A method of treating cancer using a combination of adoptive cell therapy and targeted immune cytokines.

Abstract

This disclosure relates to a method for improving the efficacy of adoptive cell therapy (ACT) and a method for treating cancer, wherein the method comprises administering a combination therapy comprising a therapeutically effective dose of ACT (e.g., immune cells comprising a modified T cell receptor (TCR) for tumor-associated antigens (TAAs) or a chimeric antigen receptor (CAR) for TAAs) and a therapeutically effective dose of a targeted immune cytokine (e.g., a fusion protein comprising an IL2 moiety and an immunoglobulin antigen-binding domain that binds to PD1) to a cancer subject in need thereof. This combination therapy demonstrates improved antitumor efficacy, improved duration of tumor control, and / or extended overall survival compared to subjects treated with ACT as monotherapy or with ACT in combination with untargeted immune cytokines.

Description

[Technical Field]

[0001] Sequence List The sequence listing for this application is submitted electronically as an XML file in ST.26 format with the filename "11195_SeqList-179227-03002", created on October 24, 2023, and measuring 65,705 bytes. The submitted sequence listing is part of this specification and is incorporated herein by reference in its entirety.

[0002] This disclosure, as a whole, relates to a combination therapy including adoptive cell therapy and targeted immune cytokines for treating cancer. [Background technology]

[0003] There are various immunotherapy strategies, including the use of adoptive cell therapy, which treats diseases such as cancer using the subject's own immune cells (or donor immune cells). Generally, adoptive cell therapy involves introducing recombinant T lymphocytes into the subject. Some examples of adoptive cell therapy include the use of engineered chimeric antigen receptors (CARs) or T cell receptors (TCRs). Generally, CARs contain a variable region of a single-chain fragment of an antibody or binding domain specific to tumor-associated antigens (TAAs), which is coupled to the cytoplasmic domain of a T cell signaling molecule via a hinge and transmembrane domain. The most common lymphocyte activation portion includes a T cell costimulatory domain linked to a T cell effector function trigger portion. With CAR-mediated adoptive cell therapy, CAR-transplanted T cells become capable of directly recognizing and attacking TAAs on target tumor cells.

[0004] Adoptive cell therapy using TCRs involves manipulating T cells to express a specific TCR, which is a heterodimer with two subunits. Each subunit contains a constant region that anchors a receptor to the cell membrane and a hypervariable region that performs antigen recognition. TCRs can recognize tumor-specific proteins both inside and outside the cell. With TCR therapy, T cells can be collected from the blood of the subject or a donor, then genetically recombined to express a novelly engineered TCR, which can then be administered to the subject to target the cancer. TCRs have been reported to mediate cell killing, enhance B cell proliferation, and limit cancer development and severity.

[0005] One reason for this is that, due to the inherent complexity of live cell culture and patient-to-patient variability, adoptive cell therapy has tended to yield limited success, resulting in unstable clinical activity. Therefore, there is a need to improve the antitumor activity of adoptive cell therapy.

[0006] Immune cytokines are antibody-cytokine complexes that preferentially localize to tumor lesions and have the potential to exert antitumor activity at the disease site. Interleukin-2 (IL-2 or IL2), a cytokine, is a pluripotent cytokine primarily produced by activated T cells. IL2 stimulates T cell proliferation and differentiation, induces the generation of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells, promotes the expression of cytokines and cytolytic molecules by T cells, facilitates B cell proliferation and differentiation and immunoglobulin synthesis by B cells, and stimulates the generation, proliferation, and activation of natural killer (NK) cells.

[0007] IL-2 is involved in the maintenance of peripheral CD4+ CD25+ regulatory T (Treg) cells, also known as suppressor T cells. Treg cells suppress effector T cells from destroying their (self) targets either by inhibiting T cell assistance and activation through cell-to-cell contact or by releasing immunosuppressive cytokines such as IL-10 or TGFβ. Depletion of Treg cells has been shown to enhance IL-2-induced antitumor immunity. However, due to its pluripotency, IL-2 is not optimal for inhibiting tumor growth. The use of IL-2 as an anti-cancer agent has also been limited by significant toxicity associated with doses required to elicit a sufficient tumor response. [Overview of the project] [Problems that the invention aims to solve]

[0008] Based on the above, there is a need for novel cancer treatments that offer improved efficacy and safety profiles. [Means for solving the problem]

[0009] The technology of this disclosure solves one or more of the aforementioned needs. In one embodiment, the technology of this disclosure is a method for improving the efficacy of adoptive cell therapy (ACT): (a) Selecting cancer targets; and (b) The present invention relates to a method comprising administering a therapeutically effective dose of ACT to a subject in combination with a therapeutically effective dose of a targeted immune cytokine, wherein the combination administration results in an improvement in the efficacy and duration of the antitumor response compared to subjects treated with ACT as monotherapy.

[0010] In another aspect, the technology of the present disclosure relates to a method for treating cancer, comprising administering a therapeutically effective dose of adoptive cell therapy (ACT) in combination with a therapeutically effective dose of targeted immune cytokine to a subject in need thereof, wherein the combination administration results in improved efficacy and duration of antitumor response compared to a subject treated with ACT as monotherapy.

[0011] Various embodiments relating to any or both aspects of the methods disclosed herein are described herein.

[0012] In some embodiments, the ACT comprises immune cells selected from T cells, tumor-infiltrating lymphocytes, and natural killer (NK) cells. In some embodiments, the immune cells comprise a modified TCR for tumor-associated antigens (TAAs) or a chimeric antigen receptor (CAR) for TAAs. In some embodiments, TAA is AFP, ALK, BAGE protein, BCMA, BIRC5 (Survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein, GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, L The following proteins are selected: MP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-Nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

[0013] In some embodiments, the targeted immune cytokine is a fusion protein comprising (a) the immunoglobulin antigen-binding domain of a checkpoint inhibitor and (b) an IL2 moiety. In some embodiments, the IL2 moiety comprises (i) IL2 receptor alpha (IL2Ra) or a fragment thereof; and (ii) IL2 or a fragment thereof. In some embodiments, the checkpoint inhibitor is an inhibitor of PD1, PD-L1, PD-L2, LAG-3, CTLA-4, TIM3, A2aR, B7H1, BTLA, CD160, LAIR1, TIGHT, VISTA, or VTCN1. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1.

[0014] In some embodiments, the antigen-binding domain includes a heavy chain variable region (HCVR) containing amino acid sequences selected from SEQ ID NOs: 1, 11, and 20, and a light chain variable region (LCVR) containing amino acid sequences selected from SEQ ID NOs: 5 and 15. In some embodiments, the antigen-binding domain includes three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences selected from: (a) SEQ ID NOs: 2, 3, 4, 6, 7, and 8, respectively; (b) SEQ ID NOs: 12, 13, 14, 16, 7, and 17, respectively; and (c) SEQ ID NOs: 21, 22, 23, 6, 7, and 8, respectively. In some embodiments, the antigen-binding domain includes HCVR / LCVR amino acid sequence pairs selected from SEQ ID NOs: 1 / 5, 11 / 15, and 20 / 5.

[0015] In some embodiments, the fusion protein includes a heavy chain containing an HIV1 isotype HCVR and a heavy chain constant region. In some embodiments, the fusion protein includes a heavy chain containing an HIV4 isotype HCVR and a heavy chain constant region. In some embodiments, the fusion protein includes a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence selected from SEQ ID NOs: 9, 18, and 24; and a light chain containing an amino acid sequence selected from SEQ ID NOs: 10, 19, and 25. In some embodiments, the fusion protein includes: (a) a heavy chain containing the amino acid sequence of SEQ ID NO: 24 and a light chain containing the amino acid sequence of SEQ ID NO: 25; (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acid sequence of SEQ ID NO: 10; or (c) a heavy chain containing the amino acid sequence of SEQ ID NO: 18 and a light chain containing the amino acid sequence of SEQ ID NO: 19.

[0016] In some embodiments, the antigen-binding domain includes a heavy chain, and the IL2 portion is attached to the C-terminus of the heavy chain via a linker containing the amino acid sequence of SEQ ID NO: 30 or 31. In some embodiments, the IL2 portion contains the amino acid sequence of SEQ ID NO: 27. In some embodiments, the L2 portion includes wild-type IL2. In some embodiments, IL2 contains the amino acid sequence of SEQ ID NO: 29. In some embodiments, the IL2 portion includes IL2 or a fragment thereof, which is linked to the C-terminus of IL2Ra or a fragment thereof via a linker. In some embodiments, IL2Ra or a fragment thereof contains the amino acid sequence of SEQ ID NO: 28. In some embodiments, the fusion protein is a dimerized fusion protein, dimerized through the constant region of the heavy chain of each monomer.

[0017] In some embodiments, the targeted immune cytokine includes a PD-1 targeting moiety and an IL2 moiety. In some embodiments, the PD-1 targeting moiety includes an immunoglobulin antigen-binding domain that specifically binds to PD-1. In some embodiments, the antigen-binding domain includes: (a) HCVR containing the amino acid sequence of SEQ ID NO: 20 and LCVR containing the amino acid sequence of SEQ ID NO: 5; (b) HCVR containing the amino acid sequence of SEQ ID NO: 1 and LCVR containing the amino acid sequence of SEQ ID NO: 5; or (c) HCVR containing the amino acid sequence of SEQ ID NO: 11 and LCVR containing the amino acid sequence of SEQ ID NO: 15. In some embodiments, the IL2 moiety includes (i) IL2Ra or a fragment thereof; and (ii) IL2 or a fragment thereof. In some embodiments, the IL2 moiety includes the amino acid sequence of SEQ ID NO: 27. In some embodiments, the targeted immune cytokine is REGN10597.

[0018] In some embodiments, cancer is selected from adrenal tumors, cholangiocarcinoma, bladder cancer, brain cancer, breast cancer, cell tumor, central or peripheral nervous system tissue cancer, cervical cancer, colon cancer, endocrine or neuroendocrine cancer or hematopoietic cancer, esophageal cancer, fibroma, gastrointestinal cancer, glioma, head and neck cancer, Lie-Fraumeni tumor, liver cancer, lung cancer, lymphoma, melanoma, meningioma, neuroendocrine type I or II tumor, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorder, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell carcinoma, parathyroid cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, kidney cancer, respiratory cancer, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, tracheal cancer, genitourinary cancer, and uterine cancer.

[0019] In some embodiments, administration of the above combination produces one or more therapeutic effects selected from delayed tumor growth, reduction in tumor cell count, tumor regression, extended survival, partial response, and complete response. In some embodiments, the therapeutically effective dose of ACT is 1 × 10⁻⁶ 6It contains one or more immune cells. In some embodiments, the therapeutically effective dose of targeted immune cytokine is 0.005 mg / kg of body weight of the subject to 10 mg / kg of body weight of the subject. In some embodiments, the targeted immune cytokine is administered intravascularly, subcutaneously, intraperitoneally, or intratumorally. In some embodiments, ACT is administered via intravenous infusion.

[0020] In some embodiments, ACT is administered before or after the administration of targeted immune cytokines. In some embodiments, ACT is administered concurrently with the administration of targeted immune cytokines. In some embodiments, targeted immune cytokines and / or ACT are administered to the subject in one or more doses.

[0021] In some embodiments, the method includes administering a further therapeutic agent or therapy to the target. In some embodiments, the further therapeutic agent or therapy is radiotherapy, surgery, chemotherapy agents, cancer vaccines, B7-H3 inhibitors, B7-H4 inhibitors, lymphocyte activator gene 3 (LAG3) inhibitors, T cell immunoglobulin and mucin domain-containing 3 (TIM3) inhibitors, galectin 9 (GAL9) inhibitors, inhibitors of T cell activation V-domain immunoglobulin (Ig)-containing inhibitor (VISTA), killer cell immunoglobulin-like receptor (KIR) inhibitors, B and T lymphocyte atenuator (BTLA) inhibitors, T cell immune receptor (TIGIT) inhibitors comprising Ig domain and ITIM domain, CD47 inhibitors, indoleamine-2, The following are selected: 3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists, angiopoietin-2 (Ang2) inhibitors, transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies against tumor-specific antigens, Calmette-Guérin vaccine, granulocyte-macrophage colony-stimulating factor (GM-CSF), cytotoxins, interleukin-6 receptor (IL-6R) inhibitors, interleukin-4 receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-12, IL-21, IL-15, antibody-drug conjugates, anti-inflammatory drugs, and combinations thereof.

