Companion drug comprising activity regulator for t cell and / or b cell

Abstract

Provided is a novel activity regulator which can regulate the activity of a T cell and / or a B cell. An activity regulator for a T cell and / or a B cell contains progesterone or a derivative thereof as an active ingredient.

Description

Combination drug containing a modulator of T cell and / or B cell activity

[0001] The present invention relates to a combination agent in which a T cell and / or B cell activity regulator containing progesterone or a derivative thereof as an active ingredient is used in combination with an anti-human PD-L1 monoclonal antibody.

[0002] The placenta possesses unique properties not found in other organs, including proliferation, invasion, and angiogenesis, as well as immune regulation to allow the mother to accept the fetus. Furthermore, the fetal tissue, the trophoblast, expresses many cancer-related genes and has a metabolic system similar to that of cancer (Non-Patent Documents 1-3). Meanwhile, despite the presence of the placenta (fetal tissue), the mother maintains a consistent immune response to infections, and the trophoblast ceases to invade within the myometrium, thereby maintaining the health of both the mother and the fetus. Therefore, by elucidating the mechanisms of pregnancy immunity and cancer immunity and utilizing the differences between them, it may be possible to halt cancer invasion and release immunosuppression without weakening the immune system, leading to groundbreaking therapies.

[0003] In cancer-bearing conditions, particularly in cancer cachexia, the inflammation associated with cancer progression, such as TNF-α and IL-6, produces the anti-inflammatory steroid hormone glucocorticoids (GCs), which suppress the overall adaptive immune system (reducing the proportion of killer cells and antibody production). However, in pregnancy-related immunity, this effect may be milder and more reversible (reducing antibody production only, recovering from GVHD, etc.). We believe that these differences are due to the presence of a concentration gradient of progesterone (PG), a pregnancy-related steroid hormone produced in large quantities in the human placenta. Among pregnancy-related hormones, PG and its derivatives inhibit tumor cell proliferation in a concentration-dependent manner (Non-Patent Documents 4 and 5), while regulating the activation of immune cells (Non-Patent Documents 6-13).

[0004] In recent years, the development of molecularly targeted cancer drugs based on immune checkpoint antibodies has led to significant advances in the treatment of refractory solid tumors. For a long time, much research has been conducted based on the hypothesis that tumor-bearing is associated with immune system suppression, and therefore that cancer-specific immune activation could eliminate cancer. However, most of these approaches have only achieved significant results through passive immunization with antibodies such as HER2. However, antibodies directed against immune checkpoint molecules, such as ipilimumab (CTLA-4 antibody), nivolumab (PD-1), and adesolizumab (PD-L1), have demonstrated groundbreaking antitumor effects, leading to new advances in cancer treatment (Non-Patent Documents 14 and 15). These results suggest a strong correlation between cancer and immune system abnormalities, and that relieving immune system suppression plays a crucial role in anticancer activity. However, due to limited success, combination checkpoint blockade (CCB) approaches using antibodies against both CTLA-4 and PD-1 have also been attempted in recent years. However, this therapy has been associated with a severe side effect, immune-related adverse events (IRAEs), which have become a major problem for this treatment. Various analyses of patient lymphocytes have been conducted to assess the risk of these side effects, but no clear indicators have been established.

[0005] Hayakawa S. No cancer in cancers: evolutionary trade-off bet weensuccessful viviparity and tumor escape from the adaptive immune system. Med Hypotheses (2006) 66:888-97.Racicot K, JY K, Aldo P, Silasi M, Mor G. Understanding the complexity of the immunesystem during pregnancy. Am J ReprodImmunol (2014) 72:107-16.Haig D. Maternal-fetal conflict, genomic imprinting and mamm alian vulnerabilities to cancer. PhilosTrans R Soc Lond B Biol Sci (2015) 370:20140178.Yahya S, Abdelhamid A, Abd-Elhalim M, Elsayed G, Eskander E. The effect of newly synthesized progesterone derivatives on apoptotic and a ngiogenic pathway in MCF-7 breast cancer cells. Steriods (2017) 126:15-23.You S, Zuo L, Li W. Optimizing the time of Doxil injectiont o increase the drug retentionin transplanted murine mammary tumors. Int J N anomedicine (2010) 5:221-9.Ndiaye K, Poole D, Walusimbi S, Cannon M, Toyokawa K, Maalou f S, et al. Progesterone effects on lymphocytes may be mediated by membrane progesterone receptors.J Reprod Immunol (2012) 95:15-26.Shah N, Imamiand N, Johnson M. Progesterone Modulation of Pr egnancy-related immune responses Front in Immunol (2018) 9:1293.Hierweger A, Engler J, Friese M, Reichardt H, Lydon J, DeMay o F, et al. Progesterone modulates the T cell response via glucocorticoid re ceptor-dependent pathways. Am J Reprod Immunol (2019) 81:e13084.Yao Y, Li H, Ding J, Xia Y, Wang L. Progesterone impairsant igen-non-specific immune protection by CD8 T memory cellsviainterferon-γ g ene hypermethylation. PLoS Pathog (2017) 13:e1006736.Monteiro C, Kasahara T, Sacramento P, Dias A, Leite S, Sil va V, et al. Human pregnancy levels of estrogen and progesterone contribute to humoralimmunity by activating TFH / B cell axis. Eur J Immunol (2020) ahea d of print.Piccinni M-P, Ciudizi M-C, Biagiotti R, Beloni L, Giannari ni L, Sampognaro S, et al.Progesterone Favors the Development of Human T He lper Cells Producing Th2-Type Cytokinesand Promotes Both 11-4 Production an d Membrane CD30 Expression in Established Thl Cell Clones. J Immunol (1995) 155:128-33.Mauvais-Jarvis F, Klein S, Ellis R. Levin E. Estradiol, Pr ogesterone, Immunomodulation, and COVID-19Outcomes. Endocriology (2020) 161:1-8.Polikarpova A, Levin I, Sigai N, Zavarzin I, Morozovc I, R ubtsov P, et al. Immunomodulatory effects of progesterone and selective liga nds of membrane progesterone receptors. Steroids (2019) 145:5-18.Patel S, Minn A. Combination CancerTherapy with ImmuneCh eckpoint Blockade: Mechanisms and Strategies. Immunity (2018) 48:417-33.He X, Xu C. Immune checkpoint signaling and cancerimmunot herapy. Cell Res (2020) 30:660-9.Miyako H, Kametani Y, Katano I, Ito R, Tsuda B, Furukawa A, et al. Antitumor effect of new HER2 peptidevaccination based on B cell ep itope. Anticancer Res (2011) 31(10):361-3368.Kametani Y, Shiina M, Katano I, Ito R, Ando K, Toyama K, et al. Development of human-human hybridoma from anti-Her-2 peptide-producing B cells in immunized NOG mice. Exp Hematol (2006) 34(9):1240-8.

[0006] An object of the present invention is to provide a novel activity modulator capable of regulating the activity of T cells and / or B cells.An object of the present invention is to provide a combination agent in which the above-mentioned T cell and / or B cell activity modulator is used in combination with an anti-human PD-L1 monoclonal antibody.

