JP7299700B2 - Dual inhibition therapy of VEGF-A/VEGFR2 pathway and pharmaceutical composition for use thereof - Google Patents

Description

The present invention is a new treatment method that uses an anti-VEGFR2 (vascular endothelial growth factor receptor 2, vascular endothelial growth factor receptor 2) antibody and an anti-VEGF-A (vascular endothelial growth factor-A, vascular endothelial growth factor) neutralizing antibody in combination. Regarding.

When cancer proliferates and metastasizes, the new formation of nutrient blood vessels called angiogenesis occurs as a nutritional supply route for maintaining proliferative ability. Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) produced by stroma and tumor cells play a major role in angiogenesis, and drugs that target these molecules for the purpose of regulating angiogenesis has been widely developed.

VEGFR, which is a receptor for VEGF, is a type of receptor tyrosine kinase, and there are three kinds of proteins, VEGFR1 to VEGFR3. In particular, although VEGFR2 has weaker binding ability to ligands than VEGFR1, it has strong tyrosine kinase activity, making a large contribution to intracellular signal transduction overall, and is considered to be greatly involved in the process of angiogenesis. ing. VEGFR2 has the ability to bind to VEGF-A, C, D and E, and is the receptor said to be most closely associated with VEGF-induced tumor angiogenesis among the three VEGFRs. .

Antibody drugs against VEGFR2, which plays an important role in angiogenesis related to cancer growth and metastasis, are being developed. Ramucirumab, a representative antibody drug, is Ramucirumab (trade name: Cyramza, manufactured by Eli Lilly). ) is a humanized monoclonal antibody of recombinant human immunoglobulin G1 (IgG1 ^b ) against VEGFR2.

By specifically binding to VEGFR2, ramucirumab inhibits the binding of ligands VEGF-A, VEGF-C, and VEGF-D, thereby inhibiting the activation of VEGFR2 and its downstream signal transduction system. As a result, it inhibits proliferation and migration of vascular endothelial cells, inhibits tumor angiogenesis, and exerts an antitumor effect.

In recent years, the usefulness of angiogenesis inhibitors has been shown also in gastric cancer, and ramucirumab is used for the second-line treatment of advanced gastric cancer. Although systemic chemotherapy has been administered to patients with unresectable advanced gastric cancer, the prognosis is not satisfactory, with an average prognosis of around 1 year. Ramucirumab significantly prolongs overall survival in the second-line treatment of advanced gastric cancer, was approved for gastric cancer in Japan in June 2015, and has since been widely used for colorectal cancer and lung cancer.

Ramucirumab, a human anti-VEGFR2 antibody, was shown to significantly prolong overall survival when used in combination with paclitaxel in the RAINBOW study, a global phase III study targeting second-line gastric cancer patients, and is also the standard treatment in Japan. (mOS (median overall survival, median overall survival): 9.6 months vs. 7.4 months, HR (Hazard Ratio) 0.81 95% CI 0.68-0.96, p = 0.017) (Non-Patent Document 1). However, the therapeutic effect was not sufficient, and according to a subset analysis of Japanese subjects in the previous RAINBOW study, the response rate of ramucirumab/paclitaxel combination therapy in second-line therapy was about 40%, and the median survival time was around 11.4 months. (Non-Patent Document 2).

Ramucirumab has few side effects and is effective in many patients with gastric cancer, but in some patients it does not show a significant therapeutic effect. For such patients with little therapeutic benefit, if it is possible to predict the therapeutic effect before the start of ramucirumab treatment or at an early stage of treatment, it will be possible to avoid unnecessary treatment for patients for whom the therapeutic effect cannot be expected. Alternative treatments can be given. In addition, from the viewpoint of proper use of pharmaceuticals, it would be advantageous to develop appropriate biomarkers for predicting therapeutic effects that would enable this. Furthermore, understanding the mechanism of resistance to ramucirumab will lead to the development of new therapeutic strategies. Therefore, the present inventors analyzed biomarkers correlated with ramucirumab therapeutic effects.