[0022] In another aspect, the art of the present disclosure relates to immune cells comprising a modified T cell receptor or chimeric antigen receptor that specifically binds to a tumor-associated antigen, for use in a method of treating or inhibiting tumor growth in combination with a targeted immune cytokine comprising (i) an antigen-binding moiety that specifically binds to human PD-1 and (ii) an IL2 moiety, wherein the method comprises administering a therapeutically effective amount of immune cells and a therapeutically effective amount of targeted immune cytokine to a subject requiring it. [Brief explanation of the drawing]

[0023] [Figure 1] As described in Example 2, this is a diagram showing an example of a MAGE-A4 TCR-T lentivirus construct for generating MAGE-A4230-239 tetramer-positive TCR-T cells. [Figure 2] The graph shows the results of in vivo studies, measured by tumor volume (mm3) of A375 tumors in mice administered with either unrelated control TCR-T cells, control TCR-T+REGN9903, control TCR-T+REGN10597, 4 × 10⁶ MAGE-A4 TCR-T cells, 4 × 10⁶ MAGE-A4 TCR-T+REGN9903 cells, or 4 × 10⁶ MAGE-A4 TCR-T+REGN10597 cells, as described in Example 2. [Figure 3] The graph shows the results of in vivo studies, measured by tumor volume (mm3) of A375 tumors in mice administered 2 × 10⁶ MAGE-A4 TCR-T, 2 × 10⁶ MAGE-A4 TCR-T + REGN9903, or 2 × 10⁶ MAGE-A4 TCR-T + REGN10597, as described in Example 2. [Figure 4] The graph shows the results of in vivo studies, measured by tumor volume (mm3) of A375 tumors in mice administered 1 × 10⁶ MAGE-A4 TCR-T, 1 × 10⁶ MAGE-A4 TCR-T + REGN9903, or 1 × 10⁶ MAGE-A4 TCR-T + REGN10597, as described in Example 2. [Figure 5] This graph shows the results of an in vivo study, measured by tumor volume (mm3) of A375 tumors in mice administered 4 × 10⁶ MAGE-A4 TCR-T cells, as described in Example 2. [Figure 6] This graph shows the results of an in vivo study, measured by tumor volume (mm3) of A375 tumors in mice administered 4 × 10⁶ MAGE-A4 TCR-T + REGN9903, as described in Example 2. [Figure 7] This graph shows the results of an in vivo study, measured by tumor volume (mm3) of A375 tumors in mice administered 4 × 10⁶ MAGE-A4 TCR-T + REGN10597, as described in Example 2. [Figure 8] This graph shows the results of an in vivo study, measured by tumor volume (mm3) of A375 tumors in mice administered 2 × 10⁶ MAGE-A4 TCR-T cells, as described in Example 2. [Figure 9] This graph shows the results of an in vivo study, measured by tumor volume (mm3) of A375 tumors in mice administered 2 × 10⁶ MAGE-A4 TCR-T + REGN9903, as described in Example 2. [Figure 10] This graph shows the results of an in vivo study, measured by tumor volume (mm3) of A375 tumors in mice administered 2 × 10⁶ MAGE-A4 TCR-T + REGN10597, as described in Example 2. [Figure 11] This graph shows the results of an in vivo study, measured by tumor volume (mm3) of A375 tumors in mice administered 1 × 10⁶ MAGE-A4 TCR-T cells, as described in Example 2. [Figure 12] This graph shows the results of an in vivo study, measured by tumor volume (mm3) of A375 tumors in mice administered 1 × 10⁶ MAGE-A4 TCR-T + REGN9903, as described in Example 2. [Figure 13] This graph shows the results of an in vivo study, measured by tumor volume (mm3) of A375 tumors in mice administered 1 × 10⁶ MAGE-A4 TCR-T + REGN10597, as described in Example 2. [Figure 14] The graph shows the results of in vivo studies, measured by the survival percentage of mice administered 4 × 10⁶ MAGE-A4 TCR-T, 4 × 10⁶ MAGE-A4 TCR-T + REGN9903, or 4 × 10⁶ MAGE-A4 TCR-T + REGN10597, as described in Example 2. [Figure 15] The graph shows the results of in vivo studies, measured by the survival percentage of mice administered 2 × 10⁶ MAGE-A4 TCR-T, 2 × 10⁶ MAGE-A4 TCR-T + REGN9903, or 2 × 10⁶ MAGE-A4 TCR-T + REGN10597, as described in Example 2. [Figure 16] The graph shows the results of in vivo studies, measured by the survival percentage of mice administered 1 × 10⁶ MAGE-A4 TCR-T, 1 × 10⁶ MAGE-A4 TCR-T + REGN9903, or 1 × 10⁶ MAGE-A4 TCR-T + REGN10597, as described in Example 2. [Figure 17-1] Figures 17A-17C are diagrams of a set of configuration diagrams showing examples of CAR constructs, as described in Example 3: Figure 17A is an anti-huCD20 CAR-T (CD20 / BBz CAR-T) having CD3z and 4-1BB signaling domains; Figure 17B is an anti-huCD20 CAR-T (CD20 / 28z CAR-T) having CD3z and CD28 signaling domains; and Figure 17C is a control CAR-T (CTRL / BBz CAR-T) having CD3z and 4-1BB signaling domains. [Figure 17-2] Same as above. [Figure 18]The graph shows the results of in vivo studies, measured by tumor volume (mm3) in C57BL / 6 mice administered 0.5 × 10⁶ CTRL / BBz CAR-T+REGN9903 0.2 mg / kg, 0.5 × 10⁶ CD20 / BBz CAR-T+REGN9903 0.2 mg / kg, 0.5 × 10⁶ CTRL / BBz CAR-T+REGN10597 0.2 mg / kg, 0.5 × 10⁶ CD20 / BBZ CAR-T+REGN10597 0.2 mg / kg, or 0.5 × 10⁶ CD20 / BBZ CAR-T+REGN10597 0.5 mg / kg, as described in Example 3. [Figure 19] The graph shows the results of in vivo studies, measured by tumor volume (mm3) in C57BL / 6 mice administered 0.5 × 10⁶ CTRL / BBz CAR-T+REGN9903 0.2 mg / kg, 0.5 × 10⁶ CD20 / CD28Z CAR-T+REGN9903 0.2 mg / kg, 0.5 × 10⁶ CTRL / BBz CAR-T+REGN10597 0.2 mg / kg, 0.5 × 10⁶ CD20 / 28Z CAR-T+REGN10597 0.2 mg / kg, or 0.5 × 10⁶ CD20 / 28z CAR-T+REGN10597 0.5 mg / kg, as described in Example 3. [Figure 20] This graph shows the results of an in vivo study, measured by tumor volume (mm3) in C57BL / 6 mice administered 0.5 × 10⁶ CTRL / BBz CAR-T + REGN9903 0.2 mg / kg, as described in Example 3. [Figure 21] The graph shows the results of an in vivo study, measured by tumor volume (mm3) in C57BL / 6 mice administered 0.5 × 10⁶ CTRL / BBz CAR-T + REGN10597 0.2 mg / kg, as described in Example 3. [Figure 22]This graph shows the results of an in vivo study, measured by tumor volume (mm3) in C57BL / 6 mice administered 0.5 × 10⁶ CD20 / BBZ CAR-T + REGN9903 0.2 mg / kg, as described in Example 3. [Figure 23] This graph shows the results of an in vivo study, measured by tumor volume (mm3) in C57BL / 6 mice administered 0.5 × 10⁶ CD20 / BBZ CAR-T + REGN10597 0.2 mg / kg, as described in Example 3. [Figure 24] The graph shows the results of an in vivo study, as described in Example 3, measuring the tumor volume (mm3) of tumors in C57BL / 6 mice administered 0.5 × 10⁶ CD20 / BBZ CAR-T + REGN10597 0.5 mg / kg. [Figure 25] This graph shows the results of an in vivo study, measured by tumor volume (mm3) in C57BL / 6 mice administered 0.5 × 10⁶ CD20 / CD28Z CAR-T + REGN9903 0.2 mg / kg, as described in Example 3. [Figure 26] This graph shows the results of an in vivo study, measured by tumor volume (mm3) in C57BL / 6 mice administered 0.5 × 10⁶ CD20 / 28Z CAR-T + REGN10597 0.2 mg / kg, as described in Example 3. [Figure 27] This graph shows the results of an in vivo study, measured by tumor volume (mm3) in C57BL / 6 mice administered 0.5 × 10⁶ CD20 / 28Z CAR-T + REGN10597 0.5 mg / kg, as described in Example 3. [Figure 28] The figure shows a pair of graphs illustrating the frequency and absolute number of peripheral blood B220+ B cells on day 7 in lymphocyte-depleted mice administered the prescribed combination therapy as described in Example 4. [Figure 29]The figure shows a pair of graphs illustrating the frequency and absolute number of peripheral blood GFP+ CAR T cells on day 7 in lymphocyte-depleted mice administered the prescribed combination therapy as described in Example 4. [Figure 30] The figure shows a pair of graphs illustrating the frequency and absolute number of peripheral blood B220+ B cells on day 7 in non-lymphocyte-depleted mice administered the prescribed combination therapy as described in Example 4. [Figure 31] The figure shows a pair of graphs illustrating the frequency and absolute number of peripheral blood GFP+ CAR T cells on day 7 in non-lymphocyte-depleted mice administered the prescribed combination therapy as described in Example 4. [Figure 32] The figure shows a pair of graphs illustrating the frequency and absolute number of peripheral blood B220+ B cells on day 21 in lymphocyte-depleted mice administered the prescribed combination therapy as described in Example 4. [Figure 33] The figure shows a pair of graphs illustrating the frequency and absolute number of peripheral blood GFP+ CAR T cells on day 21 in lymphocyte-depleted mice administered the prescribed combination therapy as described in Example 4. [Figure 34] The figure shows a pair of graphs illustrating the frequency and absolute number of peripheral blood B220+ B cells on day 21 in non-lymphocyte-depleted mice administered the prescribed combination therapy as described in Example 4. [Figure 35] The figure shows a pair of graphs illustrating the frequency and absolute number of peripheral blood GFP+ CAR T cells at day 21 in non-lymphocyte-depleted mice administered the prescribed combination therapy as described in Example 4. [Figure 36] This is a graph showing the average tumor volume in mice administered the prescribed combination therapy, as described in Example 5. [Figure 37-1]Figures 37A-D are diagrams relating to Example 6. Figure 37A is a graph showing the expression of PD-1 on anti-huMUC16 or control CAR+ T cells after co-culture in vitro with the indicated tumor cell line. Figure 37B is a schematic diagram of the in vivo study. Figure 37C is a graph showing the mean tumor growth (mean + SD) monitored over time, and statistical analysis was performed using two-way ANOVA with Bonferroni's multiple comparison test (**P≦0.01, ***P≦0.001, ****P≦0.0001). Figure 37D is a collection of individual tumor growth curves, in which case the data represent results from experiments performed using two different syngeneic tumor models. [Figure 37-2] Same as above. [Figure 37-3] Same as above. [Figure 37-4] Same as above. [Modes for carrying out the invention]

[0024] The technology of this disclosure is at least in part based on the unexpected discovery that targeted immune cytokines enhance the in vivo antitumor activity of immune cells (e.g., T cells) containing modified TCRs or CARs. Cell therapies for treating cancer (hereinafter referred to herein as “adoptive cell therapy,” ACT, or adoptive immunotherapy) involve immune cells (e.g., T cells) modified with TCRs or CARs, in which case the TCRs or CARs are targeted to TAAs. Such cell therapies exhibit moderate and non-persistent tumor control. IL2 is administered for cell proliferation and amplification; however, naked IL2 or untargeted IL2 results in toxicity in the subject. In contrast, without being bound by any particular theory, when IL2 is co-administered with a portion targeted to a checkpoint inhibitor (hereinafter referred to herein as “targeted immune cytokines”), the combination is thought to result in a targeted agent that drives the proliferation, amplification, and survival of immune cells. Enhanced survival corresponds to an improved duration of the antitumor response. As described herein, administration of targeted immune cytokines results in extended survival and longer duration of antitumor activity of T cells modified with TCR or CAR against TAAs. Non-limiting examples of such TAAs include, among others, MAGE-A4 and CD20. The aforementioned co-administration results in a greater antitumor response (e.g., greater tumor reduction) and a longer duration of response in mice. Thus, what is demonstrated by the combination therapy of targeted immune cytokines and TCR-modified or CAR-modified immune cells of this disclosure is an unexpected synergistic antitumor efficacy in inducing potent and persistent tumor control in cancer subjects.

[0025] Methods for treating cancer This disclosure includes a method for improving the efficacy of adoptive cell therapy (ACT), wherein the method includes administering a combination therapy comprising a therapeutically effective dose of ACT and a therapeutically effective dose of targeted immune cytokines to a cancer subject. This disclosure also includes a method for treating cancer, wherein the method includes administering a combination therapy comprising a therapeutically effective dose of ACT and a therapeutically effective dose of targeted immune cytokines to a subject in need thereof.

[0026] As used herein, terms such as “treating” and “treat” mean to alleviate symptoms, eliminate the causal relationship of symptoms on a temporary or permanent basis, delay or inhibit tumor growth, reduce tumor cell volume or tumor burden, promote tumor regression, cause tumor shrinkage, necrosis and / or disappearance, prevent tumor recurrence, prevent or inhibit metastasis, inhibit metastatic tumor growth, and / or extend the survival of the subject.

[0027] As used herein, the expression “subjects in need of it” refers to human or non-human mammals exhibiting one or more symptoms or signs of cancer, as well as human or non-human mammals diagnosed with cancer and human or non-human mammals in need of treatment for cancer. The term “subjects” includes subjects with primary or metastatic tumors (advanced malignancies). In certain embodiments, the expression “subjects in need of it” includes subjects with tumors that are resistant or refractory to previous therapies (e.g., chemotherapy) or are poorly controlled by previous therapies. The expression also includes subjects with tumors for which conventional chemotherapy is not prudent due to toxic side effects, for example. For example, the expression includes subjects who have undergone one or more cycles of chemotherapy and have experienced toxic side effects.

[0028] As used herein, the terms “tumor” or “cancer” refer to a disease characterized by the uncontrolled (and often rapid) proliferation of abnormal cells. Cancer cells may spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers described herein include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal cancer, autonomic ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, reproductive tract cancer, colorectal cancer, meningeal cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, upper respiratory tract and gastrointestinal cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, and others. The terms "tumor," "cancer," and "malignant tumor" are used interchangeably in this specification.

[0029] In certain embodiments, the methods of the Disclosure for treating or inhibiting tumor growth include, but are not limited to, treating or inhibiting the growth of anal cancer, bladder cancer, hematological cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, myeloma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, and uterine cancer.

