Methods and compositions for use in cell therapy of neoplastic diseases - Patents.com

Abstract

The present disclosure relates to methods of using interleukin-2 (IL2) muteins, pharmaceutical formulations thereof, and methods useful for treating human diseases in combination with adoptive cell therapy. In particular, the disclosure provides compositions and methods for using αβhIL2 muteins to selectively stimulate the proliferation of antigen-experienced T cells ex vivo, and optionally in vivo; methods of using αβhIL2 muteins for the activation and expansion of antigen-experienced T cells in an isolated cell population; ex vivo methods of using αβhIL2 muteins to prepare a cell population enriched for antigen-experienced T cells and administer the cell population to a subject; ex vivo methods of using αβhIL2 muteins to prepare a cell population enriched for antigen-experienced T cells and administer the cell population to a subject, and administer to a subject a therapeutically effective amount of αβhIL2 muteins (e.g., such that the administered cell population proliferates and has a therapeutic effect) and a composition comprising a population of T cells enriched for antigen-activated cells. TIFF2024529340000026.tif88128

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 221,857, filed July 14, 2021, the disclosure of which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] Background to the disclosure Adoptive cell therapy, particularly therapy using tumor-infiltrating lymphocytes (TILs), or "TIL therapy," is a therapeutic modality with remarkable and proven efficacy in treating neoplastic diseases in human subjects. See, for example, Rosenberg (U.S. Pat. No. 5,126,132A, issued June 30, 1992) and Spiess, et al. (1987) J Natl Cancer Inst 79:1067-1075. In a typical current approach, human TIL therapy consists of: (1) isolation of a cell population from a subject, where the cell population includes tumor-infiltrating lymphocytes (TILs); (2) ex vivo expansion and activation of the isolated cell population; and (3) reinfusion of the expanded, activated cell population. Often, patients are treated with a preparative lymphocyte-depleting regimen before reinfusion of the cells and administration of human interleukin-2 (hIL2) in combination with reinfusion of the cell population. Preparative lymphodepletion regimens deplete diverse immune cells, including Tregs, eliminate cellular "sinks," and are associated with improved antitumor efficacy. Systemic administration of IL2 supports the in vivo persistence of reinfused TILs. In typical clinical practice, immediately after TIL infusion, patients receive intravenous hIL2 at a dose of 720,000 IU / kg every 8 hours up to the maximum tolerated dose, commonly referred to as high-dose IL2 therapy. This administration of hIL2 following reinfusion of the expanded cell population is believed to further enhance TIL survival and clinical efficacy.

[0003] Subjects with metastatic melanoma treated substantially according to this regimen achieved approximately 50% objective tumor response in several Phase I / II clinical trials. Rosenberg, et al. (2011) Clin Cancer Res 17:4550-4557 (Non-Patent Document 2); Andersen, et al. (2016) Clin Cancer Res 22:3734-3745 (Non-Patent Document 3); and Besser, et al. (2013) Clin Cancer Res 19:4792-4800 (Non-Patent Document 4). Based on the success of TIL therapy observed in melanoma patients, other researchers have investigated the efficacy of TIL therapy in cervical cancer (Stevanovic, et al. (2015) J Clin Oncol 33:1543-1550 (Non-Patent Document 5)), renal cell carcinoma (Andersen, et al. (2018) Cancer Immunol Res 6:222-235 (Non-Patent Document 6)), breast cancer (Lee, et al. (2017) Oncotarget 8:113345-113359 (Non-Patent Document 7)), non-small cell lung cancer (Ben-Avi, et al. (2018) Cancer Immunol Immunotherapy 67:1221-1230 (Non-Patent Document 8)), gastrointestinal cancer (Turcotte (2013) J Immunol 191:2217-2225 (Non-Patent Document 9) and Turcotte et al (2014) Clin Cancer Res 20:331-343 (Non-Patent Document 10)), cholangiocarcinoma (Tran, et al. (2014) Science 344:641-645 (Non-Patent Document 11)), pancreatic cancer (Hall, et al. (2016) J Immunother Cancer 4:61 (Non-Patent Document 12)), head and neck cancer (Junker, et al. (2011) Cytotherapy 13:822-834 (Non-Patent Document 13)), and ovarian cancer (Fujita, et al. (1995) Clin Cancer Res 1: 501-507 (Non-Patent Document 14)).