[0007] In the present invention, immunoregulation is achieved by prescribing progesterone or its derivatives to treat pathologies induced by immune system activation, such as autoimmune disease-like side effects caused by immune checkpoint inhibitors (ICIs). The inventors suspected that progesterone could be used to control IRAEs. First, the inventors elucidated the differences in immunosuppression between progesterone and glucocorticoids, then elucidated the effects of combined use of progesterone and atezolizumab on the immune system, and further developed a conjugate of atezolizumab and liposomes encapsulating progesterone, thereby successfully achieving more efficient immunoregulation. The present invention was completed based on the above findings. Specifically, the present invention provides the following: <1> A combination agent for use in combination therapy with an anti-human PD-L1 monoclonal antibody, the active ingredient of which is a T cell and / or B cell activity modulator containing progesterone or a derivative thereof as an active ingredient. <2> The combination drug according to <1>, wherein the T cell and / or B cell activity modulator acts reversibly on T cells and / or B cells. <3> The combination drug according to <1> or <2>, wherein the T cell and / or B cell activity modulator maintains the expression of CD62L and localizes T cells in lymph nodes. <4> The combination drug according to any one of <1> to <3>, wherein the T cell and / or B cell activity modulator reduces the expression of PD-1 in T cells and / or B cells. <5> The combination drug according to any one of <1> to <4>, wherein the expression of PD-L1 in T cells and / or B cells is suppressed. <6> The combination drug according to any one of <1> to <5>, wherein the expression of PD-1 and PD-L1 in T cells and / or B cells is suppressed. <7> The combination drug according to any one of <1> to <6>, wherein the anti-human PD-L1 monoclonal antibody is a humanized anti-human PD-L1 monoclonal antibody. <8> The combination drug according to any one of <1> to <7>, wherein the humanized anti-human PD-L1 monoclonal antibody is atezolizumab. <9> A pharmaceutical composition comprising, as active ingredients, a T cell and / or B cell activity modulator containing progesterone or a derivative thereof as an active ingredient and an anti-human PD-L1 monoclonal antibody. <10> The pharmaceutical composition according to <9>, wherein the T cell and / or B cell activity modulator acts reversibly on T cells and / or B cells.<11> The pharmaceutical composition according to <9> or <10>, wherein the agent for regulating T cell and / or B cell activity maintains the expression of CD62L and localizes T cells in lymph nodes. <12> The pharmaceutical composition according to any one of <9> to <11>, wherein the agent for regulating T cell and / or B cell activity reduces the expression of PD-1 in T cells and / or B cells. <13> The pharmaceutical composition according to any one of <9> to <12>, wherein the expression of PD-L1 in T cells or B cells is suppressed. <14> The pharmaceutical composition according to any one of <9> to <13>, wherein the expression of PD-1 and PD-L1 in T cells or B cells is suppressed. <15> The pharmaceutical composition according to any one of <9> to <14>, wherein the anti-human PD-L1 monoclonal antibody is a humanized anti-human PD-L1 monoclonal antibody. <16> The pharmaceutical composition according to any one of <9> to <15>, wherein the humanized anti-human PD-L1 monoclonal antibody is atezolizumab. <17> An immunoliposome comprising a liposome containing a T cell and / or B cell activity modulator having progesterone or a derivative thereof as an active ingredient, wherein an anti-human PD-L1 monoclonal antibody is bound to the membrane of the liposome. <18> The immunoliposome according to <17>, wherein the T cell and / or B cell activity modulator acts reversibly on T cells and / or B cells. <19> The immunoliposome according to <17> or <18>, wherein the T cell and / or B cell activity modulator maintains the expression of CD62L and localizes T cells to lymph nodes. <20> The immunoliposome according to any one of <17> to <19>, wherein the T cell and / or B cell activity modulator reduces the expression of PD-1 in T cells and / or B cells. <21> The immunoliposome according to any one of <17> to <20>, which suppresses the expression of PD-L1 in T cells and / or B cells. <22> The immunoliposome according to any one of <17> to <21>, which suppresses the expression of PD-1 and PD-L1 in T cells and / or B cells. <23> The immunoliposome according to any one of <17> to <22>, wherein the anti-human PD-L1 monoclonal antibody is a humanized anti-human PD-L1 monoclonal antibody. <24> The immunoliposome according to any one of <17> to <23>, wherein the humanized anti-human PD-L1 monoclonal antibody is atezolizumab.<25> A pharmaceutical composition comprising the immunoliposome according to any one of <17> to <24>.

[0008] This specification includes the disclosure of Japanese Patent Application No. 2021-128012, from which this application claims priority.

[0009] The combination drug of the present invention can regulate the activation of T cells and / or B cells, thereby enabling efficient immunoregulation.

[0010] Figure 1 shows the relationship between P4 / COR concentration and the proliferation ability of various cell lines. The left two panels show the human choriocarcinoma cell lines JEG-3 and BeWo and the kidney-derived cell line HEK293. The right two panels show the mouse myeloma cell line P3X, the lymphoma cell line A20, and human PBMC. P4 and COR were added at concentrations ranging from 0 to 200 μM and cultured for 10 or 3 days. PBMCs were cultured for 3 days. Figure 2 shows the relationship between P4 / COR concentration and PD-1 expression (%) on activated T cells. PBMCs were analyzed by FCM after 3 days of culture. The left two panels show the CD25 and PD-1 expression patterns of T cells obtained by FCM. The upper panel shows the gate on CD4 T cells, and the lower panel shows the gate on CD8 T cells, respectively, showing the percentage of CD25- and PD-1-negative (double negative; DN) and positive (double positive; DP) fractions among CD4 T cells. The steroid concentration-dependent proportions of each cell fraction are plotted on the vertical axis of the right figure. The left two panels for CD4 and CD8, respectively, are DN cells (P4 and COR), and the right two panels are DP cells (P4 and COR). For P4, the horizontal axis represents the concentration of COR or P4 added, showing the relationship between P4 and COR concentrations. Figure 3 shows CD25 / PD-1 / PD-L1 expression in activated T cells. PBMCs were cultured for 3 days, analyzed by FCM. The upper panel shows the development of PD-1 and CD25, and the lower panel shows the development of PD-1 and PD-L1. The left two panels were cultured with 20 μM COR, and the right panel with 20 μM P4. CD4 T cells and CD8 T cells were further analyzed. The square in the upper right represents DP cells. Figure 4 shows cytokine production in PBMCs activated in the presence of P4. PBMCs were cultured for 3 days, analyzed by FCM. The vertical axis represents the concentration (pg / mL) of each cytokine in the culture supernatant under P4 0-200 μM conditions. Figure 5 shows the relationship between P4 / COR pretreatment and the percentage of activated T cells.After 6 hours of incubation with 200 μM P4 / COR, the cells were washed, and TSST-1 was added with or without P4 / COR. After 3 days of incubation, PBMCs were analyzed by FCM. The upper panel shows the percentage of PD-1 / PD-L1 DN and DP fractions in CD4 T cells, and the lower panel shows the percentage of PD-1 / PD-L1 DN and DP fractions in CD8 T cells. For each fraction, the left panel shows cells cultured with 200 μM COR, and the right panel shows cells cultured with 200 μM P4. Figure 6 shows the relationship between P4 / COR pretreatment and the percentage of B cells. After 6 hours of incubation with 200 μM P4 / COR, the cells were washed, and TSST-1 was added with or without P4 / COR. After 3 days of incubation, PBMCs were analyzed by FCM. The left two panels show PD-L1 SP cells, and the right two panels show PD-1 / PD-L1DP cells. The left panel shows the fractions cultured with 200 μM COR, and the right panel shows the fractions cultured with 200 μM P4. Figure 7 shows the human cell engraftment potential in PBMC-NOG mice treated with P4 / COR. Four weeks after transplantation of human PBMCs, mice were continuously subcutaneously administered with P4 or COR twice weekly. The graph on the left shows the average cell count, and the photograph on the right shows immunohistochemical staining of mouse lung sections (including bronchi) using an anti-human CD45 antibody (a leukocyte marker). Figure 8 shows the human cell engraftment potential in PBMC-NOG mice transplanted with PBMCs cultured briefly with P4 or COR. Mice were transplanted with human PBMCs cultured with 200 μM P4 / COR for 6 hours and immunized biweekly with CH401MAP. The left panel shows the number of spleen cells and the right panel shows the specific antibody titers in plasma after 4 weeks. Figure 9 shows CD62L expression on splenic human lymphocytes in mice transplanted with PBMCs cultured for a short period with P4 or COR, as described in Figures 7 and 8. Expression of CD62L on lymphocytes was confirmed by FCM.The top panel shows CD62LMFI (CD45 gate) expressed by human CD45-positive cells in splenocytes from NOG-hIL-4-Tg-humanized mice 4 weeks after transplantation. The bottom panel shows CD62LMFI (CD45 gate) expressed by human CD45-positive cells in splenocytes from NOG-humanized mice 4 weeks after transplantation. Numbers indicate the percentage of positive cells. Figure 10 shows the relationship between atezolizumab and PD-L1 expression on lymphocytes. Human PBMCs were cultured in medium containing 0-200 μM P4 / COR and 100 μg / mL atezolizumab for 3 days, and PD-L1 expression on lymphocytes was confirmed by FCM. The top two panels show T cells (CD3 gate), and the bottom two panels show B cells (CD19 gate). The top row of each column represents the treatment without atezolizumab (Atz(-)), and the bottom row represents the treatment with atezolizumab (Atz(+)). The vertical axis represents the number of counts, and the horizontal axis represents the expression level of PD-L1. Figure 11 shows the relationship between atezolizumab and PD-1 expression on lymphocytes. Human PBMCs were cultured for 3 days in medium containing 0-200 μM P4 / COR and 100 μg / mL atezolizumab, and PD-1 expression on lymphocytes was confirmed by FCM. The top two panels represent T cells (CD3 gate), and the bottom two panels represent B cells (CD19 gate). The top row of each column represents the treatment without atezolizumab (Atz(-)), and the bottom row represents the treatment with atezolizumab (Atz(+)). The vertical axis represents the number of counts, and the horizontal axis represents the expression level of PD-1. Figure 12 shows the regulation of PBMC activation by atezolizumab and P4: cell proliferation. Human PBMCs were cultured for 3 days in medium containing 0-200 μM P4 and 100 μg / mL atezolizumab, or in D2125 containing 20 μM P4 equivalent. Changes in cell number and the degree of aggregation due to cell activation were examined. The upper left panel shows the protocol, the lower left panel shows photographs, and the right panel shows cell counts on day 3. A significance test was performed, but no significant differences were found. Figure 13 shows the modulation of PBMC activation (T cells) by P4 and D2125. Human PBMCs were cultured for 3 days as described in Figure 12, and PD-1 / PD-L1 expression on lymphocytes was confirmed by FCM.The upper panel shows the percentages of PD-1-positive cells, PD-L1-positive cells, PD-1 / PD-L1-negative cells, and PD-1 / PD-L1-positive cells in CD3-gated cells. The bottom two panels show the expression levels (MFI) of PD-1 and PD-L1 in CD3-gated cells. By t-test, p<0.05*, p<0.01**, and p<0.005*** were obtained. Figure 14 shows the modulation of PBMC activation (CD4 / CD8 T cells) by P4 and D2125. Human PBMCs were cultured for 3 days as described in Figure 11, and the percentages of PD-1-positive cells, PD-L1-positive cells, PD-1 / PD-L1-negative cells, and PD-1 / PD-L1-positive cells in CD3-gated and CD4-gated cells or CD8-gated cells are shown. CD4 (left panel) and CD8 (right panel). p<0.05*, p<0.01**, p<0.005*** by t-test. Figure 15 shows the results of measuring CD19+ B cells, CD3+ T cells, CD4+ helper T cells, and CD8+ killer T cells in non-tumor-bearing mice (CMV-NOG-hIL-4-Tg mice) and tumor-bearing mice (CMV-NOG-hIL-4-Tg mice transplanted with the human breast cancer cell line MDA-MB-231 and human PBMCs) administered atezolizumab (Atz) or atezolizumab-bound liposome-encapsulated P4 (Lipo-P4). p<0.05*, p<0.01** by t-test. Figure 16 shows the results of histochemical staining of tumor tissues after administration of atezolizumab (Atz) or atezolizumab-bound liposome-encapsulated P4 (Lipo-P4) to tumor-bearing mice (CMV-NOG-hIL-4-Tg mice transplanted with the human breast cancer cell line MDA-MB-231 and human PBMCs). Figure 17 shows the results of measuring tumor diameters after administration of atezolizumab (ATZ) or atezolizumab-bound liposome-encapsulated P4 to tumor-bearing mice (CMV-NOG-hIL-4-Tg mice transplanted with the human breast cancer cell line MDA-MB-231 and human PBMCs).

