Combination therapy comprising a Fas axis antagonist and a Treg cell depletor antagonist for the treatment of cancer - Patents.com

Abstract

The present disclosure relates to the combination of a Fas axis antagonist, such as an anti-FasL antibody, with a Treg cell depleting therapy, such as an anti-CD25 antibody, for use in the treatment of cancer, optionally including a cancer vaccine.

Description

[Technical field]

[0001] The present invention relates to a combination of a Fas axis antagonist and a Treg cell depletion therapy for use in the treatment of cancer. [Background technology]

[0002] Depletion of regulatory T cells (Treg) with antibodies against CD25 has been shown to be effective in various mouse models of cancer, see, for example, Patent Document 1, Patent Document 2, Patent Document 3, and Non-Patent Document 1. However, some mouse models with low immunogenicity have been shown to have low response rates, and in some cases, only partial responses in reducing tumor growth have been achieved.

[0003] Combination therapy for treating cancer consisting of anti-CD25 Treg-depleting antibodies and recombinant GM-CSF-secreting tumor vaccine (GVAX) has been shown to enhance therapeutic efficacy in relation to tumor size and mouse survival in such mouse models. However, partial responses to such combination therapy have been shown to be followed in some circumstances by subsequent tumor recurrence.

[0004] Thus, although current anti-CD25 antibody-based therapies have been shown to be effective in the initial treatment of cancer, there is a need to provide additional therapeutic regimens that can help prevent tumor recurrence and further improve patient survival. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2017 / 174331 [Patent Document 2] International Publication No. 2018 / 167104 [Patent Document 3] International Publication No. 2019 / 175222 [Non-patent literature]

[0006] [Non-Patent Document 1] Solomon I et al, Nature Cancer, vol 1, p1153-1166 (2020) Summary of the Invention

[0007] The present invention relates to the combination of Treg cell depletion therapy and Fas axis binding antagonists for use in the treatment of cancer.

[0008] The inventors have found that the use of a Fas axis antagonist, such as an anti-FasL antibody, in combination with a Treg cell depleting therapy, such as an anti-CD25 antibody, optionally together with a cancer vaccine, such as GVAX, provides a cancer treatment regimen that reduces the likelihood of tumor recurrence and improves patient survival.

[0009] Thus, in one aspect of the present invention there is provided a combination of a Treg cell depleting therapy and a Fas axis antagonist for use in the treatment of cancer, wherein the Treg cell depleting therapy and the Fas axis antagonist are for separate, simultaneous or sequential administration.

[0010] In one aspect of the invention there is provided a combination of an anti-CD25 antibody and an anti-FasL antibody for use in the treatment of cancer, wherein the anti-CD25 antibody and the anti-FasL antibody are for separate, simultaneous or sequential administration.

[0011] A further aspect of the invention provides a Treg cell depleting therapy (e.g., an anti-CD25 antibody) for use in the treatment of cancer, the Treg cell depleting therapy being for use in combination with a Fas axis antagonist (e.g., an anti-FasL antibody).

[0012] A further aspect of the invention provides a Fas axis antagonist (e.g. an anti-FasL antibody) for use in the treatment of cancer, wherein the Fas axis antagonist is for use in combination with a Treg cell depleting therapy (e.g. an anti-CD25 antibody).

[0013] A further aspect of the present invention provides a cancer vaccine for use in the treatment of cancer, the cancer vaccine being for use in combination with a Fas axis antagonist (e.g. an anti-FasL antibody) and a Treg cell depletion therapy (e.g. an anti-CD25 antibody).

[0014] A further aspect of the invention provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of each of a Treg cell depleting therapy and a Fas axis antagonist.

[0015] A further aspect of the invention provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of each of an anti-CD25 antibody and an anti-FasL antibody.

[0016] A further aspect of the invention provides a Fas axis antagonist (e.g. an anti-FasL antibody) for use in the treatment or prevention of recurrence of cancer in a subject, wherein the subject has undergone Treg cell-depleting therapy for the treatment of cancer, and the Fas axis antagonist is for administration to the subject following the Treg cell-depleting therapy.

[0017] A further aspect of the invention provides a Treg cell depletion therapy for use in the treatment or prevention of recurrence of cancer in a subject, wherein the Treg cell depletion therapy is for use in combination with at least one dose of a Fas axis antagonist (e.g. an anti-FasL antibody), and the Fas axis antagonist is for administration to the subject following the Treg cell depletion therapy.

[0018] A further aspect of the invention provides a method of treating or preventing recurrence of cancer in a subject, wherein the subject has undergone Treg cell-depleting therapy for the treatment of cancer, the method comprising administering at least one dose of a Fas axis antagonist (e.g., an anti-FasL antibody) to the subject after the Treg cell-depleting therapy.

[0019] A further aspect of the invention is a method of treating or preventing recurrence of cancer in a subject, comprising the steps of: a) a priming dose of Treg cell depletion therapy; b) administering to the subject a booster dose of Treg cell depleting therapy and a dose of a Fas axis antagonist; Methods are provided in which a booster dose of the Treg cell depleting therapy is administered prior to the Fas axis antagonist.

[0020] A further aspect of the invention provides a priming dose of a Treg cell depleting therapy for use in treating or preventing recurrence of cancer in a subject, wherein the subject is further administered a booster dose of the Treg cell depleting therapy and a dose of a Fas axis antagonist, wherein the booster dose of the Treg cell depleting therapy is administered prior to the Fas axis antagonist.

[0021] A further aspect of the invention provides a combination of a) a priming dose of a Treg cell depleting therapy, b) a booster dose of a Treg cell depleting therapy, and c) a dose of a Fas axis antagonist for use in treating or preventing recurrence of cancer in a subject, wherein the priming dose of the Treg cell depleting therapy and the booster dose of the Treg cell depleting therapy are administered prior to the Fas axis antagonist.

[0022] A further aspect of the invention is a method of treating or preventing recurrence of cancer in a subject, comprising the steps of: a) a priming dose of an anti-CD25 antibody; and b) administering to the subject a booster dose of an anti-CD25 antibody and a dose of an anti-FasL antibody; Methods are provided in which a booster dose of an anti-CD25 antibody is administered prior to the anti-FasL antibody.

[0023] A further aspect of the invention provides a priming dose of an anti-CD25 antibody for use in treating or preventing recurrence of cancer in a subject, wherein the subject is further administered a booster dose of the anti-CD25 antibody and a dose of an anti-FasL antibody, wherein the booster dose of the anti-CD25 antibody is administered prior to the anti-FasL antibody.

[0024] The invention also provides embodiments of any of the above aspects, wherein the subject is further administered a cancer vaccine and / or the subject has also received cancer vaccine therapy.

[0025] The invention also provides for the use of a component or combination of components in any of the disclosed methods of treatment.

[0026] Other aspects and embodiments of the invention are described in more detail below. [Brief description of the drawings]

[0027] [Figure 1] Figure 1. Study design in in vivo mouse model. C57BL / 6 mice were injected with 50,000 B16 / BL6 melanoma cells subcutaneously (sc) in the right flank on day 0. Mice were divided into 4 groups (Figure 1A-D) and received 200 μg of anti-CD25NIB antibody, 1×106 irradiated (150 Gy) GVAX, isotype control, and / or 200 μg of anti-FasL (MFL3) antibody at the indicated time points. [Diagram 2] FIG. 1 shows an in vivo mouse model showing tumor growth after initial combination therapy containing anti-CD25 antibody and cancer vaccine, followed by further treatment with (A) isotype control, (B) anti-FasL antibody, (C) weekly anti-CD25 antibody, and (D) anti-FasL antibody and anti-CD25 antibody. [Diagram 3] FIG. 1 shows animal survival over the indicated days in a mouse cancer model after initial combination therapy including anti-CD25 antibody and cancer vaccine followed by treatment with isotype control, anti-FasL antibody, weekly anti-CD25 antibody, or a combination of anti-FasL and anti-CD25 antibodies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The present invention provides a combination of a Treg cell depleting therapy and a Fas axis binding antagonist for use in treating cancer in a subject. In particular, the present invention provides a Treg cell depleting therapy, such as an anti-CD25 antibody, as a first component, and a Fas axis binding antagonist, such as an anti-FasL antibody, as a second component (and optionally subsequent components), for use in treating cancer in a subject. The present invention also provides a method of treating cancer in a subject comprising administering a Treg cell depleting therapy, such as an anti-CD25 antibody, and administering a Fas axis binding antagonist, such as an anti-FasL antibody.

[0029] References herein to "combination" or "in combination" refer to separate, simultaneous or sequential administration, unless the context indicates otherwise.

[0030] The inventors have found that administering anti-CD25 antibodies in combination with Fas axis binding antagonists helps prevent tumor recurrence and improves the therapeutic efficacy of anti-CD25 antibody treatment. Tregs are known to contribute to the immunosuppressive tumor microenvironment (TME). In humans, high tumor infiltration by Treg cells and, more importantly, a low ratio of effector T (Teff) cells to Treg cells is associated with poor outcomes in multiple human cancers (Shang et al., 2015, Sci Rep. 5:15179). Conversely, a high Teff / Treg cell ratio is associated with a favorable response to immunotherapy in both humans and mice (Hodi et al., 2008, Proc. Natl. Acad. Sci. USA, 105, 3005-3010; Quezada et al., 2006, J Clin Invest. 116(7):1935-45). However, during anti-CD25 antibody treatment, the inventors found that in partially responsive tumors, there are changes in the tumor microenvironment (TME) over time. Characterization of the TME in partially responsive tumors revealed changes including loss of activated effector T cells, gain of resting T cells, gain of regulatory T cells (Treg), and increase in M2-like suppressor myeloid cells. This change in the TME, especially if there can be a loss of activated Teff cells and gain of Treg cells over time, may lead to recurrence of originally responsive tumors.

[0031] The present inventors found that Fas-mediated cell death of activated Teff could be inhibited by blocking the Fas / FasL axis with an inhibitor of the FasL / Fas signaling system (i.e., a Fas axis antagonist), for example, an anti-FasL antibody or an anti-Fas antibody. Fas expression was higher in activated Teff cells compared with other T cells, and the %Fas+ cells of activated Teff cells showed a tendency to increase in stable tumors before recurrence, suggesting that activated Teff may be more susceptible to Fas-mediated cell death.

[0032] In particular, the inventors have found that administration of an inhibitor of the FasL / Fas signaling pathway (i.e. a Fas axis antagonist), e.g. an anti-FasL antibody or an anti-Fas antibody, in combination with a Treg cell depletion therapy, e.g. the administration of an anti-CD25 antibody, optionally further combined with a cancer vaccine, e.g. GVAX, can maintain an immunogenic tumor microenvironment (TME), in particular a high Teff / Treg cell ratio, thereby achieving improved therapeutic outcomes.

[0033] The therapeutic regimen of the present invention provides enhanced therapeutic efficacy compared to anti-CD25 administered alone or in combination with a cancer vaccine, or a Fas axis antagonist (such as an anti-FasL antibody) administered alone.

[0034] Thus, in a first aspect of the present invention, there is provided a combination comprising a Treg cell depleting therapy and a Fas axis antagonist for use in the treatment of cancer. The Treg cell depleting therapy and the Fas axis antagonist are for separate, simultaneous or sequential administration. In one embodiment, the combination comprises a) a first component comprising a Treg cell depleting therapy and b) a second component comprising a Fas axis antagonist for separate, simultaneous or sequential use in the treatment of cancer.

[0035] The Treg cell depleting therapy may be administered in combination with one or more additional therapeutic agents. Preferably, the additional therapeutic agent is a cancer vaccine. Thus, in some embodiments, the Treg cell depleting therapy is administered in combination with a cancer vaccine, and the cancer vaccine is for separate administration, simultaneous administration, or sequential administration with the Treg cell depleting therapy.