[0004] A notable advantage of TIL therapy is that, in contrast to the monoclonal specificity of TCR or CAR T cells, it results in broad polyclonal responses against both defined and novel tumor antigens, as well as in the context of all possible MHC molecules. Additionally, "on-target / off-tumor" toxicity, a problem associated with genetically modified T cell therapies (such as CAR T cells), is less frequently observed with TIL therapy. TILs recognize neoantigens that arise as a result of tumor-specific mutations, and studies suggest that such neoantigen-reactive T cells may be responsible for inducing tumor regression after TIL therapy. As a result, TIL therapy is expected to be particularly effective against tumors with high mutation rates, such as skin and small cell lung cancer, tumors with microsatellite instability or mismatch repair deficiency, and tumors of viral origin.

[0005] Two current methods of ex vivo expansion and activation of TILs are used: the "selected TIL" method and the "young" TIL method.

[0006] The "selected TIL" method is a more traditional approach, involving ex vivo expansion of TILs in two stages: first, in which TILs from tumor fragments are maintained in the presence of high-dose IL2 for 4–5 weeks; and second, in which specific subsets of TILs that exhibit IFNγ secretion in response to autologous tumor cell challenge are expanded. This involves a "rapid expansion protocol" or "REP" using soluble anti-CD3 mAb in the presence of excess (e.g., a 200:1 ratio) irradiated PBMC feeder cells (either autologous or allogeneic feeders) for 2 days, followed by an additional 12 days of culture in the presence of IL2. A typical REP results in a 1,000- to 2,000-fold expansion of TILs during the 2-week culture period. To obtain the cell numbers required during a typical course of TIL therapy using current methods, approximately 5 × 10 7 A pre-REP TIL is required.

[0007] A recent TIL preparation protocol, known as the "young TIL" method, eliminates the initial expansion before the cells are subjected to REP, avoiding a selection step based on tumor reactivity and instead using unselected bulk TILs for REP expansion. Reports suggest that the overall responses using such "young TIL" methods in refractory melanoma patients are similar to those reported with "selected" TIL approaches, but such young TIL products are likely to have a lower percentage of tumor-reactive T cells, which may correlate with lower anti-tumor activity, and no direct side-by-side comparison of young TIL methods with selected TIL methods has been performed in clinical trials with large numbers of patients.

[0008] hIL2 is a pluripotent cytokine with a broad spectrum of effects on the immune system, playing an important role in regulating both immune activation, suppression, and homeostasis. The property of hIL2 to promote the proliferation and expansion of activated T lymphocytes makes it particularly useful in TIL therapy protocols, and it is used in both the ex vivo and in vivo phases of current TIL therapy efforts in human subjects. The consensus amino acid sequence of wild-type human IL2 is found in Genbank under the accession locator NP_000577.2.

[0009] Human IL2 exerts its intracellular signaling activity on T cells through its interaction with two IL2 receptor signaling complexes: (a) the "intermediate affinity" IL2 receptor (also referred to as "IL2Rβγ"), which contains CD122 and CD132, and (b) the "high affinity" IL2 receptor complex (also referred to as "IL2Rαβγ"), which contains the CD25, CD122, and CD132 proteins.

[0010] CD25 is a 55 kD polypeptide that is constitutively expressed on Treg cells and inducibly expressed on other T cells in response to activation (e.g., by CD3). CD25 is also referred to in the literature as the "low affinity" IL2 receptor. hIL2 binds approximately 10 -8It binds to human CD25 with a Kd of M. Human CD25 is expressed as a 272-amino acid preprotein containing a 21-amino acid signal sequence, which is post-translationally removed to yield a 251-amino acid mature protein. Amino acids 22-240 (amino acids 1-219 of the mature protein) correspond to the extracellular domain. Amino acids 241-259 (amino acids 220-238 of the mature protein) correspond to the transmembrane domain. Amino acids 260-272 (amino acids 239-251 of the mature protein) correspond to the intracellular domain. The intracellular domain of CD25 is relatively short (13 amino acids) and is not associated with any independent signaling activity. The IL2 / CD25 complex has not been observed to produce a detectable intracellular signaling response. The consensus human CD25 nucleic acid and protein sequences can be found under GenBank accession numbers NM_000417 and NP_0004Q8, respectively.

