Vegfr-2 targeted antibody-drug conjugate effective in the treatment of ovarian cancer

Abstract

The invention relates to VEGFR-2 targeted antibody-drug conjugates comprising the monoclonal antibody, Ramucirumab, conjugated to a therapeutic agent (MMAE) with a cleavable maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl linker in the presence of cathepsin, for use in the treatment of ovarian cancer. The VEGFR-2 targeted antibody drug conjugates (ADCs) of the invention can also be used for the treatment of other types of cancer other than ovarian cancer expressing target receptors.

Description

[0001] VEGFR-2 TARGETED ANTIBODY-DRUG CONJUGATE EFFECTIVE IN THE TREATMENT OF OVARIAN CANCER

[0002] Technical Field of the Invention

[0003] The invention relates to VEGFR-2 (Vascular endothelial growth factor receptor) targeted antibody-drug conjugates containing the monoclonal antibody, Ramucirumab, conjugated to a therapeutic agent (MMAE; Monomethyl auristatin E) with a cleavable linker (MC-VC-PABC; maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl) in the presence of cathepsin, for use in the treatment of ovarian cancer. The VEGFR- 2 targeted antibody drug conjugates (ADC) of the invention can also be used for the treatment of cancer types with high VEGFR-2 expression, other than ovarian cancer expressing target receptors.

[0004] State of the Art

[0005] Ovarian cancer, defined as the uncontrolled proliferation of ovarian cells and the resulting malignant tumour formation, is one of the most common causes of cancer- related deaths in women in developed countries. One to two out of every 100 women develop ovarian cancer during their lifetime. When diagnosed, ovarian cancer is usually very advanced, and therefore treatment is more challenging than many other types of female cancer. Therefore, as in all other cancers, early diagnosis is of great importance for the success of treatment.

[0006] Overexpression of VEGF is associated with overall survival in ovarian cancer, as VEGF levels in serum have been shown to be an independent prognostic factor in patients with ovarian cancer

[0001] , It is also secreted by most solid tumours and is expressed in >70% of ovarian carcinomas. The biological functions of VEGFs are mediated by VEGFR-1 (Flt-1 ), VEGFR-2 (KDR / Flk-1 ) and VEGFR-3 (Flt-4) as a tyrosine kinase receptor family, and VEGFR-2 in particular is associated with microvascular permeability, endothelial cell proliferation, invasion, migration and survival [2], Considering the aforementioned association, in vitro inhibition of VEGFR- 2 performed in the state of the art significantly reduces tumour cell migration and invasion on ovarian cancer cells [3]. Early diagnosis is necessary to avoid the high morbidity and mortality rates caused by ovarian cancer and to have a better chance of cure. The standard treatment applied in ovarian cancer includes taxane and platinum-based chemotherapy. In addition, anti- angiogenic bevacizumab and Poly(ADP-ribose) polymerase (PARP) inhibitors have gained momentum in the treatment of this gynecological malignancy in the last decade

[0007] [4], However, acquired multi-drug resistance (MDR) to chemotherapy and the toxic effects on healthy cells pose a major problem in the treatment of ovarian cancer. In addition to chemotherapeutic drugs, hormone therapy and targeted drugs are also widely used in the treatment of ovarian cancer. Hormone therapy is the use of hormones or hormone blocking drugs to fight cancer. Hormone therapy agents include luteinising hormone-releasing hormone (LHRH) agonists, tamoxifen and aromatase inhibitors. If LHRH agonists are given to ovarian cancer patients for a long time, these agents can cause bone weakness and sometimes osteoporosis. Tamoxifen, unlike LHRH agonists that completely block oestrogen hormone, does not cause bone loss because it keeps the oestrogen level in the body at a certain level, but it can cause blood clots in the legs. Aromatase inhibitors have different side effects such as joint and muscle pain and bone weakening, which result in osteoporosis and bone fractures

