 conjugate (DAR4) Anti-HER2 antibody (trastuzumab) was reduced with 5 mM TCEP in the original buffer (20 mM histidine, 150 mM sodium chloride, pH 6.0) and the reaction vial was placed in an incubator-shaker at 22° C. with a rotation speed of 60 rpm for 3 h. After reduction, TCEP was removed via a spin desalting column (40K, 2 mL).

[0233] DMSO and FL118-linker compound solution (10 mg / mL stock in DMSO, 10 eq. relative to antibody) were added to the reduced antibody solution for a final DMSO concentration of 10%. The reaction mixture was mixed appropriately and the reaction vial was left at 4° C. for 2 hours.

[0234] After conjugation, the reaction mixture was exchanged with 18 mM MES (pH 6.5) through a spin desalting column (40K, 5 mL). The product was then sterile filtered through a 0.2 μm PVDF single-use filter. The resulting immunoconjugate was characterized, and after adding 0.07 mM PS80 and 20 mM trehalose dehydrate, the produced immunoconjugate was lyophilized.

[0235] The DAR of the immunoconjugate of Example 6 was determined using HIC and MS, and the average MS-DAR value was confirmed to be 4.02 (Table 2, and FIG. 5a).

[0236] [Table 2]

[0237] Example 7: Preparation of anti-HER2 antibody-linker2-FL118 conjugates and anti-HER2 antibody-linker3-FL118 conjugates The reaction vial was placed in a thermostatic water bath set at 25°C for 3 hours to reduce the anti-HER2 antibody (trastuzumab) in the original buffer (20 mM histidine, 150 mM sodium chloride, pH 6.0) with 5 mM TCEP. After reduction, TCEP was removed through a spin desalting column (100K, 0.5 mL).

[0238] DMSO and FL118-Linker 2 compound solution or FL118-Linker 3 compound solution (each in DMSO, 5 mM stock, 20 eq. relative to antibody) were added to the reduced antibody solution with a final DMSO concentration of 10%. The reaction mixture was mixed appropriately and the reaction vial was placed in a thermostatic water bath set at 25°C for 4 hours. To terminate the reaction, cysteine ​​solution (10 mM stock in distilled water, 40 eq. relative to antibody) was added to the reaction solution, and then the mixture was left under the same conditions for 30 minutes.

[0239] The reaction product was purified through a SEC column under phosphate buffer conditions, and the production of the immune complex of Example 7 was confirmed using SDS PAGE, SEC and HIC.

[0240] Example 8: Preparation of anti-EGFR antibody-linker1-FL118 conjugate (DAR 8) Anti-EGFR antibody (cetuximab) was reduced with 5 mM TCEP in the original buffer (20 mM histidine, 150 mM sodium chloride, pH 6.0) and the reaction vial was placed in an incubator-shaker at 22° C. with a rotation speed of 60 rpm for 3 hours. After reduction, TCEP was removed via spin desalting columns (40K, 2 mL×2).

[0241] DMSO and FL118-linker compound solution (10 mg / mL stock in DMSO, 12 eq. relative to antibody) were added to the reduced antibody solution for a final DMSO concentration of 10%. The reaction mixture was mixed appropriately and the reaction vial was left at 4° C. for 2 hours.

[0242] After conjugation, the reaction mixture was exchanged with 18 mM MES (pH 6.5) through a spin desalting column (40K, 10 mL). The product was then sterile filtered through a 0.2 μm PVDF single-use filter. The resulting immunoconjugate was characterized, and after adding 0.07 mM MPS80 and 20 mM trehalose dehydrate, the produced immunoconjugate was lyophilized.

[0243] The DAR of the immunoconjugate of Example 8 was determined using HIC and MS, and the average MS-DAR value was confirmed to be 8.00 (Table 3).

[0244] [Table 3]

[0245] Example 9: Preparation of anti-Trop-2 antibody-Linker 1-FL118 conjugate (DAR 8) An anti-Trop-2 antibody-linker1-FL118 conjugate (DAR8) was prepared in the same manner as in Example 8, except that an anti-Trop-2 antibody (sacituzumab) was used instead of an anti-EGFR antibody (cetuximab). Ta.