[0011] The present invention will be described in further detail below. <T cell and / or B cell activity regulator> The T cell and / or B cell activity regulator of the present invention contains progesterone or a derivative thereof as an active ingredient.

[0012] The modulators of T cell and / or B cell activity of the present invention can act reversibly on T cells and / or B cells. The modulators of T cell and / or B cell activity of the present invention can maintain CD62L expression and localize T cells to lymph nodes. The modulators of T cell and / or B cell activity of the present invention can reduce PD-1 expression in T cells and / or B cells, and can also suppress PD-L1 expression in T cells and / or B cells.

[0013] The progesterone or derivative thereof may be a natural or synthetic compound, and may be progesterone, a progesterone metabolite (e.g., 17α-hydroxyprogesterone, etc.), or any other progestin. When a progestin is used, the synthetic progesterone is preferably selected from the group consisting of derivatives of progesterone or testosterone, or derivatives of other molecules and / or compounds with progestogenic activity. A derivative refers to a chemical compound that is derived from or results from one or more chemical reactions of the parent compound. The progestin may be natural progesterone, synthetic progesterone, a natural or synthetic derivative of progesterone and / or other progestogenic compounds, or a combination thereof.

[0014] Specific examples of progestins include, but are not limited to, 17α-hydroxyprogesterone caproate, medroxyprogesterone acetate, norethindrone, norethindrone acetate, norethindrone enanthate, desogestrel, levonorgestrel, lynestrenol, ethynodiol diacetate, norgestrel, norgestimate, norethynodrel, gestodene, drospirenone, trimegestone, levodesogestrel, gestodrin, nestrone, etonogestrel, and derivatives of 19-nortestosterone.

[0015] The method of administration of the modulator of T cell and / or B cell activity is not particularly limited and may be either oral or parenteral. Parenteral administration includes, but is not limited to, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, or intramuscular administration. The modulator of T cell and / or B cell activity of the present invention can be administered, for example, by injection. A solution for injection can be formulated using a carrier such as a salt solution, a glucose solution, a mixture of saline and glucose solution, or various buffer solutions. Alternatively, the modulator may be formulated in a powder form and mixed with the liquid carrier at the time of use to prepare an injection solution.

[0016] The modulators of T cell and / or B cell activity of the present invention can be prepared by known methods using pharmaceutically acceptable carriers depending on the dosage form, such as excipients, binders, disintegrants, lubricants, diluents, solubilizers, suspending agents, isotonicity agents, pH adjusters, buffers, stabilizers, colorants, flavorings, and odorants.

[0017] For oral administration, oral liquids, powders, pills, capsules, tablets, etc. can be used. Oral liquids can be prepared as oral liquid preparations such as suspensions and syrups using water, sugars such as sucrose, sorbitol, fructose, etc., glycols such as polyethylene glycol, oils such as sesame oil and soybean oil, preservatives such as alkyl parahydroxybenzoate, flavors such as strawberry flavor and peppermint, etc. Powders, pills, capsules, and tablets can be formulated using excipients such as lactose, glucose, sucrose, mannitol, etc., disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin, etc., surfactants such as fatty acid esters, plasticizers such as glycerin, etc.

[0018] The modulators of T cell and / or B cell activity of the present invention can be administered in a therapeutically effective amount, for example, at a daily dose of 1 μg / kg body weight to 1000 mg / kg body weight.

[0019] The T cell and / or B cell activity modulator of the present invention can be used in combination therapy with an anti-human PD-L1 monoclonal antibody. That is, the present invention provides a pharmaceutical composition containing the T cell and / or B cell activity modulator of the present invention and an anti-human PD-L1 monoclonal antibody as active ingredients.

[0020] A monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population. A monoclonal antibody may be a chimeric antibody, in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies.

[0021] The monoclonal antibody may be prepared by the hybridoma method or the recombinant DNA method. As the monoclonal antibody, its fragment (e.g., F(ab')2, Fab', Fab, Fv, sFv, etc.) may also be used.

[0022] The anti-human PD-L1 monoclonal antibody is preferably a humanized anti-human PD-L1 monoclonal antibody. Antibody humanization techniques generally involve using recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptides of an antibody molecule. A humanized non-human antibody (or fragment thereof) is a chimeric antibody or chimeric antibody chain (or a fragment thereof such as sFv, Fv, Fab, Fab', F(ab')2, or other antigen-binding portion of an antibody) that contains a portion of the antigen-binding site derived from a non-human (donor) antibody incorporated into the framework of a human (recipient) antibody.

[0023] To generate a humanized antibody, residues from one or more complementarity-determining regions (CDRs) of a recipient (human) antibody molecule are replaced with residues from one or more CDRs of a donor (non-human) antibody molecule known to have the desired antigen-binding properties (e.g., a particular level of specificity and affinity for the target antigen). In some cases, Fv framework (FR) residues of the human antibody are replaced with corresponding non-human residues. A humanized antibody may also contain residues that are not found in the recipient antibody or in the imported CDR and framework sequences. Humanized antibodies generally contain one or more amino acid residues introduced from a non-human source. Humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted with residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically the Fc of a human antibody.

[0024] Methods for humanizing non-human antibodies are known. For example, humanized antibodies can be made by following the method of Winter et al. (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)) by substituting rodent CDR(s) or CDR sequences for the corresponding sequences of a human antibody.

[0025] Examples of anti-human PD-L1 monoclonal antibodies include the human PD-L1 monoclonal antibody human atezolizumab (trade name Tecentriq), the human PD-L1 monoclonal antibody avelumab (trade name Bavencio), and the human PD-L1 monoclonal antibody durvalumab (trade name Imfinzi), with atezolizumab being particularly preferred.

[0026] The amount of anti-human PD-L1 monoclonal antibody used will vary depending on the type of anti-human PD-L1 monoclonal antibody used in combination, the administration method, the patient's symptoms, age, etc., but for example, in the case of oral administration, it can be from 0.01 mg / kg body weight (preferably 0.1 mg / kg body weight) to 1000 mg / kg body weight (preferably 100 mg / kg body weight) per dose, and in the case of intravenous administration, it can be from 0.001 mg / kg body weight (preferably 0.01 mg / kg body weight) to 1000 mg / kg body weight (preferably 100 mg / kg body weight) per dose. The number of doses can be one to several times per day.

[0027] An example of the pharmaceutical composition of the present invention is an immunoliposome composed of a liposome containing the T cell and / or B cell activity modulator of the present invention, in which an anti-human PD-L1 monoclonal antibody is bound to the membrane of the liposome, or an immunoliposome in which an anti-human PD-1 monoclonal antibody is bound to the membrane of the liposome.

[0028] Liposomes are lipid structures formed by amphiphilic vesicle-forming lipids. Liposomes are typically closed vesicles composed of a single or multiple lipid bilayer with an internal aqueous phase. A lipid bilayer is a structure in which the hydrophobic regions of polar lipid molecules associate with each other, with the hydrophobic regions facing toward the center of the bilayer and the hydrophilic regions facing toward the aqueous phase. An immunoliposome is a complex formed by a liposome and a protein (such as an antibody).