[0008] [5]. Anti-VEGF / VEGFR angiogenic inhibitors, non-VEGF angiogenic inhibitors, PARP inhibitors, EGFR inhibitors, folate receptor inhibitors, and IGFR inhibitors are used in targeted therapies. In the state of the art, two main strategies are used to inhibit the VEGFR-signaling pathway, inhibition of the ligand (VEGF) with antibodies or soluble receptors and inhibition of the receptor with tyrosine kinase inhibitors [6, 7], The use of antibodies as specific delivery vehicles to target cytotoxic drugs to tumour cells and thus reduce their systemic toxicity is a more effective approach, therefore antibodydrug conjugates (ADCs) are used in the state of the art for the treatment of ovarian cancer by conjugating high-potency cytotoxic compounds to tumour-targeting antibodies. The antibody-drug conjugate strategy not only increases the therapeutic effect of potent cytotoxic drugs, but also helps minimise chemotherapy-related side effects.

[0009] The patent application numbered EP3521314B1 in the state of the art relates to antibody-drug conjugates and their use in therapy, especially in anti-cancer or antiinflammatory therapy. In said document, targeted antibody-drug conjugates are synthesised by using a cytotoxic drug conjugated to antibodies or antibody fragments. However, although VEGFR-2 is among the target antigens in the mentioned study, ovarian cancer is not among the cancer types mentioned.

[0010] Due to the limitations and inadequacies of the solutions in the state of the art, the fact that chemotherapeutic agents used in the treatment of ovarian cancer have a toxic effect on healthy cells as well as cancerous cells, that is, they are not target specific and this causes systemic problems, and also the fact that cancer cells develop resistance to existing drugs over time and that sufficient cytotoxic effect cannot be achieved with the use of monoclonal antibodies (mAbs) alone, it has become necessary to make a development in the field of ovarian cancer treatment.

[0011] Brief Description and Aims of the Invention

[0012] The invention discloses a VEGFR-2 targeted antibody-drug conjugate comprising ramucirumab monoclonal antibody conjugated to the Monomethyl auristatin E (MMAE) therapeutic agent with maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (vc) which is a cleavable linker in the presence of cathepsin, for use in the treatment of ovarian cancer. VEGFR-2 targeted antibody drug conjugates (ADC) of the invention can be used for the treatment of breast cancer, prostate cancer, lung cancer, glioblastoma, gastrointestinal cancer, hepatocellular carcinoma, renal cell carcinoma, bladder cancer and osteosarcoma, in addition to ovarian cancer expressing target receptors.

[0013] The aim of the invention is to prepare an antibody-drug conjugate that directly targets cancerous cells without having a toxic effect on healthy cells. The antibody-drug conjugate of the invention is Ramucirumab-valine-citrulline-MMAE (R-vcMMAE). By means of the ramucirumab antibody included in the antibody-drug conjugate of the invention, the active cytotoxic drug MMAE is specifically delivered to ovarian cancer cells and the death of cancerous cells can be directly achieved without affecting healthy cells.

[0014] Another aim of the invention is to treat ovarian cancer. By means of the VEGFR-2 targeting of the ADCs of the invention, recognition of VEGFR-2, which is highly expressed in ovarian cancer cells, and subsequent death of cancerous cells is achieved with the cytotoxic agent. Description of Drawings

[0015] Figure 1. SDS-PAGE images of the synthesised R-vcMMAE under A) reducing conditions (left side of the protein marker) and non-reducing conditions (right side of the protein marker); L: light chain, H: heavy chain, HL: one heavy one light chain, H2: two heavy chains, H2L: two heavy one light chain, H2L2: two heavy two light chains.

[0016] Figure 2. HIC-UPLC chromatogram for synthesised R-vcMMAE

[0017] Figure 3. SEC-UPLC chromatograms for reference Ramucirumab and synthesised R- vcMMAE

[0018] Figure 4. Deconvoluted mass spectra for A) reference Ramucirumab and B) synthesised R-vcMMAE reduced with DTT

[0019] Figure 5. Surface Plasmon resonance (SPR) sensograms of A) Lot 1 reference antibody, B) Lot 2 reference antibody, C) Lot 3 reference antibody, and D) synthesised R-vcMMAE

