Combination Therapy and Its Use

Abstract

The present invention provides a novel combination therapy comprising radiation therapy, a bispecific immunocytokine, and optionally a Treg depleting agent. The present invention also provides methods of using said combination therapy in the treatment of cancer, preferably solid tumors.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to combination therapy of PD-1-targeted IL-2 variant immunoconjugates, radiation therapy, and optionally depletion of regulatory T cells (Tregs), preferably by using a non-IL-2 blocking anti-CD25 antibody. The present invention also relates to the use of these combination therapies for the treatment of solid tumors. [Background technology]

[0002] Background of the Invention The advent of cancer immunotherapy has revolutionized the way oncologists manage and treat cancer, increasing patient response rates and overall survival. However, the success of immunotherapies, most notably immune checkpoint inhibitors, has not been all-encompassing, with varying levels of efficacy across cancer types and even among patients with the same malignancy. 1 Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent malignancies worldwide. 2 , which may be characterized by their immunologically cold tumors and high resistance to therapy, ultimately leading to poor treatment outcomes. 3 Radiation therapy is used as the standard of care in many cases, not only directly killing tumor cells but also helping to sensitize the tumor microenvironment (TME) to immunotherapy. 4 Despite these efforts, the development of acquired resistance to combination radioimmunotherapy, the underlying mechanisms of which remain to be fully understood, continues to occur.

[0003] The immune system plays a fundamental role in disease progression and treatment response in HNSCC 4,5 Radiation therapy (RT) induces the influx of proinflammatory immune cells into the TME. 4 However, this induction of a proinflammatory TME is transient and is ultimately suppressed by the influx of immunosuppressive regulatory T cells (Tregs). 4,6Tregs have been identified as important regulators of resistance to radioimmunotherapy. 7-9 , which can indirectly suppress antitumor immunity through IL-2 sequestration. Eliminating Tregs using αCD25 can activate natural killer cells (NK), a process mediated by IL-2 signaling via CD122. 10,11 NK cells, a type of innate lymphoid cell, play a key role in tumor surveillance and direct cytotoxic cell killing both locally and peripherally. 12,13 Furthermore, NK cells are dependent on IL-2 signaling to regulate much of their homeostasis and cytotoxicity. 14,15 , making it an attractive target for IL-2-directed immunotherapy.

[0004] Immune exhaustion is another confounding factor in acquired resistance 16 High intratumoral surface expression of programmed cell death protein 1 (PD-1) on lymphocytes and upregulation of programmed cell death ligand 1 (PD-L1) on tumor cells and suppressor immune cells certainly prevent tumor-infiltrating lymphocytes from mounting a sustained antitumor response. 4,6,17 Blocking PD-1 + It has been shown to restore T effector function, but PD-1 + It also has the opposing effect of amplifying Treg-mediated immunosuppression in Tregs 18,19 Therefore, αPD-1 therapy + Reactivation of effector T cells may only be achievable by subsequent depletion of Tregs.

[0005] Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor known to construct an immunosuppressive tumor microenvironment (TME) and is known to be resistant to traditional targeted and cytotoxic therapies, such as radiation therapy (RT) (Dougan, 2017; Quinonero et al., 2019). Even in the face of immune-activating treatments, responses in this disease type are almost always transient (Molejon et al., 2015). Therefore, consideration of the biological factors contributing to immune escape during treatment failure is essential to overcome intrinsic resistance. Given that PDAC is a systemic disease with a high risk of metastasis and that only approximately 10% of patients are diagnosed at an early stage (Zhu et al., 2018), treatments aimed at improving response and outcomes must focus on systemic immunity. The practice of immunotherapy in PDAC is still in its infancy, and improved treatments for this deadly disease are needed. [Brief explanation of the drawings]

[0006] [Figure 1]Treatment with αCD25 and PD1-IL2v results in tumor growth delay in HNSCC tumors. A) Tumor growth curves for mice implanted with LY2 tumors. B) Kaplan-Meier survival curves for mice implanted with LY2 tumors. RT:RT + αCD25: Hazard Ratio = 4.943; 95% CI = 1.312-18.62. RT:RT + αCD25 + PD1-IL2v: Hazard Ratio = 11.43; 95% CI = 2.742-47.67. LY2 tumors were established and treated with (a) PD1-IL2v (0 / 7), (b) RT (0 / 7), (c) RT + aCD25 (5 / 7), (d) RT + PD1-IL2v (0 / 7), and (e) RT + aCD25 + PD1-IL2v (7 / 8). RT was administered as a single dose of 10 Gy. Antibodies were administered weekly via IP injection throughout the study period. C) Tumor growth curves for mice implanted with MOC2 tumors. D) Kaplan-Meier survival curves for mice implanted with MOC2 tumors. RT:RT + αCD25 + PD1-IL2v: Hazard Ratio = 2.388; 95% CI = 0.801-7.121. MOC2 tumors were established and treated with (a) RT (0 / 8), (b) RT + PD1-IL2v (0 / 8), or (c) RT + αCD25 + PD1-IL2v (2 / 8). RT was administered in three doses of 8 Gy, 3-4 days apart. Antibodies were administered weekly via IP injection throughout the study period. E) Tumor growth curves for mice implanted with P029 tumors. F) Kaplan-Meier survival curves for mice implanted with P029 tumors. RT: RT + αCD25 + PD1-IL2v: Hazard ratio = 2.354; 95% CI = 0.7418-7.471. P029 tumors were established and treated with (a) RT (0 / 7), (b) RT + αCD25 (1 / 7), (c) RT + PD1-IL2v (1 / 7), and (d) RT + αCD25 + PD1-IL2v (1 / 8). RT was administered in three 8 Gy fractions, 5 days apart. Antibodies were administered weekly via ip injection for the duration of the study. For all Figures 1: n = 7-8 for all in vivo experiments. All statistical analyses were performed using one-way ANOVA unless otherwise noted. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001 [Figure 2]Radiation therapy and PD1-IL2v suppress lung metastasis. A) Schematic illustrating the experimental design. P029 tumors were established in C57 Bl / 6 mice and treated with RT and a combination of αCD25, PD1-IL2v, αCD25 and PD1-IL2v, or no immunotherapy. RT was administered in three 8 Gy fractions, 5 days apart. Antibodies were administered by weekly i.p. injection starting 1 day before RT. B) Percentage of mice in each treatment group that developed macroscopic lung metastases. n=7. C) Frequency of circulating tumor cells (CTCs) in the blood of tumor-bearing P029 mice. CTCs were defined as CD45-EpCAM+ and CD45-EpCAM+pan-Cytokeratin+. D) Percentage of P029 tumor-implanted mice that developed lung metastases between treatment groups. All statistical analyses were performed using one-way ANOVA unless otherwise noted. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001 [Figure 3] Survival rates in preclinical mouse PDAC models. A) Kaplan-Meier survival analysis of mice bearing orthotopically implanted pancreatic tumors, PK5L1940, treated with (a) RT, (b) RT + aCD25, (c) RT + aPD1-IL2v, or (d) RT + aCD25 + aPD1-IL2v. B) Kaplan-Meier survival analysis of mice bearing orthotopically implanted pancreatic tumors, FC1242, treated with (a) RT, (b) RT + aCD25, (c) RT + aPD1-IL2v, or (d) RT + aCD25 + aPD1-IL2v. RT was administered on day 7 after implantation. aPD1-IL2v and aCD25 were administered weekly starting on day 7 after implantation. n≥7 / group. P values ​​were calculated using Student's t-test; * indicates p<0.05, ** indicates p<0.01. [Figure 4]Peripheral activation of antitumor immune populations reduces metastatic burden in a PDAC model. A) Flow cytometry analysis of NK cell frequency (top) and NK cell expression of functional markers DNAM1 (middle) and Gnzmb (bottom) in the blood of pancreatic tumor-bearing mice treated with (a) RT, (b) RT+aCD25, (c) RT+aPD1-IL2v, and (d) RT+aCD25+aPD1-IL2v per group. n≥5 / group. P values ​​were calculated using Student's t-test; * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. B) Kaplan-Meier survival analysis of PK5L1940-implanted mice using a unilateral splenectomy model of metastatic pancreatic cancer. Mice were (a) untreated or treated with (b) RT, (c) RT + aCD25, (d) RT + aPD1-IL2v, or (e) RT + aCD25 + aPD1-IL2v. n ≥ 7 / group. P values ​​were calculated by Student's t-test, where * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. C) Kaplan-Meier survival analysis of mice implanted with FC1242 cells using a unilateral splenectomy model of metastatic pancreatic cancer. RT was administered on day 7 after implantation. PD1-IL2v and aCD25 were administered weekly starting on day 7 after implantation. Treatment groups: (a) RT alone (n ≥ 7), (b) RT + aCD25 (n < 7), (c) RT + PD1-IL2v (n ≥ 7), and (d) RT + aCD25 + PD1-IL2v (n ≥ 7). P values ​​were calculated by Student's T-test and were (a)–(c) < 0.01 and (d) < 0.001. DETAILED DESCRIPTION OF THE INVENTION

[0007] Detailed Description of the Invention The present invention provides a combination for use in the treatment of cancer, the combination comprising: a) a first component comprising an effective amount of a Treg cell depleting agent; b) a second component comprising an effective amount of a bispecific immunocytokine; and c) a third component comprising an effective amount of radiation therapy, wherein components a)-c) are for simultaneous or sequential administration.

[0008] In one embodiment, the use of the first component a) is optional.Thus, in another embodiment, the present invention provides a combination for use in the treatment of cancer, which combination comprises an effective amount of a bispecific immunocytokine and an effective amount of radiation therapy for simultaneous or sequential administration.

[0009] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. In interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall also include the plural and vice versa.

[0010] Bispecific immunocytokines Bispecific immunocytokines used in the combination therapies described herein comprise a PD-1-targeting antigen-binding moiety and an IL-2-based effector moiety, including, for example, IL-2 variants, as described in WO 2018 / 184964. In one embodiment, the PD-1-targeting antigen-binding moiety is an antibody, or antigen-binding fragment thereof, that binds to PD-1 in an immune cell, particularly a T cell, or tumor cell environment that expresses PD-1 (compared to wild-type IL-2, e.g., human IL-2 set forth as SEQ ID NO: 4), and an IL-2 variant, particularly a variant of human IL-2, with reduced binding affinity to the α-subunit of the IL-2 receptor, for example, at one, two, or three positions selected from: i) positions corresponding to residues 42, 45, and 72 of human IL-2 set forth as SEQ ID NO: 4 or ii) an IL-2 comprising one, two, or three amino acid substitutions, e.g., three substitutions at three positions, e.g., the specific amino acid substitutions F42A, Y45A, and L72G; or ii) an amino acid substitution at a position corresponding to residue 3 of human IL-2 set forth as SEQ ID NO:4, in addition to the features set forth in i), e.g., the specific amino acid substitution T3A; or iii) four amino acid substitutions at positions corresponding to residues 3, 42, 45, and 72 of human IL-2 set forth as SEQ ID NO:4, e.g., the specific amino acid substitutions T3A, F42A, Y45A, and L72G. In one embodiment, the antibody may be an IgG antibody, particularly an IgG1 antibody. In another embodiment, the PD-1-targeting IL-2 variant immunoconjugate may comprise a single IL-2 variant with reduced binding affinity to a subunit of the IL-2 receptor (i.e., not more than one IL-2 variant moiety).

[0011] In a preferred embodiment, targeting of PD-1 by the PD-1-targeting IL-2 variant immunoconjugate can be achieved by targeting PD-1 as described in WO 2018 / 1184964. Targeting of PD-1 can be achieved with an anti-PD-1 antibody or antigen-binding fragment thereof. The anti-PD-1 antibody can comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 1 or a variant thereof that retains functionality. The anti-PD-1 antibody can comprise a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 2 or a variant thereof that retains functionality. The anti-PD-1 antibody may comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1, or a variant thereof that retains functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 2, or a variant thereof that retains functionality. The anti-PD-1 antibody may comprise the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO:2.