[0030] In some embodiments, the methods of the present disclosure result in improved efficacy and duration of the antitumor response. Methods according to these embodiments of the present disclosure include selecting a cancer subject and administering a therapeutically effective dose of targeted immune cytokines to the subject in combination with a therapeutically effective dose of adoptive cell therapy. In certain embodiments, the methods result in an improvement in tumor inhibition of, for example, about 20%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80% compared to subjects treated with ACT as monotherapy or subjects treated with ACT in combination with untargeted immune cytokines (such as untargeted IL2 cytokines).

[0031] In certain embodiments, the method results in an improvement in the duration of the antitumor response by, for example, about 20%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80% compared to subjects treated with ACT as monotherapy or in combination with ACT and a non-targeted immune cytokine (such as a non-targeted IL2 cytokine). In certain embodiments, administration of a targeted immune cytokine in combination with ACT results in an improvement in the response and duration of the response in subjects by, for example, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50% compared to untreated subjects or subjects treated with ACT as monotherapy or in combination with a non-targeted immune cytokine (such as a non-targeted IL2 cytokine).

[0032] In certain embodiments, the method of the present disclosure can delay tumor growth and development by, for example, about 3 days, more than 3 days, about 7 days, more than 7 days, more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 1 year, more than 2 years, or more than 3 years, compared to an untreated subject, a subject treated with ACT monotherapy, or a subject treated with ACT in combination with a non-targeted immune cytokine (such as a non-targeted IL2 cytokine).

[0033] In certain embodiments, administration of any of the combinations disclosed herein prevents tumor recurrence and / or extends the survival of a subject, for example, beyond the survival of an untreated subject, a subject treated with ACT as monotherapy, or a subject treated with ACT in combination with a non-targeted immune cytokine (such as a non-targeted IL2 cytokine), by only 1 to 5 days, only 5 days, only 10 days, only 15 days, more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months.

[0034] In certain embodiments, administration of targeted immune cytokines in combination with ACT to a cancer subject results in the complete disappearance of all evidence relating to tumor cells ("complete response"). In certain embodiments, administration of targeted immune cytokines in combination with ACT to a cancer subject results in a reduction of at least 30% of tumor cells or tumor size ("partial response"). In certain embodiments, administration of targeted immune cytokines in combination with ACT to a cancer subject results in the complete or partial disappearance of tumor cells / lesions, including measurable novel lesions. Tumor reduction can be measured by any method known in the art, e.g., by radiography, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analysis.

[0035] In certain embodiments, administration of targeted immune cytokines in combination with ACT to cancer subjects results in an improvement in overall response rates compared to untreated subjects, subjects treated with ACT monotherapy, or subjects treated with ACT in combination with untargeted immune cytokines (such as untargeted IL2 cytokines).

[0036] In certain embodiments, administration of a therapeutically effective dose of the ACT of the Disclosure and a therapeutically effective dose of the targeted immune cytokine of the Disclosure to a cancer subject results in an extension of the subject's overall survival (OS) and progression-free survival (PFS) compared to a subject treated with ACT as monotherapy or with ACT in combination with a non-targeted immune cytokine (such as a non-targeted IL2 cytokine).

[0037] In certain embodiments, PFS is extended by at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, or at least 3 years compared to subjects treated with ACT as monotherapy or in combination with ACT and untargeted immune cytokines (such as untargeted IL2 cytokines).

[0038] In certain embodiments, OS is extended by at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, or at least 3 years compared to subjects treated with ACT as monotherapy or in combination with ACT and untargeted immune cytokines (such as untargeted IL2 cytokines).

[0039] Adoptive cell therapy (ACT) The methods of this disclosure include the administration of targeted immune cytokines in combination with ACT. As used herein, the terms “adoptive cell therapy,” “ACT,” and “adoptive immunotherapy” are interchangeable and refer to the administration of modified immune cells to a cancer subject. “Immune cells” (also interchangeably referred to herein as “immune effector cells”) refer to cells that are part of the subject’s immune system and help fight cancer in the subject’s body. Non-exclusive examples of immune cells for use in the methods of this disclosure include T cells, tumor-infiltrating lymphocytes, and natural killer (NK) T cells. The immune cells may be autologous or heterologous to the subject receiving therapy.

[0040] As used herein, the terms "T cell" and "T lymphocyte" are used interchangeably. T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) cells or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4 + T cells), CD4 + T cells, cytotoxic T cells (CTL; CD8 + T cells), tumor infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + T cells, or other subsets of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells and memory T cells. Also included are "natural killer T (NKT) cells" or "NKT cells", which refer to a special population of T cells that express a semi-invariant ab T cell receptor but also express various molecular markers normally associated with NK cells such as NK1.1. NKT cells include NK1.1 + and NK1.G as well as CD4 + and CD4, CD8 + and CD8 cells.

[0041] The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or detrimental effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also included are "gamma-delta T cells (γδ T cells)," a special population of cells that possess a distinctly different TCR on their surface. Unlike the majority of T cells, whose TCR consists of two glycoprotein chains called α-TCR and β-TCR chains, the TCR of γδ T cells consists of γ- and d-chains. γδ T cells can play a role in immune surveillance and immunomodulation, but they are also an important source of IL-17 and robust CD8 + It was found to induce a cytotoxic T cell response. This also includes "regulatory T cells" or "Tregs," which are T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs are typically Foxp3-positive CD4 + T cells, specifically Treg cells, produce IL-10 CD4 + This may also include Foxp3-negative regulatory T cells, which are a type of T cell.

[0042] T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic problems, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. In some embodiments, T cells can be obtained from units of blood collected from a subject using several techniques known to those skilled in the art, such as FICOLL isolation. In one embodiment, T cells derived from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets.

[0043] The immune effector cells of this disclosure, such as T cells, can be isolated and then recombined (to form modified immune cells) using known methods, or the immune cells can be activated and amplified or, in the case of progenitor cells, differentiated before being recombined in vitro. In some embodiments, immune effector cells, such as T cells, can be recombined with a TCR or CAR as described herein (e.g., transduced with a viral vector containing nucleic acids encoding a TCR or CAR), and then activated and amplified in vitro. Techniques for activating and amplifying T cells are known in the art and suitable for use with the techniques of this disclosure. See, for example, US6,905,874;US6,867,041;US6,797,514;WO2012079000;US2016 / 0175358. TCR-expressing or CAR-expressing immune effector cells suitable for use in the methods of this disclosure can be produced according to known techniques described in the art.

[0044] For use in the methods of this disclosure, immune cells may be modified with a TCR for TAA or with a CAR. In other words, non-limiting examples of ACTs for use in the methods of this disclosure include modified TCRs for tumor-associated antigens (TAAs) or chimeric antigen receptors (CARs) for TAAs.

[0045] TAAs can originate from any cancer, but are not limited to, the following: adrenal tumors, cholangiocarcinomas, bladder cancers, brain cancers, breast cancers, cell tumors, central or peripheral nervous system tissue cancers, cervical cancers, colon cancers, endocrine or neuroendocrine cancers or hematopoietic cancers, esophageal cancers, fibromas, gastrointestinal cancers, gliomas, head and neck cancers, Lie-Fraumeni tumors, liver cancers, lung cancers, lymphomas, melanomas, meningiomas, neuroendocrine type I or II tumors, multiple myelomas, myelodysplastic syndromes, myeloproliferative disorders, nasopharyngeal cancers, oral cancers, oropharyngeal cancers, osteosarcomas, ovarian cancers, pancreatic cancers, islet cell carcinomas, parathyroid cancers, pheochromocytomas, pituitary tumors, prostate cancers, rectal cancers, kidney cancers, respiratory cancers, sarcomas, skin cancers, gastric cancers, testicular cancers, thyroid cancers, tracheal cancers, genitourinary cancers or uterine cancers.

[0046] In certain embodiments, TAA is selected from: AFP, ALK, BAGE protein, BCMA, BIRC5 (Survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HL A / MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p 53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA(FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-Nouvel antigen (Tn), TRP-1, TRP-2, tyrosinase, or uroplakin-3.

[0047] As used herein, “T cell receptor” refers to an isolated TCR polypeptide that specifically binds to TAA, or a TCR expressed on isolated immune cells (e.g., T cells). TCRs bind to epitopes on small antigenic determinants on the surface of antigen-presenting cells (e.g., those found in tumor-associated antigens) that associate with major histocompatibility complexes (MHC; in mice) or human leukocyte antigens (HLA; in humans). TCRs also refer to members of the immunoglobulin superfamily capable of specifically binding to MHC receptor-bound antigen peptides, possessing a variable binding domain, constant domain, transmembrane region, and short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd edition, Current Biology Publications, 1997).

[0048] As used herein, the term “polypeptide” refers to any polymer, regardless of its size, that is essentially composed of, preferably, any of 20 natural amino acids. The term “protein” is often used in relation to relatively larger proteins, and “peptide” is often used in relation to smaller polypeptides, although the use of these terms often overlaps in the art. Unless otherwise specified, the term “polypeptide” generally refers to proteins, polypeptides, and peptides. Peptides useful according to this disclosure are, as determined by standard molecular sizing techniques such as centrifugation or SDS-polyacrylamide gel electrophoresis, generally between about 0.1 and 100 KD and up to about 1000 KD, preferably between about 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 30-50 KD.

[0049] TCRs can be found on the cell surface and are generally composed of heterodimers having α and β chains (also known as TCRα and TCRβ, respectively) or γ and δ chains (also known as TCRγ and TCRδ, respectively). Similar to immunoglobulins, the extracellular portion of the TCR chain (e.g., α and β chains) contains two immunoglobulin regions, a variable region (e.g., TCR variable α or Vα and TCR variable β or Vβ; usually amino acids 1-116 based on Kabat numbering at the N-terminus), and a constant region adjacent to the cell membrane (e.g., TCR constant domain α or Cα, usually amino acids 117-259 based on Kabat, TCR constant domain β or Cβ, usually amino acids 117-295 based on Kabat). Also, similar to immunoglobulins, the variable domain contains a CDR separated by a framework region (FR). In some embodiments, TCRs are found on the surface of T cells (or T lymphocytes) and associate with the CD3 complex. The TCR source of this disclosure may be derived from various animal species, such as humans, mice, rats, rabbits, or other mammals. In some embodiments, the TCR source of this disclosure is a mouse genetically engineered to produce a TCR containing human alpha and beta chains (see, for example, WO2016 / 164492).

[0050] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid sequence within the antibody variable region that confers antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the definitions of Kabat, Chothia, ABM, and IMGT. For example, see Kabat, 1991, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al., 1997, J.Mol.Biol.273:927-948 (Chothia numbering scheme); Martin et al., 1989, Proc.Natl.Acad.Sci.USA 86:9268-9272 (ABM numbering scheme); and Lefranc et al., 2003, Dev.Comp.Immunol.27:55-77 (IMGT numbering scheme). Publicly available databases for identifying CDR sequences within antibodies are also available.

[0051] The TCRα and TCRβ polypeptides (and similarly the TCRγ and TCRδ polypeptides) are linked to each other via disulfide bonds. Each of the two polypeptides constituting the TCR contains an extracellular domain including a constant region and a variable region, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and cytoplasmic tail are also part of the constant region). The variable region of the TCR determines its antigen specificity and contains three CDRs, as with immunoglobulins. The TCR is expressed on most T cells in the body and is known to be involved in the recognition of MHC restriction antigens. The TCRα chain contains covalently linked Vα and Cα regions, while its β chain contains a Vβ region covalently linked to the Cβ region. The Vα and Vβ regions form a pocket or gap that can bind to the antigen in association with the major histocompatibility complex (MHC) (or HLA in humans).

[0052] The term "HLA" refers to the human leukocyte antigen (HLA) system or complex, which is a gene complex encoding human MHC proteins. These cell surface proteins are responsible for regulating the immune system in humans. HLAs (A, B, and C) corresponding to MHC class I present peptides from within the cell. The term "HLA-A" refers to the group of human leukocyte antigens (HLAs) encoded by the HLA-A locus. HLA-A is one of the three main types of human MHC class I cell surface receptors. The receptor is a heterodimer, consisting of a heavy α chain and a smaller β chain. The α chain is encoded by the variant HLA-A gene, and the β chain (β2-microglobulin) is the invariant β2-microglobulin molecule. * The term "HLA-A" (also known as "HLA-A1") refers to a group of specific MHC class I alleles at the HLA-A locus; the α chain is HLA-A * It is encoded by the O2 gene, and the β chain is encoded by the β2-microglobulin or B2M locus.

[0053] TCRs are detection molecules with excellent specificity and, like antibodies, exhibit great diversity. The general structure of TCR molecules, including their binding to peptides:MHC, and techniques for the production and use of such molecules are described in PCT / US98 / 04274, PCT / US98 / 20263, and WO99 / 60120.

[0054] For example, non-human animals (e.g., rodents, e.g., mice or rats) can be genetically engineered to express human or humanized TCRs containing a variable domain encoded by at least one human TCR variable region gene segment. See, for example, WO2016 / 164492. For example, genetically modified mice are provided by Veloci-T® mouse technology (Regeneron), which enables the production of fully human therapeutic TCRs against tumor and / or viral antigens and can be used to produce TCRs suitable for use in the technologies of this disclosure. Those skilled in the art can obtain modified TCR sequences through standard mutagenesis techniques in combination with the assays described herein and test them for specific binding affinity and / or specificity. Useful mutagenesis techniques known in the art include, but are not limited to, de novo gene synthesis, oligonucleotide-specific mutagenesis, region-specific mutagenesis, linker-scan mutagenesis, and PCR-based site-specific mutagenesis.