[0020] Figure 6. Cell-based ELISA results for ramucirumab and R-vcMMAE

[0021] Figure 7. Cell viability analysis of Ramucirumab, R-vcMMAE, MMAE and Paclitaxel in A) healthy ovarian cell line (OSE-SV40) and B, C and D) ovarian cancer cell lines (A2780, A2780cis and OVCAR-3)

[0022] Detailed Description of the Invention

[0023] The invention relates to VEGFR-2 targeted antibody-drug conjugate comprising ramucirumab monoclonal antibody conjugated to Monomethyl auristatin E (MMAE) therapeutic agent with maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (vc) which is a cleavable linker in the presence of cathepsin, for use in the treatment of ovarian cancer. VEGFR-2 targeted antibody drug conjugate (AIC) of the invention can also be used for the treatment of breast cancer, prostate cancer, lung cancer, glioblastoma, gastrointestinal cancer, hepatocellular carcinoma, renal cell carcinoma, bladder cancer and osteosarcoma as an alternative to ovarian cancer expressing target receptors. The antibody-drug conjugates of the invention have the general formula Ab-(L-D)n. Here, Ab is an antibody targeting a VEGFR-2 receptor; L represents a linker (mc-vc- pAB) and D represents a therapeutic agent, and n is an integer between 1 and 8. In the invention, the antibody targeting the VEGFR-2 receptor is Ramucirumab. Maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-VC-PABC), a cleavable linker in the presence of cathepsin, is used as the linker. Monomethyl auristatin E (MMAE) is used as the cytotoxic therapeutic agent.

[0024] By means of the antibodies in the ADC of the invention, the effective cytotoxic drug is specifically delivered to ovarian cancer cells and the death of cancerous cells can be directly achieved without affecting healthy cells.

[0025] According to Figure 1 , the profile comparison of Ramucirumab and the synthesised ADC (R-vcMMAE) is shown by SDS-PAGE analysis. Under reduced conditions, both mAb and ADC gave only two bands indicating the heavy (H) and light (L) chain. While only one band was observed in unconjugated Ramucirumab under non-reduced conditions, the synthesised R-vcMMAE showed six bands representing light chain (L), heavy chain (H), one heavy one light chain (HL), two heavy chains (H2), two heavy one light chain (H2L) and two heavy two light chains (H2L2). SDS-PAGE analysis showed that the drug bound heterogeneously (0-8; drug-antibody ratio) to the antibody as expected. Also, as can be seen from Figure 2, the HIC-UPLC spectrum of the synthesised R-vcMMAE showed four main peaks corresponding to zero, two, four, five and six drug combinations per antibody. The analysis showed that the amount of the reference antibody decreased as a result of the reaction with vcMMAE and therefore the cytotoxic drug could bind to the free thiol groups. The average DAR of synthesised R-vcMMAE based on the calculated areas of peaks from the HIC chromatogram is approximately 3.2.

[0026] Table 1 : Determination of drug-to-antibody ratio (DAR) based on relative peak area of each conjugated drug

[0027] 0 2 4 5 6

[0028] SEC-UPLC results in Figure 3 show that the monomer peak area of the reference antibody is 98.99%, while the monomer peak area of the R-vcMMAE conjugate is 95.58%. This shows that the impurity rate in the synthesised ADC is less than 5%. According to the mass analysis result under reduced conditions in Figure 4, the reference antibody gave a higher intensity peak at approximately 23.3 kDa and 46.6 kDa molecular weights. The R-vcMMAE synthesised after drug conjugation gave peaks at approximately 24.8 kDa and 53.3 kDa molecular weights, indicating that the drug was mostly bound to the heavy chain (Fc region) of the antibody. According to the SPR analysis results in Figure 5, the Kd value of the antibody made with three different lots is between 10.55 pM and 16.98 pM. The Kd value of the synthesised antibody drug conjugate was calculated as 9.82 pM. A low Kd value means that the binding affinity to the antigen increases. In this case, it was shown that the binding affinity of the synthesised ADC to the antigen was similar to the reference antibody and that the cytotoxic drug conjugation did not affect the affinity of the antibody to the target antigen.