[0011] CD122 is a single-pass type I transmembrane protein. Human CD122 (hCD122) is expressed as a 551-amino acid protein, the first 26 of which contain a signal sequence that is post-translationally cleaved to yield the 525-amino acid mature protein. Amino acids 27-240 (amino acids 1-214 of the mature protein) correspond to the extracellular domain, amino acids 241-265 (amino acids 225-239 of the mature protein) correspond to the transmembrane domain, and amino acids 266-551 (amino acids 240-525 of the mature protein) correspond to the intracellular domain. As used herein, the term CD122 includes naturally occurring variants of the CD122 protein, including S57F and D365E (as numbered according to the mature hCD122 protein). The consensus wild-type hCD122 nucleic acid and protein sequences can be found under GenBank accession numbers NM_000878 and NP_000869, respectively.

[0012] CD132 is a type 1 cytokine receptor shared by receptor complexes for IL-4, IL-7, IL-9, IL-15, and IL-21, and is consequently referred to in the literature as the "common" gamma chain. Human CD132 (hCD132) is expressed as a 369-amino acid preprotein containing a 22-amino acid N-terminal signal sequence. Amino acids 23-262 (amino acids 1-240 of the mature protein) correspond to the extracellular domain, amino acids 263-283 (amino acids 241-262 of the mature protein) correspond to the 21-amino acid transmembrane domain, and amino acids 284-369 (amino acids 262-347 of the mature protein) correspond to the intracellular domain. The human CD132 nucleic acid and protein sequences can be found under GenBank accession numbers NM_000206 and NP_000197, respectively.

[0013] hIL2 binds approximately 10 to the intermediate affinity CD122 / CD132 (IL2βγ) receptor complex. -9 M. The intermediate affinity receptor complex is expressed mostly on resting T cells and NK cells. In comparison, hIL2 has a Kd of approximately 10 for the high IL2 affinity receptor complex. -11 It has a Kd of M. Most cells, such as resting T cells, express only CD122 and CD132, which have relatively low affinity for IL2 compared to the CD25 / CD122 / CD132 high-affinity receptor complex, and therefore exhibit low responsiveness to IL2. The high-affinity receptor complex is primarily identified on activated lymphocytes, which inducibly express CD25, and on Treg cells, which constitutively express CD25.

[0014] Expansion of TILs, as currently performed, either in selected TIL or young TIL processes, is performed in the presence of hIL2. While the ability of wt-hIL2 to broadly and potently activate and induce proliferation of T cells makes it attractive for use in TIL therapy, its use in TIL therapy presents significant problems both ex vivo and in vivo.

[0015] Ex vivo exposure of TILs to high-dose IL2 is associated with terminal T cell differentiation. The degree of T cell differentiation after the ex vivo stimulation procedure may affect the in vivo survival, proliferation capacity, and efficacy of TILs after reinfusion, to the extent that other cytokines, such as IL-15 or IL21, have been proposed for use to circumvent the effects of IL2 in the ex vivo preparation of TILs. Li, et al. (2010) J Immunol. 2010; 184: 452-465 (Non-Patent Document 15). Furthermore, it is ideal for the final TIL product to be administered to be as enriched as possible for tumor-specific TIL clones. The non-specificity of hIL2 prevents it from providing selective support to T cell clones that have experienced tumor antigens, and the most effective T cell clones that have experienced tumor antigens may be outcompeted and diluted during the ex vivo expansion phase. Additionally, prolonged ex vivo contact with IL2 may result in overstimulation of isolated T cells, driving them toward exhaustion, such that a significant proportion of T cells to be reinfused into the subject may not be in an optimal state for anti-tumor efficacy.

[0016] In vivo support regimens involving systemic administration of hIL2 are also associated with significant toxicity as well as mediation of autoimmunity and graft rejection, among other side effects. The most common side effects resulting from the use of IL2 support therapy after adoptive cell transfer (ACT) include chills, high fever, hypotension, oliguria, and edema due to systemic inflammation and capillary leak syndrome, as well as reports of autoimmune phenomena such as vitiligo or uveitis.

[0017] Apart from the IL2-mediated issues mentioned above, other aspects of current approaches to TIL therapy present toxicity issues for patients. In current approaches to TIL therapy after ex vivo expansion, TIL cell products are produced in approximately 10 11 ~10 13This large dose of cells in patients demonstrates the utility of preparative lymphodepletion regimens prior to reinfusion of TILs. These lymphodepletion regimens are associated with additional toxicities, such as pancytopenia and febrile neutropenia, as well as supportive care with high doses of IL2 following re-administration of the enriched TIL cell population.