[0247] Example 10: Preparation of animal models and materials used to assess in vitro cytotoxicity and in vivo efficacy All procedures related to animal handling, care, and treatment in this study were performed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Model Organisms Center, Inc. Thirty female mice, 9-11 weeks old, weighing 16-18 g, were housed 3-5 per cage (300 mm × 180 mm × 150 mm) and maintained at constant temperature (18-26 °C) and humidity (40-70%) in individually ventilated cages with free access to radiation-sterilized dry granular diet and sterile dietary water.

[0248] The immune complexes of Example 5 (molecular weight 159 kDa, purity 97%), Example 6 (molecular weight 154 kDa, purity 98%), Example 8 (molecular weight 160 kDa, purity 99%) and Example 9 (molecular weight 160 kDa, purity 99.6%) were prepared in the form of freeze-dried powder and stored at -80°C.

[0249] Trastuzumab (molecular weight 148 kDa, purity 97%) supplied by BCD in a solution stored at -80°C was used as a control dissolved in buffer (20 mM histidine, 150 mM sodium chloride, pH 6.0) (concentration 10.09 mg / mL), cetuximab (molecular weight 148 kDa, purity 100%) was used dissolved in buffer (20 mM histidine, 150 mM sodium chloride, pH 6.0) (concentration 10.47 mg / mL), and sacituzumab (molecular weight 148 kDa, purity 100%) was used dissolved in buffer.

[0250] DS8201 (molecular weight 156 kDa, purity 99%) supplied by BCD in a solution stored at -80°C was used as a control in a state dissolved in a buffer solution (25 mM histidine, pH 5.5) (concentration 2.87 mg / mL). DS8201 is an immunoconjugate of trastuzumab and deruxtecan, and the anti-HER2 targeting antibody of the immunoconjugates in Examples 5 and 6 is the same as trastuzumab, and the cytotoxic drug (payload), deruxtecan, is a topoisomerase I inhibitor and has similarities in structure and function to FL118 in Examples. However, DS8201 is different from the immunoconjugates in Examples 5 and 6 in the structure and properties of the linker, using a cleavable tetrapeptide-based linker. DS8201 has the property of being internalized, cleaved, and the released drug is known to have high cell membrane permeability and to be cytotoxic to surrounding cancer cells regardless of target expression (Cancer Sci 2016;107:1039~1046). In recent years, excellent efficacy has been reported in a second clinical trial targeting HER2-positive metastatic breast cancer patients (N Engl J Med 2020;382:610~621), and it has been designated as a breakthrough treatment by the US FDA for HER2-positive recurrent and metastatic breast cancer and HER2-positive recurrent and metastatic gastric cancer in 2017 and 2020, respectively.

[0251] [ka]

[0252] Example 11: In vitro cytotoxicity evaluation Analysis of the cytotoxicity of the immunoconjugates of Examples 5, 6, 8 and 9 and DS8201 as a control against the HER2-overexpressing JIMT-1 breast cancer cell line, the EGFR-overexpressing MDA-MB-468 breast cancer cell line or the MDA-MB-468 cell line was carried out as follows.

[0253] Immunoconjugate-mediated cytotoxicity against JIMT-1 or MDA-MB-468 cells was determined by cell viability assay. On day 0, JIMT-1 was inoculated at 6 × 10 per well into a 96-well plate (Greinier, # 655090) containing 50 μl of DMEM (Gibco, # 11995-065) containing 10% FBS (HyClone, SV30087.03). 3 1 cell (after 3-day culture) or 3 x 10 cells per well 3 In the case of MDA-MB-468 cells, 6 × 10 cells were seeded per well in a 96-well plate (Greinier, #655090) containing 50 μl of RPMI (Gibco, #11875-093) containing 10% FBS (HyClone, SV30087.03) on day 0. 3 1 cell (after 3-day culture) or 3 x 10 cells per well 3 The cells were seeded at 100 μl / well (for 6-day culture). The next day, the immunoconjugate samples were serially diluted 3-fold (2000, 666.667, 222.222, 74.074, 24.6914, 8.2305, 2.7435, 0.9145, 0.3048, 0.1016 and 0 nM) and then added to the seeded cells at 50 μl / well, respectively. The cells were cultured at 37°C in a 5% CO2 incubator for 3 or 6 days.