[0029] Table 2: SPR analysis results for three lots of the reference Ramucirumab antibody and the synthesised R-vcMMAE

[0030] According to Figure 6, cell-based ELISA was performed to test whether the binding affinity of the synthesised R-vcMMAE to the VEGFR-2 antigen on the cells was affected. The results show that the synthesised ADC can bind to the VEGFR-2 receptor on the surface of ovarian cancer cells in a concentration-dependent manner. Since no significant signal could be obtained for the healthy ovarian cell line OSE-SV40, the data were not presented. In Figure 7, the in vitro cytotoxicity of the synthesised R- vcMMAE was evaluated in OSE-SV40 (Figure 7A), A2780 (Figure 7B), A2780cis (Figure 7C) and OVCAR-3 (Figure 7D) ovarian cells after 72 hours of drug treatment. According to the results obtained from the cell viability test, it is seen that the paclitaxel used in the clinic and the cytotoxic drug MMAE used in the synthesis of ADC also affect healthy cells and are not selective, and the antibody alone does not affect cell viability. On the other hand, it was observed that ADC obtained by conjugation of MMAE to Ramucirumab was more effective in primary, cis-platinum-resistant primary and metastatic ovarian cancer cell lines compared to healthy cells and affected cell viability. In addition, it is understood from Figure 7 that the IC50 value of the synthesised R- vcMMAE in ovarian cancer cell lines is approximately 6.4 nM.

[0031] Synthesis method of the antibody-drug conjugates of the invention comprises the process steps of i. Partial or complete reduction of disulphide bonds in the antibody by incubating ramucirumab monoclonal antibody with tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) in phosphate buffer containing sodium chloride (NaCI) and ethylenediaminetetraacetic acid (EDTA) or in borate buffer or Tris buffer on a heater-stirrer, ii. Determination of the concentration of the fraction containing reduced Ramucirumab after the removal of the reducing agent TCEP or DTT in the medium using a desalting column containing G25 resin or a cut-off filter, iii. Dissolving the vcMMAE linker-drug compound first in dimethyl sulphoxide (DMSO) according to the determined concentration and then treating by adding it to Ramucirumab with reduced sulphide bonds, and iv. Removing the unreacted vcMMAE binder-drug compound at the end of the treatment by centrifugation using a cut-off filter and changing the buffer to phosphate buffer.

[0032] In another embodiment of the invention, synthesis method of the antibody-drug conjugates of the invention comprises the process steps of i. Partial or complete reduction of disulphide bonds in the antibody by incubating ramucirumab monoclonal antibody and TCEP or DTT at concentrations 1 -10 molar of this monoclonal antibody in 0-100 mM phosphate buffer containing 0- 100 mM NaCI and 0-10 mM EDTA or in borate buffer or Tris buffer at pH=6- 8.5 for 30 min-5 hours at 25-50 °C and at 0-500 rpm in a heater-stirrer, ii. Determination of the concentration of the fraction containing reduced Ramucirumab after removing the reducing agent TCEP or DTT in the medium using a desalting column containing G25 resin or a cut-off filter, or alternatively without removing the reducing agents from the reaction medium, iii. Dissolving the vcMMAE binder-drug compound first in DMSO according to the determined concentration and then adding to Ramucirumab, which is diluted with 0-70% DMSO by volume and whose sulphide bonds are reduced, in a way that the concentration of said antibody is 1 -15 molar times and treating at 0-30 °C and 0-500 rpm for 30 min-24 hours, and iv. Removing the unreacted vcMMAE binder-drug compound at the end of the treatment by centrifugation using a 10-100 kDa cut-off filter and changing the buffer to phosphate buffer.