[0029] The liposome of the present invention is preferably composed of amphiphilic vesicle-forming lipids, including phospholipids, glycolipids, sphingolipids, sterols, glycols, saturated or unsaturated fatty acids, surfactants, and lipid derivatives having hydrophilic polymers.

[0030] Phospholipids are broadly classified into glycerophospholipids and sphingophospholipids. Representative glycerophospholipids include phospholipids with at least one head group, such as phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidic acid (PA). On the other hand, representative sphingophospholipids include sphingomyelin.

[0031] Among the above, examples of the phosphatidylcholines include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylcholine (DLPC), didecanoylphosphatidylcholine (DDPC), dioctanoylphosphatidylcholine (DOPC), dihexanoylphosphatidylcholine (DHPC), dibutyrylphosphatidylcholine (DBPC), dielaidoylphosphatidylcholine, dilinoleo ... oleoyl phosphatidylcholine, diarachidonoyl phosphatidylcholine, diicosenoyl phosphatidylcholine (DEPC), diheptanoyl phosphatidylcholine, dicaproyl phosphatidylcholine, diheptadecanoyl phosphatidylcholine, dibehenoyl phosphatidylcholine, eleostearoyl phosphatidylcholine, hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC), 1-palmitoyl-2-arachidonoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, 1-palmitoyl-2-linoleoyl phosphatidylcholine, 1-palmitoyl-2-myristoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1,2-dimyristoylamido-1, Examples of such phosphatidylcholine include 2-deoxyphosphatidylcholine, 1-myristoyl-2-palmitoylphosphatidylcholine, 1-myristoyl-2-stearoylphosphatidylcholine, di-0-hexadecylphosphatidylcholine, trans-dielaidoylphosphatidylcholine, dipalmiteroylphosphatidylcholine, n-octadecyl-2-methylphosphatidylcholine, n-octadecylphosphatidylcholine, 1-laurylpropanediol-3-phosphocholine, erythro-N-lignoceroylsphingophosphatidylcholine, and palmitoyl-(9-cis-octadecenoyl)-3-sn-phosphatidylcholine.

[0032] Examples of phosphatidylethanolamines (cephalins) include dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylethanolamine (DMPE), didecanoylphosphatidylethanolamine (DDPE), N-glutarylphosphatidylethanolamine (NGPE), lysophosphatidylethanolamine, N-(7-nitro-2,1,3-benzoxydiazol-4-yl)-1,2-dioleoyl-sn-phosphatidylethanolamine, eleostearoylphosphatidylethanolamine, N-succinyldioleoylphosphatidylethanolamine, and 1-hexadecyl-2-palmitoylglycerophosphatidylethanolamine.

[0033] Glycolipids that can be used include glyceroglycolipids, sphingoglycolipids, and other glycolipids. Sterols that can be used include cholesterol. Neutral lipids that can be used include diglycerides (e.g., diolein, dipalmitolein).

[0034] Examples of saturated or unsaturated fatty acids that can be used include caprylic acid, pelargonic acid, capric acid, undecylenic acid, lauric acid, tridecylenic acid, myristic acid, pentadecylenic acid, palmitic acid, margaric acid, stearic acid, nonadecylenic acid, arachidic acid, dodecenoic acid, tetradecenoic acid, oleic acid, linoleic acid, linolenic acid, eicosenoic acid, erucic acid, and docosapentaenoic acid. Examples of charged lipids that can be used include anionic lipids and cationic lipids. Examples of surfactants that can be used include cationic surfactants, anionic surfactants, amphoteric surfactants, and nonionic surfactants.

[0035] The derivative lipid having a hydrophilic polymer can be one consisting of the above-mentioned lipid and hydrophilic polymer, in which the lipid and the hydrophilic polymer are bound together by forming a covalent bond between a functional group of the lipid and a functional group of the hydrophilic polymer, either directly or via a linker.

[0036] Examples of lipid derivatives of hydrophilic polymers include, but are not limited to, polyethylene glycol-modified lipids, polyethyleneimine derivatives, polyvinyl alcohol derivatives, polyacrylic acid derivatives, polyacrylamide derivatives, dextran derivatives, polyglycerin derivatives, chitosan derivatives, polyvinylpyrrolidone derivatives, polyaspartic acid amide derivatives, poly-L-lysine derivatives, mannan derivatives, and pullulan derivatives.

[0037] In the present invention, an anti-human PD-L1 monoclonal antibody or an anti-human PD-1 monoclonal antibody can be bound to the membrane of the liposome. The binding of the anti-human PD-L1 monoclonal antibody or the anti-human PD-1 monoclonal antibody to the membrane of the liposome can be achieved by adding the anti-human PD-L1 monoclonal antibody or the anti-human PD-1 monoclonal antibody to a liposome solution and allowing it to react.

[0038] Functional agents may also be added to immunoliposome components. Examples of functional agents include membrane stabilizers, membrane surface hydrophilicity adjusters, curvature adjusters, antioxidants, charge-imparting agents, and cryoprotectants. Examples include stabilizers such as sugars, glycolipids, glycerin, and polyethylene glycol, and antioxidants such as tocopherol and ascorbic acid. Cholesterols can be used as membrane stabilizers, membrane surface hydrophilicity adjusters, or liposome curvature adjusters, while tocopherols can be used as antioxidants. Any other liposome-stabilizing compound may replace cholesterol. Other liposome-stabilizing compounds are known in the art. For example, saturated phospholipids produce liposomes with a high transition temperature. To avoid limiting electrostatic association between the antigen and the liposome, antibodies and progesterone may be sequestered inside the liposome.

[0039] The present invention also provides pharmaceutical compositions comprising a therapeutically effective amount of immunoliposomes and pharmaceutically acceptable diluents, carriers, solubilizers, emulsifiers, preservatives and / or adjuvants. The pharmaceutical compositions of the present invention may include formulation agents to alter, maintain or preserve pH, osmolality, viscosity, clarity, color, isotonicity, sterility, stability, dissolution rate, sustained-release rate, absorption rate or permeability rate.

[0040] Formulation materials can include, but are not limited to, natural lipids, synthetic lipids, sphingolipids, ether lipids, sterols, cardiolipin, cationic lipids, and lipids modified with poly(ethylene glycol) and other polymers. Synthetic lipids can include the following fatty acid moieties: lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, oleoyl, linoleoyl, erucoyl, or combinations of these fatty acids. Amino acids such as glycine, alanine, glutamine, asparagine, arginine or lysine, antibacterial agents, antioxidants such as ascorbic acid, sodium sulfate or sodium bisulfite, buffers such as phosphoric acid, citric acid, borate buffer, bicarbonate, Tris-HCl solution, bulking agents such as mannitol or glycine, chelating agents such as ethylenediaminetetraacetic acid (EDTA), complexing agents such as caffeine, polyvinylpyrrolidine, β-cyclodextrin or hydroxypropyl-β-cyclodextrin, bulking agents such as glucose, mannose or dextrin, other carbohydrates such as monosaccharides, disaccharides, glucose, mannose or dextrin, colorants, flavoring agents, diluents, emulsifiers and hydrophilic polymers such as polyvinylpyrrolidine, low molecular weight polypeptides, salt forming agents counterions, preservatives such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorexidine, sorbic acid, or hydrogen peroxide; solvents such as glycerin, propylene glycol, or polyethylene glycol; sugar alcohols such as mannitol or sorbitol; suspending agents; surfactants such as PEG, sorbitan esters, polysorbates such as polysorbitate 20 or polysorbitate 80, triton, tromethamine, lecithin, or cholesterol; stabilizing enhancers such as sucrose or sorbitol; elasticity enhancers such as sodium chloride, potassium chloride, or mannitol sorbitol; transport agents, diluents, excipients, and / or pharmaceutical adjuvants.

[0041] The amount of these pharmaceutical substances added is preferably 0.01 to 100 times, and particularly preferably 0.1 to 10 times, the weight of the immunoliposome. The excipients and carriers in the pharmaceutical composition may be liquid or solid. The excipients and carriers may be water for injection or physiological saline. Pharmaceutical compositions can be prepared as lyophilized products or liquids, with the selected composition and required purity, as appropriate drugs.

[0042] The T cell and / or B cell activity modulator, concomitant agent, and pharmaceutical composition of the present invention can be used for one or more types of cancer selected from carcinoma, sarcoma, lymphoma, leukemia, myeloma, germ cell tumor, brain tumor, carcinoid, neuroblastoma, retinoblastoma, and nephroblastoma. Specific examples of carcinoma include kidney cancer, malignant melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival cancer, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, colon cancer, rectal cancer, appendix cancer, anal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer, and vaginal cancer. Specific examples of sarcoma include liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, undifferentiated polymorphic sarcoma, and Examples of cancers include myxoid fibrosarcoma, malignant peripheral nerve sheath tumor, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, and epithelioid sarcoma. Lymphomas include B-cell lymphoma, T / NK cell lymphoma, and Hodgkin's lymphoma. Leukemias include myeloid leukemia, lymphocytic leukemia, myeloproliferative disorders, and myelodysplastic syndromes. Myelomas include multiple myeloma. Germ cell tumors include testicular cancer and ovarian cancer. Brain tumors include glioma and meningioma. In particular, breast cancer, lung cancer (especially non-small cell lung cancer and small cell lung cancer), hepatocellular carcinoma, urothelial carcinoma, and malignant melanoma are also included.