As a result, in gastric cancer cases to which ramucirumab was administered, VEGF-A in plasma exhibited the following behaviors, which correlated with prognosis. In Figure 1, two patients were administered ramucirumab alone, and the others were administered in combination with paclitaxel. survival period) and OS. A negative correlation was observed between plasma VEGF-A concentration, PFS, and OS (Fig. 1(A)). In addition, when the plasma VEGF-A concentration was divided into two groups, a high level group and a low level group, according to the optimal cutoff value (424 pg / ml), PFS and OS were analyzed. was unsatisfactory (Fig. 1(B)). A negative correlation between plasma VEGF-A concentration and PFS and OS was not observed with paclitaxel administration, which is considered to be a phenomenon characteristic of ramucirumab administration. Ramucirumab administration causes the following phenomena. (1) Plasma VEGF-A markedly increased early after administration, and a negative correlation was observed between this VEGF-A level and survival time. (2) When divided into two groups according to the optimal cutoff, the prognosis of the high-value group was significantly poorer than that of the low-value group. (3) The early increase in VEGF-A after administration is not transient, and the high level is maintained until exacerbation (Patent Document 1, Non-Patent Document 3).

According to the above results, ramucirumab administration does not produce the effect of ramucirumab in a group of patients in whom blood VEGF-A concentration increases, so there is a possibility that a response can be obtained by suppressing the increase in VEGF. . However, the treatments that have already been reported to suppress both VEGF and VEGFR have not yielded desirable results (Non-Patent Documents 4-7). Specifically, these therapies are therapies that attempt to suppress both VEGFR and VEGF using the kinase inhibitor Sorafenib and the monoclonal antibody bevacizumab against VEGF. However, according to the above reports, the combined therapy of sorafenib and bevacizumab does not show much effect, or the side effects are so strong that the treatment cannot be continued, and it has not yet become an established treatment method.

Moreover, excess VEGF-A after administration of ramucirumab may adversely affect subsequent treatment course. The reason for this is that excessive VEGF-A induces angiogenesis and tumor growth through a pathway mediated by VEGFR1. In recent years, VEGF-A has also been reported to have a suppressive effect on tumor immunity through the proliferation and infiltration of regulatory T cells (Non-Patent Document 8), and may have an adverse effect through mechanisms mediated by immunosuppression. There is

WO2018/124153

Wilke, H. et al., 2014, Lancet Oncol., Vol.15, p.1224-1235. Shitara, K. et al., 2016, Gastric Cancer, Vol.19(3), pp.927-938, doi: 10.1007/s10120-015-0559-z. https://meetinglibrary.asco.org/record/156166/abstract CastellanoD. et al., 2013, Eur. J. Cancer. Vol.49(18), pp.3780-3787. Galanis, E. et al., 2013, Clin. CancerRes., Vol.19(17), pp.4816-4823. Lee, J.M., et al., 2010, Br. J. Cancer, Vol.102(3), pp.495-459. Azad, N.S. et al., 2008, J. Clin.Oncol., Vol.26(22), pp.3709-3714. Kahn, K.A. and Kerbel, R.S., 2018, Nat. Rev. Clin. Oncol., Vol.15(5), pp. 310-324.

As already shown by the present inventors, the efficacy of administration of an anti-VEGFR2 antibody typified by ramucirumab can be determined from the VEGF-A concentration in the blood at an early stage of treatment. However, for a group of patients who do not respond to treatment with ramucirumab, there is an option of not receiving ramucirumab treatment, but there is no effective alternative treatment. An object of the present invention is to provide a therapeutic drug and a therapeutic method that are effective for a group of patients for whom administration of ramucirumab is not effective. Another object of the present invention is to provide therapeutic agents and therapeutic methods that eliminate the adverse effects of excess VEGF-A produced by anti-VEGFR2 antibody administration on treatment.