[0036] Thus, in one embodiment of the present invention, a combination is provided for use in the treatment of cancer, comprising a) a first component comprising a Treg cell depleting therapy, b) a second component comprising a Fas axis antagonist, and c) a third component comprising a cancer vaccine. The Treg cell depleting therapy, the Fas axis antagonist, and the cancer vaccine are preferably administered separately. Preferably, the administration of the Treg cell depleting therapy to the subject is followed by the administration of the cancer vaccine to the subject, and then the administration of the Fas axis antagonist to the subject.

[0037] Preferably, the treatment regimen may include administration of multiple doses of a Treg cell depleting therapy, a Fas axis antagonist, and / or a cancer vaccine.

[0038] In a preferred embodiment of the present invention, the Treg cell depletion therapy comprises administering an anti-CD25 antibody, preferably in combination with a cancer vaccine. The administration of an anti-CD25 antibody in combination with a cancer vaccine enhances the therapeutic effect of the Treg cell depletion therapy.

[0039] In a preferred embodiment of the invention, the Fas axis antagonist is an anti-FasL antibody. Thus, in one embodiment of the invention, there is provided a combination comprising a) a first component comprising an anti-CD25 antibody, b) a second component comprising an anti-FasL antibody, and c) a third component comprising a cancer vaccine for simultaneous, separate or sequential use in the treatment of cancer.

[0040] Preferably, the Fas axis antagonist (such as an anti-FasL antibody) is administered after administration of the anti-CD25 antibody and the cancer vaccine. By "administered after" it is meant that the Fas axis antagonist (e.g., an anti-FasL antibody) is administered preferably at a time sufficient for these components to achieve the desired effect after administration of the anti-CD25 antibody (and optionally after administration of the cancer vaccine). For example, the Fas axis antagonist (e.g., an anti-FasL antibody) is administered after depletion of Treg cells in the subject achieved by administration of the anti-CD25 antibody (and optionally administration of the cancer vaccine).

[0041] In a preferred embodiment, the treatment regimen involves administering to the subject an anti-CD25 antibody, followed by administration of a cancer vaccine, followed by administration of a Fas axis antagonist (such as an anti-FasL antibody).

[0042] The therapeutic regimen may include administering multiple doses of anti-CD25 antibody, cancer vaccine, and / or Fas axis antagonist (e.g., anti-FasL antibody). In one embodiment, the therapeutic regimen includes administering multiple doses of anti-FasL antibody. In one embodiment, the anti-FasL antibody is for administration in combination with a further dose of anti-CD25 antibody, and the further dose of anti-CD25 antibody is for administration before the anti-FasL antibody. In other words, the therapeutic regimen includes administering at least two doses of anti-CD25 antibody, followed by a first dose of anti-FasL antibody. Multiple doses of anti-CD25 antibody may be administered to maintain Treg depletion in the subject.

[0043] In one embodiment, the second dose of anti-CD25 antibody is administered at least 7 days, preferably at least 10 days, and more preferably 14 to 35 days after the administration of the first dose of anti-CD25 antibody.

[0044] In one embodiment, each dose of anti-FasL antibody is administered within 20 days after the second and each subsequent dose of anti-CD25 antibody. For example, anti-FasL antibody is preferably administered after depletion of Treg cells in the subject achieved by administration of anti-CD25 antibody. Preferably, each dose of anti-FasL antibody is administered at least 1 day, preferably at least 3 days, more preferably at least 7 days after the second and each subsequent dose of anti-CD25 antibody is administered.

[0045] The administration of a dose of anti-FasL antibody followed by administration of a second dose of anti-CD25 antibody is repeated multiple times, i.e. periodically. Each administration of anti-CD25 antibody in combination with anti-FasL antibody may be referred to as a treatment cycle. In one embodiment, each treatment cycle of the anti-CD25 antibody / anti-FasL antibody combination is repeated multiple times, preferably with an interval of at least 7 days between the start of each cycle, i.e. each treatment cycle comprising administration of anti-CD25 antibody and anti-FasL antibody is carried out over a period of at least 7 days.

[0046] Preferably, each treatment cycle ranges from 7 to 48 days, with the start date of each treatment cycle (i.e., day 1 of the cycle) being calculated from the administration of the anti-CD25 antibody. The treatment cycle may include a rest period, i.e., the treatment cycle may include a period during which the antibody, or optionally the cancer vaccine, is not administered. In some embodiments, each subsequent dose of the anti-CD25 antibody is administered once per week (Q1W), once per two weeks (Q2W), once per three weeks (Q3W), or once per six weeks (Q6W), with a dose of the anti-FasL antibody being administered between each dose of the anti-CD25 antibody. Each subsequent dose of the anti-CD25 antibody is administered at a frequency that maintains the depletion of Treg cells in the subject.

[0047] For example, in one embodiment, an anti-FasL antibody is administered 7 days after administration of each additional dose of an anti-CD25 antibody, and the administration of the anti-FasL antibody and the anti-CD25 antibody is repeated multiple times with an interval of at least 14 days between administration of each anti-CD25 antibody dose. For example, an anti-CD25 antibody is administered on day 1 of each treatment cycle and an anti-FasL antibody is administered on day 8 of the treatment cycle, each treatment cycle being at least 14 days long, and if a cancer vaccine is administered, it may be administered on any one or more of days 2 through 7 of each treatment cycle.

[0048] The Fas axis antagonist (eg, an anti-FasL antibody) is preferably administered while the subject has depleted Treg levels as a result of treatment with the anti-CD25 antibody (and optionally the cancer vaccine).

[0049] In embodiments in which the therapeutic regimen includes administration of a cancer vaccine, the first dose of the cancer vaccine is preferably administered prior to administration of the first dose of the Fas axis antagonist (e.g., an anti-FasL antibody) and prior to administration of the second dose of the anti-CD25 antibody.

[0050] The therapeutic regimen may also include administration of multiple doses of the cancer vaccine. One or more doses of the cancer vaccine are preferably administered after the first dose of the anti-CD25 antibody is administered. In a further embodiment, the cancer vaccine may be administered substantially simultaneously with the anti-CD25 antibody. The therapeutic regimen may include: After the first dose of anti-CD25 antibody, followed by administration of one or more doses of a cancer vaccine; This may include subsequent administration of one or more alternating doses of anti-CD25 antibody and Fas axis antagonist, with each further dose of anti-CD25 antibody being administered before a dose of the Fas axis antagonist.

[0051] Thus, in one embodiment, the treatment regimen comprises, in the following order: a) administering an anti-CD25 antibody; b) administering a cancer vaccine, preferably wherein administering the cancer vaccine comprises administering multiple doses of the cancer vaccine, preferably three doses of the cancer vaccine; c) administering an anti-CD25 antibody; and d) administering a Fas axis antagonist (such as an anti-FasL antibody); The treatment regimen includes periodically repeating step c) and step d). Each treatment cycle including step c) and step d) is preferably performed for a period of at least 7 days. For example, each dose of anti-CD25 antibody and Fas axis antagonist administered in step c) and step d) is administered Q1W, Q2W, Q3W, or Q6W. Preferably, each subsequent dose of anti-CD25 antibody is administered at a frequency to maintain the depletion of Treg cells in the subject. The Fas axis antagonist is preferably administered after the depletion of Treg cells in the subject is achieved by administering anti-CD25 antibody.

[0052] In some embodiments, an additional dose of a cancer vaccine may be administered in combination with the second or subsequent dose of an anti-CD25 antibody. In other words, the treatment regimen may include: After the first dose of anti-CD25 antibody, followed by administration of one or more doses of a cancer vaccine; Administration of one or more doses of the anti-CD25 antibody, the cancer vaccine, and the Fas axis antagonist follows, with each additional dose of the anti-CD25 antibody being for administration prior to the dose of the Fas axis antagonist, and with each additional dose of the cancer vaccine being administered between each additional dose of the anti-CD25 antibody and the Fas axis antagonist.

[0053] Thus, in one embodiment, the treatment regimen comprises, in the following order: a) administering an anti-CD25 antibody; b) administering a cancer vaccine, preferably wherein administering the cancer vaccine comprises administering multiple doses of the cancer vaccine, preferably three doses of the cancer vaccine; c) administering an anti-CD25 antibody; and d) administering a cancer vaccine; and e) administering a Fas axis antagonist (such as an anti-FasL antibody), The treatment regimen includes periodically repeating steps c) to e). Each treatment cycle including steps c) to e) is preferably performed for a period of at least 7 days. For example, each dose of the components administered in steps c) to e) is administered Q1W, Q2W, Q3W or Q6W. Preferably, each subsequent dose of the anti-CD25 antibody is administered at a frequency that maintains depletion of Treg cells in the subject. The Fas axis antagonist is preferably administered after depletion of Treg cells in the subject achieved by administration of the anti-CD25 antibody.

[0054] In some embodiments of the invention, the initial dose of a Treg cell depleting therapy, e.g., an anti-CD25 antibody, and optionally the initial one or more doses of a cancer vaccine, can be considered as a priming therapy. The second and subsequent doses of a Treg cell depleting therapy, e.g., subsequent doses of an anti-CD25 antibody, and any further doses of a cancer vaccine, can be considered as a booster therapy.

[0055] A "priming therapy" according to the present invention comprises administration of a Treg cell depleting therapy (e.g., an anti-CD25 antibody), and optionally a cancer vaccine administered after the Treg cell depleting therapy. In some embodiments, the priming therapy can comprise administering multiple doses of a cancer vaccine after administration of the Treg cell depleting therapy.

[0056] A "booster cycle", "booster therapy" or "booster treatment" or the like according to the present invention comprises administration of a Treg cell depleting therapy (e.g., an anti-CD25 antibody) and a Fas axis antagonist (e.g., an anti-FasL antibody), and optionally a cancer vaccine. Each booster cycle or booster therapy comprises administration of a Treg cell depleting therapy, then optionally one or more doses of a cancer vaccine, if used, then administration of a Fas axis antagonist. The Fas axis antagonist is preferably administered after depletion of Treg cells in the subject, achieved by administration of a Treg cell depleting therapy (and optionally a cancer vaccine). Preferably, the present invention comprises multiple rounds of booster therapy, e.g., each booster cycle is repeated multiple times, i.e., each booster cycle is performed two, three, four or more times.

[0057] Thus, in a further aspect of the invention, there is provided a priming dose of a Treg cell depleting therapy, and optionally a priming dose of a cancer vaccine, for use in treating cancer in a subject, the subject being further administered a booster dose of a Fas axis antagonist (e.g., an anti-FasL antibody) and the Treg cell depleting therapy, the booster dose of the Treg cell depleting therapy being administered prior to the Fas axis antagonist. In some embodiments, the booster dose of the Treg cell depleting therapy can be administered in combination with a further booster dose of the cancer vaccine.

[0058] By "administered prior to" it is meant that the Treg cell depleting therapy, e.g., an anti-CD25 antibody, is preferably administered sufficiently prior to administration of the Fas axis antagonist for the Treg cell depleting therapy to achieve the desired effect, e.g., depletion of Treg cells in the subject.

[0059] In one embodiment, a priming dose of an anti-CD25 antibody for use in treating cancer in a subject is provided, and the subject is further administered a dose of an anti-FasL antibody and a booster dose of the anti-CD25 antibody, the booster dose of the anti-CD25 antibody being administered before the anti-FasL antibody. In some embodiments, the booster dose of the anti-CD25 antibody may be followed by administration of a booster dose of the cancer vaccine prior to administration of the anti-FasL antibody.

[0060] In one embodiment, a dose of an anti-FasL antibody for use in treating cancer in a subject is provided, where the subject is first administered a priming dose of an anti-CD25 antibody, optionally administered a priming dose of a cancer vaccine, and then administered a booster dose of the anti-CD25 antibody.

[0061] A further embodiment of the invention is a method of treating cancer in a subject, comprising: a) a priming dose of an anti-CD25 antibody and a priming dose of a cancer vaccine, and b) administering to the subject a dose of an anti-FasL antibody and a booster dose of an anti-CD25 antibody; Methods are provided in which a booster dose of the anti-CD25 antibody is administered prior to the anti-FasL antibody.