[0043] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0044] Example 1: Cell Lines and Mice JEG-3, HEK293, BeWo, P3X, and A20 were all stored in the Department of Molecular Life Sciences, Department of Basic Medicine, Tokai University School of Medicine. JEG-3, HEK293, and BeWo were cultured in D-MEM, while P3X and A20 were cultured in RPMI1640. NOG mice were purchased from Invivo Science (Kawasaki, Japan). NOG-hIL-4-Tg mice were maintained in an isolator at the Tokai University School of Medicine Laboratory Animal Facility. From these mice, mice with human IL-4 concentrations of 100 pg / mL or higher as determined by DNA typing and ELISA were selected for transplantation.

[0045] Human peripheral blood mononuclear cells (PBMCs) were prepared as follows.

[0046] Thirty milliliters of heparinized peripheral blood was collected from healthy donors using Vacutainer ACD tubes (NIPRO Corporation, Osaka, Japan). The collected peripheral blood was immediately layered on Ficoll-Hypaque (SI GMA-ALDRICH, London, UK), and the mononuclear cell fraction was collected by density centrifugation (500 × g, 30 min, 20°C). The cells were washed with PBS by centrifugation at 300 × g, 5 min, 4°C, and then counted for use.

[0047] The various cell lines were cultured as follows.

[0048] Human choriocarcinoma cell lines JEG-3 and BeWo, and embryonic kidney cell line HEK293 were cultured in DMEM (Gibuco) supplemented with 10% fetal calf serum (FCS) with various concentrations of water-soluble progesterone (P4; water-soluble) (Sigma) or cortisol (COR) (Sigma) for 10 days at 37°C and 5% CO2. Mouse myeloma cell line P3-X63-Ag8-U1 (P3X) and mouse lymphoma cell line A20 were cultured in RPMI1640 supplemented with 10% FCS with various concentrations of P4 or COR for 3 days at 37°C and 5% CO2. Cell proliferation was assessed by cell counting.

[0049] Human peripheral blood mononuclear cells (PBMCs) were cultured as follows.

[0050] PBMCs (final concentration 1 x 106 / mL) were cultured in RPMI 1640 supplemented with 10% FCS, supplemented with various concentrations of P4 or COR, and stimulated with toxic shock syndrome toxin-1 (TSST-1) at 1 μg / mL for 3 days at 37°C and 5% CO2. These cells were harvested, washed with PBS, and then aliquoted into Fisher tubes at 1 x 106 cells / tube. For analysis of human cell surface markers, fluorescently labeled anti-human monoclonal antibodies (mAbs) were used. Cells were stained with these mAbs for 15 min at 4°C, then washed with PBS containing 1% (w / v) BSA. Analysis was then performed using a FACS Fortessa or FACS Verse (BD Bioscience, Franklin Lakes, NJ). For each analysis, cells in the leukocyte or lymphocyte gate were further gated for human CD45 expression. A list of the antibodies used is shown in Table 1. The culture supernatant was collected, and 25 μL of the supernatant was taken and cytokines were quantified using BioLegend's Legend plex™ kit.

[0051]

[0052]

[0053] For restimulation experiments, PBMCs were cultured in RPMI 1640 medium supplemented with 10% FCS and 200 μM P4 or COR at 37°C and 5% CO for 6 hours. After washing, the cells were cultured at a final concentration of 1 x 10 6 The cells were cultured again at 1 μM TSST-1 in the presence or absence of 200 μM P4 or COR at 1 μM / mL. After 72 hours, the cells were harvested and analyzed by FCM.

[0054] For the atezolizumab addition experiment, 0-200 μM P4 or COR, 1 μM TSST-1, and 100 μg / mL atezolizumab were added to PBMCs, which were then cultured at 37°C and 5% CO2 for 72 hours and analyzed by FCM.

[0055] Transplantation into PBMC-NOG-hIL-4-Tg mice and analysis of engrafted human cells were performed as follows.

[0056] PBMCs were cultured in RPMI 1640 medium supplemented with 10% FCS and 200 μM P4 or COR at 37°C and 5% CO for 6 hours. After harvesting, the cells were washed and diluted to 2.5 × 10 6 Each PBMC was intravenously transplanted into 8- to 9-week-old NOG-hIL-4-Tg mice (blood hIL-4 concentration 100 pg / mL or more).

[0057] The CH401 MAP peptide is a 20-mer MAP peptide containing a partial sequence of HER2 / neu as the epitope for anti-HER2 monoclonal antibodies (Miyako H, et al., Anticancer Res (2011) 31(10):361-368). This peptide was synthesized using T Rink amide resin (0.4-0.7 mmol / g) with an ACT357 peptide synthesizer (Advanced Chemtech, Louisville, KY). The peptide was mixed with an equal volume of Freund's complete adjuvant (CFA) (Wako Pure Chemical Industries, Ltd, Osaka, Japan) (50 g / head, 100 μl 1:1 / v:v) to form an emulsion, which was then administered intraperitoneally to PBMC-NOG-hIL-4-Tg mice. As a negative control, an equal volume of PBS was emulsified and administered to mice in the same manner. Booster immunizations were performed 2 weeks after the primary immunization using Freund's incomplete adjuvant (IFA) (Wako Pure Chemical Industries, Ltd.). Two weeks later, mice were anesthetized, heparinized blood was collected, and the mice were sacrificed. The human leukocyte fractions from the collected lymphoid tissues were analyzed by flow cytometry (FCM). Plasma components were used to measure antibody titers by ELISA, and spleen cells were used to produce hybridomas.

[0058] PBMC-NOG-hIL-4-Tg mouse spleen cell hybridomas were prepared as follows. Spleen cells were prepared from immunized humanized NOG-hIL-4-Tg mice, lysed, and washed with PBS. These cells were mixed with P3X and fused by electrofusion using BEX CFB16-HB (BEX Co. Ltd., Tokyo, Japan) and an LF497P2 electrode. The electrofusion conditions were AC 30 V, 20 s, DC 350 V, 30 μs / 500 ms, DC cycle 3, AC 30 V, 7 s, fade on, in electrofusion buffer (0.3 M mannitol, 0.1 mM calcium chloride, and / or 0.1 mM magnesium chloride). Fused cells were cultured in HAT medium for 2 weeks. Quantification of antibody levels in the culture supernatant was performed using ELISA.

[0059] Protein quantification by ELISA was performed as follows. Human IL-4 protein levels were measured using the Human IL-4 ELISA Set BD OptEIA™ (BD Biosciences). IgG antibody levels were quantified as previously reported (Kametani Y, et al., Exp Hematol (2006) 34(9):1240-1248). Wells of microtiter plates (Sumiron, Tokyo, Japan) were coated with CH401MAP peptide dissolved in carbonate buffer (pH 9.5) and incubated overnight at 4°C for antigen adsorption. The wells were then washed with PBS-Tween (0.05% v / v) and incubated with 3% BSA-PBS for 2 hours at room temperature (RT). After three PBS-Tween washes, mouse plasma was added in 10-fold serial dilutions and incubated for 2 hours at RT. The plate was washed three times and biotin-conjugated mouse anti-human IgG mAb (BD Pharmingen, San Diego, USA) (1:3,000) was added. The plate was incubated at 37°C for 2 hours, then washed three times and streptavidin-horseradish peroxidase (1:50,000 v / v; BD Pharmingen) was added. The plate was incubated at RT for 1 hour, then washed and EIA substrate kit solution (Bio-Rad Laboratories, Hercules, CA, USA) was added. The reaction was stopped with 10% HCl, and the absorbance at 450 nm was measured.

[0060] The NOG graft-versus-host disease (GVHD) model was analyzed as follows.

[0061] For analysis using a GVHD model, PBMCs prepared using the same method as above were washed with PBS and then transplanted intravenously at 2.5 × 106 aliquots into 8-9 week-old NOG mice. P4 (2 mg / head) or COR (2 mg / head), or PBS as a negative control, was administered subcutaneously twice a week (every 3-4 days). Body weight was measured at the same time as administration. After 4 weeks, the mice were euthanized by cardiac blood collection under anesthesia. Human cells were prepared from various lymphoid organs, hemolyzed, and subjected to FCM. Lungs and livers were also isolated and subjected to immunohistochemical staining as described below.