[0018] As a result, in the context of TIL therapy, there is a need in the art for agents that allow for the selective expansion and activation of tumor antigen-experienced T cell populations ex vivo without driving the desired population of these tumor antigen-experienced T cells toward differentiation and / or exhaustion, agents that provide support for activated TIL cell products without significant systemic toxicity, and agents that avoid (or minimize the need for) lymphodepletion before reinfusion of the TIL cell product. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] U.S. Patent No. 5,126,132A [Non-patent literature]

[0020] [Non-Patent Document 1] Spiess, et al. (1987) J Natl Cancer Inst 79:1067-1075 [Non-patent document 2] Rosenberg, et al. (2011) Clin Cancer Res 17:4550-4557 [Non-patent document 3] Andersen, et al. (2016) Clin Cancer Res 22:3734-3745 [Non-patent document 4] Besser, et al. (2013) Clin Cancer Res 19:4792-4800 [Non-Patent Document 5] Stevanovic, et al. (2015) J Clin Oncol 33:1543-1550

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Summary of the Invention

[0021] Disclosure Overview The present disclosure provides compositions and methods for using αβhIL2 muteins to selectively stimulate the proliferation of antigen-experienced T cells ex vivo, and optionally in vivo.

[0022] The present disclosure provides methods of using αβhIL2 muteins for the activation and expansion of antigen-experienced T cells in isolated cell populations.

[0023] In some aspects, the present disclosure is directed to the ex vivo use of αβhIL2 muteins to prepare cell populations enriched for antigen-experienced T cells and administering the cell populations to a subject.

[0024] In some aspects, the present disclosure is directed to the ex vivo use of αβhIL2 muteins to prepare cell populations enriched for antigen-experienced T cells and administering the cell populations to a subject.

[0025] In some aspects, the present disclosure is directed to the ex vivo use of αβhIL2 muteins to prepare a cell population enriched in antigen-experienced T cells, administering the cell population to a subject, and administering a therapeutically effective amount of αβhIL2 mutein to the subject (e.g., so that the administered cell population proliferates and has a therapeutic effect).

[0026] In some aspects, the present disclosure is directed to the ex vivo use of αβhIL2 muteins to prepare polyclonal cell populations enriched for antigen-experienced T cells, administering the cell population to a subject, and administering a therapeutically effective amount of the IL2 muteins of the present disclosure to the subject.

[0027] In some aspects, the present disclosure is directed to the ex vivo use of αβhIL2 muteins to prepare polyclonal cell populations enriched in tumor antigen-experienced T cells, administering the cell population to a subject, and administering a therapeutically effective amount of αβhIL2 muteins to the subject.

[0028] In some aspects, the present disclosure is directed to the ex vivo use of a first αβhIL2 mutein to prepare a polyclonal cell population enriched in tumor antigen-experienced T cells, administering the cell population to a subject, and administering a therapeutically effective amount of a second αβhIL2 mutein to the subject, wherein the first αβhIL2 mutein and the second αβhIL2 mutein are the same.

[0029] In some embodiments, the present disclosure is directed to the ex vivo use of a first αβhIL2 mutein to prepare a polyclonal cell population enriched in tumor antigen-experienced T cells, administering the cell population to a subject, and administering to the subject a therapeutically effective amount of a second αβhIL2 mutein of the disclosure, wherein the first IL2 mutein and the second αβhIL2 mutein comprise the same amino acid sequence. In some embodiments, the present disclosure is directed to the ex vivo use of a first IL2 mutein to prepare a polyclonal cell population enriched in tumor antigen-experienced T cells, administering the cell population to a subject, and administering to the subject a therapeutically effective amount of a second biased IL2 mutein, wherein the first αβhIL2 mutein and the second αβhIL2 mutein comprise the same amino acid sequence, but the second αβhIL2 mutein has been modified to provide a prolonged half-life in vivo.

[0030] In some aspects, the present disclosure is directed to the ex vivo use of a first αβhIL2 mutein to prepare a polyclonal cell population enriched in tumor antigen-experienced T cells, administering the cell population to a subject, and administering to the subject a therapeutically effective amount of a second αβhIL2 mutein having reduced binding affinity for the extracellular domain of hCD132, wherein the first αβhIL2 mutein and the second αβhIL2 mutein comprise different amino acid sequences.