[0012] The PD-1-targeted IL-2 variant immunoconjugate may comprise a polypeptide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, or a variant thereof that retains functionality. The PD-1-targeted IL-2 variant immunoconjugate may comprise a polypeptide sequence in which a PD-1-specific Fab heavy chain shares a carboxy-terminal peptide bond with an Fc domain subunit that contains a hole modification. The PD-1-targeted IL-2 variant immunoconjugate may comprise the polypeptide sequence of SEQ ID NO:5 or SEQ ID NO:6, or a variant thereof that retains functionality. The PD-1-targeted IL-2 variant immunoconjugate may comprise a PD-1-specific Fab light chain. The PD-1-targeted IL-2 variant immunoconjugate may comprise the polypeptide sequence of SEQ ID NO:7, or a variant thereof that retains functionality. The polypeptides may be covalently linked, for example, by a disulfide bond. The polypeptide chain of the Fc domain may contain the amino acid substitutions L234A, L235A, and P329G (which may also be referred to as LALA P329G).

[0013] As described in WO 2018 / 184964, the PD-1-targeted IL-2 variant immunoconjugate can be a PD-1-targeted IgG-IL-2qm fusion protein having the sequences set forth as SEQ ID NOs: 5, 6, and 7 (e.g., as described in Example 1 of WO 2018 / 184964). The PD-1-targeted IL-2 variant immunoconjugate having the sequences set forth as SEQ ID NOs: 5, 6, and 7 is referred to herein as "PD1-IL2v." The PD-1-targeted IL-2 variant immunoconjugate having the sequences set forth as SEQ ID NOs: 8, 9, and 10 is referred to herein as "muPD1-IL2v," which is the murine surrogate.

[0014] In another embodiment, the PD-1-targeted IL-2 variant immunoconjugate used in the combination therapy of the invention may comprise a) a heavy chain variable domain VH of SEQ ID NO: 1 and a light chain variable domain VL of SEQ ID NO: 2, and the polypeptide sequence of SEQ ID NO: 3, or b) the polypeptide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 and SEQ ID NO: 7, or c) the polypeptide sequences of SEQ ID NO: 8, and SEQ ID NO: 9, and SEQ ID NO: 10.

[0015] As described herein, the PD-1-targeted IL-2 variant immunoconjugates and antigen-binding molecules used in the combination therapies described herein may comprise an Fc domain that is composed of two subunits and contains a modification that promotes heterodimerization of two non-identical polypeptide chains. The PD-1-targeted IL-2 variant immunoconjugates and antigen-binding molecules used in the combination therapies described herein may comprise an Fc domain subunit that contains a knob mutation and an Fc domain subunit that contains a hole mutation.

[0016] A "heterodimerization-promoting modification" is a manipulation of the peptide backbone or a post-translational modification of a polypeptide that reduces or prevents the association of the polypeptide with an identical polypeptide to form a homodimer. As used herein, heterodimerization-promoting modifications particularly include separate modifications made to each of two polypeptides desired to form a dimer, where the modifications are complementary to each other so as to promote the association of the two polypeptides. For example, a heterodimerization-promoting modification may alter the structure or charge of one or both of the polypeptides desired to form a dimer, making their association sterically or electrostatically favorable, respectively. Heterodimerization occurs between two non-identical polypeptides, such as two subunits of an Fc domain, where additional immunoconjugate components (e.g., antigen-binding moieties, effector moieties) fused to each subunit are not identical. In the immunoconjugates and bispecific antibodies according to the present invention, the heterodimerization-promoting modification is in the Fc domain. In some embodiments, the heterodimerization-promoting modification comprises an amino acid mutation, specifically an amino acid substitution. In certain embodiments, the heterodimerization-promoting modification comprises a distinct amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain. The most extensive protein-protein interaction site between the two polypeptide chains of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain. In a specific embodiment, the modification is a knob-into-hole modification comprising a knob modification in one of the two subunits of the Fc domain and a hole modification in the other of the two subunits of the Fc domain.

[0017] Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a "protuberance" ("knob") into the interface of a first polypeptide and a corresponding "cavity" ("hole") into the interface of a second polypeptide, where the protuberance can be positioned in the cavity to promote heterodimer formation and prevent homodimer formation. The protuberance is constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A complementary depression of identical or similar size to the protrusion is created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). The protrusion and depression can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In a more specific embodiment, the Fc domain subunit that constitutes the knob modification further comprises the amino acid substitution S354C, and the Fc domain subunit that constitutes the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of disulfide bridges between the two subunits of the Fc region, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).The numbering of amino acid residues in the Fc region follows the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. A "subunit" of an Fc domain, as used herein, refers to one of the two polypeptides that form the dimeric Fc domain (i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association). For example, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0018] In another embodiment, modifications that promote heterodimerization of two non-identical polypeptide chains include modifications that mediate electrostatic steering effects, such as those described in WO 2009 / 089004. Generally, this method involves replacing one or more amino acid residues at the interface of the two polypeptide chains with a charged amino acid residue, such that homodimer formation is electrostatically disfavored, but heterodimer formation is electrostatically favored.

[0019] An IL-2 variant with reduced binding affinity for a subunit of the IL-2 receptor can be fused to the carboxy-terminal amino acid of an Fc domain subunit containing a knob modification. Without being bound by theory, fusion of an IL-2 variant to an Fc domain knob-containing subunit will further minimize the generation of homodimeric immunoconjugates containing two IL-2 variant polypeptides (steric clash of the two knob-containing polypeptides).

[0020] The Fc domain of immunoconjugates and antigen-binding molecules can be engineered to have altered binding affinity for Fc receptors, specifically Fcγ receptors, compared to unengineered Fc domains, as described in WO 2012 / 146628. The binding of the Fc domain to complement components, specifically C1q, can be altered as described in WO 2012 / 146628. The Fc domain confers favorable pharmacokinetic properties to immunoconjugates and bispecific antibodies, including a long serum half-life that contributes to favorable accumulation in target tissues and favorable tissue-to-blood distribution ratios. However, this can also lead to undesired targeting of Fc receptor-expressing cells rather than the desired antigen-bearing cells. Furthermore, simultaneous activation of the Fc receptor signaling pathway can lead to cytokine release, which, combined with the long half-life of the effector moiety and immunoconjugate, can result in excessive cytokine receptor activation and severe side effects upon systemic administration. Consistent with this, conventional IgG-IL-2 immunoconjugates have been described to be associated with infusion reactions (see, e.g., King et al., J Clin Oncol 22, 4463-4473 (2004)).

[0021] Thus, the Fc domain of immunoconjugates and antigen-binding molecules can be engineered to have reduced binding affinity to Fc receptors. In one such embodiment, the Fc domain contains one or more amino acid mutations that reduce the binding affinity of the Fc domain to Fc receptors. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to Fc receptors by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there are two or more amino acid mutations that reduce the binding affinity of the Fc domain to Fc receptors, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to Fc receptors by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, immunoconjugates and bispecific antibodies comprising engineered Fc domains exhibit less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity of immunoconjugates and bispecific antibodies comprising non-engineered Fc domains. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an Fcγ receptor, more specifically, an FcγRIIIa, FcγRI, or FcγRIIa receptor. Preferably, binding to each of these receptors is reduced. In some embodiments, the binding affinity to complement components, particularly C1q, is also reduced. In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain to the receptor, is achieved when the Fc domain (or an immunoconjugate comprising the Fc domain) exhibits greater than about 70% of the binding affinity to FcRn of the non-engineered form of the Fc domain (or an immunoconjugate comprising the non-engineered form of the Fc domain). The Fc domain, or immunoconjugates and bispecific antibodies of the invention comprising said Fc domain, may exhibit greater than about 80%, and even greater than about 90%, of such affinity. In one embodiment, the amino acid mutation is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at position P329.In more specific embodiments, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain comprises an additional amino acid substitution at a position selected from S228, E233, L234, L235, N297, and P331. In more specific embodiments, the additional amino acid substitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a specific embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235. In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A, and P329G (LALA P329G). This combination of amino acid substitutions almost completely eliminates Fcγ receptor binding to the human IgG Fc domain, as described in WO 2012 / 130831, which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function. The numbering of amino acid residues in the Fc region follows the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0022] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods known in the art and described in WO 2012 / 146628. Genetic methods can include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The exact nucleotide changes can be confirmed, for example, by sequencing.

[0023] In one embodiment, the Fc domain is engineered to have reduced effector function compared to a non-engineered Fc domain, as described in WO 2012 / 146628. Reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce apoptosis, reduced cross-linking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming.

[0024] Compared to IgG1 antibodies, IgG4 antibodies have reduced binding affinity to Fc receptors and reduced effector function. Thus, in some embodiments, the Fc domain of the antigen-binding molecule is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P. To further reduce its binding affinity to Fc receptors and / or its effector function, in one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E. In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G. In a specific embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235, and P329, specifically the amino acid substitutions S228P, L235E, and P329G. Such IgG4 Fc domain variants and their Fcγ receptor binding properties are described in European Patent Application WO 2012 / 130831, which is incorporated herein by reference in its entirety.

[0025] Radiation therapy In an embodiment of the present invention, the PD1-IL2v immunoconjugates described herein are used in combination with radiation therapy.

[0026] Radiation therapy or radiotherapy ("RT") is the use of ionizing radiation, usually delivered by a linear accelerator, to control or kill malignant cells, typically as part of cancer treatment. Radiation therapy can cure many types of cancer, provided the cancer is localized to one site in the body. Radiation therapy can also be used as an adjuvant treatment after surgery to remove a primary malignant tumor (e.g., early-stage breast cancer) to prevent tumor recurrence. Radiation therapy has a synergistic effect with chemotherapy and has been used before, during, and after chemotherapy in susceptible cancers.

[0027] Radiation therapy is commonly used for cancerous tumors due to its ability to control cell proliferation. Ionizing radiation damages the DNA of cancerous tissue, leading to cell death. Shaped radiation beams can be delivered from several angles to intersect the tumor, sparing normal tissue (such as the skin or organs through which radiation must pass to treat the tumor), providing a much higher absorbed dose than surrounding healthy tissue. In addition to the tumor itself, draining lymph nodes may also be included in the radiation field if they are clinically or radiologically involved in the tumor or are considered at risk for metastasis of occult malignancies. A margin of normal tissue surrounding the tumor must be included to account for uncertainties in daily setup and tumor movement within the body. These uncertainties can result from internal movement or the movement of external skin markers relative to the tumor's location. The response of cancer to radiation can be expressed as radiosensitivity. Radiosensitive cancer cells can be rapidly killed by moderate doses of radiation. These include leukemia, most lymphomas, and germ cell tumors. The majority of epithelial cancers are only moderately radiosensitive and require fairly high doses (60–70 Gy) of radiation to achieve a cure. Some cancer types are highly radioresistant; that is, they may require doses much higher than clinically safe to be cured. It is important to distinguish a tumor's radiosensitivity, which is partly a laboratory measurement, from the actual clinical "cure potential" of cancer. For example, leukemia is generally incurable with radiation therapy because it disseminates throughout the body. Lymphoma can be cured if it is confined to a single body site. Similarly, many common, moderately radiosensitive tumors are routinely treated with curative doses of radiation therapy if they are detected early. Cancers such as skin, head and neck, breast, non-small cell lung, cervical, and prostate cancers are often incurable because radiation therapy cannot treat the entire body.

[0028] Tumor response to radiation therapy depends on tumor size. Very large tumors respond less well to radiation than small tumors or microscopic disease. Various strategies have been employed to overcome this effect. The most common approach is surgical resection before radiation therapy. This is most commonly seen in the treatment of breast cancer with mastectomy followed by adjuvant radiation therapy. Another approach is to shrink the tumor with neoadjuvant chemotherapy before definitive radiation therapy. A third approach is to increase the cancer's radiosensitivity by administering certain drugs during the radiation therapy course. Examples of radiosensitizing drugs include cisplatin, nimorazole, and cetuximab.

[0029] Radiation therapy typically causes minimal or no side effects, although short-term pain recurrences may occur for several days after treatment due to edema compressing nerves in the treatment area. High doses can cause a variety of side effects, ranging from acute side effects during treatment (acute side effects), to long-term side effects months to years after treatment (long-term side effects), and even after retreatment (cumulative side effects). The nature, severity, and duration of side effects depend on the organ receiving radiation, the treatment itself (type of radiation, dose, and fractionation), and the patient. The most commonly reported side effects include fatigue and mild to moderate sunburn-like skin irritation. Fatigue often develops midway through a course of treatment and can persist for several weeks after treatment has ended. Irritated skin heals, but may not retain its previous elasticity. Radiation side effects are often limited to the area of ​​the patient's body receiving treatment and are dose-dependent. For example, high doses of head and neck radiation can be associated with cardiovascular complications, thyroid dysfunction, and pituitary axis dysfunction. Therefore, lower doses of radiation therapy may be preferred.