[0062] Mouse tissues were fixed in 20% formalin (Wako Pure Chemical Industries, Ltd.) and embedded in paraffin. Paraffin blocks were thinly sectioned, deparaffinized, mounted on slides, and stained with hematoxylin and eosin (HE). For immunohistochemical staining with anti-human CD45 antibodies, slides were heat-treated at 97°C for 20 minutes and then incubated with 0.3% H2O2 / MetOH at room temperature for 10 minutes to remove endogenous peroxidase. Subsequently, the slides were blocked with 1% goat serum, and anti-human CD45 (Dako) was added and incubated overnight at 4°C. The slides were washed three times for 5 minutes with 0.01M PBS and then developed with DAB solution. After 3 minutes of washing in running water, the slides were stained with hematoxylin, dehydrated, cleared, and mounted.

[0063] Conjugates of P4-encapsulated liposomes and atezolizumab were prepared as follows. 2.0 mg / mL atezolizumab (in 10 mM PBS buffer, pH 7.4) was mixed with 500 mM EDTA to a final concentration of 5 mM and 2-mercaptoethanol to a final concentration of 50 mM, and the mixture was stirred. After incubation at 37°C for 90 minutes, the mixture was ultrafiltered (Amicon Ultra 15 10 kDa) with 10 mM HEPES buffer, pH 7.2 (containing 5 mM EDTA) to remove the 2-mercaptoethanol. The mixture was then filtered through a 0.22 μm filter (13 mm diameter).

[0064] Progesterone-encapsulating liposomes were prepared as follows.

[0065] A 50 mL recovery flask was fitted with a rotor, and lipids (DSPC: 42.5 mg, DSPE-PEG2000 MA: 13.8 mg) were dissolved in 7 mL of tBuOH melted at 50 °C and stirred in a 50 °C water bath for 5 minutes. 3.5 mL of a 4.0 mg / mL progesterone solution (dissolved in tBuOH) was added, followed by stirring in a 50 °C water bath for 5 minutes, freezing in liquid nitrogen for 5 minutes, and lyophilization. 7 mL of 10 mM HEPES, 150 mM NaCl (pH 7.2) was added and stirred at 37 °C for 1 hour. The mixture was sonicated using a bath sonicator, and samples were taken before and every 30 minutes to measure particle size and zeta potential. The particle size measurement was stopped after 2 hours (4 x 30 minutes) or if the particle size measurement did not reach a single peak, the process was stopped and the next step was carried out. Extruder processing was performed (400 nm, 200 nm, 100 nm, once each).

[0066] The antibody modification to the liposome was carried out as follows.

[0067] 750 μg of reduced atezolizumab was added to 2.5 mL of liposome solution, and the mixture was stirred at room temperature for 2 hours, followed by overnight stirring at 4°C. Ultrafiltration (CAT: PBMK02510, pore size 300 Kda) was performed using a 10 mL ultrafiltration cell. This process was repeated until the wastewater volume was at least four times the sample volume used for ultrafiltration. Filtration was then performed using a 0.22 μm filter (13 mm diameter). The lipid and protein content of the resulting sample were measured, along with particle size, zeta potential, and progesterone concentration, with the following results.

[0068]

[0069] Statistical analysis was performed using Microsoft Excel (Microsoft, Redmond, WA). Data are presented as mean ± SD. Significance was tested using two-sided Student's test analysis. [Results] P4 and COR have been reported to bind to PgR and support tumor progression in steroid hormone receptor-positive luminal tumors. However, there are also reports that they have anticancer effects against epithelial cancers, including breast cancer cell lines (Yahya S, et al., Steriods (2017) 126:15-23; You S, et al., Int J Nanomedicine (2010) 5:221-229). Therefore, we first examined the effect of P4 on cancer cell lines. JEG-3, BeWo, P3X, A20, and HEK293 were cultured in the presence of progesterone (P4) or cortisol (COR), which is used to suppress the side effects of ICIs, and concentration-dependent changes in proliferation were observed (Figure 1).

[0070] The results showed that the addition of ~200 μM COR did not significantly alter cell proliferation in JEG-3, BeWo, and HEK293 cells, whereas the addition of 200 μM P4 significantly inhibited proliferation in all cell lines. In contrast, the addition of ~2 μM COR to lymphoid cells P3X and A20 cells inhibited proliferation, whereas the addition of P4 did not inhibit proliferation up to ~20 μM, and only at a high concentration of 200 μM did it become apparent. These results suggest that COR is more effective than P4 in inhibiting lymphoid cells, whereas P4 may be more effective in inhibiting epithelial cancers such as choriocarcinoma.

[0071] Therefore, we cultured peripheral blood mononuclear cells (PBMCs) from healthy individuals in the presence of the superantigens TSST-1, P4, and COR, and confirmed changes in proliferation ability.We also clarified the activation and exhaustion levels of the cells by analyzing cell membrane surface antigens using flow cytometry (FCM).

[0072] Cell counting revealed that proliferation was suppressed at high concentrations of P4, but that there was almost no difference in cell number between the control group and the control group (Figure 1). We then examined the levels of activation and exhaustion, and found that CD25-positive, PD-1-positive late-activated T cells were hardly observed in fresh PBMCs, but the proportions of both CD4 T cells and CD8 T cells increased in the control group after 3 days of culture (Figure 2).

[0073] However, in the presence of 20-200 μM P4, the number of late-activated T cells in both CD4 and CD8 T cells decreased in a P4 concentration-dependent manner, and was almost undetectable at 200 μM. Furthermore, the number of activation marker-negative T cells increased in a P4 concentration-dependent manner. On the other hand, in the presence of 20-200 μM COR, the change in the percentage of late-activated T cells in both CD4 and CD8 T cells was independent of COR concentration, indicating that PD-1 expression was not suppressed in a COR concentration-dependent manner. On the other hand, the number of unactivated T cells did not increase significantly, indicating a decrease in the number of activated cells (data not shown). These results suggest that P4 inhibits activation and exhaustion of T lymphocytes more potently than COR in a concentration-dependent manner. On the other hand, B cells barely expressed PD-1, and its expression was not upregulated by TSST-1 or P4 / COR stimulation (data not shown).

[0074] Although it is well known that COR suppresses the production of inflammatory cytokines, the extent to which P4 suppresses the production of various cytokines is unclear. Therefore, we analyzed the effect of P4 on cytokine production using some of the culture supernatants mentioned above. As a result, we observed a P4 concentration-dependent suppression of cytokine production for all 10 cytokines shown in Figure 4.

[0075] Therefore, we investigated the changes in activation markers that occur when P4 or COR is added continuously versus transiently. We examined the extent to which CD25+ PD-1+ cells express PD-L1, the PD-1 ligand. We found that the majority of CD25+ PD-1+ cells were also PD-L1 positive (Figure 3). Therefore, we analyzed the enhancement of exhaustion signaling between lymphocytes by comparing the percentage of PD-1 / PD-L1 positive cells in P4-treated and COR-treated cells. The results showed that sustained P4 treatment significantly reduced the percentage of late-activated CD4 and CD8 T cells, whereas transient treatment showed activation levels comparable to or even greater than those of the control group. In contrast, transient COR treatment significantly suppressed CD8 T cell activation compared to the control group, but continued COR treatment did not suppress the percentage of late-activated cells as much as P4 (Figure 5).

[0076] On the other hand, in B cells, the addition of COR tended to decrease PD-L1 expression, whereas continuous administration of P4 suppressed PD-L1 expression, although the effect was less than that in T cells, and transient administration tended to maintain expression (Figure 6). Furthermore, transient administration of P4 tended to enhance PD-L1 expression.

[0077] We therefore investigated the in vivo effects of P4 and COR on the immune response to xenografts using a model of GVHD in NOG mice transplanted with human PBMCs (Figure 7). When P4 was administered to NOG mice (Hu-PBMC-NOG) immediately after transplantation, the number of spleen cells was higher than in controls (Figure 7, left panel). CD8 T cell counts also tended to increase. However, GVHD-related weight loss was minimal, with only a slight initial loss. Lymphocyte infiltration into peripheral tissues such as the lungs was also suppressed, although this was different from that observed in non-transplanted mice, resulting in significant symptomatic relief (Figure 7, right panel). On the other hand, COR administration induced a decrease in lymphocytes and attenuated GVHD symptoms, but also impaired lymphocyte engraftment.

[0078] Next, to confirm the effect of P4 / COR on antibody production, PBMCs from healthy donors were cultured in vitro for 6 hours in the presence of 200 μM P4 or COR at 5% CO2 and 27°C, and then intravenously transplanted into NOG-hIL-4-Tg mice. They were then immunized with 50 μg of CH401MAP emulsified in Freund's adjuvant. Two weeks later, the mice were boosted with incomplete adjuvant, and analysis was performed two weeks later. The results showed that PBMCs cultured with P4 and transplanted showed comparable engraftment ability to the control group, and specific antibody production was maintained (Figure 8). In contrast, cells cultured with COR showed reduced engraftment ability and suppressed specific antibody production compared to the control and P4-transplanted groups.