[0031] In some embodiments, the present disclosure provides methods of using a cell population enriched in antigen-experienced T cells, comprising administering the cell population to a subject for the treatment of a disease, disorder, or condition. In some embodiments, the present disclosure provides methods of using a cell population enriched in antigen-experienced T cells, comprising administering the cell population in combination with an αβhIL2 mutein to a subject for the treatment of a disease, disorder, or condition.

[0032] In some aspects, the present disclosure provides a method of treating a subject suffering from a disease, disorder, or condition by obtaining a tissue sample (e.g., blood, tumor tissue) from the subject, isolating antigen-experienced T cells from the tissue sample, and contacting the isolated antigen-experienced T cells with αβhIL2 mutein 2 ex vivo.

[0033] In some aspects, the present disclosure provides methods for preparing a population of T cells comprising polyclonal antigen-experienced T cells, the method comprising obtaining a tissue sample (e.g., blood, tumor tissue) from a subject suffering from a disease, disorder, or condition, isolating antigen-experienced T cells from the tissue sample, and contacting the isolated antigen-experienced T cells with an αβhIL2 mutein ex vivo.

[0034] In some aspects, the present disclosure provides a population of T cells, including a population of polyclonal antigen-experienced T cells, prepared by a method of obtaining a tissue (e.g., blood, tumor tissue) sample from a subject suffering from a disease, disorder, or condition; isolating antigen-experienced T cells from the tissue sample; and contacting the isolated antigen-experienced T cells with an αβhIL2 mutein ex vivo.

[0035] In some embodiments, the present disclosure provides methods of treating a subject suffering from a disease, disorder, or condition by obtaining a tissue sample (e.g., blood, tumor tissue) from the subject, isolating antigen-experienced T cells from the tissue sample, contacting the isolated antigen-experienced T cells ex vivo with an αβhIL2 mutein to provide a cell population enriched in antigen-experienced T cells, and administering the cell population to the subject. In some embodiments, the tissue is a neoplasm. In some embodiments, the neoplasm is a solid tumor. In some embodiments, the tissue is blood.

[0036] In some aspects, the present disclosure provides for the use of αβhIL2 muteins in combination with adoptive cell therapy (eg, TIL therapy) during the ex vivo and / or in vivo phases.

[0037] In some embodiments, the present disclosure provides for the use of an αβhIL2 mutein in combination with TIL therapy during the ex vivo and in vivo phases. In some embodiments, the present disclosure provides for the use of an IL2 mutein in combination with TIL therapy during either the ex vivo TIL expansion phase or the in vivo phase, wherein the biased IL2 mutein used in the ex vivo TIL expansion phase is the same as the αβhIL2 mutein used in the in vivo phase. In some embodiments, the present disclosure provides for the use of an αβhIL2 mutein in combination with TIL therapy during either the ex vivo and in vivo phases, wherein the αβhIL2 mutein used in the ex vivo TIL expansion phase is different from the αβhIL2 mutein used in the in vivo TIL support phase.

[0038] A desirable subpopulation of isolated TILs is those recently activated by exposure to tumor antigens in the presence of TCR signals. Contact with tumor antigens and costimulation by TCR upregulates CD25 expression such that "antigen-experienced" TILs correlate with a CD8+CD25+ phenotype.

[0039] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) isolating a tissue sample from a subject, wherein the tissue sample comprises a population of TILs; (b) contacting the isolated tissue sample of step (a) ex vivo with an amount of an αβhIL2 mutein at a concentration sufficient to induce proliferation and activation of TILs to produce an expanded cell population comprising activated TILs; and (c) administering the expanded cell population containing activated TILs from step (b) to a subject. Includes.

[0040] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) isolating a tissue sample from a subject, wherein the tissue sample comprises a population of antigen-activated T cells; (b) contacting the tissue sample of step (a) ex vivo with an amount of the first αβhIL2 mutein sufficient to induce proliferation of antigen-activated T cells to produce an expanded cell population comprising antigen-activated T cells; (c) administering to a subject a population of antigen-activated T cells from the expanded cell population of step (b); and (d) administering a therapeutically effective amount of a second αβhIL2 mutein to the subject. Including, The first and second αβhIL2 muteins may be the same or different.