[0030] The radiation dose used in photon radiation therapy is measured in grays (Gy) and varies depending on the type and stage of the cancer being treated. For definitive cases, typical doses for solid epithelial tumors range from 60 to 80 Gy, while lymphomas are treated with doses ranging from 20 to 40 Gy. Prophylactic doses typically range from 45 to 60 Gy, delivered in 1.8 to 2 Gy fractions (for breast and head and neck cancers). Many factors are considered by the radiation oncologist when selecting the dose, including whether the patient has received chemotherapy, the patient's comorbidities, and whether radiation therapy is administered before or after surgery. The delivery parameters of the prescribed dose are determined during treatment planning (part of dosimetry). Treatment planning is typically performed on a dedicated computer using specialized treatment planning software. Depending on the radiation delivery method, multiple angles or sources may be used to achieve the desired total dose. Planners will attempt to deliver the prescribed dose uniformly to the tumor while minimizing the dose to surrounding healthy tissue.

[0031] The total dose is fractionated (delivered over a period of time) for several important reasons. Fractionation allows normal cells time to recover, while tumor cells typically repair less efficiently between doses. Fractionation also allows tumor cells that were in a relatively radioresistant stage of the cell cycle after one treatment to transition to a sensitive stage before the next fraction is administered. Similarly, tumor cells that have been chronically or acutely hypoxic (i.e., relatively radioresistant) can reoxygenate between fractions, improving tumor cell killing. A typical fractionation schedule for adults is 1.8–2 Gy per day, 5 days per week. For some cancer types, a longer fractionation schedule can allow tumors to begin regrowing, so for tumor types including head and neck cancer and cervical squamous cell carcinoma, it is preferable to terminate radiation therapy within a certain time frame. Smaller fraction sizes reduce the incidence and severity of late side effects in normal tissues, so a typical fraction size for children may be 1.5–1.8 Gy per day. In some cases, toward the end of the treatment series, two daily fractions are used. This schedule, known as a simultaneous boost regimen or hyperfractionation, is used for tumors that grow rapidly, especially small tumors in the head and neck.

[0032] One type of fractionation schedule that has recently become increasingly popular and continues to be studied is hypofractionation, a type of radiation therapy in which the total radiation dose is divided into large doses. Typical doses vary greatly depending on the cancer type, ranging from 1.8 Gy / fraction to 20 Gy / fraction, the latter being typical for stereotactic treatment of subcranial lesions (stereotactic ablative body radiotherapy, or SABR, also known as SBRT) or stereotactic radiosurgery (SRS) for intracranial lesions. Hypofractionation at doses in the 5-20 Gy range may be preferred. Depending on the cancer being treated, hypofractionation at doses in the 1.8-2.2 Gy range may be of particular interest. The rationale for hypofractionation is to reduce the chance of local recurrence by denying clonogenic cells the time necessary to reproduce and to take advantage of the radiosensitivity of some tumors. In particular, stereotactic therapy is intended to destroy clonogenic cells by a process of ablation, i.e., delivery of a dose intended to directly destroy clonogenic cells, rather than by repeatedly interrupting the process of clonogenic cell division (apoptosis) as in conventional radiation therapy.

[0033] There are two forms of focal radiation therapy: external beam radiation therapy and internal radiation therapy. External beam radiation therapy (EBRT) is the most common form of radiation therapy. An external source of ionizing radiation is directed at a specific part of the patient's body. In contrast to brachytherapy (brachytherapy) and non-brachytherapy, where the radiation source is internal, external beam radiation therapy delivers radiation to the tumor from outside the body. Orthovoltage ("superficial") x-rays are used to treat skin cancers and superficial structures. X-rays and electron beams are the most widely used radiation sources in external beam radiation therapy.

[0034] Internal radiation therapy (brachytherapy) is a form of radiation therapy in which a sealed radiation source is placed inside or next to the area requiring treatment. The advantage of brachytherapy is that radiation only affects a very localized area around the radiation source. This reduces exposure to healthy tissue far from the source, potentially causing unnecessary damage to surrounding healthy tissue, while allowing for a fairly high dose of localized radiation to be treated. Additionally, the radiation source remains in the correct position relative to the tumor even if the patient moves or the tumor moves internally during treatment.

[0035] In a preferred embodiment, the present invention provides a combination therapy described herein, wherein the radiation therapy comprises localized radiation therapy selected from external beam radiation or brachytherapy. In another preferred embodiment, the present invention provides a combination therapy described herein, wherein the radiation therapy comprises localized hypofractionated radiation therapy. In yet another preferred embodiment, the present invention provides a combination therapy described herein, wherein the radiation therapy comprises one or several doses of localized hypofractionated radiation in the range of 1 Gy to 20 Gy, particularly in the range of 5 Gy to 20 Gy. In yet another preferred embodiment, the present invention provides a combination therapy described herein, wherein the radiation therapy comprises one to three doses of localized hypofractionated radiation in the range of 5 Gy to 10 Gy. In yet another preferred embodiment, the present invention provides a combination therapy described herein, wherein the radiation therapy comprises one dose of localized hypofractionated radiation in the range of 8 Gy to 10 Gy, preferably 8 Gy or 10 Gy. In yet another preferred embodiment, the present invention provides a combination therapy described herein, wherein the radiation therapy comprises three doses of localized hypofractionated radiation in the range of 5 Gy to 10 Gy, preferably 8 Gy.

[0036] Radiation therapy as defined herein can be administered according to any schedule known to those skilled in the art, such as, for example, an oncologist with expertise in radiation therapy for cancer treatment. In one embodiment, radiation therapy is administered after components a) and b).

[0037] In another embodiment, components a) and b) as defined herein are administered on day 1 and radiotherapy is administered once on day 2 of a treatment cycle, for example of 21 to 28 days, preferably of a 28 day treatment cycle.

[0038] In yet another embodiment, components a) and b) as defined herein are administered on day 1 and radiotherapy is administered on day 2, followed by additional doses every 5-7 days until the end of the treatment cycle, for example for 21-28 days, preferably a 28 day treatment cycle.

[0039] Treg depletion by CD25 ligation 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 before therapy. Compounds that deplete Treg cells (i.e., "Treg cell depleting agents") are known in the art. Treg depletion can be measured by techniques known in the art, for example, as disclosed in WO 2018 / 167104 and Simpson et al. (2013) J Exp Med 210, 1695-710. In one embodiment, the "Treg cell depleting agent" is an anti-CD25 antibody as defined herein.

[0040] As used herein, "regulatory T cells" ("Tregs," "Treg cells," or "Tregs") refer to a lineage of CD4+ T lymphocytes specialized in controlling autoimmunity, allergy, and infection. Typically, they regulate the activity of T cell populations, but can also influence certain innate immune system cell types. Tregs are usually identified by expression of the biomarkers CD4, CD25, and Foxp3. Naturally occurring Treg cells typically comprise approximately 5-10% of peripheral CD4+ T lymphocytes. However, within the tumor microenvironment (i.e., tumor-infiltrating Treg cells), they can comprise 20-30% of the total CD4+ T lymphocyte population.

[0041] 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 precursors, and oligodendrocytes. CD25 associates with CD122 and CD132 to form a heterotrimeric complex that acts as a high-affinity receptor for IL-2. The consensus sequence of human CD25 is identified by Uniprot accession number P01589 (referred to herein as SEQ ID NO: 11).

[0042] As used herein, "anti-CD25 antibody" or "antibody that binds to CD25" refers to an antibody that can bind to the CD25 subunit of the IL-2 receptor. This subunit is also known as the α subunit of the IL-2 receptor. In one embodiment, an anti-CD25 antibody is an antibody that has the ability to specifically bind to the CD25 subunit (antigen) of the IL-2 receptor.

[0043] "Specific binding," "specifically binds," and "specifically binds" are understood to mean that the antibody has a dissociation constant (Kd) for the antigen of interest of less than about 10 M, 10 M, 10 M, 10 M, 10 M, 10 M, 10 M, or 10 M. In preferred embodiments, the dissociation constant is less than 10 M, for example, in the range of 10 M, 10 M, 10 M, 10 M, 10 M, or 10 M.

[0044] Anti-CD25 antibodies suitable for use in the present invention are those that are capable of depleting or reducing Treg cells.

[0045] As used herein, references to "depletion" or "depletion" (with respect to depletion of regulatory T cells with an anti-CD25 antibody agent) mean that the number, ratio, or proportion of Tregs is reduced compared to when the antibody is not administered. In certain embodiments of the invention described herein, greater than about 5%, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 99% of regulatory T cells are depleted.

[0046] Anti-CD25 antibodies capable of depleting Treg cells and suitable for use in the present invention include, for example, those described in WO 2017 / 174331, WO 2018 / 167104, WO 2019 / 008386, WO 2019 / 175215, WO 2019 / 175216, WO 2019 / 175217, WO 2019 / 175220, and WO 2019 / 17522. The contents of WO 2019 / 175223, WO 2019 / 175224, and WO 2019 / 175226 are incorporated herein by reference.

[0047] In preferred embodiments of the invention, the anti-CD25 antibody binds to FcγR with high affinity, preferably to an activating receptor. Preferably, the antibody binds to FcγRI and / or FcγRIIa and / or FcγRIIIa with high affinity. In specific embodiments, the antibody binds to at least one activating Fcγ receptor with a dissociation constant of less than about 10 M, 10 M, 10 M, 10 M, or 10 M.

[0048] In some embodiments, the antibody is an IgG1 antibody, preferably a human IgG1 antibody, capable of binding to at least one Fc-activating receptor. For example, the antibody may bind to one or more receptors selected from FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the antibody may bind to FcγRIIIa. In some embodiments, the antibody may bind to FcγRIIIa and FcγRIIa, and optionally to FcγRI. In some embodiments, the antibody may bind to these receptors with high affinity, for example, with a dissociation constant of less than about 10 M, 10 M, 10 M, or 10 M.

[0049] In some embodiments, the antibody binds to the inhibitory receptor FcγRIIb with low affinity, hi some embodiments, the antibody binds to FcγRIIb with a dissociation constant of greater than about 10 M, greater than about 10 M, or greater than about 10 M.

[0050] In some embodiments, the anti-CD25 antibody may be afucosylated. The Fc region of the antibody may be modified to change the glycosylation profile using techniques known in the art. Available techniques for producing antibodies with no or reduced fucosylation profile include commercially available techniques, such as GlyMAXX (ProBiogen), and methods such as those disclosed in WO 2011 / 035884.

[0051] 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 nonspecific cytotoxic cells expressing Fc receptors (FcR) (e.g., natural killer (NK) cells, neutrophils, macrophages) recognize bound antibodies on target cells and lyse 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 (FcR) (e.g., macrophages) recognize bound antibodies on target cells and phagocytose the target cells.

[0052] The anti-CD25 antibodies used in the present invention may function through ADCC and ADCP activities, which may be measured using available assays known in the art.

[0053] In some embodiments of the invention, an anti-CD25 antibody does not inhibit the binding of interleukin-2 (IL-2) to CD25. Reference 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 "non-blocking" antibody (with respect to not blocking 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. Reference 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 as an anti-CD25 antibody that "does not inhibit IL-2 signaling," or as an anti-CD25 NIB (NIB = non-IL-2 blocking). References to "non-blocking," "non-IL-2 blocking," "does not block," or "does not block" (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%, less than about 40%, less than about 35%, less than about 30%, and preferably less than about 25% compared to IL-2 signaling in the absence of the antibody.

[0054] Some anti-CD25 antibodies can allow IL-2 to bind to CD25 but still block signaling through the CD25 receptor. Non-IL-2 blocking anti-CD25 antibodies allow IL-2 to bind to CD25 and promote at least 50% of the level of signaling through the CD25 receptor compared to signaling in the absence of the anti-CD25 antibody.