[0055] In some embodiments, a method for generating a TCR for TAA may include immunizing a non-human animal (e.g., a rodent, e.g., a mouse or rat), for example, a genetically modified non-human animal containing a human TCR variable locus that has not been rearranged in its genome, with a specific peptide derived from TAA; enabling the animal to initiate an immune response to the peptide; isolating T cells reactive to the peptide from the animal; determining the nucleic acid sequence of the human TCR variable region expressed by the T cells; cloning the human TCR variable region into a nucleotide construct containing the nucleic acid sequence of the human TCR constant region such that the human TCR variable region is operably linked to the human TCR constant region; and expressing a human T cell receptor specific to the peptide from the construct. The steps of isolating T cells, determining the nucleic acid sequence of the human TCR variable region expressed by the T cells, cloning the human TCR variable region into a nucleotide construct containing the nucleic acid sequence of the human TCR constant region, and expressing a human T cell receptor are performed using standard techniques known to those skilled in the art.

[0056] As used herein, an HLA-presenting peptide (such as an HLA-A2-presenting peptide) may refer to a peptide bound to an HLA protein, such as an HLA protein expressed on the surface of a cell. Therefore, a TCR that binds to an HLA-presenting peptide binds to the HLA-bound peptide and, in some cases, to the HLA itself. The interaction with HLA can confer specificity for binding to peptides presented by a particular HLA. In some embodiments, the TCR can bind to an isolated HLA-presenting peptide. In some embodiments, the TCR can bind to an HLA-presenting peptide on the surface of a cell.

[0057] As used herein, “chimeric antigen receptor” or “CAR” refers to an antigen-binding protein comprising an immunoglobulin antigen-binding domain (e.g., an immunoglobulin variable domain) and a TCR constant domain or a portion thereof, which can be administered to a target as chimeric antigen receptor T cell (CAR-T) therapy. As used herein, the “constant domain” of a TCR polypeptide comprises a membrane-proximal TCR constant domain and may also include a TCR transmembrane domain and / or a TCR cytoplasmic tail. For example, in some embodiments, the CAR is a dimer comprising a first polypeptide comprising an immunoglobulin heavy chain variable domain linked to the TCRβ constant domain, and a second polypeptide comprising an immunoglobulin light chain variable domain (e.g., a κ or λ variable domain) linked to the TCRα constant domain. In some embodiments, the CAR is a dimer, which comprises a first polypeptide containing an immunoglobulin heavy chain variable domain linked to the TCRα constant domain, and a second polypeptide containing an immunoglobulin light chain variable domain (e.g., a κ or λ variable domain) linked to the TCRβ constant domain.

[0058] As used herein, “variable domain” refers to the variable region of the alpha chain or the variable region of the beta chain that is directly involved in the binding of the TCR to the antigen. As used herein, the term “constant domain” refers to the constant regions of the alpha chain and the constant regions of the beta chain that are not directly involved in the binding of the TCR to the antigen but exhibit various effector functions.

[0059] A CAR is a typically artificial, constructed hybrid protein or polypeptide containing an antigen-binding domain of scFv or other antibody agent linked to a T cell signaling domain. In the context of this disclosure, CARs are directed to tumor-associated antigens. Features of CARs include the ability to redirect the specificity and responsiveness of T cells to a selected target in an MHC-unrestricted manner using the antigen-binding properties of a monoclonal antibody. MHC-unrestricted antigen recognition gives T cells expressing a CAR the ability to recognize an antigen independently of antigen processing, thereby circumventing the primary mechanism of tumor evasion. When used in the ACTs disclosed herein, immune cells can be manipulated to express a CAR in any known manner, including, for example, by transfection using RNA and DNA, both of which are known in the art.

[0060] In some embodiments, immunoeffector cells expressing a TCR or CAR are first harvested from their culture medium and then formulated in a treatment-effective dose by washing and concentrating the cells in a system of medium and container suitable for administration ("pharmaceutically acceptable" carrier). The appropriate infusion medium may be any isotonic medium preparation, usually physiological saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but a 5% dextrose aqueous solution or Ringer's lactate solution may also be used. The infusion medium may be supplemented with human serum albumin.

[0061] The therapeutically effective number of immune cells to be administered in the method disclosed herein is typically 10 2 More than 10 cells, for example, 10 6 Including the number of cells and any number smaller than that, 10 8 Including the number of cells and any number smaller than that, 10 9 Including one or fewer cells, or 10 10 These are more than one cell. The number and / or type of cells to be administered to the target will depend on the ultimate intended use of the therapy.

[0062] The TCRs and CARs of this disclosure may be recombinant, which can be prepared, expressed, isolated, or obtained by techniques or methods known in the art, such as recombinant DNA technologies including, for example, DNA splicing and transgenic expression. Recombinant TCRs or CARs may be expressed in expression systems of non-human mammals (including transgenic non-human mammals, e.g., transgenic mice) or cells (e.g., CHO cells), or isolated from recombinant combinatorial human antibody libraries.

[0063] Targeted immune cytokines As used herein, “targeted immune cytokine” refers to a cytokine such as interleukin-2 (IL2) that is linked to a portion that binds to (i.e., “targets” the checkpoint inhibitor) a checkpoint inhibitor. Non-limiting examples of checkpoint inhibitors include inhibitors of PD1, PD-L1, PD-L2, LAG-3, CTLA-4, TIM3, A2aR, B7H1, BTLA, CD160, LAIR1, TIGHT, VISTA, or VTCN1. In some embodiments, the targeted immune cytokine comprises the immunoglobulin antigen-binding domain of the checkpoint inhibitor. In one preferred embodiment, the checkpoint inhibitor is a PD-1 inhibitor (e.g., an anti-PD-1 antibody or its antigen-binding fragment). In certain embodiments, the targeted immune cytokine is a fusion protein comprising (i) the antigen-binding domain of the checkpoint inhibitor and (ii) the IL2 portion.

[0064] In some embodiments, the antigen-binding domain specifically binds to human PD-1. In some embodiments, the antigen-binding domain is an antibody or its antigen-binding fragment.

[0065] As used herein, the term “fusion protein” means a protein comprising two or more polypeptide sequences linked by covalent or non-covalent bonds. Fusion proteins encompassed by this disclosure include the translation product of a chimeric gene construct in which a nucleic acid sequence encoding a first polypeptide is linked with a nucleic acid sequence encoding a second polypeptide to form a single open reading frame. Alternatively, a fusion protein may also be encoded by two or more gene constructs on separate vectors that can be co-expressed in a host cell. Overall, a “fusion protein” is a recombinant protein of two or more proteins linked by peptide bonds or by several peptides. In some embodiments, a fusion protein may also include a peptide linker between the two domains.

[0066] Fusion proteins disclosed herein may contain one or more conservative modifications. Fusion proteins having one or more conservative modifications may retain a desired functional property, which can be tested using functional assays known in the art. The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding properties of a protein containing the amino acid sequence in question. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). They may include one or more conserved modifications. Cas proteins with one or more conserved modifications may retain a desired functional property, which can be tested using functional assays known in the art. As used herein, the term “conservative sequence modification” refers to an amino acid modification that does not significantly affect or alter the binding properties of a protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions are those in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with non-loading side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0067] As used herein, “antibody” refers to an immunoglobulin molecule (i.e., a “complete antibody molecule”) consisting of four polypeptide chains: two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, as well as its polymer (e.g., IgM) or its antigen-binding fragment. Each heavy chain consists of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (consisting of domains CH1, CH2, and CH3). Each light chain consists of a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions called CDRs, which are separated by more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of the antibody (or its antigen-binding portion) may be identical to those of the human germline sequence, or they may be naturally or artificially modified. The amino acid consensus sequence can be defined based on parallel analysis of two or more CDRs. The term "antibody" also includes the antigen-binding fragment of a complete antibody molecule.

[0068] As used herein, “antigen” means any substance that causes the immune system to produce antibodies against that antigen or to produce a specific cellular immune response. A disease-associated antigen is any substance associated with any disease that causes the immune system to produce antibodies against that antigen or to produce a specific cellular immune response.

[0069] As used herein, the “antigen-binding fragment” of an antibody, the “antigen-binding portion” of an antibody, etc., include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen-binding fragment of an antibody can be derived from a complete antibody molecule using any appropriate standard technique, such as protein digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated by chemical or molecular biological techniques to, for example, position one or more variable and / or constant domains into appropriate configurations, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids.

[0070] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv(scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., isolated CDRs such as the CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Other manipulated molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplant antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also included within the expression “antigen-binding fragment” as used herein.

[0071] The antigen-binding fragment of an antibody will typically contain at least one variable domain. The variable domain may be of any size and have any amino acid composition, and will generally contain at least one CDR adjacent to or within a frame comprising one or more framework sequences. L V associated with the domain H In antigen-binding fragments having a domain, V H Domain and V L Domains can be positioned in any appropriate arrangement relative to each other. For example, the variable region may be a dimer, V H -V H , V H -V L or V L -V L It may also contain a dimer of the antibody. Alternatively, the antigen-binding fragment of the antibody may be a monomer V H Domain or V L It may contain a domain.

[0072] In some embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of the variable domain and constant domain that may be found within the antigen-binding fragment of the antibody of this disclosure include: (i)V H -C H 1;(ii)V H -C H 2; (iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv)V L -C L These include any of the exemplary configurations described above, in any configuration of the variable domain and the constant domain, the variable domain and the constant domain may be directly linked to each other, or they may be linked by a full hinge, a partial hinge, or a linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible linkage between adjacent variable domains and / or adjacent constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of this disclosure may consist of one or more monomers that are non-covalently associated with each other. H Domain or V L The material may include homodimers or heterodimers (or other polymers) of any of the variable and constant domain configurations described above, associated non-covalently with the domain (e.g., by disulfide bonds).

[0073] In some embodiments, the antigen-binding domain comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), where: HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, 12, or 21; HCDR2 comprises the amino acid sequence of SEQ ID NO: 3, 13, or 22; HCDR3 comprises the amino acid sequence of SEQ ID NO: 4, 14, or 23; LCDR1 comprises the amino acid sequence of SEQ ID NO: 6 or 16; LCDR2 comprises the amino acid sequence of SEQ ID NO: 7; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 8 or 17.

[0074] In some embodiments, the antigen-binding domain includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each comprising the respective amino acid sequences of (i) SEQ ID NOs: 2, 3, 4, 6, 7, and 8; (ii) SEQ ID NOs: 12, 13, 14, 16, 7, and 17; or (iii) SEQ ID NOs: 21, 22, 23, 6, 7, and 8.

[0075] In some embodiments, the antigen-binding domains include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NOs. 21, 22, 23, 6, 7, and 8, respectively.

[0076] In some embodiments, the antigen-binding domain includes an HCVR containing the amino acid sequences of SEQ ID NOs. 1, 11, and 20, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NOs. 1, 11, and 20, and an LCVR containing the amino acid sequence of SEQ ID NOs. 5 or 15, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NOs. 5 or 15. Sequence identity can be calculated using an algorithm, for example, the Needleman-Wunsch algorithm for global alignment (Needleman et al., J.Mol.Biol. 48: pp. 443-453 (1970)), or the Smith-Waterman algorithm for local alignment (Smith et al., J.Mol.Biol., 147: pp. 195-197 (1981)). A suitable alternative algorithm is described by Dufresne et al., Nature Biotechnology, 20:1269-1271 (2002), and is used in the GenePAST software (GQ Life Sciences, Inc.; Boston, MA).

[0077] In some embodiments, the antigen-binding domain includes an HCVR / LCVR amino acid sequence pair selected from SEQ ID NOs: 1 / 5, 11 / 15, and 20 / 5.

[0078] In some embodiments, the fusion protein further comprises the heavy chain constant region of SEQ ID NO: 26.

[0079] In some embodiments, the fusion protein comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 9, 18, or 24, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 9, 18, or 24; and the light chain comprises the amino acid sequence of SEQ ID NO: 10, 19, or 25, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 10, 19, or 25.

[0080] In some embodiments, the fusion protein includes a heavy / light chain sequence pair containing the amino acid sequences of SEQ ID NOs. 9 / 10, 18 / 19, or 24 / 25. In some embodiments, the fusion protein includes a heavy / light chain sequence pair containing the amino acid sequences of SEQ ID NOs. 24 and 25.

[0081] In some embodiments, the IL2 portion comprises (i) IL2 or a fragment thereof; and (ii) IL2 receptor alpha ("IL2Rα" or "IL2Ra") or a fragment thereof.

[0082] In some embodiments, the IL2 portion may include a wild-type (e.g., human wild-type) or variant IL2 domain fused to the IL2-binding domain of IL2Ra, possibly via a linker. In some embodiments, the IL2-binding domain of the IL2Ra fragment is linked at its C-terminus to an IL2 (wild-type or variant) domain or fragment via a linker.

[0083] As used herein, the “wild-type” form of IL2 is a form of IL2 that is otherwise identical to the mutant IL2 polypeptide, except that the wild-type form has wild-type amino acids at each amino acid position of the mutant IL2 polypeptide. For example, if the IL2 mutant is full-length IL2 (i.e., IL2 that is not fused or conjugated to any other molecule), then the wild-type form of this mutant is full-length native IL2.

[0084] In some embodiments, IL2 or a fragment thereof includes the amino acid sequence of SEQ ID NO: 29. In some embodiments, the IL2 portion includes the amino acid sequence of SEQ ID NO: 27.

[0085] Targeted immune cytokines may include one or more linkers (e.g., peptide linkers or non-peptide linkers) that link various components of the molecule. In some embodiments, two or more components of a targeted immune cytokine are linked to each other by peptide linkers. In non-limiting examples, linkers can be used to link (a) the IL2 moiety and the antigen-binding domain of a checkpoint inhibitor, (b) different domains within the IL2 moiety (e.g., the IL2 domain and the IL2Ra domain), or (c) different domains within the antigen-binding moiety (e.g., different components of the anti-PD-1 antigen-binding domain).