TECHNICAL FIELD The present invention relates to novel therapeutic methods and therapeutic agents for improving therapeutic effects on patients for whom therapeutic effects have not been obtained by administration of anti-VEGFR antibodies such as ramucirumab.
(1) A concomitant drug that is used in combination with an anti-VEGFR2 antibody drug for a disease in which angiogenesis is a cause of aggravation, comprising an anti-VEGF antibody as an active ingredient.
(2) The combination drug according to (1), wherein the anti-VEGF antibody is an anti-VEGF-A antibody.
(3) The concomitant drug according to (1) or (2), wherein the disease is a tumor.
(4) The concomitant drug according to (3), wherein the tumor is gastric cancer, colon cancer, or small cell lung cancer.
(5) The concomitant drug according to any one of (1) to (4), which is used simultaneously or separately with the anti-VEGFR2 antibody drug.
(6) The concomitant drug according to any one of (1) to (5), wherein the anti-VEGFR2 antibody drug is ramucirumab and the concomitant drug containing the anti-VEGF antibody as an active ingredient is bevacizumab.
(7) A method for testing patients who respond to the combined use of an anti-VEGFR2 antibody drug and an anti-VEGF antibody drug, wherein the sample collected from the patient before the start of treatment with the anti-VEGFR2 antibody drug and in the early stage after the start of treatment A test method characterized by measuring and comparing the VEGF-A concentrations of
(8) The test method according to (7), wherein the sample is blood, plasma, or serum.
(9) A kit for testing a patient who responds to the combined use of an anti-VEGFR2 antibody drug and an anti-VEGF antibody drug, the test comprising a reagent for measuring the VEGF-A concentration in a patient sample. kit.
(10) A method of treatment comprising combined administration of an anti-VEGFR2 antibody drug and an anti-VEGF antibody drug for diseases exacerbated by angiogenesis.
(11) The treatment method according to (10), wherein the anti-VEGFR2 antibody drug and the anti-VEGF antibody drug are administered in combination at the same time or at separate timings.
(12) Measure blood VEGF concentration before administration of anti-VEGFR2 antibody drug and early after administration, and if significant increase in blood VEGF concentration is observed after administration of anti-VEGFR2 antibody drug, anti-VEGFR2 antibody drug and the anti-VEGF antibody drug are administered in combination (10) or (11).

FIG. 2 shows an analysis of the correlation between plasma VEGF-A concentration and prognosis. (A) A diagram showing the relationship between VEGF-A concentration in patient plasma and PFS (left) and OS (right) on day 8 after administration of ramucirumab. (B) PFS (left) and OS (right) in plasma VEGF-A high level group and low level group on day 8 of ramucirumab administration. A diagram showing the amount of VEGF-A produced in human stomach cancer cells. The figure which shows the analysis result in a mouse|mouth transplantation tumor model. (A) Diagram showing the administration schedule of DC101 antibody, a mouse surrogate antibody of ramucirumab. (B) Changes in mouse VEGF-A in plasma after DC101 antibody administration. (C) Changes in human VEGF-A in plasma after DC101 antibody administration. The figure which shows the analysis result in a mouse|mouth transplantation tumor model. (A) Changes in human or mouse PlGF (Placental Growth factor) in plasma after DC101 antibody administration, (B) Changes in human or mouse VEGF-C in plasma after DC101 antibody administration, ( C) Changes in human or mouse VEGF-D in plasma after DC101 antibody administration. A diagram showing the effect of combined administration of an anti-VEGFR2 antibody (DC101) and an anti-VEGF-A antibody (mVEGF) in a mouse transplanted tumor model. (A) A diagram showing an antibody administration schedule. (B) A diagram showing changes in tumor volume due to administration of each antibody. (C) A photograph showing the appearance of tumors transplanted to mice in each antibody administration 14 days after the start of antibody administration.

As shown in Figure 1, after administration of ramucirumab, in the group of patients in which plasma VEGF-A did not increase significantly, the therapeutic effect of ramucirumab was obtained. I thought it would be possible to get it. As mentioned above, treatment with small-molecule drugs targeting VEGFR is hampered by their inherent side effects. It was thought that it might be effective. Therefore, the following analysis was performed.