[0055] IL-2 signaling through CD25 can be measured, for example, by methods described in International Publication No. WO 2018 / 167104 and known in the art. Comparison of IL-2 signaling in the presence and absence of an anti-CD25 antibody agent can be performed under the same or substantially the same conditions. 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 an anti-CD25 antibody at a concentration of 10 μg / ml for 30 minutes, followed by the addition of various concentrations of IL-2 (e.g., 10 U / ml, or various 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 then be measured using a fluorescently labeled antibody against phosphorylated STAT5 peptide analyzed by flow cytometry. The blocking rate of IL-2 signaling can be calculated as follows: Blocking rate (%)=100×[(%Stat5+ cells no antibody group−%Stat5+ cells 10 μg / ml antibody group) / (%Stat5+ cells no antibody group).

[0056] Examples of non-blocking anti-CD25 antibodies are described in WO 2018 / 167104, WO 2019 / 175215, WO 2019 / 175216, WO 2019 / 175217, WO 2019 / 175220, and WO 2019 / 17522. The contents of WO 2019 / 175223, WO 2019 / 17524, and WO 2019 / 17526 are incorporated herein by reference in their entireties.

[0057] 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 IL-2 binding to CD25.

[0058] As used herein, "epitope" refers to the portion of an antigen bound by an antibody or antigen-binding fragment. As is well known in the art, epitopes can be formed from both adjacent amino acids (linear epitopes) or non-adjacent amino acids juxtaposed by tertiary folding of a protein (conformational epitopes). Epitopes formed from adjacent amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes are conformational in that they comprise portions of an antigen that are not covalently adjacent within 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 conformational epitope is one that comprises non-contiguous amino acid residues in the CD25 extracellular domain. A linear epitope is an epitope that comprises consecutive amino acid residues in the CD25 extracellular domain. Means for determining the precise sequence and / or specific amino acid residues of the epitope of an anti-CD25 antibody are known in the literature, including peptide competition, binding to CD25 sequences from different species from the antigen sequence, truncation and / or mutagenesis (e.g., by alanine scanning or other site-directed mutagenesis), phage display-based screening, yeast display technology, or (co)crystallography techniques. Methods for determining the spatial conformation of epitopes are also well known in the art, including, for example, X-ray crystallography and 2D nuclear magnetic resonance. See, for example, "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.

[0059] In one embodiment, the anti-CD25 antibodies according to the invention are those disclosed in WO2019 / 175216, WO2019 / 175217 and WO2019 / 1175222.

[0060] In another embodiment, the anti-CD25 antibody according to the invention is the antibody disclosed in WO 2019 / 1175222 as "aCD25-a-686" or "αCD25MabGlyMAXX." This antibody may also be referred to as RG6292.

[0061] In yet another embodiment, the anti-CD25 antibody according to the invention is a fucosylated human IgG1 monoclonal antibody having a heavy chain (HC) sequence having the sequence of SEQ ID NO: 12 and a light chain (LC) sequence having the sequence of SEQ ID NO: 13. Such antibodies are known to be "non-IL-2 blocking," i.e., do not inhibit the binding of IL-2 to CD25 (see, e.g., WO 2019 / 1175222).

[0062] Variants of the above-defined antibodies may also be used. Antibody variants also include antibodies in which the sequences for each of the light and heavy chains contain amino acid sequences that are at least 80% identical thereto. The term "percent identity (%)" as known in the art refers to 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 refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Although there are many methods for measuring the identity between two polypeptide or two polynucleotide sequences, commonly used methods for determining identity are codified in computer programs. Preferred computer programs for determining the 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 (for example, gaps can be introduced into the first sequence for best alignment) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is the alignment of two sequences that results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., percent identity = number of identical positions / total number of positions x 100). Generally, reference to percent identity in this specification refers to the percent identity along the entire length of the molecule, unless the context dictates or implies otherwise.

[0063] As used herein, the term "antibody" refers to both intact immunoglobulin molecules and fragments thereof containing an antigen-binding site, and includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including, but not limited to, chimeric antibodies, humanized antibodies, heteroconjugates, and / or multispecific antibodies (e.g., bispecific antibodies, diabodies, tribodies, and tetrabodies), as well as antigen-binding fragments of antibodies, including, for example, Fab', F(ab')2, Fab, Fv, rlgG, polypeptide-Fc fusions, single chain variants (scFv fragments, VHHs, Trans-bodies®, Affibodies®, shark single domain antibodies, single chain or tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTEs®, bicyclic peptides, and other alternative immunoglobulin protein scaffolds. In some embodiments, the antibody may lack covalent modifications (e.g., glycosylation) that it has when produced naturally. In some embodiments, the antibody may include covalent modifications (e.g., glycosylation, detectable moieties, therapeutic moieties, catalytic moieties, or other chemical groups (e.g., polyethylene glycol) that improve the stability or administerability of the antibody). In some embodiments, the antibody may be in the form of a masked antibody (e.g., Probodies®). Masked antibodies may include 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 linker that is cleavable (e.g., by a protease). Selective cleavage of the linker in the desired environment, i.e., the tumor environment, releases the masking / blocking peptide, allowing antigen binding to occur 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 anti-cullins (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 to a single antibody sequence and bind to more or fewer different epitopes within the antigen (e.g., different epitopes within the human CD25 extracellular domain that bind to different reference anti-human CD25 antibodies). Polyclonal or oligoclonal antibodies can be provided in a single preparation for medical use, as described in the literature (Kearns JD et al., 2015. Mol Cancer Ther. 14:1625-36).

[0064] The antibodies used in the present invention can be monospecific, bispecific, or multispecific. A "multispecific antibody" can be specific for different epitopes of a single target antigen or polypeptide, or can 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 monovalently to CD25. In some embodiments, the antibody is a TCB, as further defined herein.

[0065] In some embodiments of the invention, the antibody is monoclonal. The antibody may additionally or alternatively be a humanized or human antibody. In further embodiments, the antibody is human, or in either case, has a format and characteristics that allow for its use and administration in human subjects.

[0066] 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.

[0067] 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 antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation).

[0068] Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with the same structural characteristics. Immunoglobulins can be derived from any class, such as IgA, IgD, IgG, IgE, or IgM. Immunoglobulins can be from any subclass, such as IgG1, IgG2, IgG3, or IgG4. In a preferred embodiment of the present invention, the anti-CD25 antibody is derived from the IgG class, preferably the IgG1 subclass. In one embodiment, the anti-CD25 antibody is derived from the human IgG1 subclass.

[0069] The combination therapy according to the present invention has beneficial pharmaceutical properties, particularly in the treatment of cancer. The term "cancer" may refer to a blood cancer or a solid cancer. In some embodiments, the cancer includes a solid tumor (e.g., a solid tumor cancer), and the treatment is for treating the cancer or preventing the recurrence of the solid tumor. In another embodiment, the treatment according to the present invention is for reducing metastatic spread. As used herein, a "solid tumor" is an abnormal growth or mass of tissue that usually does not contain cystic or liquid areas, particularly tumors and / or metastases (wherever located) other than leukemia or non-solid lymphoid cancers. Solid tumors can 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 cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic, or fibrous connective tissue), carcinomas (including tumors arising from epithelial cells), mesothelioma, neuroblastoma, retinoblastoma, etc. In one embodiment, the solid cancer (or tumor) is selected from breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, kidney cancer, renal cancer, liver cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, or prostate cancer. In some embodiments, the cancer is selected from acute myeloid leukemia, diffuse large B-cell lymphoma, multiple myeloma, melanoma, non-small cell lung cancer, kidney cancer, ovarian cancer, bladder cancer, pancreatic cancer, sarcoma, and / or colon cancer.

[0070] In yet another embodiment there is provided a combination for use as defined herein, wherein the solid tumor is selected from head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), melanoma, lung cancer, kidney cancer, breast cancer, colon cancer, ovarian cancer, cervical cancer, liver cancer, prostate cancer, bladder cancer, gastric cancer, glioblastoma and sarcoma, preferably head and neck squamous cell carcinoma (HNSCC), pancreatic cancer and pancreatic ductal adenocarcinoma (PDAC).

[0071] As used herein, the term "treatment" of cancer, "treat" or "treating" cancer refers to the achievement of at least one positive therapeutic effect, such as a reduction in the number of cancer cells, a reduction in tumor size, a reduction in the rate of cancer cell invasion into peripheral organs, or a reduction in the rate of tumor metastasis or tumor growth. A positive therapeutic effect in cancer can be measured in several ways (e.g., Weber (2009) J Nucl Med 50, 1S-10S). For example, with regard to tumor growth inhibition, according to the National Cancer Institute (NCI) criteria, a T / C% ratio of 42% is the minimum level of anti-tumor activity. A T / C<10% is considered a high level of anti-tumor activity, where T / C(%)=median treated tumor volume / median control tumor volume×100.

[0072] As used herein, "combination" or "in combination" means separate, simultaneous or sequential administration, unless the context indicates otherwise.

[0073] As used herein, a "therapeutically effective amount" (or effective amount) is the amount of each compound or combination that elicits the biological or medical response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, physician, or other clinician. In some embodiments, 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 in accordance with a treatment regimen. Those skilled in the art will appreciate that a "therapeutically effective amount" need not actually achieve successful treatment in a particular subject. In some embodiments, the treatment achieved by a therapeutically effective amount is either progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS). PFS, also known as "time to tumor progression," refers to the length of time during and after treatment during which the cancer does not grow, and includes the amount of time the patient experiences a complete or partial response, as well as the amount of time the patient experiences stable disease. DFS refers to the length of time during and after treatment during which the patient remains disease-free. OS refers to the extension of life expectancy compared with a naive or untreated individual or patient.

[0074] One aspect of the present invention is to provide a combination therapy that enables, improves, or enhances "effective treatment" of patients with cancer. According to the present invention, one condition for such "effective treatment" is the depletion of Treg cells in said patient. Thus, in one embodiment, a combination for use as defined herein is provided, wherein the combination is used to reduce overall tumor burden. In another embodiment, a combination for use as defined herein is provided, wherein the combination is used to treat acquired resistance to a previous therapy for the same cancer in the same patient. In yet another embodiment, a combination for use as defined herein is provided, wherein the combination is used to prevent the formation of metastases, preferably lung metastases, or to reduce metastatic spread.

[0075] In another embodiment, the present invention discloses a pharmaceutical product comprising a combination for use as defined herein, together with instructions on how to apply it.

[0076] The following is a series of clauses that define the present invention and its preferred aspects and embodiments:

[0077] 1. A combination for use in the treatment of cancer, the combination comprising: a) a first component comprising an effective amount of a Treg cell depleting agent; b) a second component comprising an effective amount of a bispecific immunocytokine; and c) a third component comprising an effective amount of radiation therapy, wherein components a)-c) are for simultaneous or sequential administration.

[0078] 2. The combination for use according to clause 1, wherein the Treg cell depleting agent is an anti-CD25 antibody.

[0079] 3. The combination for use according to clause 2, wherein the anti-CD25 antibody is a monoclonal antibody that does not inhibit the binding of IL2 to CD25.

[0080] 4. The combination for use according to any one of clauses 1 to 3, wherein the bispecific immunocytokine is an antibody that blocks the PD-1 / PD-L1 signaling axis and simultaneously binds to the IL2 receptor on the same cells (PD1-IL2v).

[0081] 5. The combination for use according to clause 4, wherein the IL2 binding domain (IL2v) is a mutated variant specific for CD122.

[0082] 6. The combination for use according to any one of clauses 1 to 5, wherein the radiotherapy comprises local radiotherapy selected from external beam radiation or brachytherapy.

[0083] 7. The combination for use according to any one of clauses 1 to 6, wherein radiotherapy comprises focal hypofractionated radiotherapy.

[0084] 8. The combination for use according to any one of clauses 1 to 7, wherein the radiotherapy comprises local hypofractionated irradiation in one or several doses in the range of 1 Gy to 20 Gy, in particular in the range of 5 Gy to 20 Gy.

[0085] 9. The combination for use according to clause 8, wherein the radiotherapy comprises local hypofractionated irradiation in 1 to 3 fractions ranging from 5 to 10 Gy.

[0086] 10. The combination for use according to clause 8 or clause 9, wherein the radiotherapy comprises local hypofractionated irradiation in the range of 8 Gy to 10 Gy, preferably 8 Gy or 10 Gy in a single dose.