[0086] Examples of flexible linkers that can be used in the targeted immune cytokines of this disclosure include those disclosed in Chen et al., Adv Drug Deliv Rev., 65(10):1357-69 (2013) and Klein et al., Protein Engineering, Design & Selection, 27(10):325-30 (2014). Particularly useful flexible linkers are monomers or polymers of glycine and serine repeats, e.g., GnS or SGn (wherein n is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). In some embodiments, the linker is a monomer or polymer of repeating G4S (GGGGS; SEQ ID NO: 32), e.g., (GGGGS)n or thereof.

[0087] In some embodiments, the IL2 portion and the antigen-binding portion are linked via a linker containing one or more repeating amino acid sequences of GGGGS (SEQ ID NO: 32). In some embodiments, the linker contains the amino acid sequence of SEQ ID NO: 30 or 31. In some embodiments, the IL2 portion is linked to the C-terminus of the antigen-binding portion via a peptide linker. In some embodiments, the linker contains the amino acid sequence of SEQ ID NO: 30.

[0088] In some embodiments, the targeted immune cytokine comprises a dimeric fusion protein. In some embodiments, the dimeric fusion protein is a homodimeric fusion protein, in which case each constituent monomer constitutes the fusion protein described herein. In some embodiments, the monomers of the dimeric fusion protein are dimerized from each other through the heavy chain constant region of each monomer. In one preferred embodiment, IL2 of the first monomer component is bound to IL2Ra contained in the second monomer component of the dimeric protein.

[0089] The targeted immune cytokines of this disclosure exhibit reduced binding to IL2Rα, IL2Rβ, and IL2Rγ. In some embodiments, the targeted immune cytokines do not compete with REGN2810, pembrolizumab, or nivolumab. In some embodiments, the targeted immune cytokines exhibit reduced activity in activating human IL2Rα / β / γ trimer and IL2Rβ / γ dimer receptor complexes compared to IL2, and increased activity in activating human IL2Rα / β / γ trimer and IL2Rβ / γ dimer receptor complexes compared to untargeted IL2Rα-IL2 constructs. In some embodiments, the targeted immune cytokines exhibit increased activity in stimulating antigen-activated T cells, as measured by IFN-γ release levels, compared to wild-type human IL2.

[0090] In some embodiments, the targeted immune cytokine is an anti-PD1-IL2Ra-IL2 fusion protein.

[0091] Combination therapy Overall, the methods of the present disclosure involve administering a combination therapy comprising a therapeutically effective dose of ACT and a therapeutically effective dose of targeted immune cytokines to a cancer subject. In some embodiments, the combination therapy of the present disclosure enhances the efficacy of ACT administered to a cancer subject compared to a subject treated with ACT as monotherapy or with ACT in combination with untargeted immune cytokines, thereby more effectively treating the cancer.

[0092] With respect to pharmaceutical compositions, the ACTs and / or targeted immune cytokines of this disclosure can be formulated with one or more carriers, excipients, and / or diluents. In some embodiments, the targeted immune cytokines can be formulated with one or more carriers, excipients, and / or diluents in the form of a fusion protein (e.g., a dimeric fusion protein). Pharmaceutical compositions comprising the ACTs and / or targeted immune cytokines of this disclosure can be formulated for specific applications, such as veterinary use or human pharmaceutical use. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers used will depend on the intended therapeutic application and the desired mode of administration of the ACTs and / or targeted immune cytokines.

[0093] The pharmaceutical compositions of this disclosure may contain either or both ACT and / or targeted immune cytokines. Such pharmaceutical compositions can be administered to a subject by various routes, such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, intrathecally, topically, or locally. In some embodiments, the pharmaceutical composition is administered to a subject intravenously or subcutaneously. The pharmaceutical compositions may be presented as appropriate in unit dosage forms containing a predetermined amount of the ACT and / or targeted immune cytokines of this disclosure per single dose.

[0094] In some embodiments, the methods of the present disclosure further include the administration of a further therapeutic agent or therapy. Non-limiting examples of further therapeutic agents or therapies include radiation, surgery, cancer vaccines, PD-L1 inhibitors (e.g., anti-PD-L1 antibodies), LAG-3 inhibitors, CTLA-4 inhibitors (e.g., ipilimumab), TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, antagonists of another T cell co-inhibitor or ligand (e.g., antibodies against LAIR1, CD160, g, or VISTA), indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., US7,087]"VEGF-Traps" such as aflibercept or other VEGF inhibitory fusion proteins as described in 411, or anti-VEGF antibodies or their antigen-binding fragments (e.g., bevacizumab or ranibizumab), or small molecule kinase inhibitors of the VEGF receptor (e.g., sunitinib, sorafenib or pazopanib), Ang2 inhibitors (e.g., nesbakumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), agonists against costimulatory receptors (e.g., agonists against glucocorticoid-induced TNFR-related proteins), antibodies against tumor-specific antigens (e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor M2-PK, prostate) Gland-specific antigens (PSA, mucin-1, MART-1, and CA19-9), vaccines (e.g., Calmette-Guérin vaccine, cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), radiotherapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines (e.g., IL-2, IL-7, IL-21, and IL-15), antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4) Examples include ADCs and anti-DS6-DM4 ADCs, anti-inflammatory drugs (e.g., corticosteroids and non-steroidal anti-inflammatory drugs), nutritional supplements such as antioxidants, and combinations thereof.

[0095] In some embodiments, further therapeutic agents or therapies include anticancer drugs. As used herein, “anticancer drug” means any agent useful for treating cancer, and this includes, but is not limited to, cytotoxins, as well as agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazines, hydroxyureas, asparaginases, corticosteroids, mitotanes (O,P'-(DDD)), biopharmaceuticals (e.g., antibodies and interferons), and radiopharmaceuticals. As used herein, “cytotoxin or cytotoxic agent” also means any agent that is harmful to cells, including chemotherapeutic agents. Examples include Taxol® (paclitaxel), temozolomide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologues.

[0096] As used herein, “therapeutic agent” means a molecule or compound that, upon administration to a subject, imparts any beneficial effect. Beneficial effects include: the availability of diagnostic determination; remission of a disease, symptom, disorder or pathological condition; mitigation or prevention of the onset of a disease, symptom, disorder or condition; and generally, weakening of a disease, symptom, disorder or pathological condition.

[0097] In some embodiments, the combined administration of ACT and targeted immune cytokines with further therapeutic agents or therapies results in improved antitumor efficacy, reduced side effects of one or both of those first-line therapies, and / or reduced dosages of one or both of those first-line therapies.

[0098] The Disclosure also provides kits comprising the ACTs of the Disclosure (e.g., immune cells modified with anti-TAA TCR or CAR) and targeted immune cytokines (e.g., fusion proteins comprising the immunoglobulin antigen-binding domain and IL-2 moiety of a checkpoint inhibitor). The kits typically include labels and instructions for use that direct the intended use of the kit contents. As used herein, the term “labels” includes any written or documentary material on, in, or supplied with the kit, or otherwise accompanying the kit. In some embodiments, the Disclosure provides a kit for treating a subject with cancer, in which case the kit includes: a therapeutically effective dose of the ACTs disclosed; a therapeutically effective dose of the targeted immune cytokines disclosed; and (b) instructions for use of the combination of doses in any of the methods disclosed herein.

[0099] Administration regimen This disclosure includes a method comprising administering a combination of the ACTs and / or targeted immune cytokines of the Disclosure to a cancer subject at a dosing frequency that achieves a therapeutic response. In some embodiments, the ACTs of the Disclosure are administered to a subject in one or more doses, at a frequency of approximately four times per week, twice per week, once per week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less, as long as a therapeutic response is achieved.

[0100] In some embodiments, the targeted immune cytokines of the Disclosure are administered to a subject in one or more doses, at a frequency of approximately four times per week, twice per week, once per week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently, as long as a therapeutic response is achieved.

[0101] In the methods described herein, the disclosed ACT is administered to a subject in combination with the disclosed targeted immune cytokine. As used herein, the expression “in combination with” means that the ACT is administered before, after, or concurrently with the targeted immune cytokine. This expression includes sequential or simultaneous administration of the ACT and the targeted immune cytokine.

[0102] In some embodiments, when ACT is administered "before" targeted immune cytokines, ACT may be administered more than 12 weeks, about 12 weeks, about 11 weeks, about 10 weeks, about 9 weeks, about 8 weeks, about 7 weeks, about 6 weeks, about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, about 1 week, about 150 hours, about 100 hours, about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes before administration of targeted immune cytokines.

[0103] In some embodiments, when ACT is administered "after" targeted immune cytokines, ACT may be administered approximately 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or more than 12 weeks after the administration of the targeted immune cytokines.

[0104] As used herein, “simultaneous” administration means that ACT and the targeted immune cytokine are administered to a subject in a single dosage form (e.g., a co-compound), or that they are administered to a subject in separate dosage forms within approximately 30 minutes of each other (i.e., before, after, or simultaneously), for example, within approximately 15 minutes, or within approximately 5 minutes. If administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both intravenously, subcutaneously, etc.); or alternatively, each dosage form may be administered via a different route. In any case, administering the components in a single dosage form, in separate dosage forms via the same route, or in separate dosage forms via different routes is all considered “simultaneous” administration for the purposes of this disclosure.

[0105] As used herein, “sequential” administration means that each dose of the selected therapy is administered to the subject at different times separated by a predetermined interval (e.g., hours, days, weeks, or months), for example, on different days. For illustrative purposes only, sequential administration may include administering an initial dose of ACT (or targeted immune cytokine), followed by one or more secondary doses of the targeted immune cytokine (or ACT), and optionally followed by one or more tertiary doses of ACT (or targeted immune cytokine). For illustrative purposes only, sequential administration may include administering to the subject an initial dose of ACT (or targeted immune cytokine), followed by one or more secondary doses of the targeted immune cytokine (or ACT), and optionally followed by one or more tertiary doses of the targeted immune cytokine (or ACT).

[0106] As used herein, “initial” dose, “secondary” dose, and “tertiary” dose refer to the time sequence of administration. Thus, the “initial” dose is the dose administered at the beginning of a treatment regimen (also referred to as the “baseline dose”); the “secondary” dose is administered after the initial dose; and the “tertiary” dose is administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount with respect to the selected therapy, or they may contain different amounts with respect to the selected therapy.

[0107] Dosage Overall, the amount of ACT and / or targeted immune cytokines administered to a subject according to the methods of this disclosure is a therapeutically effective dose. As used herein, “therapeutically effective dose” means the amount of targeted immune cytokines in combination with ACT, which results in one or more of the following, compared to an untreated subject or a subject treated with ACT as monotherapy: (a) reduction in the severity or duration of cancer symptoms; (b) enhanced inhibition of tumor growth, or increased tumor necrosis, tumor shrinkage, and / or tumor disappearance; (c) delay in tumor growth and development; (d) inhibition, delay, or cessation of tumor metastasis; (e) prevention of recurrence of tumor growth; (f) extension of survival of the cancer subject; and / or (g) reduction in the use or need for conventional anticancer therapies (e.g., reduction or elimination of the use of chemotherapeutic agents or cytotoxic agents).

[0108] In some embodiments, the therapeutically effective dose of ACT is approximately 1 × 10⁻⁶ 6 pcs or more, 2×10 6 pcs or more, 3×10 6 pcs or more, 4×10 6 More than 5×10 6 pcs or more, 6×10 6 7×10 pieces or more 6 pcs or more, 8×10 6 pcs or more, 9×10 6 pcs or more, 1×10 7 pcs or more, 2×10 7 pcs or more, 3×10 7 pcs or more, 4×10 7 More than 5×10 7 pcs or more, 6×107 or more than, 7 × 10 7 or more than, 8 × 10 7 or more than, 9 × 10 7 or more than, 1 × 10 8 or more than, 2 × 10 8 or more than, 3 × 10 8 or more than, 4 × 10 8 or more than, 5 × 10 8 or more than, 6 × 10 8 or more than, 7 × 10 8 or more than, 8 × 10 8 or more than, 9 × 10 8 or more than, 1 × 10 9 or more than, 2 × 10 9 or more than, 3 × 10 9 or more than, 4 × 10 9 or more than, 5 × 10 9 or more than, 6 × 10 9 or more than, 7 × 10 9 or more than, 8 × 10 9 or more than, 9 × 10 9 It can include immune effector cells expressing a modified TCR or CAR against a tumor-associated antigen, which are administered in an amount of or more than cells. In some embodiments, the amount of ACT administered to the subject is 1 × 10 6 or more immune cells containing.

[0109] In some embodiments, the therapeutically effective dose of targeted immune cytokines ranges from approximately 0.05 mg to approximately 600 mg, for example, approximately 0.05 mg, approximately 0.1 mg, approximately 1.0 mg, approximately 1.5 mg, approximately 2.0 mg, approximately 10 mg, approximately 20 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 110 mg, approximately 120 mg, approximately 130 mg, approximately 140 mg, approximately 150 mg, approximately 160 mg, approximately 170 mg, approximately 180 mg, approximately 190 mg, approximately 200 mg, approximately 210 mg, approximately 220 mg, approximately 230 mg, approximately 240 mg, approximately 250 mg, approximately 260 mg, approximately Target-directed cytokines can be 270 mg, approximately 280 mg, approximately 290 mg, approximately 300 mg, approximately 310 mg, approximately 320 mg, approximately 330 mg, approximately 340 mg, approximately 350 mg, approximately 360 mg, approximately 370 mg, approximately 380 mg, approximately 390 mg, approximately 400 mg, approximately 410 mg, approximately 420 mg, approximately 430 mg, approximately 440 mg, approximately 450 mg, approximately 460 mg, approximately 470 mg, approximately 480 mg, approximately 490 mg, approximately 500 mg, approximately 510 mg, approximately 520 mg, approximately 530 mg, approximately 540 mg, approximately 550 mg, approximately 560 mg, approximately 570 mg, approximately 580 mg, approximately 590 mg, or approximately 600 mg.