[0041] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) administering to a subject a therapeutically effective amount of a first αβhIL2 mutein; (b) isolating a tissue sample from the subject, wherein the tissue sample comprises a population of antigen-activated T cells; (c) contacting the tissue sample of step (b) ex vivo with an amount of a second αβhIL2 mutein sufficient to induce proliferation of antigen-activated T cells to produce an expanded cell population comprising antigen-activated T cells; and (d) administering to a subject a population of antigen-activated T cells from the expanded cell population of step (c). Includes.

[0042] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) administering to a subject a therapeutically effective amount of a first αβhIL2 mutein; (b) isolating a tissue sample from the subject, wherein the tissue sample comprises a population of antigen-activated T cells; (c) contacting the tissue sample of step (b) ex vivo with an amount of a second αβhIL2 mutein sufficient to induce proliferation of antigen-activated T cells to produce an expanded cell population comprising antigen-activated T cells; (d) administering to a subject a population of antigen-activated T cells from the expanded cell population of step (c); and (e) administering to the subject a therapeutically effective amount of a third αβhIL2 mutein. Including, The first, second and third αβhIL2 muteins are the same; the first and third αβhIL2 muteins are the same; the first and second αβhIL2 muteins are the same, or the first, second and third αβhIL2 muteins are each different αβhIL2 muteins.

[0043] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) isolating a tissue sample from a subject, wherein the tissue sample comprises a population of antigen-activated T cells; (b) applying an ex vivo cell selection process to the isolated tissue sample of step (a) to generate a subpopulation of antigen-activated T cells enriched for antigen-activated T cells bearing activation marker antigens; (c) expanding the subpopulation of antigen-activated T cells enriched for one or more marker antigens produced from step (b) by ex vivo contacting the subpopulation of antigen-activated T cells enriched for one or more marker antigens with an amount of a second αβhIL2 mutein sufficient to induce proliferation of the antigen-activated T cells, or optionally, for a time sufficient to expand the amount of antigen-activated T cells bearing the one or more marker antigens, to produce an expanded cell population comprising antigen-activated T cells enriched for one or more marker antigens; and (d) administering to a subject an amount of antigen-activated T cells enriched for activation marker antigens from the expanded cell population containing antigen-activated T cells enriched for activation marker antigens of step (c). Includes.

[0044] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) isolating a tissue sample from a subject, wherein the tissue sample comprises a population of antigen-activated T cells; (b) applying an ex vivo cell selection process to the isolated tissue sample of step (a) to generate a subpopulation of antigen-activated T cells enriched for antigen-activated T cells bearing one or more marker antigens; (c) expanding the subpopulation of antigen-activated T cells enriched for the one or more marker antigens produced from step (b) by ex vivo contacting the subpopulation of antigen-activated T cells enriched for the one or more marker antigens with an amount of a second αβhIL2 mutein sufficient to induce proliferation of the antigen-activated T cells, or optionally, for a time sufficient to expand the amount of antigen-activated T cells bearing the one or more marker antigens, to produce an expanded cell population comprising antigen-activated T cells enriched for the one or more marker antigens; (d) administering to the subject an amount of antigen-activated T cells enriched for one or more marker antigens from the expanded cell population comprising antigen-activated T cells enriched for activation marker antigens of step (c); and (e) administering a therapeutically effective amount of a second αβhIL2 mutein to the subject. Including, The first and second αβhIL2 muteins may be the same or different.

[0045] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) administering to a subject a therapeutically effective amount of a first αβhIL2 mutein; (b) isolating a tissue sample from the subject, wherein the tissue sample comprises a population of antigen-activated T cells; (c) applying an ex vivo cell selection process to the isolated tissue sample of step (a) to generate a subpopulation of antigen-activated T cells enriched for antigen-activated T cells bearing activation marker antigens; (d) expanding the subpopulation of antigen-activated T cells enriched for one or more marker antigens produced from step (c) by ex vivo contacting the subpopulation of antigen-activated T cells enriched for one or more marker antigens with an amount of a second αβhIL2 mutein sufficient to induce proliferation of the antigen-activated T cells, or optionally, for a time sufficient to expand the amount of antigen-activated T cells bearing the one or more marker antigens, to produce an expanded cell population comprising antigen-activated T cells enriched for one or more marker antigens; and (e) administering to a subject an amount of antigen-activated T cells enriched for activation marker antigens from the expanded cell population containing antigen-activated T cells enriched for activation marker antigens of step (d). Including, The first and second αβhIL2 muteins may be the same or different.