[0087] 11. The combination for use according to clause 8 or clause 9, wherein the radiotherapy comprises local hypofractionated irradiation in the range of 5 Gy to 10 Gy, preferably 8 Gy in three fractions.

[0088] 12. The combination for use according to any one of clauses 1 to 11, wherein radiotherapy is administered after components a) and b).

[0089] 13. The combination for use according to clause 12, wherein components a) and b) are administered on day 1 and radiotherapy is administered once on day 2 of the treatment cycle.

[0090] 14. The combination for use according to clause 12, wherein components a) and b) are administered on day 1 and radiotherapy is administered on day 2, with subsequent booster doses every 5 to 7 days until the end of the treatment cycle.

[0091] 15. The combination for use according to any one of clauses 1 to 14, wherein the cancer is a solid tumor.

[0092] 16. The combination for use according to clause 15, wherein the solid tumor is selected from head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), melanoma, lung cancer, kidney cancer, breast cancer, colon cancer, ovarian cancer, cervical cancer, liver cancer, prostate cancer, bladder cancer, gastric cancer, glioblastoma and sarcoma.

[0093] 17. The combination for use according to clause 15, wherein the solid tumor is selected from head and neck squamous cell carcinoma (HNSCC), pancreatic cancer and pancreatic ductal adenocarcinoma (PDAC).

[0094] 18. The combination for use according to any one of clauses 1 to 17, wherein the combination is used to reduce the overall tumor burden.

[0095] 19. The combination for use according to any one of clauses 1 to 17, wherein the combination is used to treat acquired resistance to a previous therapy for the same cancer in the same patient.

[0096] 20. The combination for use according to any one of clauses 1 to 17, wherein the combination is used to prevent the formation of metastases, preferably lung metastases, or to reduce the spread of metastases.

[0097] 21. A pharmaceutical product comprising a combination for use according to any one of clauses 1 to 20, together with instructions on how to apply it.

[0098] 22. A method for treating a patient with cancer, comprising administering an effective amount of a combination according to any one of clauses 1 to 20.

[0099] 23. A combination for use according to any one of clauses 1 to 20, a pharmaceutical product according to clause 21 or a method according to clause 22, wherein component a) is optional.

[0100] 24. A combination for use according to any one of clauses 1 to 20, a pharmaceutical product according to clause 21 or a method according to clause 22, which does not contain component a). [Example]

[0101] material and method research design The objective of this study was to clarify how PD1-IL2v, anti-CD25, and radiation therapy (RT) modulate antitumor immune responses and their subsequent impact on (i) primary and (ii) distant tumor development. These objectives were first utilized publicly available ssRNA-seq datasets (n = 44) and RNA-seq and CyTOF analyses of a recently completed phase 1 trial (n = 16) to validate immunological targets. The primary techniques used were flow cytometry (n = 5–6), histology (n = 4), proteomics / metabolomics (n = 3–5), and ELISA (n = 6 / sample). Mice for in vivo studies were age- and sex-matched and randomly assigned to treatment groups. Sample size was determined to achieve statistical significance, and endpoints were determined by IACUC protocol.

[0102] cell line The MOC2, LY2, and P029 mouse squamous cell carcinoma cell lines were used for in vivo studies. Cell lines were cultured in the appropriate media: LY2 and P029 in DMEM-F12 containing 10% FBS and 1% primocin / fungin, and MOC2 in a 1:2 mixture of DMEM-F12 / IMDM supplemented with 10% FBS, 1% primocin / fungin, 1.75 μg EGF, 20 μg hydrocortisone, and 0.1% insulin, as previously described. The P029 cell line was provided in collaboration with X.J. Wang, Department of Pathology, University of Colorado Anschutz Medical Campus, and was generated as follows:

[0103] Establishment of the P029 cell line: K15-CrePR1, LSL-Kras on a C57BL / 6J background G12D and Smad4 f / f Mice were bred and tail-cut genotyped to express the oncogenic mutant Kras in stratified epithelium, as previously described. G12DWe established trigenic mice carrying Cre recombinase driven by the keratin 15 promoter, which activates expression of and deletes the Smad4 tumor suppressor. 70 Female mice, P029, developed spontaneous skin lesions in the neck region, at which point they were sacrificed and tumors harvested for histological evaluation and cell line generation. Tumors were minced with a scalpel, dissociated using a gentleMACS Tissue Dissociator with a C-tube (Miltenyi), and incubated in 1 mg / mL type II collagenase (Worthington) for 40 minutes at 37°C. The tissue suspension was rinsed in PBS using centrifugation between washes and initially cultured for 7 days in complete medium (DMEM / F12 medium containing 10% FBS and 1x Primocin antibiotic). To promote epithelial cell growth and reduce fibroblast growth, cells were cultured in serum-free keratinocyte medium (Gibco) supplemented with 2 ng / mL EGF and 1x Primocin. After 2 weeks of culture and four passages to expand epithelial cells and eliminate fibroblasts, a stable proliferating cell line, P029, was established. To verify tumor establishment and metastatic potential, the P029 cell line was implanted into the flanks of recipient female C57BL / 6J mice using 50,000 cells in 50% Matrigel / 50% PBS (Corning) and monitored for 6 weeks when tumors reached 2 cm in diameter. Complete necropsy and histological evaluation demonstrated that P029 cells metastasized to the lungs, liver, and lymph nodes.

[0104] The murine KPC pancreatic cancer cell lines PK5L1940 and FC1242 were passaged in RPMI1640 supplemented with 10% FBS. Cells were passaged every 2–3 days at a density of 1:4–1:10. Cells were not expanded beyond passage 20.

[0105] Mouse models and tumor studies C57Bl / 6 and Balb / c mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and Charles River (Wilmington, MA, USA), respectively, and used for in vivo studies of MOC2, P029, and LY2. Mice were housed up to five per cage, and all animal model protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz Medical Campus. MOC2, LY2, and P029 mice were orthotopically implanted into the buccal mucosa as previously described. 7 Mice were appropriately age-matched and randomly assigned to each group until tumor volume reached approximately 150 mm 3 Treatment was initiated when tumor volume reached 100%. Tumors were measured twice weekly using digital calipers, and tumor volume was calculated as V = (A x B 2 ) / 2. A is the short diameter and B is the long diameter. For tumor studies, the following cell numbers were implanted into the buccal mucosa: 1 × 10 for the MOC2 cell line; 5 LY2 cells were transplanted into each mouse at 1 × 10 6 Cells were transplanted into each mouse, 5 × 10 for P029 4 Cells were implanted into each mouse, and primary tumors, serum, and lungs were collected at the time of sacrifice.

[0106] Female C57BL6 mice (6 weeks old) were purchased from Jackson Laboratories (Indianapolis, IN, USA). All mice were maintained in accordance with ethical guidelines and conditions established and overseen by the Animal Care and Use Committee of the University of Colorado, Anschutz Medical Campus. The protocols used for animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Colorado, Anschutz Medical Campus.

[0107] Mice were first anesthetized using isoflurane, and local orthotopic transplantation was performed by making a 1 cm incision in the left subchondral region. The mouse pancreas was located and exposed, and 200,000 PK5L1940 or FC1242 KPC cells suspended 1:1 in Matrigel (Corning, Corning, NY) were injected. The pancreas was then reintroduced into the abdomen, and the mouse's peritoneum and skin were closed. The protocol has been described in further detail (Qiu and Su, 2013). In vivo studies of survival and flow cytometry were performed and analyzed separately.

[0108] Metastatic orthotopic transplantation was performed as described above, with the spleen exposed after a subcostal incision. The spleen was first ligated with horizon clips, and one hemisphere was injected with 200,000 KPC cells suspended in 50 μl of 10% RPMI, followed by a washout injection of 50 μl of PBS. The pancreatic duct was then ligated with horizon clips, and the hemispleen was excised before closing the peritoneum and skin. Metastatic transplantation has been described in further detail (Soares et al., 2014). For cancer-specific mortality, mice determined to have died from other causes were excluded from the analysis.

[0109] Tumor rechallenge, 5 × 10 5 PK5L1940 cells or 1 x 10 cells / 0.1 ml final concentration 6 The tumor size was measured twice weekly with a digital caliper and calculated using the formula (V = A × B). 2 / 2mm 3 ) was used to estimate tumor volume, where A is the longer diameter of the tumor and B is the shorter diameter of the tumor.

[0110] Prior to adoptive transfer experiments, CD8 T cells were sorted using a CD8 negative selection kit (StemCell Technologies catalog number 19853) according to the manufacturer's guidelines. Cells were counted, resuspended in 100 μL of PBS, and injected into restrained mice via the tail vein.

[0111] Antibodies and Drugs aCD25 (anti-CD25), PD1-IL2v, and DP47-IL2v were provided in collaboration with Roche Pharmaceuticals. aCD25 is RG6292 or a murine surrogate antibody of RG6292, as defined herein. PD1-IL2v or its murine surrogate is used as defined herein (see, e.g., SEQ ID NOS: 5-10). DP47-IL2v is a non-targeted version of the IL2v moiety used as a control. aCD25 was administered at a concentration of 3 mg / kg, and PD1-IL2v and DP47-IL2v were administered at a concentration of 0.5 mg / kg. Antibodies were administered weekly by IP injection, beginning one day before the start of RT. In studies without radiotherapy, IP injections were administered at a time point after tumor implantation equivalent to one day before RT. aPD-1 (clone: ​​29F.1A12) and αNK1.1 (clone: ​​PK136) were administered twice weekly by IP injection at 10 mg / kg. NK cell depletion was verified using flow cytometry analysis. All dilutions were performed using sterile DPBS (Gibco).

[0112] irradiation Irradiation was performed using a PXi-225Cx image-guided irradiator at 225 kV, 20 mA, and a 0.3 mm Cu filter. Mice were anesthetized with vaporized isoflurane and irradiated in the prone position with RT at a dose rate of 5.6 Gy / min. Dose rates were checked monthly using an ionization chamber, and CBCT scans were acquired for accurate mouse positioning. The irradiation plan was based on Monte Carlo simulations of the mouse model.

[0113] Specifically, for the PDAC model (Examples 3 and 4): Image-guided radiation therapy was performed using an X-Rad SmART small animal irradiator (Precision X-ray, North Bradford CT) at 225 kVp, 20 mA, and a 0.3 mm Cu filter. Mice were placed in a prone position, and CT scans were acquired. Radiation was delivered at a dose rate of 5.6 Gy / min. A single 8 Gy dose of X-ray radiation was delivered to the mouse pancreas using a 10 mm square beam with field edges at the mouse's midline and below the left rib cage. To determine the appropriate time and current, Monte Carlo simulations were performed using SmART-ATP software (SmART Scientific Solutions, Maastricht, Netherlands) with CBCT scans of one mouse. All mice received identical treatment after fluoroscopic repositioning. For all in vivo experiments, radiation was administered 7 days after implantation.