[0110] In some embodiments, the amount of targeted immune cytokine administered to the subject is 0.005 mg / kg of control body weight to 10 mg / kg of subject body weight, for example, 0.01 mg / kg of subject body weight to 10 mg / kg of subject body weight, 0.02 mg / kg of subject body weight to 10 mg / kg of subject body weight, 0.03 mg / kg of subject body weight to 10 mg / kg of subject body weight, 0.04 mg / kg of subject body weight to 10 mg / kg of subject body weight, 0.05 mg / kg of subject body weight to 10 mg / kg of subject body weight, 0.06 mg / kg of subject body weight to 10 mg / kg of subject body weight 1kg, 0.07mg / Target weight 1kg~10mg / Target weight 1kg, 0.08mg / Target weight 1kg~10mg / Target weight 1kg, 0.09mg / Target weight 1kg~10mg / Target weight 1kg, 0.1mg / Target weight 1kg~10mg / Target weight 1kg, 0.2mg / Target weight 1kg~10mg / Target weight 1kg, 0.3mg / Target weight 1kg~10mg / Target weight 1kg, 0.4mg / Target weight 1kg~10mg / Target weight 1kg, 0.5mg / Target weight 1kg~10mg / Target weight 1kg, 0.6mg / Target body weight 1kg~10mg / target body weight 1kg, 0.7mg / target body weight 1kg~10mg / target body weight 1kg, 0.8mg / target body weight 1kg~10mg / target body weight 1kg, 0.9mg / target body weight 1kg~10mg / target body weight 1kg, 1mg / target body weight 1kg~10mg / target body weight 1kg, 0.005mg / target body weight 1kg~5mg / target body weight, for example, 0.01mg / kg~5mg / kg, 0.02mg / kg~5mg / kg, 0.03mg / kg~5mg / kg, 0.04mg / kg~5mg / kg, 0.05mg Includes 10 mg / kg, 0.06 mg / kg to 5 mg / kg, 0.07 mg / kg to 5 mg / kg, 0.08 mg / kg to 5 mg / kg, 0.09 mg / kg to 5 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.2 mg / kg to 5 mg / kg, 0.3 mg / kg to 5 mg / kg, 0.4 mg / kg to 5 mg / kg, 0.5 mg / kg to 5 mg / kg, 0.6 mg / kg to 5 mg / kg, 0.7 mg / kg to 5 mg / kg, 0.8 mg / kg to 5 mg / kg, 0.9 mg / kg to 5 mg / kg, or 1 mg / kg to 5 mg / kg.

[0111] Where used herein, the singular forms “a,” “an,” and “the” include multiple references unless otherwise explicitly indicated by the context. Where used herein, the terms “including,” “comprising,” “containing,” and “having,” and their variations, mean to encompass the items and their equivalents listed therein, as well as any further subject matter, unless otherwise specified. Where used herein, phrases such as “in one embodiment,” “in various embodiments,” and “in some embodiments” are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but may refer to the same embodiment unless the context indicates otherwise. Where used herein, the terms “and / or” or “ / ” mean any one of the items to which the term relates, any combination of items, or all of the items.

[0112] As used herein, the terms “approximately” or “about” refer to a value that is similar to the stated reference value when applied to one or more of the values ​​of interest. In some embodiments, unless otherwise specified or evident from the context (unless such a number appears to exceed 100% of the possible values), the terms “approximately” or “about” refer to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the stated reference value.

[0113] This disclosure is merely illustrative of the principles of the art of this disclosure. None of the examples described herein are intended to be limiting, but only to describe some of the many possible embodiments relating to the appended claims. Those skilled in the art will readily recognize the various modifications and changes that can be made without following the examples of embodiments and uses described herein and without departing from the true spirit and scope of the following claims. All references cited and / or discussed herein are incorporated herein by reference, in whole, and to the same extent as each reference is incorporated by reference individually. [Examples]

[0114] The art of this disclosure will now be illustrated by the following examples. Any use of these and other examples within this specification is illustrative only and does not in any way limit the scope and spirit of the invention or any exemplified form. Similarly, the invention is not limited to any particular preferred embodiment described herein. In fact, modifications and variations of the invention may be obvious to those skilled in the art by reading this specification and can be made without departing from its spirit and scope. The invention is therefore limited only by the claims, along with the entire range of equivalents enjoyed by the claims. Furthermore, although efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), some experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is the average molecular weight, temperature is degrees Celsius, room temperature is approximately 25°C, and pressure is atmospheric pressure or near atmospheric pressure. [Examples]

[0115] Generation of anti-PD1-IL2Ra-IL2 fusion protein Three types of anti-PD1-IL2Ra-IL2 fusion proteins were generated by expressing in host cells a first polynucleotide sequence encoding the heavy chain of an anti-PD-1 antibody ligated to the N-terminus of the IL2 moiety, and a second polynucleotide sequence encoding the light chain of the anti-PD-1 antibody. The IL2 moiety contains IL2 ligated to the C-terminus of IL2Ra. The first and second polynucleotide sequences may be carried by the same expression vector or by different expression vectors. See U.S. Patent Application No. 17 / 806,566.

[0116] Table 1 lists the amino acid sequence identifiers for three types of anti-PD1-IL2Ra-IL2 fusion proteins.

[0117] [Table 1]

[0118] The IL2 portion (SEQ ID NO: 27) contains IL2 (SEQ ID NO: 29) linked to the C-terminus of IL2Ra (SEQ ID NO: 28). The IL2 portion (SEQ ID NO: 27) is linked to the C-terminus of the heavy chain constant region (SEQ ID NO: 26) of the anti-PD-1 antibody via a linker containing the amino acid sequence of SEQ ID NO: 30.

[0119] In the case of REGN10595, the heavy chain (HC) (SEQ ID NO: 9) contains the amino acid sequence of HCVR (SEQ ID NO: 1) and the heavy chain constant region (SEQ ID NO: 26) linked to the IL2 portion (SEQ ID NO: 27) via a linker (SEQ ID NO: 30).

[0120] In the case of REGN10486, the heavy chain (HC) (SEQ ID NO: 18) contains the amino acid sequence of HCVR (SEQ ID NO: 11) and the heavy chain constant region (SEQ ID NO: 26) linked to the IL2 moiety (SEQ ID NO: 27) via a linker (SEQ ID NO: 30).

[0121] In the case of REGN10597, the heavy chain (HC) (SEQ ID NO: 24) contains the amino acid sequence of HCVR (SEQ ID NO: 20) and the heavy chain constant region (SEQ ID NO: 26) linked to the IL2 moiety (SEQ ID NO: 27) via a linker (SEQ ID NO: 30).

[0122] Table 2 lists the amino acid sequences of three types of anti-PD1-IL2Ra-IL2 fusion proteins.

[0123] [Table 2-1] [Table 2-2] [Table 2-3] [Examples]

[0124] In vivo antitumor efficacy of combination therapy with MAGE-A4 TCR-T cells and REGN10597 TCR-T cell generation: HLA-A2 / MAGE-A4 230-239 A human TCR (derived from VelociT mice) (WO2020 / 257288) targeting (PN45545) was cloned into a pLVX lentiviral vector containing an EF1a promoter and a T2A:eGFP sequence to facilitate tracking of transduced T cells. A VSV pseudotyped lentivirus was constructed for transduction of primary human T cells (Figure 1). Table 3 lists the amino acid sequences of an example of a MAGE-A4 TCR-T lentiviral construct.

[0125] [Table 3-1] [Table 3-2] [Table 3-3]

[0126] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) and stimulated with CD3 / CD28 microbeads + 100 U / ml recombinant human IL-2. Three days post-stimulation, the endogenous TCR was deleted via CRISPR / Cas9 targeting, followed by transduction with a lentivirus at MOI=5. Transduced cells were amplified with CD3 / CD28 microbeads + 100 U / ml recombinant human IL-2 for 14 days and then cryopreserved for in vivo experiments.

[0127] Xenotumor transplantation and measurement - Day 10, immunodeficiency NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mouse, 5x10 6 HLA-A2 + MAGEA4 + Human melanoma tumor cells A375 were subcutaneously injected. Using mass spectrometry techniques, the A375 melanoma cells were identified as MAGEA4. 230-239 It was determined that approximately 450 cell surface copies of the peptide were expressed. On day 0 (10 days after tumor transplantation), mice were randomized and intravenously injected with MAGE-A4 TCR-T at the following three dose levels: MAGE-A4 230-239 Tetramer-positive TCR-T cells 4.0×10 6 pieces, 2.0×10 6 individual or 1.0 × 10 6 The control group consisted of 4.0 × 10⁶ unrelated tetramer-positive TCR-T (control TCR-T). 6 The following drugs were administered: REGN10597 (0.5 mg / kg) was administered intraperitoneally on days 7, 14, and 21 after T-cell administration. A non-targeted control anti-MUC16-IL2Ra-IL2 (REGN9903) was administered as an isotype control. Tumor growth was evaluated up to 49 days after T-cell dose. Mice were euthanized according to the IACUC protocol if the tumor diameter exceeded 20 mm.

[0128] Calculation of the growth and inhibition of xenotumors To determine the tumor volume using an external caliper, the maximum vertical diameter (length in mm) and maximum horizontal diameter (width in mm) were determined. The tumor volume based on the caliper measurement was given by the formula: Volume (mm 3 ) = (length × width) 2 It was calculated using ) / 2.

[0129] A375 tumors grew progressively in untreated or unrelated control TCR-T-treated mice (Figure 2, Tables 4-16). MAGE-A4 TCR-T monotherapy demonstrated dose-dependent antitumor activity (Figures 2-4, Tables 4-16). Addition of REGN10597 0.5 mg / kg, initiated 7 days after T-cell administration, increased antitumor activity at each dose level (Figures 2-16, Tables 4-17). 4×10 6 MAGE-A4 TCR-T alone induced transient, early tumor regression, but in this case, most tumors recurred within one month of treatment (2 out of 8 mice were tumor-free on day 31) (Table 11). The addition of REGN10597 significantly enhanced tumor control, with 8 out of 9 mice remaining tumor-free for the remainder of the study. 4×10 6 One mouse administered with MAGE-A4 TCRT + REGN10597 was euthanized on day 34 due to weight loss; there was no indication that this death was treatment-related. Similarly, 2 × 10 6 While MAGE-A4 TCR-T alone demonstrated very slight and transient antitumor activity, this activity was significantly enhanced by REGN10597 (6 out of 9 mice were tumor-free on day 20) (Table 9). The increased tumor control was 2 × 10⁶ compared to animals treated with MAGE-A4 TCR-T alone or REGN9903. 6 This is also reflected in the significant improvement in the probability of survival of mice treated with MAGE-A4 TCR-T+REGN10597 (p=<0.0001 (log-rank (Mantel-Cox) test)). Finally, 1×10 6 Individual MAGE-A4 TCR-T alone did not show a difference in tumor growth compared to control-treated animals, but 1 × 10⁻¹⁶ MAGE-A4 TCR-T alone did not show a difference in tumor growth compared to control-treated animals. 6By combining these MAGE-A4 TCR-T units, 1 × 10 6 Compared to MAGE-A4 TCR-T alone, tumor growth was significantly delayed (p=0.023) and survival was significantly enhanced (p=0.0051, log-rank (Mantel-Cox) test) (Figures 4, 13, and 16). Neither unrelated TCR-T + REGN10597 nor MAGE-A4 TCR-T + non-targeted IL2Ra-IL2 REGN9903 mediated any further effect on antitumor efficacy (Figure 2).

[0130] In summary, these data indicate that REGN10597 enhances the in vivo antitumor activity of engineered human MAGE-A4 TCR-T cells. Therefore, this representative example supports the expectation that administering ACT in combination with targeted immune cytokines to cancer patients will result in improved efficacy and duration of the antitumor response compared to patients treated with ACT as monotherapy.

[0131] [Table 4]

[0132] [Table 5]

[0133] [Table 6]

[0134] [Table 7]

[0135] [Table 8]

[0136] [Table 9]

[0137] [Table 10]

[0138] [Table 11]

[0139] [Table 12]

[0140] [Table 13]

[0141] [Table 14]

[0142] [Table 15]

[0143] [Table 16]

[0144] [Table 17] [Examples]

[0145] Synergistic antitumor efficacy of combination therapy with anti-huCD20 CAR-T cells and anti-PD1-IL2Ra-IL2 (REGN10597) CD3+ T cells were isolated from the spleen of C57BL / 6 mice (PD-1 humanized mice) that express human PD-1 instead of mouse PD-1. These cells were stimulated with CD3 / CD28 microbeads + recombinant human IL-2, and then transduced with retroviruses expressing various CAR constructs. The cells were then cultured with IL7 and IL15, further amplified, and subsequently cryopreserved. The T cells were engineered to express one of the following three CARs: (1) anti-huCD20 CAR-T (CD20 / BBz CAR-T) with CD3z and 4-1BB signaling domains; (2) anti-huCD20 CAR-T (CD20 / 28z CAR-T) with CD3z and CD28 signaling domains; and (3) control CAR-T (CTRL / BBz CAR-T) with CD3z and 4-1BB signaling domains. Schematic diagrams of these CAR constructs are shown in Figures 17A-17C.

[0146] Table 18 lists the amino acid sequences of the CAR constructs CD20 / BBz CAR-T and CTRL / BBz CAR-T.