[0046] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) administering to a subject a therapeutically effective amount of a first αβhIL2 mutein; (b) isolating a tissue sample from the subject, wherein the tissue sample comprises a population of antigen-activated T cells; (c) applying an ex vivo cell selection process to the isolated tissue sample of step (a) to generate a subpopulation of antigen-activated T cells enriched for antigen-activated T cells bearing activation marker antigens; (d) expanding the subpopulation of antigen-activated T cells enriched for the one or more marker antigens produced from step (c) by ex vivo contacting the subpopulation of antigen-activated T cells enriched for the one or more marker antigens with an amount of a second αβhIL2 mutein sufficient to induce proliferation of the antigen-activated T cells, or optionally, for a time sufficient to expand the amount of antigen-activated T cells bearing the one or more marker antigens, to produce an expanded cell population comprising antigen-activated T cells enriched for the one or more marker antigens; (e) administering to a subject an amount of antigen-activated T cells enriched for activation marker antigens from the expanded cell population comprising antigen-activated T cells enriched for one or more marker antigens of step (d); and (f) administering to the subject a therapeutically effective amount of a third αβhIL2 mutein. Including, The first, second and third αβhIL2 muteins are the same; the first and third αβhIL2 muteins are the same; the first and second αβhIL2 muteins are the same, or the first, second and third αβhIL2 muteins are each different αβhIL2 muteins.

[0047] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) isolating a tissue sample from a subject, wherein the tissue sample comprises a population of antigen-activated T cells; (b) contacting the tissue sample of step (a) ex vivo with an amount of αβhIL2 mutein sufficient to induce proliferation of antigen-activated T cells to produce an expanded cell population comprising antigen-activated T cells; (c) contacting the expanded cell population containing the antigen-activated T cells of step (b) with a T cell activating agent; and (d) administering to a subject a population of antigen-activated T cells from the expanded cell population of step (c). Includes.

[0048] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) isolating a tissue sample from a subject, wherein the tissue sample comprises a population of antigen-activated T cells; (b) contacting the tissue sample of step (a) ex vivo with an amount of the first αβhIL2 mutein sufficient to induce proliferation of antigen-activated T cells to produce an expanded cell population comprising antigen-activated T cells; (c) contacting the expanded cell population containing the antigen-activated T cells of step (b) with a T cell activating agent; (d) administering to a subject a population of antigen-activated T cells from the expanded cell population of step (c); and (e) administering a therapeutically effective amount of a second αβhIL2 mutein to the subject. Including, The first and second αβhIL2 muteins may be the same or different.

[0049] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) administering to a subject a therapeutically effective amount of a first αβhIL2 mutein; (b) isolating a tissue sample from the subject, wherein the tissue sample comprises a population of antigen-activated T cells; (c) contacting the tissue sample of step (b) ex vivo with an amount of a second αβhIL2 mutein sufficient to induce proliferation of antigen-activated T cells to produce an expanded cell population comprising antigen-activated T cells; (d) contacting the expanded cell population containing the antigen-activated T cells of step (c) with a T cell activating agent; and (e) administering to a subject a population of antigen-activated T cells from the expanded cell population of step (d). Includes.

[0050] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) administering to a subject a therapeutically effective amount of a first αβhIL2 mutein; (b) isolating a tissue sample from the subject, wherein the tissue sample comprises a population of antigen-activated T cells; (c) contacting the tissue sample of step (b) ex vivo with an amount of a second αβhIL2 mutein sufficient to induce proliferation of antigen-activated T cells to produce an expanded cell population comprising antigen-activated T cells; (d) contacting the expanded cell population containing the antigen-activated T cells of step (c) with a T cell activating agent; (e) administering to a subject a population of antigen-activated T cells from the expanded cell population of step (d); and (f) administering to the subject a therapeutically effective amount of a third αβhIL2 mutein. Including, The first, second and third αβhIL2 muteins are the same; the first and third αβhIL2 muteins are the same; the first and second αβhIL2 muteins are the same, or the first, second and third αβhIL2 muteins are each different αβhIL2 muteins.