[0114] Flow cytometry Tumors, blood, and tumor-draining lymph nodes were harvested and processed for flow cytometry analysis. Tumor tissue was minced and incubated in collagenase III (Worthington) for 30 minutes at 37°C. After incubation, the tissue was passed through a 70µm nylon cell strainer to generate a single-cell suspension. After centrifugation, red blood cells were lysed using RBC lysis buffer (Invitrogen), neutralizing the lysis buffer with HBSS. Lymph nodes were similarly processed by mechanical dissociation into a single-cell suspension. Immediately after collection, blood was centrifuged and resuspended in RBC lysis buffer as described above. Cells were transferred to 24-well plates, incubated with monensis and brefeldin to prevent cytokine release, and stimulated with a PMA / ionomycin cocktail for 4 hours at 37°C. After incubation, cells were incubated in FC block (CD16 / CD32 antibody, Tonbo Bioscience) for 15 minutes at 4°C. Cells were then incubated in the Live / Dead Fixable Aqua Viability Stain Kit (Invitrogen) in the dark for 20 minutes at 4° C. Cells were then stained for surface markers and incubated for 20 minutes at 4° C. For immune cell analysis, the following antibodies were used: PerCP-CD45 (clone: ​​30-F11, Biolegend), BUV805-CD3 (clone: ​​17A2, BD Biosciences), BUV 496-CD4 (clone: ​​GK1.5, BD Biosciences), BB515-CD8 (clone: ​​53-6.7, BS Biosciences), BV570-CD44 (clone: ​​IM7, Biolegend), Pe-Cy7-NKp46 (clone: ​​29A1.4, Biolegend), Superbright 436-CD69 (clone: ​​H1.2F3, eBioscience), and BV605-DNAM-1 (clone: ​​TX42).1, Biolegend), BV786-CD25 (clone: ​​3C7, BD Biosciences), BUV395-PD-1 (clone: ​​J43, BD Biosciences), BV650-PD-L1 (clone: ​​MIH5, BD BioSciences), PE-Cy5-CD11c (clone: ​​N418, Biolegend), PE / Dazzle 594-MHC II (clone: ​​M5 / 114.15.2, Biolegend), PerCP-Cy5.5-CD80 (clone: ​​16-10A1, Biolegend), BUV661-CD11b (clone: ​​M1 / 70, BD Biosciences), Alexa Fluor 647-Ly6C (clone: ​​HK1.4, Biolegend), BV421-Ly6G (clone: ​​1A8, Biolegend), Alexa Fluor 700-CD19 (clone: ​​6D5, Biolegend), BV480-F4 / 80 (clone: ​​T45-2342), eFluor 450-iNOS (clone: ​​CXNFT, eBioscience), PE-CD163 (clone: ​​S15049F, Biolegend), APC-eFluor 780-Ki-67 (clone: ​​SolA15, eBioscience), Alexa Fluor 532-Foxp3 (clone: ​​FjK-16s, eBioscience), APC-IL-2 (clone: ​​JES6-5H4, eBioscience), BUV737-IFNγ (clone: ​​XMG1.2, BD Biosciences), FITC-Granzyme B (clone: ​​QA16A02, Biolegend), BV750-TNFα (clone: ​​MP6-XT22, Biolegend), BV711-IL-10 (clone: ​​JES5-16E3, BD Bioscience), PE-pan-cytokeratin (clone: ​​AE-1 / AE-3, Novus Biologicals), BV650-EpCAM (clone: ​​G8.8, Biolegend), Alexafluor 488-pan cytokeratin (clone: ​​AE1 / AE3, Invitrogen), Alexafluor 700-NK1.1 (clone: ​​S17016D, Biolegend), eFluor450-CD122 (clone: ​​TM-b1, eBioscience), PE / Dazzle594-CD25 (clone: ​​C37, Biolegend), Alexafluor 647-CD11b (clone: ​​M1 / 70, Biolegend), BV711-NKG2D (clone: ​​CX5, BD Bioscience), PerCP-eFluor 710-NKG2A (clone: ​​20d5, eBioscience), BUV661-Ly49H (clone: ​​3D10, BD Bioscience), PerCP-Cy5.5-KLRG1 (clone: ​​2F1, BD Bioscience), BV750-OX40 (clone: ​​OX-40, BD Bioscience), PE-LFA-1 (clone: ​​H155-78, Biolegend), Superbright The following antibodies were used: 436-CXCR3 (clone: ​​CXCR3-173, eBioscience), APC-CD137 (clone: ​​17B5, eBioscience), BV421-NKG2I (clone: ​​854929, BD Bioscience), FITC-Ly49G2 (clone: ​​4D11, eBioscience), and BV786-Ly49A (clone: ​​A1, BD Bioscience). After surface staining, cells were fixed and permeabilized overnight using the Foxp3 perm / fix kit (Invitrogen). After incubation, cells were stained for intracellular markers and incubated for 30 minutes at 4°C. Samples were then run on a Cytek Aurora spectral cytometer at the University of Colorado Diabetes Research Center Flow Cytometry Core. Fluorescence-minus-one controls were used to determine the gating strategy. Data were analyzed using Flowjo analysis software.

[0115] Immunohistochemistry and H&E staining At the time of sacrifice, lungs were harvested, embedded in OCT medium, and frozen at -80°C for tissue preservation. Frozen tissue was delivered to the University of Colorado Anschutz Medical Campus, Gates Center for Regenerative Medicine Histology Core, where it was sectioned onto slides, mounted, and stained with H&E. Tissue was mounted in triplicate.

[0116] NK cytotoxicity assay NK cells were collected and isolated from the blood of tumor-bearing C57BL / 6 mice using an NK negative selection isolation kit (Stemcell). P029 tumor cells were stained with calcein (Thermofisher) at a concentration of 2 μg / mL in RPMI medium containing 10% FBS. NK cells and P029 tumor cells were incubated together at a 2:1 ratio of NK to tumor cells and incubated at 37°C for 4 hours. After incubation, the plate was centrifuged, and the supernatant was removed and transferred to a 96-well plate. The plate was read using a Tecan Infinite M plex fluorescence plate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Cytolysis was calculated using the following formula: [(test release - spontaneous release) / (maximum release - spontaneous release)] x 100. Maximum release was calculated by incubating P029 tumor cells with 1% Triton x 100 in RPMI, and spontaneous release was calculated by incubating cells in incubation medium without added stimuli.

[0117] Image Stream Cytometry NK cells were harvested and isolated from the spleens of tumor-bearing mice. They were processed according to the protocol described above to obtain a single-cell suspension, and NK cells were isolated using an NK isolation kit (Stemcell) according to the manufacturer's instructions. Isolated NK cells were then stained with the following antibodies: APC-EpCAM, PE / Dazzle 594-NKp 46 (Biolegend), and FITC-Granzyme B (Biolegend). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). After staining, cells were washed, resuspended in PBS, and processed on an Amnis Imagestream X Mk II imaging flow cytometer (Amnis, Seattle, WA). Analysis was performed using IDEAS 6.2 software (Amnis, Seattle, WA).

[0118] Metabolomics and Proteomics Blood from tumor-bearing mice was collected and processed as described above to obtain single-cell suspensions. After processing, CD8 and NK cells were isolated using a CD8 T cell (Stemcell) and NK cell (Militenyi) negative isolation kit according to the manufacturer's instructions. Isolated cells were stained with PE-CD8 and APC-NKp46 and then cell sorted to obtain pure single-cell populations. Cell sorting was performed using a Beckman Coulter MoFlo XDP750 (Beckman Coulter). High-throughput metabolomics analysis was performed on mouse serum samples at the University of Colorado School of Medicine Metabolomics Facility. Samples were thawed on ice, and chilled 5:3:2 methanol:acetonitrile:water (v / v / v) was added to each tube, 1:24 serum:buffer (v / v), followed by centrifugation. 72Metabolites were extracted from serum by vortexing for 30 minutes at 4°C and centrifuging for 10 minutes, both as described in

[2014] . All supernatants were analyzed in duplicate (10 μL injection each) by ultra-high performance liquid chromatography using a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive mass spectrometer in negative and positive polarity modes. For each method, UHPLC was performed for 5 minutes at a flow rate of 0.45 mL / min using a Phenomenex C18 column. Samples were introduced into the MS by electrospray ionization. Technical details have been previously described. 73 Data were analyzed using Maven (1.4.20-dev-772) and quality control was maintained as described. 72,74 Data were sum-normalized and autoscaled to generate heatmaps for PCA, PLS-DA, and MetaboAnalyst (5.0).

[0119] Samples were applied to a 10 kDa molecular weight cutoff filter and digested according to the FASP protocol. Briefly, samples were mixed in a filter unit containing 8 M urea, 0.1 M ammonium bicarbonate (AB) (pH 8.0) and centrifuged at 14,000 g for 15 min. Proteins were reduced with 10 mM DTT for 30 min at room temperature, centrifuged, and alkylated with 55 mM iodoacetamide for 30 min at room temperature in the dark. After centrifugation, samples were washed three times with urea solution and three times with 50 mM AB, pH 8.0. Protein digestion was performed overnight at 37°C using sequencing-grade modified trypsin (Promega) at a 1 / 50 protease / protein (wt / wt) ratio. Peptides were recovered from the filter using 50 mM AB. 20 μl of each sample was loaded onto individual Evotips for desalting, then washed with 20 μL of 0.1% FA, followed by the addition of 100 μL of storage solvent (0.1% FA) to keep the Evotips moist until analysis. Peptides were separated on a Pepsep column (150 μm diameter, 15 cm) packed with ReproSil C18 1.9 μm, 120A resin using an Evosep One system (Evosep, Odense, Denmark). The system was coupled to a timsTOF Pro mass spectrometer (Bruker Daltonics, Bremen, Germany) via a nanoelectrospray ion source (Captive Spray, Bruker Daltonics). The mass spectrometer was operated in PASEF mode. The ramp time was set to 100 ms, and 10 PASEF MS / MS scans were acquired per top N acquisition cycle. MS and MS / MS spectra were recorded from m / z 100 to 1700. Ion mobilities were adjusted from 0.7 to 1.50 Vs / cm. 2Scans were performed up to 1000 counts. Low-abundance precursor ions above a threshold of 500 counts and below a target of 20,000 counts were scheduled for repetition for 0.4 min, while others were dynamically excluded. Identification settings were as follows: trypsin, specific, up to two missed cleavages, up to two isotopic errors allowed in precursor selection, MS1 tolerance of 10.0 ppm, MS2 tolerance of 0.4 Da, fixed modifications: carbamidomethylation of C (+57.021464 Da), variable modifications: oxidation of M (+15.994915 Da), acetylation of protein N-terminus (+42.010565 Da), pyrrolidone derived from peptide N-terminus Q or C (-17.026549 Da). Graphs were rendered using Graphpad Prism (9.3.1).

[0120] statistical analysis All statistical analyses were performed using GraphPad Prism v9. Statistical analysis was completed using one-way analysis of variance (ANOVA) with Tukey's correction for comparisons with three or more groups and unpaired t-tests for comparisons using only two groups. Kaplan-Meier curves were used for survival analysis, with the log-rank (Mantel-Cox) test for comparisons of all groups.

[0121] Example 1: Treatment with αCD25 and PD1-IL2v results in tumor growth delay in HNSCC tumors This example tests the hypothesis that simultaneously targeting both PD-1 and IL-2 signaling increases therapeutic efficacy. We used the murine immunocytokine PD1-IL2v (muPD1-IL2v), which blocks the PD-1 / PD-L1 signaling axis and simultaneously binds to the IL-2 receptor on the same cells. The mutated IL-2v binding domain is unique in that it is specific for CD122, allowing it to bypass CD25-mediated Treg activation. 31~33However, Tregs can also express CD122, and previous studies have demonstrated that depletion of Tregs is essential for overcoming radioresistance and therapeutic response. Taking this into consideration, we developed a novel αCD25 antibody optimized to interact with the activating Fc region to promote enhanced depletion of intratumoral Tregs while leaving IL-2 signaling intact. 34 This approach was used to identify CD25-deficient cells, namely CD8 T cells and NK cells. 34 This allows for greater depletion of tumor-infiltrating Tregs while selectively expanding and activating Tregs.

[0122] The combination of RT with αCD25 eradicated LY2 tumors in approximately 80% of mice (Figure 1A, Figure 1B). Interestingly, in the Treg-dependent LY2 model, muPD1-IL2v, with or without radiation therapy, showed no significant therapeutic effect on local tumor progression, further demonstrating the need for Treg depletion in this model. In the MOC2 tumor model, the addition of muPD1-IL2v to RT significantly reduced tumor growth, and the addition of aCD25 further improved survival (Figure 1C, Figure 1D). These treatments were further tested in a newly developed Kras / Smad4 / p53-mutated metastatic P029 tumor model. All combinations of RT with αCD25 and muPD1-IL2v showed tumor growth delay (Figure 1E), and all combinations showed increased survival compared to RT alone (Figure 1F).

[0123] Example 2: Reduction of pulmonary metastases Given the increased systemic activation of circulating lymphocytes and NK cells, this example explores the functional implications of this increase for systemic tumor spread. The P029 model was used due to its high propensity for widespread metastasis, particularly to the lungs. Lung tissues examined were harvested from orthotopically implanted P029 tumors harvested at the time of sacrifice (Figure 2A). A reduction in the percentage of mice exhibiting overall metastatic lung lesions was observed in the muPD1-IL2v-treated group (Figure 2B). Cancer cells detached from primary tumors can enter the circulation and seed tumors at distant sites. 24Analysis of circulating tumor cells (CTCs) revealed that they express EpCAM or cytokeratin, both markers of circulating tumor cells, and CD45. - The results showed that the percentage of cells was significantly reduced in the groups receiving muPD1-IL2v, in contrast to RT alone and RT + αCD25 (Figure 2C, D). These data suggest that muPD1-IL2v helps prevent metastatic spread and distant lesion formation by reducing the amount of tumor cells in the circulation, even in the setting of local tumor progression.