[0254] Cell viability was detected on day 4 or 7 using CellTiter-Glo (Promega, # G7573) according to the manufacturer's instructions. Plates were equilibrated to room temperature for approximately 15 minutes before adding 50 μl / well of CellTiter-Glo solution. Plates were gently shaken for approximately 1 minute and then incubated at room temperature for 10 minutes. Luminescence was then read using Envision (PerkinElmer).

[0255] The cytotoxic effect obtained for each immunoconjugate was calculated relative to the luminescence value obtained in the control wells (cell only) according to the following formula: Cytotoxicity%=100×(V cell only -V sample ) / V cell on ly

[0256] IC for cytotoxicity was measured using GraphPad Prism 6.0 software. 50 To obtain the values, a four parameter nonlinear regression analysis was used.

[0257] result The immune complex of Example 5 was added and cultured for 3 or 6 days in both cases, exhibiting a cytotoxic effect and inducing apoptosis. Surprisingly, the immune complex of Example 5 exhibited a significantly superior cytotoxic effect than DS8201 (positive control group). When cultured for 3 days, DS8201 exhibited only 27.2% cytotoxicity, whereas the immune complex of Example 5 exhibited 88.6% cytotoxicity, demonstrating a more than three-fold superior effect. In addition, when cultured for 6 days, the immune complex of Example 5 had a significantly higher IC than the positive control DS8201. 50 It was confirmed that the effect was more than 100 times better in terms of the value (Figure 6 and Table 4 ) 。 [Table 4]

[0258] In the case of the immunoconjugate of Example 6, when added to JIMT-1 cells and cultured for 3 or 6 days, all of them showed cytotoxic effects and induced apoptosis. As in Example 5, the immunoconjugate of Example 6 also showed a significantly superior cytotoxic effect to DS8201 (positive control group) (Figure 7). and Table 5 ) 。 [Table 5]

[0259] In addition, when the immune complex of Example 8 was added to MDA-MB-468 cells and cultured for 3 or 6 days, all of them showed cytotoxic effects and induced apoptosis. That is, it was confirmed that the immune complex of Example 8 also showed a relatively high level of cytotoxicity, similar to Examples 5 and 6 (FIG. 9). and Table 6 ) 。 [Table 6]

[0260] In addition, the immunoconjugate of Example 9 also showed cytotoxicity and induced apoptosis when added to MDA-MB-468 cells and cultured for 3 or 6 days. That is, it was confirmed that the immunoconjugate of Example 9 also showed a considerably high level of cytotoxicity, similar to Examples 5, 6, and 8 (FIG. 11).

[0261] Example 12: In vivo efficacy evaluation 12-1.Cell culture JIMT-1 breast cancer cells were cultured in DMEM containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin at 5% CO2 and 37°C and routinely subcultured twice weekly by trypsin-EDTA treatment. Cells growing in exponential growth phase were harvested and counted for tumor inoculation. MDA-MB-468 breast cancer cells were cultured in RPMI containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin at 5% CO2 and 37°C and routinely subcultured twice weekly by trypsin-EDTA treatment. Cells growing in exponential growth phase were harvested and counted for tumor inoculation.

[0262] 12-2. Tumor inoculation JIMT-1 tumor cells (1 × 10) in 0.2 mL of DPBS with Matrigel 7 ) or MDA-MB-468 cells (1 × 10 7 ) was inoculated subcutaneously into the right front flank of each mouse. The average tumor volume was approximately 215 mm 3 When the animals reached the target age, they were randomly assigned to groups ( Tables 7 and 8 ), treatment to study drug efficacy was then initiated.

[0265] [Table 7]

[0266] Observation During routine monitoring, animals were checked daily for normal behavior such as mobility, food and water consumption (observation only), weight change (weighed twice weekly), eye / hair matting, tumor growth and efficacy of treatment. Signs of inflammatory bowel disease were recorded based on the number of animals in each subset.