[0079] Furthermore, the expression of CD62L, a lymph node localization marker for lymphocytes, was upregulated in the spleens of mice transplanted with P4-treated PBMCs compared with CTRL mice (Fig. 9, top). This trend was also confirmed by continuous administration of P4 to humanized NOG mice, a GVHD model (Fig. 9, bottom). This indicates that P4 maintains CD62L expression in vivo for as long as 4 weeks.

[0080] These results suggest that COR treatment reduces the engraftment ability of PBMCs and reduces cytotoxic activity and B cell function, whereas P4 maintains the number and function of immune cells.

[0081] Atezolizumab, an ICI, is an anti-PD-L1 antibody that has been shown to bind to PD-L1-expressing tumor cells and inhibit the exhaustion of PD-1-expressing T cells in the tumor. PD-L1 is also expressed on activated T and B lymphocytes, and therefore inhibits lymphocyte-to-lymphocyte exhaustion signaling. However, this often promotes the activation of autoreactive T cells, resulting in autoimmune-like side effects. First, we cultured human PBMCs in the presence of the superantigen TSST-1 for 3 days at 5% CO2 and 37°C, and analyzed the effect of the PD-L1 antibody on lymphocyte PD-L1 expression using flow cytometry.

[0082] The results showed that TSST-1-stimulated PBMCs cultured with atezolizumab had reduced PD-L1 expression on T cells compared with PBMCs cultured without atezolizumab (Figure 10, second column on the left).

[0083] Furthermore, when P4 or COR was added to these culture systems, PD-1 expression in T cells decreased in a P4 concentration-dependent manner, along with a corresponding decrease in PD-L1 expression. The addition of atezolizumab further reduced PD-L1 expression, and with 200 μM P4, almost no T cells expressed PD-L1. On the other hand, B cells rarely expressed PD-1, but PD-L1 expression was confirmed even in the presence of P4 and COR (Figure 10, bottom panel). PD-L1 expression was particularly high at 20 μM P4, and this expression was reduced by the addition of atezolizumab. These results suggest that P4 has a weak inhibitory effect on PD-L1 expression on B cells, but that at the physiological concentration of 20 μM, it can be suppressed by the combination of atezolizumab.

[0084] On the other hand, PD-1 expression on T cells and B cells was shown to be upregulated by atezolizumab administration (Figure 11). This was also shown to be reduced by the combination of atezolizumab and P4. In vivo, a trend toward reduced PD-L1 expression and increased PD-1 expression was observed in humanized mice treated with atezolizumab (data not shown).

[0085] These results demonstrate that P4 dose-dependently reduces PD-1 / PD-L1 expression on T cells, but has little effect on reducing PD-L1 expression on B cells, and that the combination of the two drugs effectively suppresses PD-L1 expression on T and B cells.

[0086] Next, liposome-encapsulated P4 was conjugated with atezolizumab to create a conjugate (D2125). This conjugate was added to PBMC cultures at a concentration equivalent to 20 μM P4 and compared with P4 / atezolizumab cocultures. No significant differences in cell counts were detected in PBMCs (Figure 12). However, when comparing PD-1 and PD-L1 expression on T cells (% positive cells and MFI), D2125 reduced the percentage of PD-1 / PD-L1-expressing cells and MFI, demonstrating a significant suppression of T cell activation compared with 20 μM P4. Furthermore, D2125 demonstrated comparable or even greater suppression of T cell activation than 10-fold higher concentrations of P4 (Figure 13). Furthermore, although the MFI of PD-1 was slightly higher than that of 200 μM P4, the difference was not significant, demonstrating efficient suppression. Furthermore, the number of CD25-positive CD4 T cells tended to increase (Fig. 14, left panel), but IL-10 production was suppressed (data not shown). The activation of CD8 T cells tended to be more strongly suppressed than that of CD4 T cells (Fig. 14, right panel).

[0087] These results demonstrate that D2125 significantly inhibits T lymphocyte activation at concentrations close to the physiological P4 concentration in placental villous luminal blood.

[0088] In conclusion, P4 potently inhibits T cell activation at higher concentrations than COR, and the effect is reversible. It also suppresses T cell exhaustion and contributes to the survival of cytotoxic T cells and B cell function. It also maintains the expression of CD62L, which induces lymphocyte localization to lymph nodes. As a result, it suppresses GVH D while maintaining specific antibody production. It also downregulates PD-1 and PD-L1 expression on T cells, but does not inhibit the atezolizumab-induced downregulation of PD-L1 expression on B cells. Combination of the two drugs can suppress PD-1 / PD-L1 signaling at high concentrations. Furthermore, liposome-encapsulated P4, which combines P4 with atezolizumab, can efficiently inhibit lymphocyte activation at the physiologically high P4 concentration present in the placental lumen. Thus, P4 and its conjugates are capable of transiently suppressing T cell function and reactivating T cells while maintaining a balanced Th1 / Th2 ratio, and are therefore considered to have excellent functions as regulators of immune system overactivation caused by ICIs and the like in autoimmune diseases, transplantation immunology, and cancer treatment. [Example 2]

[0089] [Materials and Methods] (1) Tumor Cells and Mice. The breast cancer cell line MDA-MB231 was stored in the Department of Molecular Life Sciences, Department of Basic Medicine, Tokai University School of Medicine. It was cultured in Leibovitz's L-15 medium with 15% FCS at 37°C in a CO2-free environment. NOG mice were purchased from INVIVO Science. CMV-NOG-hIL-4-Tg mice were generated by the Central Institute for Experimental Animals and maintained in an isolator at the Laboratory Animal Facility of Tokai University School of Medicine, or maintained at the Central Institute for Experimental Animals. Human IL-4 levels were measured by DNA typing and ELISA before transplantation.

[0090] CMV-NOG-hIL-4-Tg mice are transgenic NOG mice transfected with an expression vector containing a CMV promoter, human IL-4 cDNA, and SV40 poly(A). (2) ELISA. Human IL-4 protein was quantified using the Human IL-4 ELISA Set BD OptEIATM (BD Biosciences, Catalog No. 555194, Rod No. 9189127). IgG antibody quantification was performed as previously reported (Kametani Y, et al., Exp Hematol (2006) 34(9): 1240-1248). Wells of microtiter plates (Sumiron) were coated with CH401MAP peptide dissolved in carbonate buffer (pH 9.5), and the antigen was allowed to adsorb to the plate overnight at 4°C. The wells were then washed with PBS-Tween (0.05% v / v) and incubated with 3% BSA-PBS for 2 hours at room temperature. After three PBS-Tween washes, mouse plasma was added in 10-fold serial dilutions and incubated for 2 hours at room temperature. The plate was washed three times, and biotin-conjugated mouse anti-human IgG mAb (BD Pharmingen) (1:3,000) was added. The plate was incubated for 2 hours at 37°C, then washed three times and streptavidin-horseradish peroxidase (1:50,000 v / v; BD Pharmingen) was added. The plate was incubated for 1 hour at room temperature, washed, and EIA substrate kit solution (Bio-Rad Laboratories) was added. The reaction was stopped with 10% HCl, and the absorbance at 450 nm was measured. For quantification, a calibration curve was prepared using standard samples with human IL-4 concentrations of 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.3 pg / ml, 15.6 pg / ml, and 7.8 pg / ml. (3) Preparation of human PBMCs. 30 mL of heparinized peripheral blood was collected from healthy donors using Vacutainer ACD tubes (NIPRO Corporation).Immediately after collection, the peripheral blood was layered on Ficoll-Hypaque (Sigma-Aldrich), and the mononuclear cell fraction was collected by density centrifugation (500 × g, 30 min, 20°C). The cells were washed with PBS by centrifugation at 300 × g, 5 min, 4°C, and then counted for use. (4) Transplantation into PBMC-NOG-hIL-4-Tg mice and analysis of engrafted human cells. 6-7 week-old NOG-hIL-4-Tg mice (blood hIL-4 concentration ≥ 100 pg / mL) were injected with 5 × 10 MDA-MB231 cells into the ventral region. 6 The tumor was injected subcutaneously. One week later, the diameter of the tumor was measured every two days using a vernier caliper, and the product of these measurements was used to determine the tumor size. Two weeks after tumor implantation, 5 × 10 6 PBMCs were transplanted intravenously. Tumor diameter measurements were continued after PBMC transplantation. Non-tumor-bearing mice were divided into two groups and administered PBS or atezolizumab (450 μg / head / dose / 3 times (every 10 days)). Tumor-bearing mice were divided into three groups and administered PBS, atezolizumab (450 μg / head / dose / 3 times (every 10 days)), or liposomes (atezolizumab 225 μg / P4 equivalent 40 μg / dose / 6 times (every 5 days)).