[0062] Preferably, booster doses of anti-FasL antibody and anti-CD25 antibody are administered periodically after administration of a priming dose of anti-CD25 antibody, i.e., step b) is repeated twice, three times, four times or more. Preferably, each treatment cycle is at least 7 to 48 days, and the start date of each cycle (i.e., day 1 of each cycle) is calculated from the administration of the booster dose of anti-CD25 antibody. For example, each booster dose of anti-CD25 antibody is administered Q1W, Q2W, Q3W or Q6W, and a dose of anti-FasL antibody is administered between each dose of anti-CD25 antibody. The booster dose of anti-CD25 antibody is administered at a frequency that maintains the depletion of Treg cells in the subject. The anti-FasL antibody is preferably administered after the depletion of Treg cells in the subject achieved by administration of the anti-CD25 antibody.

[0063] Preferably, the anti-FasL antibody is administered within 20 days after each booster dose of the anti-CD25 antibody. More preferably, each dose of the anti-FasL antibody is administered at least 1 day, preferably at least 3 days, more preferably at least 7 days after each booster dose of the anti-CD25 antibody.

[0064] For example, in one embodiment, a booster dose of an anti-CD25 antibody is administered on day 1 of each booster cycle, and an anti-FasL antibody is administered on or after day 7 of each cycle, with each cycle being at least a 14 day cycle.

[0065] In one embodiment, the initial priming regimen of anti-CD25 antibody and cancer vaccine may include multiple doses of the cancer vaccine.

[0066] Preferably, multiple doses of the cancer vaccine are administered after a priming dose of the anti-CD25 antibody and prior to administration of subsequent doses of the anti-CD25 antibody. Preferably, the first dose of the cancer vaccine is administered at least one day after administration of the anti-CD25 antibody.

[0067] In one embodiment, the first booster cycle begins at least 7 days, preferably at least 10 days, more preferably 14 to 35 days after the administration of the priming dose of anti-CD25 antibody. Each booster cycle is preferably at least 7 days, more preferably 7 to 48 days. Preferably, anti-CD25 antibody and anti-FasL antibody are each administered once per cycle. In some embodiments, a booster dose of a cancer vaccine can be administered in combination with a booster dose of an anti-CD25 antibody during each cycle of anti-CD25 antibody / anti-FasL antibody. Preferably, a booster dose of a cancer vaccine is administered between the administration of each dose of anti-CD25 antibody and anti-FasL antibody in each booster cycle.

[0068] A further aspect of the invention relates to the treatment or prevention of recurrence of cancer in a subject. One embodiment of the invention provides a method of treating or preventing recurrence of cancer in a subject, the subject having undergone a Treg cell-depleting therapy for the treatment of cancer, the method comprising administering at least one dose of a Fas axis antagonist, e.g., an anti-FasL antibody, to the subject after the Treg cell-depleting therapy.

[0069] One embodiment of the present invention is a method of treating or preventing recurrence of cancer in a subject, comprising: a) administering a priming dose of Treg cell depleting therapy; b) administering one or more priming doses of a cancer vaccine; and c) administering a booster dose of Treg cell depleting therapy; and d) optionally administering a booster dose of a cancer vaccine; e) administering a dose of a Fas axis antagonist; The method includes periodically repeating steps c) to e). Each treatment cycle including steps c) to e) is preferably performed for a period of at least 7 days. For example, each dose of the components administered in steps c) to e) is administered Q1W, Q2W, Q3W or Q6W. Preferably, each subsequent dose of the anti-CD25 antibody is administered at a frequency that maintains depletion of Treg cells in the subject. The Fas axis antagonist is preferably administered after depletion of Treg cells in the subject achieved by administration of the anti-CD25 antibody (and optionally, the cancer vaccine).

[0070] A further embodiment of the invention provides a Fas axis antagonist, such as an anti-FasL antibody, for use in the treatment or prevention of recurrence of cancer in a subject, wherein the subject has undergone Treg cell-depleting therapy for the treatment of cancer, and the Fas axis antagonist is for administration to the subject following the Treg cell-depleting therapy.

[0071] Another embodiment of the invention provides a Treg cell-depleting therapy for use in the treatment or prevention of cancer recurrence in a subject, wherein the Treg cell-depleting therapy is for use in combination with at least one dose of a Fas axis antagonist, such as an anti-FasL antibody, and wherein the Fas axis antagonist is for administration to the subject following the Treg cell-depleting therapy.

[0072] A further embodiment of the present invention provides a combination of a) a priming dose of a Treg cell depleting therapy, b) a booster dose of a Treg cell depleting therapy, and c) a dose of a Fas axis antagonist for use in treating or preventing recurrence of cancer in a subject, wherein the priming dose of the Treg cell depleting therapy and the first booster dose of the Treg cell depleting therapy are administered before the Fas axis antagonist.

[0073] A further embodiment of the present invention provides a combination of a) a priming dose of a Treg cell depleting therapy, b) a priming dose of a cancer vaccine, c) a booster dose of a Treg cell depleting therapy, and d) a dose of a Fas axis antagonist for use in treating or preventing recurrence of cancer in a subject, wherein the priming dose of the Treg cell depleting therapy, the priming dose of the cancer vaccine, and the booster dose of the Treg cell depleting therapy are administered before the Fas axis antagonist, and the priming dose of the cancer vaccine is administered between the priming dose of the Treg cell depleting therapy and the first booster dose of the Treg cell depleting therapy.

[0074] In some embodiments, the treatment comprises repeated administration of a booster dose of the Treg cell depleting therapy and a Fas axis antagonist, where each booster dose of the Treg cell depleting therapy is administered before each dose of the Fas axis antagonist. The repeated administration of each booster dose of the Treg cell depleting therapy and the dose of the Fas axis antagonist is repeated periodically.

[0075] Preferably, the combination further comprises e) a booster dose of the cancer vaccine, and each booster dose of the cancer vaccine is administered between each booster dose of the Treg cell depleting therapy and each dose of the Fas axis antagonist. The repeated administration of each booster dose of the Treg cell depleting therapy, each booster dose of the cancer vaccine, and each dose of the Fas axis antagonist is repeated periodically.

[0076] Preferably, each treatment cycle lasts for at least 7 days. Preferably, each cycle ranges from 7 to 48 days, with the start date of each cycle (i.e., day 1 of each cycle) being calculated from the administration of the booster dose of the anti-CD25 antibody. The booster dose of the Treg cell depletion therapy (e.g., anti-CD25 antibody) is administered at a frequency that maintains Treg cell depletion in the subject. The Fas axis antagonist (e.g., anti-FasL antibody) is preferably administered after depletion of Treg cells in the subject achieved by administration of the Treg cell depletion therapy.

[0077] Treating or preventing cancer recurrence means avoiding the recurrence, relapse or re-emergence of cancer after an initial response or partial response to a prior cancer treatment. For example, in the context of treatment of solid tumors, cancer recurrence may be accompanied by an increase in tumor volume.

[0078] The Treg cell depletion therapy preferably comprises the administration of an anti-CD25 antibody. In some embodiments, the anti-CD25 antibody is administered in combination with a further therapeutic agent, such as a cancer vaccine. The combination of the anti-CD25 antibody and the cancer vaccine may be administered as described for the first aspect of the invention.

[0079] The Fas axis antagonist, such as an anti-FasL antibody, is preferably administered in combination with a further dose of an anti-CD25 antibody, which is administered after the further dose of the anti-CD25 antibody. The combination of anti-FasL antibody and anti-CD25 antibody, and optionally the cancer vaccine, can be administered cyclically as described for the first aspect of the invention.

[0080] A further aspect of the invention is a method of treating or preventing recurrence of cancer in a subject comprising administering to the subject a therapeutically effective amount of a Treg cell depleting therapy and a therapeutically effective amount of a Fas axis antagonist.

[0081] A further aspect of the invention is the use of a Treg cell depletion therapy and a Fas axis antagonist in the manufacture of a medicament for the treatment of cancer.

[0082] A further aspect of the invention provides an anti-CD25 antibody for use in the treatment of cancer, wherein the anti-CD25 antibody is for use in combination with an anti-FasL antibody.

[0083] A further aspect of the invention provides the use of an anti-CD25 antibody in the manufacture of a medicament for the treatment of cancer, wherein the anti-CD25 antibody is for use in combination with an anti-FasL antibody.

[0084] A further aspect of the invention provides an anti-FasL antibody for use in the treatment of cancer, wherein the anti-FasL antibody is for use in combination with an anti-CD25 antibody.

[0085] A further aspect of the invention provides an anti-FasL antibody in the manufacture of a medicament for the treatment of cancer, wherein the anti-FasL antibody is for use in combination with an anti-CD25 antibody.

[0086] A further aspect of the invention provides a cancer vaccine for use in the treatment of cancer, the cancer vaccine being for use in combination with an anti-FasL antibody and an anti-CD25 antibody.

[0087] A further aspect of the invention provides the use of a cancer vaccine in the manufacture of a medicament for the treatment of cancer, the cancer vaccine being for use in combination with an anti-FasL antibody and an anti-CD25 antibody.

[0088] Some aspects and embodiments of the invention are further described with reference to a Fas axis antagonist that is an anti-FasL antibody, and a Treg cell depleting therapy that includes administration of an anti-CD25 antibody, although other Fas axis antagonists and Treg cell depleting therapies may also be used in the present invention. The present invention relates to the treatment of cancer. In one embodiment, the cancer is a CD25-positive cancer. The cancer may be a hematological cancer or a solid cancer.

[0089] In some embodiments, the cancer is selected from acute myeloid leukemia, diffuse large B-cell lymphoma, melanoma, non-small cell lung cancer, renal cancer, ovarian cancer, bladder cancer, sarcoma, colorectal cancer, or more generally, any human cancer in which the B16 cell line can serve as a preclinical model to validate compounds as useful for therapeutic management.

[0090] In some embodiments, the cancer comprises a solid tumor (e.g., a solid tumor cancer) and the treatment is for the treatment of the cancer or prevention of recurrence of the solid tumor. As used herein, a "solid tumor" is an abnormal growth or mass of tissue that does not usually contain cysts or liquid areas, particularly tumors and / or metastases (which may occur anywhere) other than leukemia or non-solid lymphoid cancers. Solid tumors may be benign or malignant. Various types of solid tumors are named according to the type of cells that form them and / or the tissue or organ in which they are located. Examples of solid tumors are sarcomas (including cancers arising from transformed cells of mesenchymal origin in tissues such as cancerous bone, cartilage, fat, muscle, blood vessels, hematopoietic, or fibrous connective tissue), carcinomas (including tumors arising from epithelial cells), mesotheliomas, neuroblastomas, retinoblastomas, etc.

[0091] As used herein, references to "treatment" of cancer, "treating" cancer, or "treating" cancer define the achievement of at least one positive therapeutic effect, such as, for example, reducing the number of cancer cells, reducing tumor size, reducing the rate of cancer cell invasion into peripheral organs, or reducing the rate of tumor metastasis or tumor growth.

[0092] Positive therapeutic effects in cancer can be measured in several ways (e.g., Weber (2009) J Nucl Med 50, 1S-10S). For example, with respect to inhibition of tumor growth, according to the criteria of the National Cancer Institute (NCI), a T / C% ratio of 42% is the lowest level of antitumor activity. T / C(%)=median treated tumor volume / median control tumor volume×100, and T / C<10% is considered to be a high level of antitumor activity. In some embodiments, the treatment achieved by the therapeutically effective amount is either progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS). PFS, also referred to as "time to tumor progression," refers to the length of time during and after treatment that the cancer does not grow, including the time the patient experiences a complete or partial response, as well as the time the patient experiences stable disease. DFS refers to the length of time the patient remains disease-free during and after treatment. OS refers to an extension of life expectancy compared to a naive or untreated individual or patient.