[0267] Tumor measurements and endpoints The primary endpoint was to determine whether tumor growth could be delayed and whether mice could be cured. The bidimensional size of the tumor was measured twice a week using a caliper, and the volume was calculated according to the formula (V = 0.5 a × b 2 , where a and b are the long and short diameters of the tumor, respectively) in mm 3 This is expressed as:

[0268] The TGI (tumor growth inhibition rate) was calculated for each group. TGI(%)=[1-(T i / T0) / (V i / V0)] × 100 (T i is the mean tumor volume of the treatment group on a given day, T0 is the mean tumor volume of the treatment group on day 1 of treatment, V i is T i V is the mean tumor volume of the vehicle control group on the same day, V0 is the mean tumor volume of the vehicle group on the first day of treatment) 。 [Table 8]

[0269] Body weight loss ≥15% or tumor volume ≥3000mm 3 Animals that were above this were euthanized at a humane endpoint.

[0270] 12-5.Statistical analysis Results are expressed as mean and standard error (mean ± SEM). Data were analyzed using two-way RM ANOVA Dunnett's multiple comparison test using Graphpad Prism 6.0 software, and p < 0.05 was considered statistically significant.

[0271] 12-6. Body weight of JIMT-1 or MDA-MB-468 tumor models The body weight of JIMT-1 or MDA-MB-468 tumor model mice was monitored regularly. 9As shown in Table 10, Figures 8, 10 and 12, no significant weight loss was observed in any of the treatment groups.

[0273] [Table 9]

[0274] [Table 10]

[0275] Unlike humans, mouse plasma has a high level of esterase activity and can easily cleave acid-sensitive bonds such as ester and carbonate functional groups, so it was predicted that side effects such as weight loss would occur when the immunoconjugate of the present invention was administered to tumor model mice, but surprisingly, no such side effects were observed.The above results indicate that the immunoconjugate of the present invention does not show toxicity to normal cells even if it separates and releases FL118 in the blood.

[0276] 12-7.Tumor growth inhibition assay in JIMT-1 or MDA-MB-468 tumor model mice The average tumor volume of JIMT-1 or MDA-MB-468 tumor model mice (n = 3) in each group is shown in Table 11. and Table 12 are shown in the table below.

[0277] [Table 11]

[0278] [Table 12]

[0280] Next, the TGI value of JIMT-1 or MDA-MB-468 tumor model mice (n=3) was calculated based on the tumor size (n=3) on the 21st day after treatment. The results are shown in Table13 ~table 15 The 。 [Table 13]

[0281] [Table 14]

[0283] [Table 15]

[0284] The results of the tumor growth curves of JIMT-1 or MDA-MB-468 tumor model mice (n=3) are shown in Figures 8, 10, and 12, respectively. As a result of the study, the average tumor size of Group 1, the saline control group, was 1336.32±361.41 mm on the 21st day after treatment. 3 and 390.24 ± 45.45 mm 3 Trastuzumab, Treatment with either cetuximab or sacituzumab only demonstrated a partial antitumor effect without statistical significance (p>0.05) compared with the saline control group, with mean tumor volumes of 671.06±92.85mm, respectively. 3 (TGI=59.41%), 363.07±44.38mm 3 (TGI=15.92%) and 433.08±126.82mm 3 (TGI=43.6%). However, the immunoconjugate of Example 5 had a TGI of 370.14±71.86, 102.20±18.12 and 73.87±12.01 mmHg at doses of 1 mg / kg, 5 mg / kg and 10 mg / kg (Groups 3, 4 and 5), respectively. 3 The mean tumor volumes of the immunoconjugate of Example 8 were 24.59±12.8 and 7.56±7.56 mm at doses of 5 mg / kg and 10 mg / kg (Groups 4 and 5), respectively. 3The mean tumor volumes of the immunoconjugate of Example 9 were 77.45±1.46 and 74.61±11.55 mm at doses of 5 mg / kg and 7 mg / kg (Groups 5 and 6), respectively. 3 It was confirmed that the mean tumor volume of each of the immunoconjugates of the present invention showed a significantly excellent antitumor effect (TGI of the immunoconjugate of Example 5 = 86.31, 110.23, 112.73%, p<0.01, 0.001, 0.001; TGI of the immunoconjugate of Example 8 = 207.18, 216.60%, p<0.001, 0.0001; TGI of the immunoconjugate of Example 9 = 129.70, 130.53%, p<0.01, 0.05). This demonstrates that the immunoconjugate of the present invention has a more excellent effect than trastuzumab, cetuximab, and sacituzumab, which are already known to have excellent antitumor effects by themselves. In addition, since it was confirmed that the immunoconjugates of the present invention all showed a dose-dependent response, they can be administered in a patient-specific manner / dosage to show maximum efficacy and minimum toxicity for each patient.