[0033] In another embodiment of the invention, synthesis method of the antibody-drug conjugates of the invention comprises the process steps of i. Partial or complete reduction of disulphide bonds in the antibody by incubating ramucirumab monoclonal antibody and TCEP or DTT at concentrations 2.5 molar of this monoclonal antibody in 0-100 mM phosphate buffer containing 50 mM NaCI and 2 mM EDTA or in borate buffer or Tris buffer at pH=7.3±0.1 for 2 hours at 37 °C and at 250 rpm in a heater-stirrer, ii. Determination of the concentration of the fraction containing reduced Ramucirumab at 280 nm wavelength after removing the reducing agent TCEP or DTT in the medium using a desalting column containing G25 resin, or alternatively without removing the reducing agents from the reaction medium, iii. Dissolving the vcMMAE binder-drug compound first in DMSO according to the determined concentration and then adding to Ramucirumab, which is diluted with 30% DMSO by volume and reduced, in a way that the concentration of said antibody is 7.5 molar times and treating at +4 °C and 250 rpm for 5 hours, and iv. Removing the unreacted vcMMAE binder-drug compound at the end of the treatment by centrifugation using a 10 kDa cut-off filter and changing the buffer to phosphate buffer.

[0034] After obtaining the antibody-drug conjugates of the invention with the method of the invention, the concentration of the synthesised antibody-drug conjugate was calculated from the standard curve obtained according to the absorbance values taken in the nanodrop against the changing concentration of commercial Ramucirumab and the physicochemical characterisation of the synthesised Ramucirumab-vcMMAE antibody-drug conjugate was performed with SDS-PAGE, UPLC-SEC, UPLC-HIC and LC-MS / MS, and its biological characterisation was performed with SPR, cell-based ELISA and cell viability test. In addition, VEGFR-2 mRNA levels in ovarian cell lines were determined using reverse transcription polymerase chain reaction (RT-qPCR).

[0035] In the invention, it was shown by qPCR that VEGFR-2 expression was expressed higher at the mRNA level in ovarian cancer cells compared to healthy cells. For this, first, total RNA from the cells was isolated using TRIzol Reagent and cDNA synthesis was performed using the kit. To evaluate VEGFR-2 expression in ovarian cell lines, qPCR analysis was performed using VEGFR-2 primers. VEGFR-2 mRNA expression was expressed higher in A2780 (primer), A2780cis (cis-platinum resistant primer) and OVCAR-3 (metastatic) cell lines compared to OSE-SV40 (healthy) cell line. Binding affinity of antibody and synthesised ADC in ovarian cancer cell lines compared to healthy cell line was shown by cell-based ELISA. For this purpose, cells (OSE-SV40, A2780, A2780cis, OVCAR-3) were grown in 96-well plates, then antibody (Ramucirumab) and ADC (R-vcMMAE) were applied at varying concentrations. The results in Figure 6 were determined by measuring the colorimetric signal formed after the TMB reagent of the HRP-conjugated secondary antibody. According to the results, both the antibody and the synthesised ADC bind to ovarian cell lines even at low concentrations. Cell viability assay was performed for the activity of Ramucirumab, ADC and MMAE used in the invention in ovarian cells. For this purpose, cells (OSE- SV40, A2780, A2780cis, OVCAR-3) were grown in 96-well plates, then antibody (Ramucirumab), ADC (R-vcMMAE) and the cytotoxic drug MMAE included in the ADC composition were applied at varying concentrations. Cell viability was measured luminometrically after 72 hours using the kit. According to Figure 7, the use of Ramucirumab alone did not affect cell viability for each cell line. While MMAE alone was effective at low concentrations, it also showed activity in healthy cell lines. As expected, since the cytotoxic drug is not effective alone and binds to the antibody, it was determined that ADC showed potency in cancer cell lines at low concentrations for targeting and did not affect healthy cells.

[0036] SPR (surface plasmon resonance) was used to compare the affinity of the synthesised ADC, which is the subject of the invention, to VEGFR-2 ligand with the affinity of Ramucirumab to VEGFR-2 ligand. For this analysis, VEGFR-2 ligand was immobilised on a CM5 chip surface. Ramucirumab and synthesised ADC with 3 different lots were injected onto the ligand surface. Binding kinetics, Kd (M), was calculated using a kinetic binding model in Biacore T200 software. According to the results: The range of Kd values for 3 different lots of Ramucirumab is between 10.55 pM and 16.98 pM. For the synthesised ADC, the Kd value was calculated as 9.8 pM. A low Kd value indicates an increase in affinity. In this case, it can be said that the cytotoxic drug bound to the antibody is similar to the affinity of the antibody to the target antigen.