As shown in the examples below, the combined use of antibodies against VEGF or VEGFR can provide therapeutic effects even in a group of patients in whom anti-VEGFR antibodies alone are ineffective. Anti-VEGF-A antibodies currently in clinical use include bevacizumab, and anti-VEGFR2 antibodies include ramucirumab, but not limited to these, anti-VEGFR antibody drugs and anti-VEGF antibody drugs that will be developed in the future can be used. . Also, a drug containing an antibody fragment as an active ingredient, such as ranibizumab, which is a Fab fragment against VEGF-A, can be used. Furthermore, a drug containing as an active ingredient a bispecific antibody that targets both VEGFR2 and VEGF-A may be used.

Diseases to which the therapeutic agents and treatment methods shown in this example are applied are diseases in which angiogenesis is involved in the onset and exacerbation of the disease. Tumors are such diseases, and gastric cancer, colon cancer, and non-small cell lung cancer, for which ramucirumab administration has been found to be particularly effective, are typical diseases. Ramucirumab is specifically indicated for unresectable advanced/recurrent gastric cancer, unresectable advanced/recurrent colorectal cancer, and unresectable advanced/recurrent non-small cell lung cancer. be done. In addition, not only the above, but for any disease for which an anti-VEGFR antibody drug is effective, there is a possibility that a higher therapeutic effect can be obtained by using an anti-VEGF antibody drug as a concomitant drug.

The therapeutic agents may be administered at the same time, or they may be administered separately according to separate schedules in consideration of the half-life of each therapeutic agent.

Concomitant administration of ramucirumab with paclitaxel is the standard treatment, but it is not administered in combination with other antibody drugs. As an antibody drug, it does not show an effect when administered alone with an anti-VEGFR antibody drug, but in a group of patients who are effective by using an anti-VEGF antibody drug in combination, after anti-VEGFR antibody administration, early blood VEGF-A This is a group of patients in which an increase in Therefore, in addition to the current standard treatment of ramucirumab and paclitaxel, bevacizumab should be administered in combination.

Alternatively, after the standard treatment of paclitaxel and anti-VEGFR antibody drug treatment, the blood VEGF-A concentration is measured early, and if the value is high, an anti-VEGF antibody drug is used as a concomitant drug, and if the value is low, it is used as an antibody drug. Treatment by administration of an anti-VEGFR antibody drug alone may be performed.

Here, the blood VEGF-A concentration is measured on the 8th day, but it may be measured as early as the 4th to 10th day after the start of administration of the anti-VEGFR2 antibody. Moreover, plasma, blood, and serum can be used as the sample. With blood, plasma, and serum, not only can samples be obtained from patients for unresectable, advanced or recurrent cancers, but the physical burden on patients can be reduced.

In addition, as shown in the examples below, even when anti-VEGFR antibody administration is less effective, administration in combination with anti-VEGF-A antibody is clearly effective. became. Considering the mechanisms of action of the two antibodies, it is possible that combined administration of an anti-VEGFR antibody may be effective for patients who are ineffective to anti-VEGF-A antibody administration.

[Example]
The present inventors considered that the excess VEGF-A increased after administration of ramucirumab adversely affects the subsequent survival period, and that neutralization of this excess VEGF-A may improve the therapeutic effect. rice field. However, as described above, a therapy that suppresses VEGFR activation with the kinase inhibitor sorafenib and inhibits the binding of VEGF to VEGFR with bevacizumab is a therapy that suppresses both ligands and receptors. , Although some effects have been obtained, combination therapy has been abandoned due to increased toxicity. Furthermore, there are no examples of treatment by suppressing ligands and receptors simultaneously with antibody drugs.

Therefore, an analysis was conducted to see if therapeutic effects could be obtained by suppressing both VEGF-A/VEGFR2 with antibody preparations. First, nude mice were inoculated with human gastric cancer cell lines to construct model mice that exhibit the same reaction as human gastric cancer patients to anti-VEGFR antibodies.

(1) Establishment of mouse model As a disease model, it is necessary to prepare a model in which an increase in VEGF-A is observed after anti-VEGFR antibody administration. When constructing a model by transplanting human tumor cells into mice, it is desirable that the tumor cells to be transplanted have sufficient VEGF-A secretion ability. First, in order to determine the tumor cells to be transplanted, the amount of VEGF-A in human gastric cancer cell lines was measured by ELISA (Fig. 2).