[0285] In summary, the FL118-linker conjugate of the present invention can bind to an antibody or an antigen-binding fragment thereof against a target antigen to form an immune complex. The immune complexes of Examples 5 and 6 prepared according to one embodiment of the present invention were confirmed to exhibit overwhelmingly superior cytotoxicity against cancer cells compared to DS8201 in vitro, despite containing the same antibody (trastuzumab) as the known immune complex DS8201 and containing a drug (payload) similar in structure and function. The immune complex of Example 8 containing cetuximab prepared according to one embodiment of the present invention was also confirmed to exhibit strong cytotoxicity in vitro. The immune complex of Example 9 containing sacituzumab prepared according to one embodiment of the present invention was also confirmed to exhibit strong cytotoxicity in vitro.

[0286] In addition, the anti-cancer activity of the immune complex of Example 5 and trastuzumab, the immune complex of Example 8 and cetuximab, and the immune complex of Example 9 and sacituzumab was tested in tumor model mice. As a result, it was confirmed that, although trastuzumab, cetuximab, and sacituzumab also showed some anti-cancer effects, the immune complex of the present invention showed significantly superior anti-cancer activity at all doses administered, and no side effects such as weight loss were observed.

[0287] Therefore, the immune complex of the present invention can be very useful for preventing or treating cancer, and the FL118-linker complex of the present invention can be very useful for producing the immune complex.

[0288] From the above description, it should be understood by those skilled in the art to which the present invention pertains that the present invention can be embodied in other specific forms without changing the technical idea or essential features thereof. In this regard, the above-described embodiments should be understood to be illustrative in all respects and not limiting. The scope of the present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims below, rather than the above detailed description, and the equivalent concepts thereof.

Claims

1. An immunoconjugate comprising: [FL118 drug of formula 1]-[acid-sensitive linker]-[antibody or a fragment thereof containing an antigen-binding site], or a pharma- ceutically acceptable salt thereof, one or more FL118 drugs of formula 1 are linked to an antibody or a fragment thereof containing an antigen-binding site via an acid-labile linker; After targeting the cancer cell by the antigen-binding site that targets the antigen of the cancer cell, the acid-sensitive linker is decomposed in the acidic environment (pH≦7) around the cancer, and the FL118 drug of formula 1 is at least partially released, and the free FL118 drug of formula 1 is transported through the cell membrane into the cell; The FL118 drug of formula 1 inhibits the action of the efflux pump, resulting in enrichment of the free FL118 drug of formula 1 in the cells; Optionally, an immunoconjugate having an FL118 drug of formula 1 bound thereto is internalized into cells, and the FL118 drug of formula 1 is released from lysosomes, characterized in that the immunoconjugate or a pharma- ceutically acceptable salt thereof: 【Chemistry 1】

2. Targeting the cancer cells with antigen-binding sites that target the antigens on the cancer cells, and then The immunoconjugate or immunoassay composition according to claim 1, characterized in that the acid-sensitive linker is decomposed in an acidic environment (pH≦7) to at least partially liberate the FL118 drug of formula 1, and the free FL118 drug of formula 1 penetrates deep into cancer tissues, passing through cell membranes and transporting into cells. and a pharma- ceutically acceptable salt thereof.