[0093] As used herein, references to "prevention," "preventing" (or prophylactic treatment) refer to delaying or preventing the onset of cancer symptoms. Prevention may be absolute (resulting in the disease not occurring) or may only be effective in some individuals or for a limited period of time.

[0094] The present invention includes the use of Fas axis binding antagonists. As used herein, the term "Fas axis binding antagonist" refers to a molecule that blocks the Fas / FasL signaling pathway (also referred to as the CD95 / CD95L pathway) and inhibits the interaction of Fas binding partners with FasL, so as to inhibit Fas-mediated cell death. In particular, the Fas axis binding antagonist for use in the present invention blocks FasL / Fas-induced apoptosis. Approaches targeting FasL / Fas signaling are discussed in Risso V et al (2022) Cell Death and Disease vol 13 A248.

[0095] FasL, also called Fas ligand, CD95L or CD178, is a type II membrane-bound protein belonging to the tumor necrosis factor (TNF) family. FasL is a protein that binds to its receptor Fas (also called FasR, APO-1 or CD95) and induces apoptosis. FasL is mainly expressed on activated T cells and natural killer (NK) cells. The Fas-FasL signaling pathway is involved in the regulation of immune responses by inducing cell apoptosis.

[0096] Fas axis binding antagonists include inhibitors of Fas, such as anti-Fas antibodies or antigen-binding fragments thereof, and inhibitors of FasL, such as anti-FasL antibodies or antigen-binding fragments thereof. Non-antibody inhibitors of FAS axis binding antagonists are also contemplated, for example, the FasL inhibitor asnercept (APG101), a glycosylated fusion protein comprising the extracellular domain of human CD95 linked to the Fc domain of human IgG1.

[0097] In one embodiment of the present invention, the Fas axis binding antagonist is an anti-FasL antibody. As used herein, "anti-FasL antibody" refers to an antibody or antibody-binding fragment thereof capable of binding to FasL. The anti-FasL antibody is a "neutralizing" or "antagonizing" antibody, meaning that the binding of the antibody to FasL results in the inhibition of the biological activity induced by the FasL polypeptide. The antibody can specifically bind to the binding site of FasL that binds to Fas and prevent FasL from binding to Fas. Preferably, the ability of the antibody to neutralize or antagonize FasL activity can be assessed by its ability to inhibit Fas-FasL mediated apoptosis. The anti-FasL antibody can suppress Fas ligand-induced apoptosis of Fas antigen expressing cells. The activity of the anti-FasL antibody can be assessed by assays and methods known in the art.

[0098] Anti-FasL antibodies are known in the art. Examples of anti-FasL antibodies are disclosed, for example, in WO 2003 / 079750, WO 1996 / 029350, EP 0842948, and EP 0872488, the contents of which are incorporated herein by reference.

[0099] Subjects who have undergone Treg cell depletion therapy are administered a Fas axis binding antagonist.

[0100] As used herein, "Treg cell depletion therapy" or "Treg depletion therapy" refers to a treatment regimen that results in a reduction in Tregs in a subject compared to the level of Tregs in the subject prior to the treatment. Compounds that deplete Treg cells are known in the art. Treg depletion can be measured by techniques known in the art, for example, as disclosed in US Pat. No. 6,399,433 and Simpson et al (2013) J Exp Med 210, 1695-710, the contents of which are incorporated herein by reference.

[0101] As used herein, "regulatory T cells" ("Treg", "Treg cells" or "Tregs") refer to a lineage of CD4+ T lymphocytes specialized in controlling autoimmunity, allergy, and infection. Typically, Tregs regulate the activity of T cell populations, but may also affect certain innate immune system cell types. Tregs are usually identified by the expression of biomarkers CD4, CD25, and Foxp3. Naturally occurring Treg cells typically represent approximately 5%-10% of peripheral CD4+ T lymphocytes. However, within the tumor microenvironment (i.e., tumor-infiltrating Treg cells), they can represent as much as 20%-30% of the total CD4+ T lymphocyte population.

[0102] In a preferred embodiment of the invention, the Treg cell depletion therapy comprises administering to a subject an anti-CD25 antibody alone or in combination with another therapeutic agent, preferably a cancer vaccine.

[0103] CD25 is the alpha chain of the IL-2 receptor and is found on activated T cells, regulatory T cells, activated B cells, some NK T cells, some thymocytes, myeloid progenitor cells, and oligodendrocytes. CD25 associates with CD122 and CD132 to form a heterotrimeric complex that functions as a high affinity receptor for IL-2. The consensus sequence for human CD25 is shown below and is identified as SEQ ID NO:1 (Uniprot Accession No. P01589, underlined corresponding to the extracellular domain of mature human CD25, amino acids 22-240):

[0104] 10 20 30 40 50 MDSYLLMWGL LTFIMVPGCQ A ELCDDDPPE IPHATFKAMA YKEGTMLNCE 60 70 80 90 100 CKRGFRRIKS GSLYMLCTGN SSHSSWDNQC QCTSSATRNT TKQVTPQPEE 110 120 130 140 150 QKERKTTEMQ SPMQPVDQAS LPGHCREPPP WENEATERIY HFVVGQMVYY 160 170 180 190 200 QCVQGYRALH RGPAESVCKM THGKTRWTQP QLICTGEMET SQFPGEEKPQ 210 220 230 240 250 ASPEGRPESE TSCLVTTTDF QIQTEMAATM ETSIFTTEYQ VAVAGCVFLL 260 270 ISVLLLSGLT WQRRQRKSRR TI

[0105] As used herein, an "anti-CD25 antibody" or an "antibody that binds CD25" refers to an antibody that can bind to the CD25 subunit of the IL-2 receptor, which is also known as the alpha subunit of the IL-2 receptor.

[0106] An anti-CD25 antibody is an antibody capable of specifically binding to the CD25 subunit (antigen) of the IL-2 receptor.

[0107] "Specific binding," "bind specifically," and "specifically bind" mean that an antibody binds to a target molecule at a concentration of approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 It is understood to mean having a dissociation constant (Kd) for the antigen of interest of less than M. In a preferred embodiment, the dissociation constant is less than 10 -8 Less than M, for example 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 It is in the range of M.

[0108] Anti-CD25 antibodies suitable for use in the present invention are those that have the ability to deplete or reduce Treg cells.

[0109] As used herein, references to "depleted" or "deplete" (with respect to depletion of regulatory T cells with an anti-CD25 antibody agent) refer to a decrease in the number, ratio or proportion of Tregs relative to when the antibody is not administered. As described herein, in certain embodiments of the invention, greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of regulatory T cells are depleted.

[0110] Anti-CD25 antibodies capable of depleting Treg cells and suitable for use in the present invention include, for example, those described in Patent Document 1, Patent Document 2, WO 2019 / 008386, WO 2019 / 175215, WO 2019 / 175216, WO 2019 / 175217, WO 2019 / 175220, WO 2019 / 17522, WO 2019 / 175223, WO 2019 / 17524, and WO 2019 / 17526, the contents of which are incorporated herein by reference.

[0111] In a preferred embodiment of the invention, the anti-CD25 antibody binds FcγR with high affinity, and preferably binds an activating receptor with high affinity. Preferably, the antibody binds FcγRI and / or FcγRIIa and / or FcγRIIIa with high affinity. In a specific embodiment, the antibody binds FcγRI and / or FcγRIIa and / or FcγRIIIa with high affinity. -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 It binds to at least one activating Fcγ receptor with a dissociation constant less than M.

[0112] In some embodiments, the antibody is an IgG1 antibody, preferably a human IgG1 antibody, and can bind to at least one Fc activating receptor. For example, the antibody can bind to one or more receptors selected from FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the antibody can bind to FcγRIIIa. In some embodiments, the antibody can bind to FcγRIIIa and FcγRIIa, optionally FcγRI. In some embodiments, the antibody binds to these receptors with high affinity, e.g., about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 It can bind with a dissociation constant less than M.

[0113] In some embodiments, the antibody binds the inhibitory receptor FcγRIIb with low affinity. -7 Higher than M, about 10 -6 Higher than M or about 10 -5 It binds FcγRIIb with a dissociation constant higher than M.

[0114] In some embodiments, the anti-CD25 antibody may be afucosylated. The Fc region of the antibody may be modified to alter the glycosylation profile using techniques known in the art. Techniques available for generating antibodies with deleted or reduced fucosylation profiles include commercially available technologies such as GlyMAXX (ProBiogen) and methods such as those disclosed in WO 2011 / 035884.

[0115] In some embodiments, anti-CD25 antibodies induce ADCC activity. Anti-CD25 antibodies exhibit ADCC activity against CD25+ target cells. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, thereby causing lysis of the target cells. In some embodiments, anti-CD25 antibodies induce ADCP activity. "Antibody-dependent cell-mediated phagocytosis" (ADCP) refers to a cell-mediated reaction in which phagocytes expressing Fc receptors (e.g., macrophages) recognize bound antibodies on target cells, thereby causing phagocytosis of the target cells.

[0116] The anti-CD25 antibodies used in the present invention may function through ADCC and ADCP activities. ADCC and ADCP can be measured using assays known and available in the art.

[0117] In some embodiments of the invention, the anti-CD25 antibody does not inhibit the binding of interleukin-2 (IL-2) to CD25. References herein to "does not inhibit the binding of interleukin-2 to CD25" may alternatively be expressed as the anti-CD25 antibody being a non-IL-2 blocking antibody or a "non-blocking" antibody (with respect to non-blocking of IL-2 binding to CD25 in the presence of the anti-CD25 antibody), i.e., the antibody does not block the binding of interleukin-2 to CD25 and in particular does not inhibit interleukin-2 signaling in CD25-expressing cells. References herein to a non-IL-2 blocking antibody may alternatively be expressed as an anti-CD25 antibody that "does not inhibit the binding of interleukin-2 to CD25" or an anti-CD25 antibody that "does not inhibit IL-2 signaling." References such as "non-blocking," "non-IL-2 blocking," "not blocking," or "without blocking" (with respect to non-blocking of IL-2 binding to CD25 in the presence of an anti-CD25 antibody) include embodiments in which the anti-CD25 antibody of the invention does not block IL-2 signaling through CD25. That is, the anti-CD25 antibody inhibits IL-2 signaling by less than 50% compared to IL-2 signaling in the absence of the antibody. In certain embodiments of the invention described herein, the anti-CD25 antibody inhibits IL-2 signaling by less than about 50%, 40%, 35%, 30%, and preferably less than about 25% compared to IL-2 signaling in the absence of the antibody.

[0118] Some anti-CD25 antibodies may allow IL-2 to bind to CD25, yet block signaling through the CD25 receptor, and such antibodies may be referred to as "non-blocking," "non-IL-2 blocking," "non-blocking," or "without blocking" (with respect to non-blocking of IL-2 signaling through CD25 in the presence of the anti-CD25 antibody). A non-IL-2 blocking anti-CD25 antibody allows IL-2 to bind to CD25 and promotes at least 50% of the level of signaling through the CD25 receptor compared to signaling in the absence of the anti-CD25 antibody. In certain embodiments of the invention described herein, the anti-CD25 antibody inhibits IL-2 signaling by less than about 50%, 40%, 35%, 30%, and preferably less than about 25%, compared to IL-2 signaling in the absence of the antibody.

[0119] IL-2 signaling through CD25 can be measured, for example, by methods such as those discussed in U.S. Patent No. 5,999,633 and by methods known in the art. Comparison of IL-2 signaling in the presence and absence of an anti-CD25 antibody agent can occur under the same or substantially the same conditions.

[0120] In some embodiments, IL-2 signaling can be determined by measuring the level of phosphorylated STAT5 protein in cells using a standard Stat-5 phosphorylation assay. For example, a Stat-5 phosphorylation assay for measuring IL-2 signaling can involve culturing PMBC cells in the presence of anti-CD25 antibody at a concentration of 10 μg / ml for 30 minutes, followed by the addition of varying concentrations of IL-2 (e.g., 10 U / ml, or varying concentrations of 0.25 U / ml, 0.74 U / ml, 2.22 U / ml, 6.66 U / ml, or 20 U / ml) for 10 minutes. The cells can then be permeabilized and the level of STAT5 protein can be measured using a fluorescently labeled antibody against phosphorylated STAT5 peptide, followed by analysis by flow cytometry. The blocking rate of IL-2 signal can be calculated as follows: % blocking = 100 x [(% Stat5+ cells in no antibody group - % Stat5+ cells in 10 μg / ml antibody group) / (% Stat5+ cells in no antibody group)].