[0124] Example 3: aPD1-IL2v+RT+aCD25 produces superior responses in a PDAC mouse model In this example, we use an orthotopic mouse model of Kras-driven PDAC and the PK5L1940 and FC1242 KPC cell lines to examine the effects of the mouse variant antibody fusion protein aPD1-IL2v in combination with radiation therapy and Treg depletion on pancreatic cancer growth. Using the PK5L1940 cell line, the addition of aCD25 to RT and aPD1-IL2v antibody treatment slightly improved survival (Figure 3A). Using the Kras-driven FC1242 KPC cell line, aPD1-IL2v also enhanced response to treatment (Figure 3B).

[0125] Example 4: RT+aPD1-IL2v combination treatment results in activation of peripheral CTLs and reduced metastatic burden in a PDAC model This example investigates the metastatic burden after RT + aPD1-IL2v treatment by flow cytometry analysis of circulating immune populations. NK cells play a role in controlling metastasis. 22,38 We analyzed peripheral NK cells. Although the frequency of circulating NK cells decreased after aCD25 and aPD1-IL2v treatment, the expression of functional markers DNAM1 and Gnzmb increased with the addition of aPD1-IL2v (Figure 4A), suggesting that aPD1-IL2v may also function to reduce metastatic burden by activating NK cells in the periphery.

[0126] To directly test the effect of this immunomodulation on metastasis and disease dissemination, this example next examined tumor cells injected through the splenic vasculature and into the liver. 76 We utilized a unilateral splenectomy metastasis model of pancreatic cancer, which reproducibly forms metastatic lesions in tumors. Data using this model with the PK5L1940 cell line are shown in Figure 4B. Furthermore, complete responses were observed in 1 / 8 mice treated with RT + aPD1-IL2v and 2 / 8 mice treated with RT + aCD25 + aPD1-IL2v, demonstrating the robust and systemic nature of the aPD1-IL2v-induced antitumor immune response. Complete responses with the combination of these therapies were also achieved using the KPC FC1242 cell line in the unilateral splenectomy metastasis model (Figure 4C).

[0127] References 1.Sambi M,Bagheri L,Szewczuk MR.Current challenges in cancer immunotherapy:Multimodal approaches to improve efficacy and patient response rates.J Oncol.2019;2019.doi:10.1155 / 2019 / 4508794 2.Nor JE, Gutkind JS.Head and Neck Cancer in the New Era of Precision Medicine.J Dent Res.2018;97(6):601-602.doi:10.1177 / 0022034518772278 3.Mandal R, Senbabaoglu Y, Desrichard A, et al.The head and neck cancer immune landscape and its immunotherapeutic implications.JCI Insight.2016;1(17).doi:10.1172 / jci.insight.89829 4.Karam SD,Raben D.Radioimmunotherapy for the treatment of head and neck cancer.Lancet Oncol.2019;20(8):e404-e416.doi:10.1016 / S1470-2045(19)30306-7 5.Waldman AD,Fritz JM,Lenardo MJ.A guide to cancer immunotherapy:from T cell basic science to clinical practice.Nat Rev Immunol.2020;20(11):651-668.doi:10.1038 / s41577-020-0306-5 6.Botticelli A,Mezi S,Pomati G,et al.The 5-Ws of immunotherapy in head and neck cancer.Crit Rev Oncol Hematol.2020;153:103041.doi:10.1016 / J.CRITREVONC.2020.103041 7.Oweida A,Hararah MK,Phan A,et al.Resistance to radiotherapy and PD-L1 blockade is mediated by TIM-3 upregulation and regulatory T-cell infiltration.Clin Cancer Res.2018;24(21):5368-5380.doi:10.1158 / 1078-0432.CCR-18-1038 8.Oweida AJ,Darragh L,Phan A,et al.STAT3 Modulation of Regulatory T Cells in Response to Radiation Therapy in Head and Neck Cancer.J Natl Cancer Inst.2019;111(12):1339-1349.doi:10.1093 / jnci / djz036 9.Ihara F,Sakurai D,Horinaka A,et al.CD45RA-Foxp3high regulatory T cells have a negative impact on the clinical outcome of head and neck squamous cell carcinoma.Cancer Immunol Immunother.2017;66(10):1275-1285.doi:10.1007 / S00262-017-2021-Z 10.Bickett TE,Knitz M,Darragh LB,et al.FLT3L Release by Natural Killer Cells Enhances Response to Radioimmunotherapy in Preclinical Models of HNSCC.Clin Cancer Res.2021;27(22):6235-6249.doi:10.1158 / 1078-0432.ccr-21-0971 11.Martin JF,Perry JSA,Jakhete NR,Wang X,Bielekova B.An IL-2 Paradox:Blocking CD25 on T Cells Induces IL-2-Driven Activation of CD56 bright NK Cells.J Immunol.2010;185(2):1311-1320.doi:10.4049 / jimmunol.0902238 12.Navarro AG,Bjorklund AT,Chekenya M.Therapeutic potential and challenges of natural killer cells in treatment of solid tumors.Front Immunol.2015;6(APR).doi:10.3389 / fimmu.2015.00202 13.Vivier E,Tomasello E,Baratin M,Walzer T,Ugolini S.Functions of natural killer cells.Nat Immunol.2008;9(5):503-510.doi:10.1038 / ni1582 14.Sim GC,Radvanyi L.The IL-2 cytokine family in cancer immunotherapy.Cytokine Growth Factor Rev.2014;25(4):377-390.doi:10.1016 / J.CYTOGFR.2014.07.018 15.Wu Y,Tian Z,Wei H.Developmental and functional control of natural killer cells by cytokines.Front Immunol.2017;8(AUG).doi:10.3389 / fimmu.2017.00930 16.Koyama S,Akbay EA,Li YY,et al.Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints.Nat Commun.2016;7.doi:10.1038 / ncomms10501 17.Sharpe AH,Pauken KE.The diverse functions of the PD1 inhibitory pathway.Nat Rev Immunol.2018;18(3):153-167.doi:10.1038 / nri.2017.108 18.Kumagai S,Togashi Y,Kamada T,et al.The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies.doi:10.1038 / s41590-020-0769-3 19.Kamada T,Togashi Y,Tay C,et al.PD-1+regulatory T cells amplified by PD-1 blockade promote hyperprogression of cancer.Proc Natl Acad Sci U S A.2019;116(20):9999-10008.doi:10.1073 / PNAS.1822001116 / - / DCSUPPLEMENTAL 20.Umana P,Deak LC,Ahmed R,et al.Differentiating PD-1+stem-like CD8 T cells towards distinct effectors with enhanced therapeutic potential by an engineered IL-2 cis-targeted to PD-1.doi:10.21203 / rs.3.rs-329812 / v1 21.Chinen T,Kannan AK,Levine AG,et al.An essential role for the IL-2 receptor in T reg cell function.Nat Immunol.2016;17(11):1322-1333.doi:10.1038 / ni.3540 22.Lopez-Soto A,Gonzalez S,Smyth MJ,Galluzzi L.Control of Metastasis by NK Cells.Cancer Cell.2017;32(2):135-154.doi:10.1016 / J.CCELL.2017.06.009 23.Sharma P,Kumar P,Sharma R.Natural killer cells-Their role in tumour immunosurveillance.J Clin Diagnostic Res.2017;11(8):BE01-BE05.doi:10.7860 / JCDR / 2017 / 26748.10469 24.Lo HC,Xu Z,Kim IS,et al.Resistance to natural killer cell immunosurveillance confers a selective advantage to polyclonal metastasis.Nat Cancer.2020;1(7):709-722.doi:10.1038 / s43018-020-0068-9 25.Puram S V.,Tirosh I,Parikh AS,et al.Single-Cell Transcriptomic Analysis of Primary and Metastatic Tumor Ecosystems in Head and Neck Cancer.Cell.2017;171(7):1611-1624.e24.doi:10.1016 / j.cell.2017.10.044 26.Cillo AR,Kurten CHL,Tabib T,et al.Immune Landscape of Viral-and Carcinogen-Driven Head and Neck Cancer.Immunity.2020;52(1):183-199.e9.doi:10.1016 / j.immuni.2019.11.014 27.Boyman O,Sprent J.The role of interleukin-2 during homeostasis and activation of the immune system.Nat Rev Immunol.2012;12(3):180-190.doi:10.1038 / nri3156 28.Zimmer J,Andres E,Hentges F.NK cells and Treg cells:A fascinating dance of cheek to cheek.Eur J Immunol.2008;38(11):2942-2945.doi:10.1002 / eji.200838813 [ PubMed ] 29.Ralainirina N,Iie Poli A,Michel T,et al.Control of NK cell functions by CD4 CD25 regulatory T cells.doi:10.1189 / jlb.0606409 doi:10.1146 / annurev.immunol.24.021605.090616 30.Wang X,Lupardus P,Laporte SL,Garcia KC.Structural Biology of Shared Cytokine Receptors 31.Ben-Shmuel A,Biber G,Barda-Saad M.Unleashing Natural Killer Cells in the Tumor Microenvironment-The Next Generation of Immunotherapy?Front Immunol.2020;11.doi:10.3389 / fimmu.2020.00275 32.Waldhauer I,Gonzalez-Nicolini V,Freimoser-Grundschober A,et al.Simlukafusp alpha(FAP-IL2v)immunocytokine is a versatile combination partner for cancer immunotherapy.MAbs.2021;13(1).doi:10.1080 / 19420862.2021 33.Klein C,Waldhauer I,Nicolini VG,et al.Cergutuzumab amunaleukin(CEA-IL2v),a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy:Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines.Oncoimmunology.2017;6(3).doi:10.1080 / 2162402X.2016.1277306 34.Solomon I,Amann M,Goubier A,et al.CD25-Treg-depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity.Nat Cancer.2020;1(12):1153-1166.doi:10.1038 / s43018-020-00133-0 35.Knitz MW,Bickett TE,Darragh LB,et al.Targeting resistance to radiation-immunotherapy in cold HNSCCs by modulating the Treg-dendritic cell axis.J Immunother Cancer.2021;9(4).doi:10.1136 / jitc-2020-001955 36.Overacre-Delgoffe AE,Chikina M,Dadey RE,et al.Interferon-γ Drives Treg Fragility to Promote Anti-tumor Immunity.Cell.2017;169(6):1130-1141.e11.doi:10.1016 / J.CELL.2017.05.005 37.Mempel TR,Henrickson SE,Von Andrian UH.T-Cell Priming by Dendritic Cells in Lymph Nodes Occurs in Three Distinct Phases.;2004.www.nature.com / nature 38.Nakamura K,Smyth MJ.Immunoediting of cancer metastasis by NK cells.Nat Cancer.2020;1(7):670-671.doi:10.1038 / s43018-020-0081-z 39.Cong J.Metabolism of Natural Killer Cells and Other Innate Lymphoid Cells.Front Immunol.2020;11.doi:10.3389 / fimmu.2020.01989 40.O’Brien KL,Finlay DK.Immunometabolism and natural killer cell responses.Nat Rev Immunol.2019;19(5):282-290.doi:10.1038 / s41577-019-0139-2 41.Choi C,Finlay DK.Optimising NK cell metabolism to increase the efficacy of cancer immunotherapy.Stem Cell Res Ther.2021;12(1).doi:10.1186 / s13287-021-02377-8 42.Waldhauer I,Steinle A.NK cells and cancer immunosurveillance.Oncogene.2008;27(45):5932-5943.doi:10.1038 / onc.2008.267 43. Paul S, Lal G. The molecular mechanism of natural killer cells function and its importance in cancer immunotherapy. Front Immunol. 2017;8(SEP). doi:10.3389 / fimmu.2017.01124 44. Fiegler N, Textor S, Arnold A, et al. Downregulation of the activating NKp30 ligand B7-H6 by HDAC inhibitors impairs tumor cell recognition by NK cells. Published online in 2013. doi:10.1182 / blood-2013-02 45. Lopez-Soto A, Huergo-Zapico L, Acebes-Huerta A, Villa-Alvarez M, Gonzalez S. NKG2D signaling in cancer immunosurveillance. Int J Cancer. 2015;136(8):1741-1750. doi:10.1002 / ijc.28775 46. Duan S, Guo W, Xu Z, et al. Natural killer group 2D receptor and its ligands in cancer immune escape. Mol Cancer. 2019;18(1). doi:10.1186 / s12943-019-0956-8 47. Somanchi SS, McCulley KJ, Somanchi A, Chan LL, Lee DA. A novel method for assessment of natural killer cell cytotoxicity using image cytometry. PLoS One. 2015;10(10). doi:10.1371 / journal.pone.0141074 48.Nishimura H,Nose M,Hiai H,Minato N,Honjo T.Development of Lupus-like Autoimmune Diseases by Disruption of the PD-1 Gene Encoding an ITIM Motif-Carrying Immunoreceptor.Immunity.1999;11(2):141-151.doi:10.1016 / S1074-7613(00)80089-8 49.Simon S,Labarriere N.PD-1 expression on tumor-specific T cells:Friend or foe for immunotherapy?Oncoimmunology.2018;7(1).doi:10.1080 / 2162402X.2017.1364828 50.Miller AM,Lundberg K,Ozenci V,et al.CD4+CD25 high T Cells Are Enriched in the Tumor and Peripheral Blood of Prostate Cancer Patients.J Immunol.2006;177(10):7398-7405.doi:10.4049 / jimmunol.177.10.7398 51.TAKENAKA M,SEKI N,TOH U,et al.FOXP3 expression in tumor cells and tumor-infiltrating lymphocytes is associated with breast cancer prognosis.Mol Clin Oncol.2013;1(4):625-632.doi:10.3892 / mco.2013.107 52.O’callaghan DS,Rexhepaj E,Gately K,et al.Tumour islet Foxp3+T-cell infiltration predicts poor outcome in nonsmall cell lung cancer.Eur Respir J.2015;46:1762-1772.doi:10.1183 / 09031936.00159515 53.Sun D-S,Miao-Qing Zhao·,Xia M,Li·,Jiang L·Y-H.The correlation between tumor-infiltrating Foxp3+regulatory T cells and cyclooxygenase-2 expression and their association with recurrence in resected head and neck cancers.doi:10.1007 / s12032-011-9903-2 54.Arce Vargas F,Furness AJS,Solomon I,et al.Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors.Immunity.2017;46(4):577-586.doi:10.1016 / J.IMMUNI.2017.03.013 55.Pisani P,Airoldi M,Allais A,et al.Metastatic disease in head&neck oncology.Acta Otorhinolaryngol Ital.2020;40(2):S1-S86.doi:10.14639 / 0392-100X-suppl.1-40-2020 Br J Cancer.2019;121(11):897-903.doi:10.1038 / s41416-019-0601-8 56.Beckham TH,Leeman JE,Xie P,et al.Long-term survival in patients with metastatic head and neck squamous cell carcinoma treated with metastasis-directed therapy [ PMC free article ] [ PubMed ] [ Cross Ref ] 57.Merzoug L Ben,Marie S,Satoh-Takayama N,et al.Conditional ablation of NKp46+ cells using a novel Ncr1greenCre mouse strain:Eur J Immunol.2014;44(11):3380–3391.doi:10.1002 / eji.201444643 58.Glasner A,Ghadially H,Gur C,et al.Recognition and Prevention of Tumor Metastasis by the NK Receptor NKp46 / NCR1.J Immunol.2012;188(6):2509-2515.doi:10.4049 / jimmunol.1102461 Sci Rep.2017;7(1).doi:10.1038 / s41598-017-12998-w 59.Glasner A,Isaacson B,Viukov S,et al.Increased NK cell immunity in a transgenic mouse model of NKp46 overexpression 60.Guerra N,Tan YX,Joncker NT,et al.NKG2D-Deficient Mice Are Defective in Tumor Surveillance in Models of Spontaneous Malignancy.Immunity.2008;28(4):571-580.doi:10.1016 / J.IMMUNI.2008.02.016 61.Pasero C,Gravis G,Guerin M,et al.Inherent and tumor-driven immune tolerance in the prostate microenvironment impairs natural killer cell antitumor activity.Cancer Res.2016;76(8):2153-2165.doi:10.1158 / 0008-5472.CAN-15-1965 62.Pearce EL,Pearce EJ.Metabolic pathways in immune cell activation and quiescence.Immunity.2013;38(4):633-643.doi:10.1016 / j.immuni.2013.04.005 63.Wang Z,Guan D,Wang S,Chai LYA,Xu S,Lam KP.Glycolysis and Oxidative Phosphorylation Play Critical Roles in Natural Killer Cell Receptor-Mediated Natural Killer Cell Functions.Front Immunol.2020;11.doi:10.3389 / fimmu.2020.00202 64.Gardiner CM.NK cell metabolism.doi:10.1002 / JLB.MR0718-260R 65.Sheppard S,Santosa EK,Lau CM,et al.Lactate dehydrogenase A-dependent aerobic glycolysis promotes natural killer cell anti-viral and anti-tumor function.Cell Rep.2021;35(9).doi:10.1016 / j.celrep.2021.109210 66.Sun Y,Xu Z,Jiang J,Xu T,Xu J,Liu P.High Expression of Succinate Dehydrogenase Subunit A Which Is Regulated by Histone Acetylation Acts as a Good Prognostic Factor in Multiple Myeloma Patients.Front Oncol.2020;10.doi:10.3389 / fonc.2020.563666 67.Satoh-Takayama N,Vosshenrich CAJ,Lesjean-Pottier S,et al.Microbial Flora Drives Interleukin 22 Production in Intestinal NKp46+Cells that Provide Innate Mucosal Immune Defense.Immunity.2008;29(6):958-970.doi:10.1016 / j.immunity.2008.11.001 [ PMC free article ] [ PubMed ] [ Cross Ref ] 68.Verrier T,Satoh-Takayama N,Serafini N,Marie S,Di Santo JP,Vosshenrich CAJ Immunol.2016;196(11):4731–4738.doi:10.4049 / gymmunol.1502673 69.Hudspeth K,Silva-Santos B,Mavilio D.Natural cytotoxicity receptors:Broader expression patterns and functions in innate and adaptive immune cells.Front Immunol.2013;4(MAR).doi:10.3389 / fimmu.2013.00069 70.White RA,Neiman JM,Reddi A,et al.Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.J Clin Invest.2013;123.doi:10.1172 / JCI65856 71.Sun D,Wang J,Han Y,et al.TISCH:A comprehensive web resource enabling interactive single-cell transcriptome visualization of tumor microenvironment.Nucleic Acids Res.2021;49(D1):D1420-D1430.doi:10.1093 / nar / gkaa1020 72.Nemkov T,Hansen KC,D’Alessandro A.A three-minute method for high-throughput quantitative metabolomics and quantitative tracing experiments of central carbon and nitrogen pathways.Rapid Commun Mass Spectrom.2017;31(8):663-673.doi:10.1002 / rcm.7834 73. Nemkov T, Reisz JA, Gehrke S, Hansen KC, D’Alessandro A. High-Throughput Metabolomics: Isocratic and Gradient Mass Spectrometry-Based Methods. In:; 2019: 13-26. doi: 10.1007 / 978-1-4939-9236-2_2 74. Gehrke S, Rice S, Stefanoni D, et al. Red Blood Cell Metabolic Responses to Torpor and Arousal in the Hibernator Arctic Ground Squirrel. Published online in 2019. doi: 10.1021 / acs.jproteome.9b00018 75. Akhmedov M, Martinelli A, Geiger R, Kwee I. Omics Playground: a comprehensive self-service platform for visualization, analytics and exploration of Big Omics Data. NAR Genomics Bioinforma. 2020; 2(1). doi: 10.1093 / nargab / lqz019 76. Soares, K.C., Foley, K., Olino, K., Leubner, A., Mayo, S.C., Jain, A., Jaffee, E., Schulick, R.D., Yoshimura, K., Edil, B., and Zheng, L. (2014). A preclinical murine model of hepatic metastases. J Vis Exp, 51677. 10.3791 / 51677. TIFF2026505817000001.tif 255170 TIFF2026505817000002.tif 254170 TIFF2026505817000003.tif 254170 TIFF2026505817000004.tif 59170