[0147] [Table 18-1] [Table 18-2]

[0148] To determine the synergistic antitumor efficacy of CD20 CAR-T cells + anti-PD1-IL2Ra-IL2 (REGN10597), a syngeneic tumor study was conducted. On day 3, PD-1 humanized C57BL / 6 mice were lymphocyte-depleted with cyclophosphamide 250 mg / kg, and subsequently, on day 0, 1 × 10⁶ human CD20-expressing MC38 mouse colon cancer cells (MC38 / hCD20 cells) were introduced into the mice. 6 The cells were injected subcutaneously. Four days after tumor transplantation, fresh thawed CAR-T cells were intravenously injected into the mice. 0.5 × 10⁶ cells were administered to the mice. 6Mice were administered either CD20 / BBz CAR-T cells, CD20 / 28z CAR-T cells, or CTRL / BBz CAR-T cells. Mice were then treated intraperitoneally on days 7, 11, 14, and 18 with either anti-PD1-IL2Ra-IL2 (REGN10597) or untargeted CTRL-IL2Ra-IL2 (REGN9903) at either 0.2 or 0.5 mg / kg. Tumor volume was measured twice weekly using calipers, with the formula: Volume = (Length × Width). 2 The tumor diameter was calculated using the formula ) / 2. If the tumor diameter exceeded 20 mm, the mouse was euthanized according to the IACUC protocol.

[0149] As shown in Figures 18-27 and Tables 19-24, MC38 / hCD20 tumors grew progressively in mice treated with CTRL / BBz CAR-T + CTRL-IL2Ra-IL2 (REGN9903; 0.2 mg / kg), CD20 / BBz CAR-T + CTRL-IL2Ra-IL2 (0.2 mg / kg), or CD20 / 28z CAR-T + CTRL-IL2Ra-IL2 (0.2 mg / kg) (Figures 18-19). In mice treated with CTRL / BBz CAR-T + PD1-IL2Ra-IL2 (REGN10597; 0.2 mg / kg), tumor growth was only slightly reduced (Figures 18-19). However, tumor growth in mice administered CD20 / BBz CAR-T + anti-PD1-IL2Ra-IL2 (0.2 mg / kg and 0.5 mg / kg) was significantly suppressed compared to mice administered CD20 / BBz CAR-T + CTRL-IL2Ra-IL2 (0.2 mg / kg; p<0.0001 and p<0.001, respectively, at day 25, by two-way ANOVA analysis) (Figures 18-27). Tumor growth in mice administered CD20 / 28z CAR-T + anti-PD1-IL2Ra-IL2 (0.2 mg / kg and 0.5 mg / kg) was also significantly suppressed compared to mice administered CD20 / 28z CAR-T + CTRL-IL2Ra-IL2 (0.2 mg / kg; p<0.0001 and p<0.001, respectively, at day 25, by two-way ANOVA analysis) (Table 24).

[0150] These data demonstrate that combining anti-PD1-IL2Ra-IL2 (REGN10597) with CAR-T cell therapy induces more potent and persistent tumor control compared to CAR-T cell therapy alone. Therefore, this representative example further supports the expectation that administering ACT in combination with targeted immune cytokines to cancer patients will result in improved efficacy and duration of the antitumor response compared to patients treated with ACT as monotherapy.

[0151] [Table 19]

[0152] [Table 20] [Table 21]

[0153] [Table 22]

[0154] [Table 23]

[0155] [Table 24] [Examples]

[0156] Synergistic efficacy of anti-huCD20 CAR T cells in combination with PD1-IL2Ra-IL2 drives superior and more persistent depletion of target cells. This embodiment relates to in vivo studies performed to demonstrate the ability of PD1-targeted IL-2 immune cytokines (PD1-IL2Ra-IL2), in combination with anti-huCD20 CAR T cell therapy, to drive superior and more persistent depletion of target cells, compared to CAR T cells alone, both against a background of lymphocyte depletion and without lymphocyte depletion.

[0157] Lymphocyte depletion via chemotherapy is commonly used in the CAR T field to facilitate transplantation of introduced cells by creating physical space and removing cell sinks to make excess growth / survival factors (such as cytokines) available. However, lymphocyte depletion is associated with side effects that may prevent patients in poor health from being eligible for CAR T therapy. Consequently, therapies that enable efficient transplantation / activation of CAR T cells without requiring lymphocyte depletion are desirable. Therefore, in this study, we tested the ability of PD1-IL2Ra-IL2 to drive superior and more persistent depletion of target cells in vivo in combination with CAR T cells, both against the background of lymphocyte depletion (mediated by cyclophosphamide treatment) and without lymphocyte depletion.

[0158] This study was performed in immunocompetent C57BL / 6 mice humanized for CD20 expression, in which case CAR T cell-mediated B cell depletion can be measured. In this model, CAR T-mediated endogenous B cell depletion is huCD20 + These animals act as surrogates for tumor cell depletion. Since these animals express mouse PD1, a surrogate PD1-IL2Ra-IL2 reagent that binds to mouse PD-1 (i.e., REGN9899, ​​Table 25) was used. The mouse PD1 binding moiety is derived from the rat anti-mPD-1 clone RMP1-14, and the corresponding non-target-directed NT-IL2Ra-IL2 reagent (i.e., REGN9901, Table 26) was used.

[0159] Table 25 provides a description of REGN9899.

[0160] [Table 25]

[0161] Table 26 provides a description of REGN9901.

[0162] [Table 26]

[0163] To generate mouse anti-huCD20 CAR T cells, CD3 + T cells were isolated from the spleen of huCD3 / huCD20 knock-in mice using an Untouched Mouse T-cell Isolation Kit (Invitrogen #11413D) and subsequently activated with CD3 / CD28 Dynabeads (Invitrogen #11161D) and recombinant human IL-2 (20 U / ml; Peprotech #200-02). After 16 hours, these T cells were transduced via spin infection on plates coated with Retronectin (Takara #T100B) with a retrovirus encoding an anti-huCD20 CAR containing the mouse CD3z intracellular signaling domain and the mouse 4-1BB intracellular signaling domain. CAR T cells binding to unrelated antigens were used as controls. CAR T cells contained a GFP reporter (via a P2A cleavage site) so that they could be identified in vivo. The CAR T cells used in this study were anti-huCD20 CAR T cells (CD20 / BBz CAR-T, Figure 17A; Table 18) possessing CD3z and 4-1BB signaling domains, and control CAR T cells (CTRL / BBz CAR-T, Figure 17C, Table 18) also possessing CD3z and 4-1BB signaling domains.

[0164] CD20 humanized mice were either lymphocyte-depleted on day -7 with intraperitoneal doses of cyclophosphamide (250 mg / kg) or left untreated, and then on day 0, 3 × 10 6individual CAR + Anti-huCD20 CAR T cells or control CAR T cells were intravenously injected into the mice. On day 1, the mice received a first dose intraperitoneally administered either PD1-IL2Ra-IL2 (i.e., REGN9899) or the control non-target-directed NT-IL2Ra-IL2 (i.e., REGN9901) (0.4 mg / kg for the lymphocyte-depleted group, or 1 mg / kg for the non-lymphocyte-depleted group). Subsequently, the mice were administered the same dose of REGN9899 or REGN9901 every 3-4 days throughout the study. Blood was collected from the mice and analyzed using immunofluorescence staining with flow cytometry analysis to determine CD45 levels on days 7 and 21. + B220 + B cells and CD45 + CD90.2 + GFP + The frequency and absolute number of CAR T cells were evaluated.

[0165] Results: On day 7, treatment with anti-huCD20 CAR T cells showed a higher peripheral blood B220 level compared to CTRL CAR T cells, regardless of whether REGN9899 was administered. +Both the frequency and absolute number of B cells were efficiently depleted (Tables 27 and 28; Figures 28-31). B cell depletion was also efficient regardless of whether the mice were lymphocyte-depleted (Tables 27 and 28; Figures 28 and 30). In lymphocyte-depleted mice, the frequency and absolute number of peripheral blood CAR T cells were increased compared to mice treated with CTRL CAR T cells (Table 27; Figure 29), and antigen-specific recognition and activation / amplification of CAR T cells were consistent. In these lymphocyte-depleted mice, the frequency (p=0.0001) and absolute number (p=0.0019) of peripheral blood CAR T cells were significantly increased in mice treated with REGN9899 compared to mice treated with REGN9901, as assessed by a two-sided, unpaired t-test, demonstrating that treatment with REGN9899 drives superior amplification / sustaining of peripheral CAR T cells. In non-lymphocyte-depleted mice, peripheral CAR T cell amplification was observed to be inferior; however, according to REGN9899, ​​a two-sided, unpaired t-test was performed to drive an increase in CAR T cell frequency compared to REEGN9901-treated mice (p=0.0305).

[0166] Summary of Day 7: At this initial stage, blood huCD20 CAR T-mediated B cell depletion was efficient regardless of whether the mice were lymphocyte-depleted or administered REGN9899. However, co-treatment with REGN9899 drove enhanced peripheral CAR T cell amplification compared to REGN9901-treated mice, particularly against the background of lymphocyte depletion.

[0167] In mice administered lymphocyte depletion, by day 21, the frequency and absolute number of B220+ B cells in mice administered huCD20 CAR T+REGN9899 returned to levels comparable to those in mice administered CTRL CAR T (Table 27; Figure 32). However, B220 +The frequency (p = 0.0005) and absolute number (p = 0.0003) of B cells were significantly decreased in mice administered with huCD20 CAR T+REGN9899 compared to mice administered with huCD20 CAR T+REGN9901 (two-sided, unpaired T-test; Table 27). These results demonstrate that the combination of REGN9899 with huCD20 CAR T cells drives the prolongation of B cell depletion in the context of lymphopenia.

[0168] In mice not administered with lymphopenia, on day 21, B220 + The frequency (p < 0.0001) and absolute number (p = 0.0056) of B cells were also significantly decreased in mice administered with huCD20 CAR T+REGN9899 compared to mice administered with huCD20 CAR T+REGN9901 (two-sided, unpaired T-test; Table 28). These results demonstrate that the combination of REGN9899 with huCD20 CAR T cells drives the prolongation of B cell depletion even when lymphopenia is not administered.

[0169] Furthermore, on day 21, the frequency (p = 0.0385) and absolute number (p = 0.0685) of peripheral blood huCD20 CAR T cells were increased in non-lymphopenic mice administered with huCD20 CAR T+REGN9899 compared to mice administered with huCD20 CAR T+REGN9901 (Table 28). Therefore, even when lymphopenia was not administered, co-treatment with REGN9899 drove enhanced peripheral CAR T cell amplification compared to REGN9901-treated mice.

[0170] Summary on Day 21: At this late stage, regardless of whether the mice were in a lymphocytopenia state, huCD20 CAR T-mediated B cell depletion was superior in mice co-treated with REGN9899 compared to mice co-treated with the control REGN9901. Furthermore, co-treatment with REGN9899 drove enhanced peripheral CAR T cell amplification in non-lymphocytopenic mice compared to REGN9901-treated mice. These results demonstrate that the combination of REGN9899 with CAR T cells drives an extension of the functional activity (measured by B cell depletion) and amplification / persistence of CAR T cells in vivo compared to CAR T alone.

[0171] In Table 27, peripheral blood B220 + in lymphocytopenic mice compared to CTRL GFP + The frequency and absolute number of B cells are described.

[0172]

Table 27

[0173] In Table 28, the frequency and absolute number of peripheral blood B220+ B cells compared to CTRL GFP+ CAR T in non-lymphocytopenic mice are described.

[0174]

Table 28

Example

[0175] Synergistic antitumor efficacy of PD1-targeted IL-2 immunocytokine (PD1-IL2Ra-IL2) treatment in combination with anti-huMUC16 CAR T cell therapy This example relates to an in vivo study conducted to demonstrate the antitumor efficacy of PD1-targeted IL-2 immunocytokine (PD1-IL2Ra-IL2) in combination with anti-huMUC16 CAR T cell therapy.

[0176] Syngeneic tumor studies were performed in immunocompetent C57BL / 6 mice humanized for MUC16 expression. Since these animals express mouse PD1, a surrogate PD1-IL2Ra-IL2 reagent that binds to mouse PD-1 (i.e., REGN9899, ​​Table 25) was used. The mouse PD1 binding moiety is derived from the rat anti-mPD-1 clone RMP1-14, for which the corresponding non-targeting NT-IL2Ra-IL2 reagent (i.e., REGN9901, Table 26) was used.

[0177] To generate mouse anti-huMUC16 CAR T cells, CD3 + T cells were isolated from the spleen of huCD3 / huMUC16 knock-in mice using the Untouched Mouse T-cell Isolation Kit (Invitrogen #11413D) and subsequently activated with CD3 / CD28 Dynabeads (Invitrogen #11161D) and recombinant human IL-2 (20 U / ml; Peprotech #200-02). After 16 hours, these T cells were transduced via spin infection on plates coated with Retronectin (Takara #T100B) with a retrovirus encoding an anti-huCD16 CAR containing the mouse CD3z intracellular signaling domain and the human 4-1BB intracellular signaling domain. CAR T cells binding to unrelated antigens were used as controls.

[0178] Table 29 lists the amino acid sequences of the anti-huMUC16 and unrelated antigen-controlling CAR constructs used in this study.

[0179] [Table 29-1] [Table 29-2]

[0180] MUC16 humanized mice were subjected to a sublethal dose of whole-body irradiation (400 cGy) to deplete lymphocytes, and one day later, 10 x 10 cells were implanted in the right flank. 6 Individual ID8 / VEGF / huMUC16 tumor cells were subcutaneously transplanted. One day after tumor transplantation, 4 × 10⁶ mice were given a 2x10⁶ dose. 6 individual CAR + Anti-huMUC16 CAR T cells or control CAR T cells were intravenously injected into the mice. On the same day, the mice were intraperitoneally administered either PD1-IL2Ra-IL2 (REGN9899, ​​Table 25) or control non-targeted NT-IL2Ra-IL2 (REGN9901, Table 26) at a dose of 1 mg / kg. Two days after CAR T cell injection, the mice were administered a single additional dose of PD1-targeted or control immune cytokines at a dose of 1 mg / kg. Tumor growth was assessed over 43 days via caliper measurements twice weekly using the following formula: (length × width) 2 It was calculated using ) / 2.