[0121] Examples of non-blocking anti-CD25 antibodies are described in WO 2019 / 175215, WO 2019 / 175216, WO 2019 / 175217, WO 2019 / 175220, WO 2019 / 17522, WO 2019 / 175223, WO 2019 / 17524, and WO 2019 / 17526, the contents of all of which are incorporated herein by reference.

[0122] The anti-CD25 antibody may specifically bind to an epitope within the extracellular region of human CD25. In some embodiments, the antibody binds to an epitope that is distinct from the IL-2 binding site and does not block the binding of IL-2 to CD25.

[0123] As used herein, "epitope" refers to a portion of an antigen that is bound by an antibody or antigen-binding fragment. As is well known in the art, epitopes can be formed from both contiguous amino acids (linear epitopes) or non-contiguous amino acids juxtaposed by tertiary folding of a protein (structural epitopes). Epitopes formed from contiguous amino acids are typically retained when exposed to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.

[0124] Epitopes are structural in that they are composed of parts of an antigen that are not covalently contiguous in the antigen, but are close to each other in three-dimensional space when the antigen is in the relevant conformation. For example, in the case of CD25, a structural epitope is an epitope composed of amino acid residues that are not contiguous in the extracellular domain of CD25, and a linear epitope is an epitope composed of amino acid residues that are contiguous in the extracellular domain of CD25. Means for determining the exact sequence and / or particularly the amino acid residues of an epitope for an anti-CD25 antibody are known in the literature and include competition with peptides from the antigen sequence, binding of truncated and / or mutated (e.g., by alanine scanning or other site-directed mutagenesis) forms to different species of CD25 sequences, phage display-based screening, yeast display techniques, or (co)crystallographic techniques. Methods for determining the spatial structure of an epitope are well known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). Thus, in some embodiments, an anti-CD25 antibody may recognize a conformational epitope.

[0125] In some embodiments, the anti-CD25 antibody binds to an epitope comprising one or more amino acid residues contained in one or more amino acid stretches selected from amino acids 150 to 163 of SEQ ID NO:1 (YQCVQGYRALHRGP) (SEQ ID NO:52), amino acids 166 to 186 of SEQ ID NO:1 (SVCKMTHGKTRWTQPQLICTG) (SEQ ID NO:53), amino acids 42 to 56 of SEQ ID NO:1 (KEGTMLNCECKRGFR) (SEQ ID NO:54), and amino acids 70 to 88 of SEQ ID NO:1 (NSSHSSWDNQCQCTSSATR) (SEQ ID NO:55). Preferably, the epitope comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or more amino acid residues contained in one or more amino acid stretches selected from amino acids 150 to 163 of SEQ ID NO:1 (YQCVQGYRALHRGP) (SEQ ID NO:52), amino acids 166 to 186 of SEQ ID NO:1 (SVCKMTHGKTRWTQPQLICTG) (SEQ ID NO:53), amino acids 42 to 56 of SEQ ID NO:1 (KEGTMLNCECKRGFR) (SEQ ID NO:54), and / or amino acids 70 to 88 of SEQ ID NO:1 (NSSHSSWDNQCQCTSSATR) (SEQ ID NO:55).

[0126] In some embodiments, the anti-CD25 antibody binds to an epitope on human CD25 that includes at least one sequence selected from amino acids 150-158 of SEQ ID NO: 1 (YQCVQGYRA) (SEQ ID NO: 56), amino acids 176-180 of SEQ ID NO: 1 (RWTQP) (SEQ ID NO: 57), amino acids 42-56 of SEQ ID NO: 1 (KEGTMLNCECKRGFR) (SEQ ID NO: 54), and amino acids 74-84 of SEQ ID NO: 1 (SSWDNQCQCTS) (SEQ ID NO: 58). Such antibodies do not inhibit binding of IL-2 to CD25.

[0127] In one embodiment, the anti-CD25 antibody binds to an epitope comprising the sequence of amino acids 70 to 84 of SEQ ID NO:1 (NSSHSSWDNQCQCTS) (SEQ ID NO:59).

[0128] Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain contains a variable domain (V H ) followed by a number of constant domains. Each light chain has a variable domain (V L ), which has a constant domain at the carboxy terminus.

[0129] The variable regions are capable of interacting with structurally complementary antigen targets and are characterized by different amino acid sequences from antibodies with different antigen specificities. The variable regions of either the heavy or light chains contain amino acid sequences capable of specifically binding to antigen targets. Among these sequences, there are small sequences that are called "hypervariable" due to the extreme variability between antibodies with different specificities. Such hypervariable regions are also called "complementarity determining regions" or "CDR" regions.

[0130] These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant. Although CDRs represent non-contiguous stretches of amino acids within the variable region, the positional arrangement of these important amino acid sequences within the variable heavy and light chain regions is known to have similar arrangements within the amino acid sequences of the variable chains, regardless of species. The variable heavy and light chains of all antibodies each have three CDR regions, which are non-contiguous with the other regions (referred to as H1, H2, H3, L1, L2, L3) for each heavy (H) and light (L) chain. The CDR regions as defined herein are defined according to Kabat (Kabat et al., 1977. J Biol Chem 252, 6609-6616).

[0131] In some embodiments, the anti-CD25 antibody is selected from the group consisting of: (a) a heavy chain variable region comprising a CDR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 2 to 5, a CDR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 6 to 11, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; (b) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25; and An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:26, CDR-L2 comprising the amino acid sequence of SEQ ID NO:27, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:28; and (c) a heavy chain variable region comprising a CDR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 31 to 33, a CDR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 34 to 38, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42.

[0132] In some embodiments, the anti-CD25 antibody is selected from the group consisting of: (a) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; (b) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 7, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; (c) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; (d) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; (e) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; and An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and (f) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:5, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:11, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:12; and An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

[0133] In some embodiments, the anti-CD25 antibody is selected from the group consisting of: (a) an antibody comprising a heavy chain variable region comprising any one of the amino acid sequences of SEQ ID NO: 16 to SEQ ID NO: 21, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; (b) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; and (c) An antibody comprising a heavy chain variable region comprising any one of the amino acid sequences of SEQ ID NO: 43 to SEQ ID NO: 48, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 49.

[0134] In some embodiments, the anti-CD25 antibody is selected from the group consisting of: (a) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; (b) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; (c) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; (d) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light variable region comprising the amino acid sequence of SEQ ID NO: 22; (e) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; (f) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; and (g) An antibody comprising a heavy chain variable region comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 16 to 21, and a light chain variable region comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22.

[0135] The sequence numbers of the complementarity determining regions (HCDR1 to HCDR3 and LCDR1 to LCDR3) and the heavy and light chain variable regions of exemplified antibodies are shown in the table below.

[0136] [Table 1]

[0137] Such antibodies are further described in WO 2019 / 175216, WO 2019 / 175217, and WO 2019 / 1175222, the contents of which are incorporated herein by reference.

[0138] The antibody referred to herein as aCD25-a-686 may also be referred to as RG6292. In a preferred embodiment, the anti-CD25 antibody is RG6292. The anti-CD25 antibody referred to as "RG6292" is an afucosylated human IgG1 monoclonal antibody. RG6292 has a heavy chain sequence having the sequence of SEQ ID NO:50 and a light chain sequence having the sequence of SEQ ID NO:51.

[0139] Such antibodies are known as "non-IL-2 blocking" antibodies and are known not to inhibit the binding of IL-2 to CD25.

[0140] Variants of the antibodies defined above can also be used. The antibody variants include those in which the sequence for each CDR sequence is: (i) at least 85% identical thereto; and / or (ii) Antibodies comprising amino acid sequences having one, two, or three amino acid substitutions relative to SEQ ID NO:2 to SEQ ID NO:15, SEQ ID NO:23 to SEQ ID NO:28, or SEQ ID NO:31 to SEQ ID NO:42.

[0141] In addition, as the antibody variant, the sequences of the light chain and the heavy chain are as follows: (i) at least 80% identical thereto; and / or (ii) Examples also include antibodies comprising an amino acid sequence having one, two, three, four, or five amino acid substitutions relative to SEQ ID NO:16 to SEQ ID NO:22, SEQ ID NO:29 to SEQ ID NO:30, or SEQ ID NO:43 to 51.

[0142] For example, one embodiment of the present invention provides an anti-CD25 antibody for use in the treatment of a cancer selected from the group comprising: a) A heavy chain variable region comprising: CDR-H1 comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 2 to 5; CDR-H2 comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 6 to 11; and CDR-H3 comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 12; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising: CDR-L1 comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 13; CDR-L2 comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 14; and CDR-L3 comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 15; b) A heavy chain variable region comprising: CDR-H1 comprising an amino acid sequence having one, two or three amino acid substitutions with respect to any one of SEQ ID NOs: 2 to 5; CDR-H2 comprising an amino acid sequence having one, two or three amino acid substitutions with respect to any one of SEQ ID NOs: 6 to 11; and CDR-H3 comprising an amino acid sequence having one, two or three amino acid substitutions with respect to SEQ ID NO: 12; and An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising: CDR-L1 comprising an amino acid sequence having one, two or three amino acid substitutions with respect to SEQ ID NO: 13; CDR-L2 comprising an amino acid sequence having one, two or three amino acid substitutions with respect to SEQ ID NO: 14; and CDR-L3 comprising an amino acid sequence having one, two or three amino acid substitutions with respect to SEQ ID NO: 15; and c) i) an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 16 to 21, or ii) a heavy chain variable region comprising an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions compared to SEQ ID NO: 16 to SEQ ID NO: 21; and i) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 22, or ii) An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence having one, two, three, four or five amino acid substitutions compared to SEQ ID NO:22.

[0143] Percent (%) identity, as known in the art, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide sequences or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. There are several methods to measure the identity between two polypeptide sequences or two polynucleotide sequences, but the commonly used methods to determine identity are codified in computer programs. Recommended computer programs for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)). The percent identity of two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the initial sequence to best align the sequences) and comparing the amino acid residues or nucleotides at corresponding positions. The term "best alignment" refers to the alignment of two sequences between two amino acids or two nucleic acid sequences. A "generic alignment" is the alignment of two sequences that results in the highest percent identity. Percent identity is determined by the number of identical amino acid residues or identical nucleotides in the sequences being compared (i.e., % identity = number of identical positions / total number of positions x 100). Generally, references to percent identity herein refer to the percent identity along the length of the entire molecule, unless the context specifies or implies otherwise.