Analysis was performed using human gastric cancer cells St4, MKN1, MKN7, MKN28, MKN45, and MKN74 (St4 was obtained from the Japan Medical Cell Resource Center/Cell Bank, and other cells were obtained from the JCRB Cell Bank).

Each cell was seeded in a petri dish under subconfluent conditions, and 3 days later, the amount of VEGF-A was measured using a human VEGF-A assay kit (R&D). The amount of VEGF-A secreted per 10,000 cells in the culture supernatant was calculated. As shown in FIG. 2, among the analyzed cells, VEGF-A secreted from MKN45 cells was the most abundant, so we decided to construct a mouse model using MKN45 cells.

nude mice, CANN. Cg-Foxn1nu/CrlCrlj (6-week-old, female, obtained from Charles River Japan Inc.) was subcutaneously transplanted with 2,000,000 human gastric cancer cell line MKN45, and 14 days after transplantation, the tumor size was measured and randomized. grouped into The tumor volume at the time of grouping was 80-150 mm ³ . A ramucirumab mouse surrogate antibody DC101 (Bio X Cell) was administered, and VEGF-A secretion was measured. Cyramza's package insert discloses that MKN45 cells are cells in which the tumor regression effect of the DC101 antibody has already been examined.

DC101 antibody was intraperitoneally administered twice a week at 5, 10 and 20 mg/kg for 2 weeks (Fig. 3(A)). Blood was collected on day 14 of administration, and human and mouse VEGF-A in plasma was measured. For the measurement, a human VEGF-A measurement kit and a mouse VEGF-A measurement kit (both manufactured by R&D) were used. It was previously confirmed that the human VEGF-A measurement kit detects only human VEGF-A, and the mouse VEGF-A measurement kit detects only mouse VEGF-A. As a result, the induction of mouse VEGF-A was confirmed when DC101 antibody was administered at doses of 10 and 20 mg/kg (FIG. 3(B)). On the other hand, no increase in human VEGF-A was observed (Fig. 3(C)).

In FIG. 3, the notation tumor (+) indicates mice transplanted with MKN45 or MKN74, which are gastric cancer cell lines. In addition, the notation of tumor (-) indicates the result of analysis of the effect of the DC101 antibody in mice not transplanted with cancer cell lines as a control.

Next, changes in other angiogenesis-related factors PlGF, VEGF-C, and VEGF-D were also analyzed. As with VEGF-A, human and mouse PlGF, VEGF-C and VEGF-D levels in plasma on day 14 of DC101 antibody administration were measured by ELISA (FIG. 4).

As a result, PlGF was markedly increased by DC101 administration, and VEGF-C and VEGF-D were weakly induced. This result was similar to that of ramucirumab-administered patient plasma (Patent Document 1, Non-Patent Document 3). In addition, all cytokines were induced from mice, and no human-derived ones were induced.

As shown above, in mice constructed by subcutaneously transplanting MKN45 cells into nude mice, the behavior of vascular growth factors produced after administration of DC101 antibody is very similar to the behavior when ramucirumab is administered to patients. , is considered to be a good model.

(2) Effect of combined use of anti-VEGFR2 antibody and anti-VEGF-A antibody In a mouse model transplanted with gastric cancer cell line MKN45, it was revealed that VEGF-A derived from the host mouse increased after administration of DC101 antibody. . Therefore, in the same mouse model, the tumor inhibitory effect of combined administration of DC101 antibody and anti-mouse VEGF-A antibody was analyzed.

Nude mice were subcutaneously implanted with MKN45 cells in the same manner as above, and 14 days later the tumor size was measured and grouped between 80 and 150 mm ³ . DC101 antibody alone (n = 3, 10 mg / kg), mouse VEGF-A neutralizing antibody 2G11-2A05 antibody alone (n = 2, 5 mg / kg), DC101 antibody (10 mg / kg) and 2G11-2A05 Antibody (5 mg/kg) combined administration (n=3) was intraperitoneally administered twice a week for 2 weeks, and tumor size and mouse body weight were measured over time (Fig. 5(A)). As a control, only the solvent was administered (n=3).