3. The immunoconjugate or pharma- ceutically acceptable salt thereof according to claim 1, characterized in that the FL118 drug of formula 1 and the acid-sensitive linker are linked by a carbonate or ester bond so that the free FL118 drug of formula 1 is released upon decomposition of the acid-sensitive linker.

4. The immunoconjugate or its pharma- ceutical acceptable salt according to claim 1, characterized in that the cells into which the free form of the FL118 drug of formula 1 is delivered are targeted cancer cells and / or their surrounding cells.

5. 2. The immunoconjugate or a pharma- ceutically acceptable salt thereof according to claim 1, wherein the antibody or its antigen-binding portion binds to a receptor on a cell surface.

6. 2. The immunoconjugate or a pharma- ceutical acceptable salt thereof according to claim 1, characterized in that the antibody or its antigen-binding site targets an antigen that is selectively distributed on the surface of a cancer cell or an antigen that is overexpressed in cancer cells and is also distributed in small amounts in normal tissues.

7. The immunoconjugate or a pharma- ceutical acceptable salt thereof according to claim 1, wherein the antibody or antigen-binding site thereof targets one of the human epidermal growth factor receptors (HER / EGFR / ERBB), namely, HER2, FolR, PSMA, or Trop-2.

8. 2. The immunoconjugate of claim 1, wherein the acid-sensitive linker is derived from a compound of formula 2: 【Chemistry 2】 Here, X １ and X ２ are each independently -H or -halogen; Y is -NH-, -NR Ａ - or null; Z is -C １ -C ４ Alkyl-, -C ３ -C ６ Cycloalkyl-, -(C １ -C ２ alkyl)-(C ３ -C ６ Cycloalkyl)-, -(C ３ -C ６ Cycloalkyl)-(C １ -C ２ alkyl)- or -(C １ -C ２ alkyl)-(C ３ -C ６ Cycloalkyl)-(C １ -C ２ alkyl)-; W is -R Ｂ -, -M- -R Ｂ -M-, -M-R Ｂ -or-R Ｂ -M-R Ｃ - and R Ａ ~R Ｃ are each independently １ -C ４ is alkyl; M is, 【Chemistry 3】 and n is an integer from 5 to 9.

9. In the formula 2, X １ and X ２ are each independently -H or -halogen; Y is -NR Ａ - or null; Z is -C １ -C ４ Alkyl-, -(C １ -C ２ alkyl)-(C ３ -C ６ cycloalkyl)- or -(C ３ -C ６ Cycloalkyl)-(C １ -C ２ alkyl)-; W is -R Ｂ -or-R Ｂ -M-R Ｃ - and R Ａ ~R Ｃ are each independently １ -C ４ is alkyl; M is, 【Chemistry 4】 and and n is an integer from 5 to 9; 9. The immunoconjugate of claim 8 or a pharma- ceutically acceptable salt thereof.

10. The immunoconjugate or a pharma- ceutically acceptable salt thereof according to claim 8, characterized in that the alcohol group site of the FL118 drug of formula 1 is linked to the alcohol group site of the acid-sensitive linker of formula 2.

11. The immunoconjugate according to claim 1, wherein [FL118 drug of formula 1]-[acid-sensitive linker] is derived from any one selected from the group consisting of compounds represented by the following formulas 3 to 5, or a pharma- ceutically acceptable salt thereof: 【Chemistry 5】 【Chemistry 6】 and 【Chemistry 7】 (wherein each n is independently an integer from 5 to 9).

12. The immune complex or a pharma- ceutical acceptable salt thereof according to claim 1, wherein the bond between the acid-sensitive linker and the antibody or a fragment thereof containing an antigen-binding site is such that a thiol group contained in the antibody or the antigen-binding fragment is bound to a maleimide group or a maleic hydrazide group of the acid-sensitive linker.

13. The immunoconjugate of claim 1, wherein the antibody or antigen-binding portion thereof is capable of targeting cancer cells resistant to the drug SN-38 of formula 6: 【Chemistry 8】

14. The immunoconjugate or a pharma- ceutically acceptable salt thereof according to claim 1, wherein the immunoconjugate has an average drug-to-antibody ratio (DAR) of 2 to 12.