[0144] As used herein, the term "antibody" refers to both intact immunoglobulin molecules and fragments thereof that contain an antigen-binding site, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies, including, but not limited to, chimeric, humanized, heteroconjugate and / or multispecific antibodies (e.g., bispecific antibodies, diabodies, tribodies, and tetrabodies), and antigen-binding fragments of antibodies, such as Fab', F(ab')2, and / or F(ab')3. 2, Fab, Fv, rIgG, polypeptide-Fc fusions, single chain variants (scFv fragments, VHH, Trans-body™, Affibody™, shark single domain antibodies, single chain or tandem diabodies (TandAb™), VHH, Anticalin™, Nanobody™, minibodies, BiTE™, bicyclic peptides, and other alternative immunoglobulin protein scaffolds). In some embodiments, the antibody may lack covalent modifications (such as glycosylation) that would occur if produced naturally. In some embodiments, the antibody may include covalent modifications (e.g., attachment of a glycosylation, detectable moiety, therapeutic moiety, catalytic moiety, or other chemical group that improves antibody stability or administration, such as polyethylene glycol). In some embodiments, the antibody may be in the form of a masked antibody (e.g., Probody™). A masked antibody may contain a blocking or "masking" peptide that specifically binds to the antigen-binding surface of the antibody and prevents the antibody from binding to the antigen. The masking peptide is linked to the antibody by a cleavable linker (e.g., a protease). Selective cleavage of the linker in the desired environment, i.e., the tumor environment, releases the masking / blocking peptide and allows antigen binding within the tumor, thereby limiting potential toxicity issues. "Antibody" may also refer to antibody-like molecules such as camelid antibodies (heavy chain only antibodies) and anticalins (Skerra (2008) FEBS J 275, 2677-83). In some embodiments, the antibodies are polyclonal or oligoclonal, generated as a panel of antibodies that each bind more or less different epitopes within an antigen (such as different epitopes within the human CD25 extracellular domain associated with different reference anti-human CD25 antibodies, or different epitopes of FasL), associated with a single antibody sequence. Polyclonal or oligoclonal antibodies can be provided for medical use in a single preparation, as described in the literature (Kearns JD et al., 2015. Mol Cancer Ther.14:1625-36).

[0145] The antibodies used in the present invention can be either monospecific, bispecific or multispecific. A "multispecific antibody" may be specific for different epitopes of one target antigen or polypeptide or may contain antigen binding domains specific for two or more target antigens or polypeptides. In some embodiments of the present invention, the antibody is monospecific. In some embodiments, the antibody binds CD25 or FasL in a monovalent manner (i.e., in a ratio of one antibody to one CD25 molecule or one FasL molecule, respectively). In further embodiments, the antibody is a monospecific bivalent antibody, i.e., the antibody binds CD25 in a ratio of one antibody to two CD25 molecules or binds FasL in a ratio of one antibody to two FasL molecules.

[0146] In some embodiments of the invention, the antibody is monoclonal. The antibody may additionally or alternatively be humanized or human. In further embodiments, the antibody is human, or in any case, is an antibody with a format and characteristics that allow for its use and administration in human subjects.

[0147] As used herein, "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0148] As used herein, a "human antibody" refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibody of the present invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0149] Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. Immunoglobulins may be from any class, such as IgA, IgD, IgG, IgE, or IgM. Immunoglobulins may be from any subclass, such as IgG1, IgG2, IgG3, or IgG4. In a preferred embodiment of the invention, the anti-CD25 antibody is from the IgG class, preferably the IgG1 subclass. In one embodiment, the anti-CD25 antibody is from the human IgG1 subclass. In a preferred embodiment of the invention, the anti-FasL antibody is from the IgG class, preferably the IgG1 subclass. In one embodiment, the anti-FasL antibody is from the human IgG1 subclass.

[0150] Treg cell depletion therapy can be administered in combination with cancer vaccine. Cancer vaccine is known in the art. As used herein, "cancer vaccine" refers to a therapeutic cancer vaccine that is administered to cancer patients and is designed to eradicate cancer cells by enhancing the patient's own immune response. In particular, cancer vaccine can generate tumor antigen-specific response. The cancer vaccine selected depends on the type of cancer to be treated. Cancer vaccines include, but are not limited to, peptide-based vaccines, whole cell vaccines, dendritic cell vaccines, and DNA vaccines.

[0151] In one embodiment, the cancer vaccine administered in accordance with the present invention is a cytokine-expressing cellular immunotherapy.

[0152] As used herein, the term "cytokine-expressing cellular immunotherapy" refers to a composition comprising a population of cells genetically engineered to express a cytokine, such as GM-CSF. The cells of such "cytokine-expressing cellular immunotherapy" comprise a DNA sequence encoding a cytokine operably linked to expression and control elements such that the cytokine is expressed by the cells. The cells are typically tumor cells that have been irradiated to prevent further cell division. The cells may be autologous (patient-specific) or allogeneic (non-patient-specific) to the patient receiving the treatment.

[0153] In one embodiment of the invention, the cytokine-expressing cellular immunotherapy comprises tumor cells that express GM-CSF and may be, for example, a cancer vaccine known as GVAX, which comprises irradiated GM-CSF-secreting tumor cells that activate and mature myeloid cells.

[0154] In the uses and methods described herein, the Fas axis antagonist, anti-FasL antibody, Treg cell depletion therapy, anti-CD25 antibody, and cancer vaccine are each administered in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to an amount (e.g., of a drug or pharmaceutical composition) sufficient to treat a disease and / or condition when administered to a population suffering from or susceptible to a disease and / or condition according to a therapeutic dosing regimen. A therapeutically effective amount is an amount that reduces the incidence and / or severity of, stabilizes, and / or delays the onset of one or more symptoms of a disease, disorder, and / or condition according to a therapeutic regimen. Those skilled in the art will understand that a "therapeutically effective amount" need not actually achieve therapeutic success in a particular subject.

[0155] The selection of an appropriate dosage of the antibody would be within the ability of one of skill in the art. For example, 0.01 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg. In some embodiments, such amounts are unit dosages (or whole fractions thereof) appropriate for administration according to a dosing regimen determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., by a therapeutic dosing regimen). Dosages may also vary as a result of route of administration, cycles of treatment, or dose escalation protocols that can be used to determine the maximum tolerated dose and the dose that limits toxicity (if any) in connection with administration of the antibody or cancer vaccine at increasing doses.

[0156] In some embodiments of the invention, each component is administered multiple times over the course of the treatment regimen. In some embodiments, the dosing regimen may include a first dose of a component at a first dosage, followed by one or more additional doses at a second dosage that is the same as the first dosage. Alternatively, the different doses of each component within the dosing regimen are different amounts of doses. In some embodiments, the dosing regimen includes a first dose at a first dosage, followed by one or more additional doses at a second dosage that is different from the first dosage. In some embodiments, different antibodies may be administered at different dosages. Alternatively, different antibodies are administered at substantially the same dosages. In some embodiments, the same anti-CD25 antibody, anti-FasL antibody, and cancer vaccine are used per dose. Alternatively, different anti-CD25 antibodies and anti-FasL antibodies, such as different Fas axis antagonists, may be used as part of the treatment regimen.

[0157] The antibodies and cancer vaccines according to any aspect of the invention described herein may be in the form of a pharmaceutical composition additionally comprising a pharma- ceutically acceptable carrier, diluent or excipient. These compositions include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, tablets, pills, or liposomes. In some embodiments, the preferred form may depend on the intended mode of administration and / or therapeutic application. Pharmaceutical compositions comprising antibodies may be administered by any suitable method known in the art, including, but not limited to, oral, mucosal, by inhalation, topical, buccal, nasal, rectal, or parenteral (e.g., intravenous, infusion, intratumoral, intranodal, subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal, or other types of administration involving physically disrupting a subject's tissue and administering the pharmaceutical composition through the break in the tissue). Such formulations may be in the form of injection or infusion solutions suitable for, for example, intradermal, intratumoral or subcutaneous administration, or intravenous infusion. Administration may involve continuous dosing (such as perfusion) for at least a selected period of time.

[0158] In some embodiments, the antibodies can be prepared with carriers that will protect the antibodies against rapid release and / or degradation, such as a controlled release formulation, e.g., implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used.

[0159] Those skilled in the art will appreciate that, for example, the route of delivery (oral vs. intravenous vs. subcutaneous vs. intratumoral, etc.) may affect the dosage and / or the required dosage may affect the delivery route. For example, if a particularly high concentration of the agent in a particular site or location (such as intratumoral) is desired and / or useful, focused delivery (e.g., in this example, intratumoral delivery) may be desirable and / or useful. Other factors to consider when optimizing the route and / or dosing schedule for a given treatment regimen may include, for example, the particular cancer being treated (type, stage, location, etc.), the clinical condition of the subject (age, general condition, etc.), and other factors known to medical practitioners.

[0160] Each component of the combination for use according to the present invention should be formulated for separate administration. The pharmaceutical composition of each component typically needs to be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the required amount of antibody with one or a combination of the components listed above in a suitable solvent, followed by sterilization by filtration as necessary. Preparations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release preparations or biodegradable preparations discussed herein. Sterile injectable preparations can be prepared using non-toxic parenterally acceptable diluents or solvents. Each pharmaceutical composition for use according to the invention may contain pharma- ceutically acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coating agents, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers, and are non-toxic to subjects at the dosages and concentrations employed. Preferably, such compositions may further comprise a pharma- ceutically acceptable carrier or excipient for use in the treatment of cancer that is compatible with a given method and / or site of administration, e.g., parenteral (subcutaneous, intradermal, or intravenous injection), intratumoral, or peritumor administration.

[0161] A further aspect of the invention provides a kit for treating cancer, the kit comprising at least two active ingredients, the kit comprising: a) a first composition comprising a Treg cell depleting therapy; b) a second composition comprising a Fas axis antagonist; and optionally instructions for using the composition in a combination therapy for the treatment of cancer. The combination therapy may comprise administering a Treg cell depleting therapy and a Fas axis antagonist according to the administration regimen described for the first and further aspects of the invention. In particular, the administration regimen may comprise administering a Fas axis antagonist after a Treg cell depleting therapy. The kit may further comprise a third composition comprising a further active ingredient c) a cancer vaccine.

[0162] The Treg cell depletion therapy, Fas axis antagonist, anti-CD25 antibody, anti-FasL antibody, cancer vaccine, and administration and treatment regimens for all further aspects of the invention may be as described for the first aspect of the invention.

[0163] Aspects and embodiments described herein with the term "comprising" may include other features or steps within their scope. Aspects and embodiments described with "comprising" are also understood to describe aspects and embodiments in which the term "comprising" is replaced with the term "consisting essentially of" or "consisting of."

[0164] The phrase "selected from the group comprising" anywhere in this specification may be replaced with the phrase "selected from the group consisting of" and vice versa.

[0165] The following is a series of paragraphs that define the present invention and its preferred aspects and embodiments.

[0166] 1. A method for the treatment of cancer in a subject, comprising administering to the subject a therapeutically effective amount of each of a Treg cell depleting therapy and a Fas axis antagonist.

[0167] 2. The method of paragraph 2, wherein the Treg depletion therapy comprises administration of a therapeutically effective amount of an anti-CD25 antibody.

[0168] 3. The method of paragraph 1 or 2, wherein the method further comprises administering a cancer vaccine.

[0169] 4. The method according to any one of items 1 to 3, wherein the Fas axis antagonist is an anti-FasL antibody.

[0170] 5. The method according to any one of items 1 to 4, which comprises administering to a subject a therapeutically effective amount of each of an anti-CD25 antibody, an anti-FasL antibody, and a cancer vaccine.

[0171] 6. The method according to item 5, wherein the anti-CD25 antibody, the anti-FasL antibody and the cancer vaccine are administered separately.

[0172] 7. The method according to item 6, comprising administering an anti-FasL antibody after administration of an anti-CD25 antibody and a cancer vaccine.

[0173] 8. The method according to any one of items 5 to 7, comprising administering an anti-FasL antibody after depletion of Treg cells in a subject by administration of an anti-CD25 antibody.

[0174] 9. The method of any one of paragraphs 5 to 8, wherein the method comprises administering to the subject an anti-CD25 antibody, followed by administration of a cancer vaccine, followed by administration of an anti-FasL antibody.

[0175] 10. The method according to any one of items 5 to 9, comprising administering multiple doses of an anti-CD25 antibody and an anti-FasL antibody.

[0176] 11. The method of paragraph 10, comprising administering at least two doses of an anti-CD25 antibody followed by a first dose of an anti-FasL antibody.

[0177] 12. The method of any one of clauses 10 or 11, wherein each dose of anti-FasL antibody is administered within 20 days of the dose of anti-CD25 antibody.

[0178] 13. The method according to any one of items 10 to 12, wherein a dose of anti-CD25 antibody is administered followed by a dose of anti-FasL antibody.