A weak tumor growth inhibitory effect was observed in the DC101 antibody (10 mg/kg) single administration group and the mouse VEGF-A neutralizing antibody 2G11-2A05 (5 mg/kg) single administration group. On the other hand, a statistically significant tumor inhibitory effect was observed in the DC101 antibody (10 mg/kg) and 2G11-2A05 antibody (5 mg/kg) combination group (FIG. 5(B)). In addition, in the appearance of the tumor by macroscopic observation, clear tumor regression was observed in the combination group (Fig. 5(C)).

Bevacizumab is considered to be minimally toxic even when used in combination, as serious side effects are relatively rare. In fact, no serious side effects were observed in the mouse model either.

As described above, in preclinical studies using mouse models, combination therapy with an anti-mouse VEGF-A neutralizing antibody and an anti-mouse VEGFR2 antibody, DC101, was found to have a significant tumor reduction effect compared to monotherapy.

Based on the above results, it is considered that antibody drugs targeting VEGFR2, which are thought to have the same mechanism of action, can be widely applied not only to ramucirumab when used for treatment in clinical practice. As for the anti-VEGF antibody, not only the existing antibody bevacizumab but also any antibody drug that exhibits a similar mechanism of action may be used.

As shown in the above examples, the combination therapy of anti-VEGFR antibody and anti-VEGF-A neutralizing antibody, that is, dual inhibition therapy of the VEGF-A/VEGFR2 pathway, anti-VEGF-A in blood early after administration of anti-VEGFR antibody It shows efficacy in patients with markedly elevated VEGF-A levels, or high VEGF-A levels when divided into two groups according to the optimal cutoff. However, since dual inhibition therapy of the VEGF-A/VEGFR2 pathway is not considered to have serious side effects, it is recommended to start concomitant administration from the beginning without examining the increase in VEGF-A levels in patients due to anti-VEGFR antibody administration. you can go

Since the present invention can provide an effective treatment method even for patients who did not respond to anti-VEGFR2 antibody drugs, specifically ramucirumab administration, it is an effective treatment for unresectable and recurrent gastric cancer. can provide the law.

In addition, as shown in this example, the ligand-receptor dual blockade therapy, which obtains a therapeutic effect by suppressing the ligand and receptor simultaneously with an antibody drug, is not a therapeutic method that has been tried so far, but a completely new therapeutic method. is.

Claims (6)

Treatment of gastric cancer, colon cancer, or small cell lung cancer in which neovascularization causes exacerbation, targeting patients with high VEGF-A concentration early after starting anti-VEGF-A antibody therapy that binds to VEGFR2 and suppresses its activation in
A pharmaceutical composition to be used in combination with an anti-VEGFR2 antibody drug that binds to VEGFR2 and suppresses its activation ,
A pharmaceutical composition comprising an anti-VEGF-A antibody that binds to and neutralizes VEGF-A as an active ingredient. the anti-VEGFR2 antibody pharmaceutical,
2. A pharmaceutical composition according to claim 1, used simultaneously or separately. wherein the anti-VEGFR2 antibody drug is ramucirumab,
3. The pharmaceutical composition according to claim 1, wherein the concomitant drug containing the anti-VEGF-A antibody as an active ingredient is bevacizumab. Determine gastric cancer, colorectal cancer, or small cell lung cancer patients who respond to combined use of an anti-VEGFR2 antibody drug that binds to VEGFR2 and suppresses its activation and an anti-VEGF-A antibody drug that binds to and neutralizes VEGF-A A method of obtaining data for
Before starting treatment with an anti-VEGFR2 antibody drug that binds to and suppresses activation of VEGFR2 collected from the patient, and measuring the VEGF-A concentration in the sample early after the start of treatment,
A method of obtaining data characterized by comparing. A data acquisition method according to claim 4 ,
A data acquisition method, wherein the sample is blood, plasma, or serum. Examination of gastric cancer, colorectal cancer, or small cell lung cancer patients who respond to combined use of an anti-VEGFR2 antibody drug that binds to VEGFR2 and suppresses its activation and an anti-VEGF-A antibody drug that binds to and neutralizes VEGF-A. is a kit,
A test kit comprising reagents for measuring VEGF-A concentration in a patient sample.

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