15. A pharmaceutical composition for preventing or treating cancer, comprising the immunoconjugate or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 14 as an active ingredient.

16. A drug-linker conjugate, or a pharma- ceutically acceptable salt thereof, in which a FL118 drug of formula 1 below is linked to an acid-sensitive linker of formula 2 below: 【Chemistry 9】 【Chemistry 10】 {where X １ and X ２ are each independently -H or -halogen; Y is -NH-, -NR Ａ - or null; Z is -C １ -C ４ Alkyl-, -C ３ -C ６ Cycloalkyl-, -(C １ -C ２ alkyl)-(C ３ -C ６ Cycloalkyl)-, -(C ３ -C ６ Cycloalkyl)-(C １ - C ２ alkyl)- or -(C １ -C ２ alkyl)-(C ３ -C ６ Cycloalkyl)-(C １ -C ２ alkyl)-; W is -R Ｂ -, -M- -R Ｂ -M-, -M-R Ｂ -or-R Ｂ -M-R Ｃ - and R Ａ ~R Ｃ are each independently １ -C ４ is alkyl, M is, 【Chemistry 11】 and and n is an integer from 5 to 9.

17. In the formula 2, X １ and X ２ are each independently -H or -halogen; Y is -NR Ａ - or null; Z is -C １ -C ４ Alkyl-, -(C １ -C ２ alkyl)-(C ３ -C ６ cycloalkyl)- or -(C ３ -C ６ Cycloalkyl)-(C １ -C ２ alkyl)-; W is -R Ｂ -or-R Ｂ -M-R Ｃ - and R Ａ ~R Ｃ are each independently １ -C ４ is alkyl; M is, 【Chemistry 12】 and and n is an integer from 5 to 9; 17. The drug-linker conjugate of claim 16, or a pharma- ceutically acceptable salt thereof.

18. The drug-linker conjugate or a pharma- ceutically acceptable salt thereof according to claim 16, characterized in that the FL118 drug of formula 1 and the acid-sensitive linker are linked by a carbonate or ester bond so that the free form of the FL118 drug of formula 1 is released upon decomposition of the acid-sensitive linker.

19. The drug-linker conjugate or a pharma- ceutically acceptable salt thereof according to claim 16, characterized in that the alcohol group site of the FL118 drug of formula 1 is linked to the alcohol group site of the acid-sensitive linker of formula 2.

20. The drug-linker conjugate according to claim 16, wherein the drug-linker conjugate is any one selected from the group consisting of compounds represented by the following formulas 3 to 5, or a pharma- ceutically acceptable salt thereof: 【Chemistry 13】 【Chemistry 14】 and 【Chemistry 15】 (wherein each n is independently an integer from 5 to 9).

21. A drug-linker conjugate or a pharma- ceutically acceptable salt thereof, characterized in that the acid-sensitive linker of formula 2 is decomposed in an acidic environment (pH ≦ 7) to release the FL118 drug of formula 1, as described in any one of claims 16 to 20.

22. (a) the drug-linker conjugate of any one of claims 16 to 20, or a pharma- ceutically acceptable salt thereof; and (b) an antibody or a fragment thereof containing an antigen-binding site thereof Including, An immunoconjugate or a pharma- ceutically acceptable salt thereof, characterized in that one or more FL118 drugs of formula 1 are linked to an antibody or a fragment thereof containing an antigen-binding site via an acid-labile linker of formula 2.

23. 23. The immunoconjugate or a pharma- ceutically acceptable salt thereof of claim 22, wherein the antibody is trastuzumab, cetuximab, or sacituzumab.

24. A method for preparing a carrier-drug conjugate, comprising binding one or more FL118 drugs of formula 1 to a carrier via an acid-sensitive linker of formula 2 using the drug-linker conjugate or a pharma- ceutically acceptable salt thereof according to any one of claims 16 to 20.

25. The method for preparing a carrier-drug complex according to claim 24, characterized in that the carrier is an antibody, a lipibody, and / or an aptamer.

Images