[0179] 14. The method of clause 13, wherein the administration of a dose of anti-CD25 antibody followed by an anti-FasL antibody is repeated multiple times with at least one week between doses of anti-CD25 antibody.

[0180] 15. The method of any one of clauses 5 to 14, comprising administering multiple doses of the cancer vaccine.

[0181] 16. The method of paragraph 15, wherein administering multiple doses of the cancer vaccine comprises administering a dose of the cancer vaccine between each dose of the anti-CD25 antibody and the anti-FasL antibody.

[0182] 17. a) administering an anti-CD25 antibody; b) administering a cancer vaccine, optionally wherein administering the cancer vaccine comprises administering multiple doses of the cancer vaccine; c) administering an anti-CD25 antibody; and d) optionally administering a cancer vaccine; e) administering a Fas axis antagonist; Item 17. The method according to item 16, wherein the method comprises repeating steps c) to e) in a treatment cycle over a period of at least 7 days.

[0183] 18. The method of any one of clauses 5 to 17, wherein the same anti-CD25 antibody is used for all doses of the anti-CD25 antibody.

[0184] 19. The method according to any one of items 5 to 18, wherein the cancer is a CD25-positive cancer.

[0185] 20. The method according to any one of items 5 to 19, wherein the cancer is a hematological cancer.

[0186] 21. The method according to any one of items 5 to 20, wherein the cancer is a solid tumor.

[0187] 22. The method according to any one of items 5 to 21, wherein the cancer is selected from acute myeloid leukemia, diffuse large B-cell lymphoma, melanoma, non-small cell lung cancer, renal cancer, ovarian cancer, bladder cancer, sarcoma and colorectal cancer.

[0188] 23. The method according to item 22, wherein the cancer is acute myeloid leukemia or diffuse large B-cell lymphoma.

[0189] 24. The method according to item 22, wherein the cancer is melanoma.

[0190] 25. The method according to any one of items 5 to 24, wherein the anti-CD25 antibody inhibits IL-2 signaling via CD25 by less than 50% compared to the IL-2 signaling in the absence of the antibody.

[0191] 26. The method of item 25, wherein the anti-CD25 antibody inhibits IL-2 signaling via CD25 by less than 25% compared to IL-2 signaling in the absence of the antibody.

[0192] 27. The method according to any one of clauses 5 to 26, wherein the anti-CD25 antibody is selected from the group consisting of: (a) a heavy chain variable region comprising a CDR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 2 to 5, a CDR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 6 to 11, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; and An antibody comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; (b) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25; and An antibody comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28; and (c) a heavy chain variable region comprising a CDR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 31 to 33, a CDR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 34 to 38, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and An antibody comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42.

[0193] 28. The method according to any one of items 5 to 27, wherein the anti-CD25 antibody is RG6292.

[0194] 29. The method according to any one of items 5 to 28, wherein the anti-CD25 antibody binds to an epitope comprising at least one sequence selected from amino acids 150 to 158 of SEQ ID NO:1, amino acids 176 to 180 of SEQ ID NO:1, amino acids 42 to 56 of SEQ ID NO:1, and amino acids 74 to 84 of SEQ ID NO:1.

[0195] 30. The method of claim 29, wherein the anti-CD25 antibody binds to an epitope comprising amino acids 70 to 84 of SEQ ID NO:1.

[0196] 31. The method according to any one of items 5 to 30, wherein the cancer vaccine is a cytokine-expressing cellular immunotherapy.

[0197] 32. The method of any one of clauses 31, wherein the cytokine-expressing cellular immunotherapy comprises irradiated GM-CSF-expressing tumor cells.

[0198] 33. A Treg cell depleting therapy for use in the treatment of cancer, wherein the Treg cell depleting therapy antibody is for use in combination with a Fas axis antagonist and optionally a cancer vaccine.

[0199] 34. An anti-CD25 antibody for use in the treatment of cancer, wherein the anti-CD25 antibody is for use in combination with an anti-FasL antibody and optionally a cancer vaccine.

[0200] 35. A Fas axis antagonist for use in the treatment of cancer, wherein the Fas axis antagonist is for use in combination with a Treg cell depletion therapy and optionally a cancer vaccine.

[0201] 36. An anti-FasL antibody for use in the treatment of cancer, wherein the anti-FasL antibody is for use in combination with an anti-CD25 antibody and optionally a cancer vaccine.

[0202] 37. A cancer vaccine for use in the treatment of cancer, wherein the cancer vaccine is for use in combination with a Fas axis antagonist and a Treg cell depletion therapy.

[0203] 38. A cancer vaccine for use according to clause 37, wherein the Fas axis antagonist is an anti-FasL antibody and the Treg cell depleting therapy is an anti-CD25 antibody.

[0204] 39. A combination of a Treg cell depleting therapy and a Fas axis antagonist for use in the treatment of cancer, wherein the Treg cell depleting therapy and the Fas axis antagonist are for separate, simultaneous or sequential administration.

[0205] 40. The combination for use according to clause 39, wherein the Treg cell depleting therapy comprises an anti-CD25 antibody.

[0206] 41. The combination for use according to clause 40, further comprising a cancer vaccine.

[0207] 42. The combination for use according to clause 41, wherein the combination comprises a) a first component comprising an anti-CD25 antibody, b) a second component comprising a Fas axis antagonist, and c) a third component comprising a cancer vaccine, for separate, simultaneous or sequential administration.

[0208] 43. A combination for use according to clause 42, wherein the Fas axis antagonist is for administration subsequent to the anti-CD25 antibody and the cancer vaccine.

[0209] 44. The combination for use according to any one of clauses 42 and 43, wherein the Fas axis antagonist is for administration following depletion of Treg cells in a subject by administration of an anti-CD25 antibody.

[0210] 45. A combination for use according to any one of clauses 42 to 44, wherein administration of an anti-CD25 antibody to a subject is followed by administration of a cancer vaccine, which is then followed by administration of a Fas axis antagonist.

[0211] 46. ​​The combination for use according to any one of clauses 42 to 45, wherein the treatment comprises administration of multiple doses of an anti-CD25 antibody.

[0212] 47. The combination for use according to any one of clauses 42 to 46, wherein the treatment comprises administration of multiple doses of a Fas axis antagonist.

[0213] 48. The combination for use according to any one of clauses 42 to 47, wherein the treatment comprises administration of multiple doses of a cancer vaccine.

[0214] 49. Administration After the first dose of anti-CD25 antibody, followed by administration of one or more doses of a cancer vaccine; 49. The combination for use according to any one of clauses 42 to 48, comprising the subsequent administration of one or more alternating doses of anti-CD25 antibody and Fas axis antagonist, wherein a further dose of anti-CD25 antibody is administered prior to the Fas axis antagonist.

[0215] 50. A combination for use according to clause 49, wherein the administration of the combination of a dose of the Fas axis antagonist and a further dose of the anti-CD25 antibody is repeated in a treatment cycle over a period of at least 7 days.

[0216] 51. The combination for use according to any one of clauses 49 or 50, wherein the administration of one or more alternating doses of anti-CD25 antibody and Fas axis antagonist also comprises the administration of one or more doses of a cancer vaccine, wherein each further dose of anti-CD25 antibody and cancer vaccine is for administration prior to a dose of the Fas axis antagonist.

[0217] 52. The combination for use according to any one of clauses 42 to 51, wherein the anti-CD25 antibody inhibits the IL-2 signal via CD25 by less than 50% compared to the IL-2 signal in the absence of the antibody.

[0218] 53. The combination for use according to any one of clauses 42 to 52, wherein the anti-CD25 antibody is as defined in any one of clauses 25 to 30.

[0219] 54. The combination for use according to any one of clauses 39 to 53, wherein the cancer vaccine is a cytokine-expressing cellular immunotherapy, preferably the cytokine-expressing cellular immunotherapy comprises GM-CSF-expressing tumor cells.

[0220] 55. A combination for use according to any one of clauses 42 to 54, wherein the Fas axis antagonist is an anti-FasL antibody.

[0221] 56. A combination of an anti-CD25 antibody, an anti-FasL antibody and a cancer vaccine for use in the treatment of cancer, wherein the anti-CD25 antibody, the anti-FasL antibody and the cancer vaccine are for separate, simultaneous or sequential administration.

[0222] 57. A method of treating or preventing recurrence of cancer in a subject, wherein the subject has undergone Treg cell-depleting therapy for the treatment of cancer, the method comprising administering at least one dose of a Fas axis antagonist to the subject after the Treg cell-depleting therapy.

[0223] 58. The method of clause 57, wherein the Fas axis antagonist is an anti-FasL antibody.

[0224] 59. The method of paragraph 57 or 58, wherein the Treg cell depletion therapy comprises administration of an anti-CD25 antibody.

[0225] 60. The method of any one of paragraphs 57 to 59, wherein the method further comprises administration of a cancer vaccine.

[0226] 61. The method according to any one of clauses 57 to 60, comprising administering a Fas axis antagonist in combination with a further dose of an anti-CD25 antibody.

[0227] 62. The method of clause 61, wherein each further dose of anti-CD25 antibody is administered followed by each dose of the Fas axis antagonist.

[0228] 63. The method comprises the steps of: a) administering an anti-CD25 antibody; b) administering a cancer vaccine, optionally wherein administering the cancer vaccine comprises administering multiple doses of the cancer vaccine; c) administering an anti-CD25 antibody; and d) administering a cancer vaccine; and e) administering a Fas axis antagonist; Item 63. The method according to item 62, wherein the method comprises repeating steps c) to e) in a treatment cycle of at least 7 days duration.

[0229] 64. A method of treating cancer in a subject, comprising: a) a priming dose of Treg cell depletion therapy; b) administering to the subject a dose of a Fas axis antagonist and a booster dose of a Treg cell-depleting therapy; The method, wherein a booster dose of the Treg cell depleting therapy is administered prior to the Fas axis antagonist.

[0230] 65. The method of clause 64, wherein the Fas axis antagonist is an anti-FasL antibody.

[0231] 66. The method of any one of clauses 64 or 65, comprising administering multiple doses of a Fas axis antagonist and an anti-CD25 antibody, each dose of the Fas axis antagonist being administered after a booster dose of the Treg cell depleting therapy.

[0232] 67. The method of any one of clauses 64 to 66, comprising administering multiple doses of the Fas axis antagonist and booster doses of the Treg cell depleting therapy, with an interval of at least 7 days between booster doses of the Treg cell depleting therapy.

[0233] 68. The method of any one of clauses 64 to 67, comprising administering a priming dose of a cancer vaccine in combination with a priming dose of a Treg cell depleting therapy.

[0234] 69. The method of clause 68, wherein a priming dose of the cancer vaccine is administered after a priming dose of the Treg cell depleting therapy and before a booster dose of the Treg cell depleting therapy.

[0235] 70. The method of clause 69, comprising administering booster doses of the cancer vaccine, wherein each booster dose of the cancer vaccine is administered in combination with each booster dose of the Treg cell depleting therapy.

[0236] 71. The method is a) administering a priming dose of Treg cell depleting therapy; b) administering one or more priming doses of a cancer vaccine; and c) administering a booster dose of Treg cell depleting therapy; and d) optionally administering a booster dose of a cancer vaccine; e) administering a dose of a Fas axis antagonist; Item 71. The method according to item 70, wherein the method comprises repeating steps c) to e) in a treatment cycle of at least 7 days duration.

[0237] 72. The method of any one of clauses 64 to 71, wherein the Treg cell depletion therapy comprises an anti-CD25 antibody.

[0238] 73. The method of claim 72, wherein the anti-CD25 antibody is as defined in any one of claims 25 to 30.

[0239] 74. The method of any one of clauses 60 to 63, or 68 to 73, wherein the cancer vaccine is as defined in any one of clauses 31 and 32.

[0240] 75. A Fas axis antagonist for use in the treatment or prevention of recurrence of cancer in a subject, wherein the subject has undergone Treg cell-depleting therapy for the treatment of cancer, and the Fas axis antagonist is for administration to the subject following the Treg cell-depleting therapy.