[0181] Results: Similar tumor growth was observed in animals treated with CTRL CAR T cells and either REGN9901 or REGN9899, ​​as well as in animals treated with anti-huMUC16 CAR T cells and REGN9901 (Table 30; Figure 36). However, tumor growth was significantly inhibited in mice treated with anti-huMUC16 CAR T cells combined with REGN9899 (Table 30; Figure 36).

[0182] The p-values ​​for the two-way ANOVA of anti-huMUC16 CAR T+REGN9899 versus CTRL CAR T+REGN9901 were as follows: Day 13: p=0.003; Day 21: p<0.0001; Day 24: p<0.0001; Day 28: p<0.0001. The p-values ​​for the two-way ANOVA of anti-huMUC16 CAR T+REGN9899 versus CTRL CAR T+REGN9901 were as follows: Day 21: p=0.0368; Day 24: p=0.0001; Day 28: p<0.0001. Note: Two mice from the "anti-huMUC16 CAR T+REGN9899" group died after measurement on day -7 due to circumstances unrelated to the study or the treatment.

[0183] Table 30 describes the tumor volume + / - SEM and the number of surviving mice with a specific antibody treatment on a specific day.

[0184]

Table 30-1

Table 30-2

Table 30-3

Example

[0185] Synergistic antitumor efficacy of PD1-targeted IL-2 immune cytokine (PD1-IL2Ra-IL2) treatment in combination with anti-huMUC16 CAR T cell therapy This example relates to an in vivo study conducted to demonstrate the synergistic antitumor efficacy of PD1-targeted IL-2 immune cytokine (PD1-IL2Ra-IL2) treatment in combination with anti-huMUC16 CAR T cell therapy.

[0186] Despite being an effective therapy for some hematological malignancies, the therapeutic activity of CAR-T cells has been limited in most solid tumors, partly due to their poor persistence and functionality in vivo. Many combination strategies are being explored to overcome these limitations of CAR-T cells in solid tumors (Young et al., Cancer Discovery, 12: pp. 1625-1633 (2022)); Al-Haider et al., Cancer Cell International, 22: p. 365 (2022). To test whether PD1-IL2Ra-IL2 improves the antitumor activity of CAR-T cells in solid tumors, we evaluated the combinatorial efficacy of anti-huMUC16 CAR-T cells + mPD1-IL2Ra-IL2 in controlling syngeneic ID8-VEGF / huMUC16-delta tumors because anti-huMUC16 CAR-T cells upregulate PD-1 expression when co-cultured with huMUC16-expressing target cells (Figure 37A). CD3 / MUC16 double-humanized mice were lymphocyte-depleted, and these mice were transplanted with ID8-VEGF / huMUC16-delta tumor cells. These mice were then treated on the indicated days with either anti-huMUC16 or control CAR-T cells in combination with mPD1-IL2Ra-IL2 or a control molecule (Figure 37B). Compared to control CAR-T cells + isotyped mAb, treatment with huMUC16 CAR-T cells + isotyped mAb moderately delayed tumor growth. This monotherapy efficacy of huMUC16 CAR-T cells was not further enhanced when these cells were combined with either NT-IL2Ra-IL2 or high-dose anti-mPD1. In contrast, the combination of huMUC16 CAR-T cells with mPD1-IL2Ra-IL2 resulted in a significant enhancement of antitumor efficacy, and tumor regression was observed in all mice in this treatment group. In mice administered control CAR-T cells, there was no therapeutic effect of mPD1-IL2Ra-IL2. This suggests that in such lymphocyte-depleted mice, the activity of mPD1-IL2Ra-IL2 is dependent on the introduced huMUC16 CAR-T cells (Figures 37C and 37D).In summary, these results demonstrate that PD1-IL2Ra-IL2 enhances the in vivo antitumor activity of CAR-T cells.

[0187] This disclosure is not limited in scope by the specific embodiments described herein. In fact, various modifications of this disclosure, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are included within the scope of the accompanying claims.

Claims

1. A method for improving the efficacy of adoptive cell therapy (ACT): (a) Selecting cancer targets; and (b) Administer a therapeutically effective dose of ACT in combination with a therapeutically effective dose of targeted immune cytokines. Includes, The administration of the aforementioned combination results in improved efficacy and duration of the antitumor response compared to subjects treated with ACT as monotherapy. method.

2. A method for treating cancer, To those who require it, administer a therapeutically effective dose of adoptive cell therapy (ACT) in combination with a therapeutically effective dose of targeted immune cytokines. Includes, The administration of the aforementioned combination results in improved efficacy and duration of the antitumor response compared to subjects treated with ACT as monotherapy. method.

3. ACT includes immune cells selected from T cells, tumor-infiltrating lymphocytes, and natural killer (NK) cells. The method according to claim 1 or 2.

4. Immune cells contain modified T cell receptors (TCRs) for tumor-associated antigens (TAAs), or chimeric antigen receptors (CARs) for TAAs. The method according to claim 3.

5. TAAs include AFP, ALK, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, and CYP1. B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein, GD2, GD3, GloboH, Glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, M AGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX 5. Selected from PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-Nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3. The method according to claim 4.

6. Targeted immune cytokines are fusion proteins containing (a) the immunoglobulin antigen-binding domain of a checkpoint inhibitor and (b) the IL2 moiety. The method according to any one of claims 1 to 5.

7. The IL2 portion comprises (i) IL2 receptor alpha (IL2Ra) or a fragment thereof; and (ii) IL2 or a fragment thereof. The method according to claim 6.

8. Checkpoint inhibitors are inhibitors of PD1, PD-L1, PD-L2, LAG-3, CTLA-4, TIM3, A2aR, B7H1, BTLA, CD160, LAIR1, TIGHT, VISTA, or VTCN1. The method according to claim 6 or 7.

9. Checkpoint inhibitors are inhibitors of PD-1. The method according to any one of claims 6 to 8.

10. The antigen-binding domain comprises a heavy chain variable region (HCVR) containing an amino acid sequence selected from SEQ ID NOs: 1, 11, and 20; and a light chain variable region (LCVR) containing an amino acid sequence selected from SEQ ID NOs: 5 and 15. The method according to any one of claims 6 to 9.

11. The antigen-binding domain comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), Here, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are: (a) Sequence IDs 2, 3, 4, 6, 7, and 8, respectively; (b) Sequence IDs 12, 13, 14, 16, 7 and 17, respectively; and (c) Sequence IDs 21, 22, 23, 6, 7 and 8, respectively Includes an amino acid sequence selected from, The method according to any one of claims 6 to 10.

12. The antigen-binding domain comprises an HCVR / LCVR amino acid sequence pair selected from SEQ ID NOs: 1 / 5, 11 / 15, and 20 / 5. The method according to any one of claims 6 to 11.

13. The fusion protein contains a heavy chain comprising the IgG1 isotype's heavy chain variable region (HCVR) and heavy chain constant region. The method according to any one of claims 6 to 12.

14. The fusion protein contains a heavy chain comprising the IgG4 isotype's heavy chain variable region (HCVR) and heavy chain constant region. The method according to any one of claims 6 to 12.

15. The fusion protein contains a heavy chain constant region that includes the amino acid sequence of SEQ ID NO:

26. The method according to any one of claims 6 to 12.

16. The fusion protein comprises a heavy chain containing amino acid sequences selected from SEQ ID NOs: 9, 18, and 24; and a light chain containing amino acid sequences selected from SEQ ID NOs: 10, 19, and 25. The method according to any one of claims 6 to 15.

17. Fusion protein is: (a) A heavy chain containing the amino acid sequence of SEQ ID NO: 24, and a light chain containing the amino acid sequence of SEQ ID NO: 25; (b) A heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acid sequence of SEQ ID NO: 10; or (c) Heavy chain containing the amino acid sequence of SEQ ID NO: 18, and light chain containing the amino acid sequence of SEQ ID NO: 19 including, The method according to any one of claims 6 to 16.

18. The antigen-binding domain includes a heavy chain, and the IL2 portion is attached to the C-terminus of the heavy chain via a linker containing the amino acid sequence of SEQ ID NO: 30 or 31. The method according to any one of claims 6 to 17.

19. The IL2 portion contains the amino acid sequence of SEQ ID NO:

27. The method according to any one of claims 6 to 18.

20. The L2 portion includes wild-type IL2. The method according to any one of claims 6 to 19.

21. The aforementioned IL2 includes the amino acid sequence of SEQ ID NO: 29, The method according to claim 20.

22. The IL2 portion includes IL2 or a fragment thereof connected to the C-terminus of IL2Ra or a fragment thereof via a linker. The method according to any one of claims 6 to 21.

23. IL2Ra or a fragment thereof contains the amino acid sequence of SEQ ID NO:

28. The method according to claim 22.

24. Fusion proteins are dimerized fusion proteins that dimerize through the constant heavy chain region of each monomer. The method according to any one of claims 6 to 23.

25. Targeted immune cytokines include a PD-1 targeting portion and an IL-2 portion. The method according to any one of claims 1 to 5.

26. The PD-1 targeting portion includes an immunoglobulin antigen-binding domain that specifically binds to PD-1. The method according to claim 25.

27. The aforementioned antigen-binding domain is: (a) Heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 20, and light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 5; (b) HCVR containing the amino acid sequence of SEQ ID NO: 1, and LCVR containing the amino acid sequence of SEQ ID NO: 5; or (c) HCVR containing the amino acid sequence of SEQ ID NO: 11, and LCVR containing the amino acid sequence of SEQ ID NO: 15 including, The method according to claim 26.

28. The IL2 portion includes (i) IL2Ra or a fragment thereof; and (ii) IL2 or a fragment thereof. The method according to any one of claims 25 to 27.

29. The IL2 portion contains the amino acid sequence of SEQ ID NO:

27. The method according to any one of claims 25 to 28.

30. The targeted immune cytokine is REGN10597. The method according to any one of claims 1 to 29.

31. Cancers are selected from adrenal tumors, bile duct cancer, bladder cancer, brain cancer, breast cancer, cell tumors, central or peripheral nervous system tissue cancers, cervical cancer, colon cancer, endocrine or neuroendocrine cancers or hematopoietic cancers, esophageal cancer, fibromas, gastrointestinal cancers, gliomas, head and neck cancers, Lie-Fraumeni tumors, liver cancer, lung cancer, lymphoma, melanoma, meningioma, neuroendocrine type I or II tumors, multiple myeloma, myelodysplastic syndromes, myeloproliferative disorders, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell carcinoma, parathyroid cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, kidney cancer, respiratory cancers, sarcomas, skin cancers, gastric cancer, testicular cancer, thyroid cancer, tracheal cancer, genitourinary cancers, and uterine cancers. The method according to any one of claims 1 to 30.

32. Administration of the aforementioned combination produces one or more therapeutic effects selected from among delayed tumor growth, reduction in tumor cell count, tumor regression, extended survival, partial response, and complete response. The method according to any one of claims 1 to 31.

33. The effective therapeutic dose (ACT) is 1 × 10⁻⁶ 6 Contains more than one immune cell, The method according to any one of claims 1 to 32.

34. The therapeutically effective dose of targeted immune cytokines is 0.005 mg / kg of target body weight to 10 mg / kg of target body weight. The method according to any one of claims 1 to 33.

35. Targeted immune cytokines are administered intravascularly, subcutaneously, intraperitoneally, or intratumorally. The method according to any one of claims 1 to 34.

36. ACT is administered via intravenous infusion. The method according to any one of claims 1 to 35.

37. ACT is administered before or after the administration of targeted immune cytokines. The method according to any one of claims 1 to 36.

38. ACT is administered simultaneously with the administration of targeted immune cytokines. The method according to any one of claims 1 to 36.

39. Targeted immune cytokines and / or ACTs are administered to the target in one or more doses. The method according to any one of claims 1 to 37.

40. Further including administering to target additional therapeutic agents or therapies, The method according to any one of claims 1 to 39.

41. Further therapeutic agents or therapies include radiation, surgery, chemotherapy agents, cancer vaccines, B7-H3 inhibitors, B7-H4 inhibitors, lymphocyte activator gene 3 (LAG3) inhibitors, T cell immunoglobulin and mucin domain-containing 3 (TIM3) inhibitors, galectin 9 (GAL9) inhibitors, T cell activation V-domain immunoglobulin (Ig)-containing inhibitor (VISTA) inhibitors, killer cell immunoglobulin-like receptor (KIR) inhibitors, B and T lymphocyte atenuator (BTLA) inhibitors, T cell immune receptor (TIGIT) inhibitors with Ig domain and ITIM domain, CD47 inhibitors, indoleamine-2,3- Selected from dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists, angiopoietin-2 (Ang2) inhibitors, transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies against tumor-specific antigens, Calmette-Guérin vaccine, granulocyte-macrophage colony-stimulating factor (GM-CSF), cytotoxins, interleukin-6 receptor (IL-6R) inhibitors, interleukin-4 receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-7, IL-12, IL-21, IL-15, antibody-drug conjugates, anti-inflammatory drugs, and combinations thereof. The method according to claim 40.

42. Immune cells comprising a modified T cell receptor or chimeric antigen receptor that specifically binds to a tumor-associated antigen, for use in a method of treating or inhibiting tumor growth in combination with targeted immune cytokines, Targeted immune cytokines include (i) an antigen-binding moiety that specifically binds to human PD-1 and (ii) an IL2 moiety; The method involves administering a therapeutically effective amount of immune cells and targeted immune cytokines to a target that requires it. immune cells.

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