[0091] Four weeks later, the mice were anesthetized, heparinized blood was collected, and the mice were euthanized. Cells were collected from the collected lymphoid tissues, red blood cells were removed with hemolysis buffer, and a cell suspension was prepared. The cell count was then counted, and the human leukocyte fraction was analyzed by flow cytometry (FCM). Tumor tissue was used for immunohistochemical staining. (5) Flow Cytometry: The same antibodies as in Example 1 were used for human immune cell staining. Cells were incubated with an appropriate amount of various fluorescently labeled antibodies for 15 minutes at 4°C and then washed with PBS containing 1% BSA. These cells were analyzed using a FACS Fortessa or Verse (BD Bioscience). After gating on live cells, a further gate was placed on human CD45-positive cells to identify the human leukocyte fraction. These cells were further separated by cell surface markers to identify lymphocyte subsets. Data analysis was performed using FlowJo (BD). (6) Histochemical Staining. Mouse tissues were fixed in 20% formalin (Wako Pure Chemical Industries) and embedded in paraffin. Paraffin blocks were thinly sectioned, deparaffinized, mounted on slides, and stained with hematoxylin and eosin (HE). For immunohistochemical staining with anti-human CD8 antibody, slides were heat-treated at 120°C for 20 minutes and then incubated with 0.3% H2O2 / MetOH at room temperature for 10 minutes to remove endogenous peroxidase. Subsequently, the slides were blocked with 1% goat serum, anti-human CD8 (Dako) was added, and the slides were incubated at 25°C for 60 minutes. The slides were then washed three times for 5 minutes with 0.01M PBS. After incubation with Simple Stain MAX-PO (Nichirei Biosciences) for 60 minutes at 25°C, the slides were washed three times for 5 minutes with 0.01M PBS, and the slides were developed with DAB solution. After rinsing in running water for 3 minutes, the sections were stained with hematoxylin, dehydrated, cleared, and mounted. (7) Statistical Processing Statistical processing was performed using Microsoft Excel (Microsoft Corporation). Data are presented as mean ± SD. Significance tests were performed using one-way ANOVA or two-sided Student's t-test analysis.[Results] In NOG-hIL-4-Tg mice carrying the CMV promoter (IL-4 concentration 100-500 pg / ml, CMV-NOG-hIL-4-Tg mice), transplantation with human PBMCs has been shown to result in a high B cell engraftment rate and a balanced engraftment of CD4+ helper T cells and CD8+ killer T cells (data not shown). Furthermore, tumor-bearing immunodeficient mice transplanted with the human breast cancer cell line MDA-MB-231 and human PBMCs have been shown to exhibit a decreased proportion of T cells and an increased proportion of B cells among CD45+ cells, as observed in peripheral blood mononuclear cells (PBMCs) from breast cancer patients. Thus, the MDA-MB231-transplanted CMV-NOG-hIL-4-Tg mice used in this study accurately reproduced the in vivo lymphocyte profile of breast cancer patients when transplanted with human peripheral blood mononuclear cells.

[0092] In this example, tumor-bearing immunodeficient mice were CMV-NOG-hIL-4-Tg mice transplanted with the human breast cancer cell line MDA-MB-231 and human PBMCs. They were then administered atezolizumab, atezolizumab-conjugated liposome-encapsulated P4, or PBS (control). The engrafted CD19+ B cells, CD3+ T cells, CD4+ helper T cells, and CD8+ killer T cells in the spleen were measured. Furthermore, the tumor tissues of the tumor-bearing immunodeficient mice administered atezolizumab, atezolizumab-conjugated liposome-encapsulated P4, or PBS (control) were examined by histochemical staining to determine the degree of lymphocyte infiltration.

[0093] The results of flow cytometry analysis of CD19+ B cells, CD3+ T cells, CD4+ helper T cells, and CD8+ killer T cells engrafted in splenocytes are shown in Figure 15 , and the results of histochemical staining of tumor tissue are shown in Figure 16 . As shown in Figure 15 , administration of atezolizumab to CMV-NOG-hIL-4-Tg mice transplanted with the breast cancer cell line MDA-MB231 reduced B cell engraftment and increased the proportion of T cells, particularly CD8+ killer T cells. Furthermore, administration of atezolizumab-bound liposome-encapsulated P4 resulted in a further reduction in B cell engraftment and a further increase in the proportion of T cells, particularly CD8+ killer T cells, compared to administration of atezolizumab alone. Furthermore, as shown in Figure 16, histochemical staining of tumor tissue revealed no lymphocyte infiltration into the tumor mass in the control PBS-treated group, whereas clear human T cell infiltration into the tumor mass was observed in the atezolizumab-treated group. Furthermore, administration of atezolizumab-conjugated liposome-encapsulated P4 resulted in more pronounced tumor regression and similar or greater infiltration of human killer T cells into the tumor mass compared to administration of atezolizumab alone. Although data are not shown, CD4+ helper T cells also infiltrated the tumor mass, and more pronounced CD4+ helper T cell infiltration was observed in the atezolizumab-conjugated liposome-encapsulated P4-treated group compared to administration of atezolizumab alone.

[0094] Furthermore, tumor diameters were measured in CMV-NOG-hIL-4-Tg mice implanted with the breast cancer cell line MDA-MB231 after administration of atezolizumab, atezolizumab-bound liposome-encapsulated P4, or PBS (control). The results are shown in Figure 17. As shown in Figure 17, the implanted breast cancer grew over time, as seen in the tumor-bearing, immunodeficient mice treated with PBS. In contrast, tumor growth was suppressed in tumor-bearing, immunodeficient mice treated with atezolizumab or atezolizumab-bound liposome-encapsulated P4 compared to the PBS-treated group.

[0095] These results demonstrate that liposome-encapsulated P4 conjugated with atezolizumab exhibits significantly superior antitumor effects to those of atezolizumab, an immune checkpoint inhibitor, in human breast cancer patients.

[0096] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A combination drug used in combination therapy with an anti-human PD-L1 monoclonal antibody, which contains as an active ingredient a T cell and / or B cell activity regulator containing as an active ingredient progesterone or a derivative thereof.

2. The combination drug according to claim 1, wherein the modulator of T cell and / or B cell activity acts reversibly on T cells and / or B cells.

3. The combination drug according to claim 1, wherein the modulator of T cell and / or B cell activity maintains the expression of CD62L and localizes T cells in lymph nodes.

4. The combination drug according to claim 1, wherein the modulator of T cell and / or B cell activity reduces the expression of PD-1 in T cells and / or B cells.

5. The combination drug according to claim 1, which suppresses the expression of PD-L1 in T cells and / or B cells.

6. The combination drug according to claim 1, which suppresses the expression of PD-1 and PD-L1 in T cells and / or B cells.

7. The combination drug according to claim 1, wherein the anti-human PD-L1 monoclonal antibody is a humanized anti-human PD-L1 monoclonal antibody.

8. The combination drug according to claim 7, wherein the humanized anti-human PD-L1 monoclonal antibody is atezolizumab.

9. A pharmaceutical composition comprising as active ingredients a T cell and / or B cell activity regulator containing progesterone or a derivative thereof and an anti-human PD-L1 monoclonal antibody.

10. The pharmaceutical composition according to claim 9, wherein the modulator of T cell and / or B cell activity acts reversibly on T cells and / or B cells.

11. The pharmaceutical composition according to claim 9, wherein the modulator of T cell and / or B cell activity maintains the expression of CD62L and localizes T cells to lymph nodes.

12. The pharmaceutical composition according to claim 9, wherein the modulator of T cell and / or B cell activity reduces the expression of PD-1 in T cells and / or B cells.

13. The pharmaceutical composition according to claim 9, which suppresses the expression of PD-L1 in T cells or B cells.

14. The pharmaceutical composition according to claim 9, which suppresses the expression of PD-1 and PD-L1 in T cells or B cells.

15. The pharmaceutical composition according to claim 9, wherein the anti-human PD-L1 monoclonal antibody is a humanized anti-human PD-L1 monoclonal antibody.

16. The pharmaceutical composition according to claim 15, wherein the humanized anti-human PD-L1 monoclonal antibody is atezolizumab.

17. An immunoliposome comprising a liposome containing a T cell and / or B cell activity regulator having progesterone or a derivative thereof as an active ingredient, wherein an anti-human PD-L1 monoclonal antibody is bound to the membrane of the liposome.

18. The immunoliposome according to claim 17, wherein the modulator of T cell and / or B cell activity acts reversibly on T cells and / or B cells.

19. The immunoliposome according to claim 17, wherein the modulator of T cell and / or B cell activity maintains the expression of CD62L and localizes T cells to lymph nodes.

20. The immunoliposome according to claim 17, wherein the modulator of T cell and / or B cell activity reduces the expression of PD-1 in T cells and / or B cells.

21. The immunoliposome according to claim 17, which suppresses the expression of PD-L1 in T cells and / or B cells.

22. The immunoliposome according to claim 17, which suppresses the expression of PD-1 and PD-L1 in T cells and / or B cells.

23. The immunoliposome according to claim 17, wherein the anti-human PD-L1 monoclonal antibody is a humanized anti-human PD-L1 monoclonal antibody.

24. The immunoliposome according to claim 23, wherein the humanized anti-human PD-L1 monoclonal antibody is atezolizumab.

25. A pharmaceutical composition comprising the immunoliposome according to any one of claims 17 to 24.