[0241] 76. A Fas axis antagonist for use according to clause 75, wherein the Fas axis antagonist is an anti-FasL antibody.

[0242] 77. A Treg cell-depleting therapy for use in the treatment or prevention of recurrence of cancer in a subject, wherein the Treg cell-depleting therapy is for use in combination with at least one dose of a Fas axis antagonist, and the Fas axis antagonist is for administration to the subject following the Treg cell-depleting therapy.

[0243] 78. The Treg cell depletion therapy for use according to paragraph 77, wherein the subject has already undergone an initial Treg cell depletion therapy for the treatment of cancer.

[0244] 79. The Treg cell depletion therapy for use according to any one of clauses 77 or 78, wherein the Fas axis antagonist is an anti-FasL antibody.

[0245] 80. A combination of a) a priming dose of a Treg cell depleting therapy, b) a booster dose of a Treg cell depleting therapy, and c) a dose of a Fas axis antagonist for use in treating or preventing recurrence of cancer in a subject, wherein the priming dose of the Treg cell depleting therapy and a first booster dose of the Treg cell depleting therapy are administered before the first dose of the Fas axis antagonist.

[0246] 81.d) The combination for use according to clause 80, further comprising a priming dose of a cancer vaccine, wherein the priming dose of the cancer vaccine is administered between the Treg cell depleting therapy and the first booster dose of the Treg cell depleting therapy.

[0247] 82. The combination for use according to any one of clauses 80 or 81, wherein the treatment comprises booster doses of the Treg cell depleting therapy and repeated administration of the Fas axis antagonist, wherein each booster dose of the Treg cell depleting therapy is administered before each dose of the Fas axis antagonist.

[0248] 83.e) The combination for use according to clause 82, further comprising a dose of a cancer vaccine booster, wherein each booster dose of the cancer vaccine is administered between each booster dose of the Treg cell depleting therapy and each dose of the Fas axis antagonist.

[0249] 84. A combination for use according to any one of clauses 80 to 83, wherein the Treg cell depleting therapy comprises an anti-CD25 antibody and the Fas axis antagonist is an anti-FasL antibody.

[0250] 85. The combination for use according to any one of clauses 81 or 83, wherein the cancer vaccine is a cytokine-expressing cellular immunotherapy, preferably the cytokine-expressing cellular immunotherapy comprises GM-CSF-expressing tumor cells.

[0251] 86. The T cell depletion therapy for use according to any one of clauses 33 or 77 to 79, the anti-CD25 antibody for use according to clause 34, the anti-Fas axis antagonist for use according to any one of clauses 35, 75 and 76, the anti-FasL antibody for use according to clause 36, the cancer vaccine for use according to any one of clauses 37 or 38, or the combination for use according to any one of clauses 39 to 56 or 80 to 85, wherein the cancer is a solid tumor.

[0252] 87. A kit comprising: a) a first composition comprising a Treg cell depleting therapy; b) a second composition comprising a Fas axis antagonist; and optionally instructions for using the composition in a combination therapy for the treatment of cancer.

[0253] 88.c) The kit of paragraph 87, further comprising a third composition comprising a cancer vaccine.

[0254] Also, the present application is to be understood as disclosing all combinations of any of the above aspects and embodiments described above with each other, unless the context requires otherwise. Similarly, the present application discloses all combinations of preferred and / or optional features alone or with any of the other aspects, unless the context requires otherwise.

[0255] The present invention will now be further illustrated by the following examples, which, with reference to the figures, are meant to assist the skilled artisan in the practice of the invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[0256] mouse C57BL / 6 mice (female, 6-8 weeks old) were obtained from Charles River Laboratories. All animal studies were performed under ethical approval and regulation from University College London and the UK Home Office.

[0257] Cell lines and tissue culture The highly tumorigenic and poorly immunogenic cell line B16 / BL6 mouse melanoma cells were obtained from Dr. IJ Fidler at MD Anderson Cancer Center (Houston, TX). B16 / BL6 expressing GM-CSF (GVAX) was used as therapy. Both cell lines have been described previously (Van Elsas et al., 1999, J Exp Med vol. 190(3) p355-366). B16 / BL6 and GVAX cells were grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal calf serum (FCS, Sigma), 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM L-glutamine (all from Gibco) at 4 °C for 1 h at 25 °C. 2 The cells were cultured at 37°C in a 5% humidified atmosphere.

[0258] therapeutic antibodies anti-CD25 NIBAntibody production was outsourced to Evitria AG (Switzerland). Anti-CD25 NIB The antibody is a murine non-IL-2 blocking anti-CD25 antibody (comprising the variable regions of clone 7D4 in a murine IgG2a backbone as described in Solomon I et al (2020) Nature Cancer, vol 1, p1153-1166, US Pat. No. 5,399,423).

[0259] Isotype control antibody (InVivoMAb polyclonal Armenian hamster IgG) (catalog number BE0091) and anti-FasL antibody (MFL3) (catalog number BE0319) were purchased from BioXcell.

[0260] In vivo tumor experiments On day 0, C57BL / 6 mice were injected with 50,000 B16 / BL6 melanoma cells subcutaneously (sc) into the right flank. On day 5, 200 μg of anti-CD25 NIB Antibodies were administered intraperitoneally (ip) at 1 × 10 6 irradiated (150 Gy) GVAX cells were injected intradermally (id) into the contralateral flank.

[0261] Tumor size was measured rigorously 3 to 4 times a week, and the volume was calculated as the product of three perpendicular diameters. Tumor volume (mm) versus days after inoculation 3 ) was used to plot tumor growth curves.

[0262] Tumor growth was closely monitored after the initial combinatorial therapy, and only tumors that responded to the initial combinatorial treatment were included for further treatment.

[0263] When most tumors had shrunk or remained at a stable size, isotype control antibody (200 μg ip) and anti-FasL (MFL3) mAb (200 μg ip) were given on day 28. A second dose (also 200 μg ip) was given one week later. In the combination treatment group, anti-CD25 mAb was given on days 25 and 35 to maintain Treg depletion. NIB(200 μg ip) was administered.

[0264] In summary, the treatment groups were as follows: Group 1: 200 μg anti-CD25 on day 5 NIB , 1 x 10 on days 6, 9, and 12 6 GVAX cells, 200 µg of isotype control on days 28 and 36 (Figure 1A) Group 2: 200 μg anti-CD25 on day 5 NIB , 1 x 10 on days 6, 9, and 12 6 GVAX cells, 200 μg of anti-FasL antibody on days 28 and 36 (Figure 1B). Group 3: 200 μg anti-CD25 on day 5 NIB , 1 x 10 on days 6, 9, and 12 6 GVAX cells, 200 μg of anti-CD25 on days 11, 17, and 24 NIB , as well as weekly injections until day 56 (Figure 1C). Group 4: 200 μg anti-CD25 on day 5 NIB , 1 x 10 on days 6, 9, and 12 6 GVAX cells, 200 μg of anti-CD25 on days 25 and 36 NIB , 200 μg of anti-FasL antibody on days 28 and 36 (Figure 1D).

[0265] Tumor size was measured two to three times a week, and mice were euthanized when any diameter reached 150 mm.

[0266] result The results showing tumor growth and survival over time are shown in Figures 2 and 3. The results show that the use of anti-FasL antibody in combination therapy with anti-CD25 antibody significantly prevented recurrent tumor growth and improved survival.

[0267] In particular, these experiments demonstrate that the addition of a combination of anti-FasL and anti-CD25 antibodies to initial combination therapy of anti-CD25 antibody and GVAX prevents recurrent tumor growth and improves survival compared to isotype control, anti-FasL antibody treatment alone, and anti-CD25 antibody treatment alone.

[0268] Due to the loss of activated Teff and acquisition of Treg over time, it is believed that treating stable tumors with an intensified therapeutic regimen (multiple cycles of combined anti-FasL and anti-CD25 treatment) may provide more antigen-specific T cells and keep the number of Tregs low in the TME, thereby preventing relapse. Blockade of the Fas / FasL axis with anti-FasL antibodies is believed to inhibit Fas-mediated cell death of activated Teff. This inhibition of Fas-mediated cell death of activated Teff combined with depletion of Tregs helps maintain an immunogenic TME, thereby achieving better therapeutic efficacy.

[0269] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described uses and methods of the invention will become apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention is not to be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology, cellular immunology, or related fields are intended to be within the scope of the following claims.

[0270] A summary of the sequences referenced in this application is provided in the table below.

[0271] [Table 2] TIFF2025514847000004.tif250170TIFF2025514847000005.tif251170TIFF2025514847000006.tif212170

Claims

1. 13. A combination of a Treg cell depleting therapy and a Fas axis antagonist for use in the treatment of cancer, wherein said Treg cell depleting therapy and said Fas axis antagonist are for separate, simultaneous or sequential administration.

2. The combination for use according to claim 1 , wherein the Treg cell depleting therapy comprises an anti-CD25 antibody.

3. The combination for use according to claim 1 or 2, further comprising a cancer vaccine.

4. The combination for use according to claim 3, wherein the combination comprises: a) a first component comprising said anti-CD25 antibody; b) a second component comprising said Fas axis antagonist; and c) a third component comprising said cancer vaccine, for separate, simultaneous or sequential administration.

5. The combination for use according to claim 4, wherein said Fas axis antagonist is for administration subsequent to said anti-CD25 antibody and said cancer vaccine.

6. 6. The combination for use according to claim 4 or 5, wherein said Fas axis antagonist is for administration following depletion of Treg cells in said subject by administration of said anti-CD25 antibody.

7. The combination for use according to any one of claims 4 to 6, wherein said administration of said anti-CD25 antibody to said subject is followed by administration of said cancer vaccine, which is then followed by administration of said Fas axis antagonist.

8. The combination for use according to any one of claims 4 to 7, wherein said treatment comprises the administration of multiple doses of said anti-CD25 antibody.

9. The combination for use according to any one of claims 4 to 8, wherein said treatment comprises the administration of multiple doses of said Fas axis antagonist.

10. The combination for use according to any one of claims 4 to 9, wherein said treatment comprises the administration of multiple doses of said cancer vaccine.

11. The administration, Following the initial administration of the anti-CD25 antibody, followed by administration of one or more doses of said cancer vaccine; 11. The combination for use according to any one of claims 8 to 10, comprising the subsequent administration of one or more alternating doses of said anti-CD25 antibody and said Fas axis antagonist, each further dose of said anti-CD25 antibody being for administration prior to a dose of said Fas axis antagonist.

12. The combination for use according to claim 11, wherein the administration of said combination of said dose of Fas axis antagonist and said further dose of said anti-CD25 antibody is repeated in a therapeutic cycle of at least 7 days.

13. The combination for use according to claim 11 or 12, wherein the administration of the one or more alternating doses of the anti-CD25 antibody and the Fas axis antagonist also comprises the administration of one or more doses of the cancer vaccine, wherein a further dose of each of the anti-CD25 antibody and the cancer vaccine is for administration prior to a dose of the Fas axis antagonist.

14. The combination for use according to any one of claims 4 to 13, wherein said anti-CD25 antibody inhibits IL-2 signalling via CD25 by less than 50% compared to the IL-2 signalling in the absence of said antibody.

15. The combination for use according to any one of claims 4 to 14, wherein said cancer vaccine is a cytokine-expressing cellular immunotherapy.

16. The combination for use according to claim 15, wherein said cytokine-expressing cellular immunotherapy comprises GM-CSF-expressing tumor cells.

17. The combination for use according to any one of claims 1 to 16, wherein said Fas axis antagonist is an anti-FasL antibody.

18. The combination for use according to any one of claims 1 to 17, wherein the cancer is a solid tumor.

19. A kit for treating cancer, comprising: a) a first composition comprising a Treg cell depletion therapy; b) a second composition comprising a Fas axis antagonist; and optionally instructions for use in combination therapy.

20. 20. The kit of claim 19, wherein the kit further comprises c) a third composition comprising a cancer vaccine.

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