[0037] REFERENCES

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Claims

CLAIMS1. A VEGFR-2-targeting antibody-drug conjugate, comprising ramucirumab monoclonal antibody conjugated to the therapeutic agent Monomethyl auristatin E (MMAE) with a cleavable linker, maleimidocaproyl-valine-citrulline-p- aminobenzoyloxycarbonyl (vc), in the presence of cathepsin.

2. An antibody-drug conjugate according to claim 1 for use in the treatment of ovarian cancer.

3. Pharmaceutical composition comprising an antibody-drug conjugate according to claim 1 or 2.

4. Synthesis method of a VEGFR-2-targeting antibody-drug conjugate, comprising the process steps of: i. Partial or complete reduction of disulphide bonds in the antibody by incubating ramucirumab monoclonal antibody with tris(2- carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) in phosphate buffer containing sodium chloride (NaCI) and ethylenediaminetetraacetic acid (EDTA) or in borate buffer or Tris buffer on a heater-stirrer, ii. Determination of the concentration of the fraction containing reduced Ramucirumab after the removal of the reducing agent TCEP or DTT in the medium using a desalting column containing G25 resin or a cut-off filter, iii. Dissolving the vcMMAE linker-drug compound first in dimethyl sulphoxide (DMSO) according to the determined concentration and then treating by adding it to Ramucirumab with reduced sulphide bonds, and iv. Removing the unreacted vcMMAE binder-drug compound at the end of the treatment by centrifugation using a cut-off filter and changing the buffer to phosphate buffer.

5. A method according to claim 4, comprising the process steps of:i. Partial or complete reduction of disulphide bonds in the antibody by incubating ramucirumab monoclonal antibody and TCEP or DTT at concentrations 1 -10 molar of this monoclonal antibody in 0-100 mM phosphate buffer containing 0-100 mM NaCI and 0-10 mM EDTA or in borate buffer or Tris buffer at pH=6-8.5 for 30 min-5 hours at 25-50 °C and at 0-500 rpm in a heater-stirrer, ii. Determination of the concentration of the fraction containing reduced Ramucirumab after removing the reducing agent TCEP or DTT in the medium using a desalting column containing G25 resin or a cut-off filter, or alternatively without removing the reducing agents from the reaction medium, iii. Dissolving the vcMMAE binder-drug compound first in DMSO according to the determined concentration and then adding to Ramucirumab, which is diluted with 0-70% DMSO by volume and whose sulphide bonds are reduced, in a way that the concentration of said antibody is 1 -15 molar times and treating at 0-30 °C and 0-500 rpm for 30 min-24 hours, and iv. Removing the unreacted vcMMAE binder-drug compound at the end of the treatment by centrifugation using a 10-100 kDa cut-off filter and changing the buffer to phosphate buffer.

6. A method according to claim 5, comprising the process steps of: i. Partial or complete reduction of disulphide bonds in the antibody by incubating ramucirumab monoclonal antibody and TCEP or DTT at concentrations 2.5 molar of this monoclonal antibody in 0-100 mM phosphate buffer containing 50 mM NaCI and 2 mM EDTA or in borate buffer or Tris buffer at pH=7.3±0.1 for 2 hours at 37°C and at 250 rpm in a heater-stirrer, ii. Determination of the concentration of the fraction containing reduced Ramucirumab at 280 nm wavelength after removing the reducing agent TCEP or DTT in the medium using a desalting column containing G25 resin, or alternatively without removing the reducing agents from the reaction medium, iii. Dissolving the vcMMAE binder-drug compound first in DMSO according to the determined concentration and then adding to Ramucirumab, whichis diluted with 30% DMSO by volume and reduced, in a way that the concentration of said antibody is 7.5 molar times and treating at +4°C and 250 rpm for 5 hours, and iv. Removing the unreacted vcMMAE binder-drug compound at the end of the treatment by centrifugation using a 10 kDa cut-off filter and changing the buffer to phosphate buffer.

7. Ramucirumab-vcMMAE antibody-drug conjugate synthesised by a method according to any one of claims 4 to 6.

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