Claims

1. 1. A combination for use in the treatment of cancer, said combination comprising: a) a first component comprising an effective amount of a Treg cell depleting agent; b) a second component comprising an effective amount of a bispecific immunocytokine; and c) a third component comprising an effective amount of radiation therapy, wherein said components a)-c) are for simultaneous or sequential administration.

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

3. The combination for use according to claim 2, wherein the anti-CD25 antibody is a monoclonal antibody that does not inhibit the binding of IL2 to CD25.

4. 4. The combination for use according to any one of claims 1 to 3, wherein said bispecific immunocytokine is an antibody that blocks the PD-1 / PD-L1 signaling axis and simultaneously binds to the IL2 receptor on the same cells (PD1-IL2v).

5. The combination for use according to claim 4, wherein the IL2 binding domain (IL2v) is a mutated variant specific for CD122.

6. The combination for use according to any one of claims 1 to 5, wherein said radiotherapy comprises local radiotherapy selected from external beam radiation or brachytherapy.

7. The combination for use according to any one of claims 1 to 6, wherein said radiotherapy comprises focal hypofractionated radiotherapy.

8. The combination for use according to any one of claims 1 to 7, wherein said radiotherapy comprises local hypofractionated irradiation in one or several doses in the range of 1 Gy to 20 Gy, in particular in the range of 5 Gy to 20 Gy.

9. The combination for use according to claim 8, wherein said radiotherapy comprises local hypofractionated irradiation in the range of 5 to 10 Gy in 1 to 3 fractions.

10. The combination for use according to claim 8 or claim 9, wherein said radiotherapy comprises local hypofractionated irradiation in the range of 8 Gy to 10 Gy, preferably 8 Gy or 10 Gy in a single dose.

11. The combination for use according to claim 8 or claim 9, wherein said radiotherapy comprises local hypofractionated irradiation in the range of 5 Gy to 10 Gy, preferably 8 Gy in three fractions.

12. The combination for use according to any one of claims 1 to 11, wherein said radiotherapy is administered after components a) and b).

13. 13. The combination for use according to claim 12, wherein components a) and b) are administered on day 1 and said radiotherapy is administered once on day 2 of a treatment cycle.

14. 13. The combination for use according to claim 12, wherein components a) and b) are administered on day 1 and said radiotherapy is administered on day 2, with subsequent booster doses every 5 to 7 days until the end of the treatment cycle.

15. The combination for use according to any one of claims 1 to 14, wherein the cancer is a solid tumor.

16. 16. The combination for use according to claim 15, wherein the solid tumor is selected from head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), melanoma, lung cancer, kidney cancer, breast cancer, colon cancer, ovarian cancer, cervical cancer, liver cancer, prostate cancer, bladder cancer, gastric cancer, glioblastoma and sarcoma.

17. 16. The combination for use according to claim 15, wherein the solid tumor is selected from head and neck squamous cell carcinoma (HNSCC), pancreatic cancer and pancreatic ductal adenocarcinoma (PDAC).

18. The combination for use according to any one of claims 1 to 17, wherein said combination is used to reduce overall tumor burden.

19. The combination for use according to any one of claims 1 to 17, wherein said combination is used to treat acquired resistance to a previous therapy for the same cancer in the same patient.

20. The combination for use according to any one of claims 1 to 17, wherein said combination is used to prevent the formation of metastases, preferably lung metastases, or to reduce the spread of said metastases.

21. A pharmaceutical product comprising a combination for use according to any one of claims 1 to 20 together with instructions on how to apply it.

22. A method for treating a patient with cancer, comprising administering an effective amount of a combination according to any one of claims 1 to 20.

23. The combination for use according to any one of claims 1 to 20, the pharmaceutical product according to claim 21 or the method according to claim 22, wherein component a) is optional.

24. The combination for use according to any one of claims 1 to 20, the pharmaceutical product according to claim 21, or the method according to claim 22, which does not comprise component a).

Images