[0051] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) isolating a tissue sample from a subject, wherein the tissue sample comprises a population of antigen-activated T cells; (b) applying an ex vivo cell selection process to the isolated tissue sample of step (a) to generate a subpopulation of antigen-activated T cells enriched for antigen-activated T cells bearing activation marker antigens; (c) expanding the subpopulation of antigen-activated T cells enriched for the one or more marker antigens produced from step (b) by ex vivo contacting the subpopulation of antigen-activated T cells enriched for the one or more marker antigens with an amount of a second αβhIL2 mutein sufficient to induce proliferation of the antigen-activated T cells, or optionally, for a time sufficient to expand the amount of antigen-activated T cells bearing the one or more marker antigens, to produce an expanded cell population comprising antigen-activated T cells enriched for the one or more marker antigens; (d) contacting the expanded cell population containing the antigen-activated T cells of step (c) with a T cell activating agent; and (e) administering to a subject an amount of antigen-activated T cells enriched for activation marker antigens from the expanded cell population containing antigen-activated T cells enriched for activation marker antigens of step (d). Includes.

[0052] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) isolating a tissue sample from a subject, wherein the tissue sample comprises a population of antigen-activated T cells; (b) applying an ex vivo cell selection process to the isolated tissue sample of step (a) to generate a subpopulation of antigen-activated T cells enriched for antigen-activated T cells bearing one or more marker antigens; (c) expanding the subpopulation of antigen-activated T cells enriched for the one or more marker antigens produced from step (b) by ex vivo contacting the subpopulation of antigen-activated T cells enriched for the one or more marker antigens with an amount of a second αβhIL2 mutein sufficient to induce proliferation of the antigen-activated T cells, or optionally, for a time sufficient to expand the amount of antigen-activated T cells bearing the one or more marker antigens, to produce an expanded cell population comprising antigen-activated T cells enriched for the one or more marker antigens; (d) contacting the expanded cell population containing the antigen-activated T cells of step (c) with a T cell activating agent; (e) administering to a subject an amount of antigen-activated T cells enriched for one or more marker antigens from the expanded cell population comprising antigen-activated T cells enriched for activation marker antigens of step (d); and (f) administering a therapeutically effective amount of a second αβhIL2 mutein to the subject. Including, The first and second αβhIL2 muteins may be the same or different.

[0053] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: a. administering to a subject a therapeutically effective amount of a first αβhIL2 mutein; b. isolating a tissue sample from a subject, wherein the tissue sample comprises a population of antigen-activated T cells; c. applying an ex vivo cell selection process to the isolated tissue sample of step (a) to generate a subpopulation of antigen-activated T cells enriched for antigen-activated T cells bearing activation marker antigens; d. expanding the subpopulation of antigen-activated T cells enriched for the one or more marker antigens produced from step (c) by contacting ex vivo the subpopulation of antigen-activated T cells enriched for the one or more marker antigens with an amount of a second αβhIL2 mutein sufficient to induce proliferation of the antigen-activated T cells, optionally for a time sufficient to expand the amount of antigen-activated T cells bearing the one or more marker antigens, to produce an expanded cell population comprising antigen-activated T cells enriched for the one or more marker antigens; e. contacting the expanded cell population containing the antigen-activated T cells of step (d) with a T cell activating agent; and f. administering to a subject an amount of antigen-activated T cells enriched for activation marker antigens from the expanded cell population containing antigen-activated T cells enriched for activation marker antigens of step (e). Including, The first and second αβhIL2 muteins may be the same or different.

[0054] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: a. administering to a subject a therapeutically effective amount of a first αβhIL2 mutein; b. isolating a tissue sample from a subject, wherein the tissue sample comprises a population of antigen-activated T cells; c. applying an ex vivo cell selection process to the isolated tissue sample of step (a) to generate a subpopulation of antigen-activated T cells enriched for antigen-activated T cells bearing activation marker antigens; d. expanding the subpopulation of antigen-activated T cells enriched for the one or more marker antigens produced from step (c) by contacting ex vivo the subpopulation of antigen-activated T cells enriched for the one or more marker antigens with an amount of a second αβhIL2 mutein sufficient to induce proliferation of the antigen-activated T cells, optionally for a time sufficient to expand the amount of antigen-activated T cells bearing the one or more marker antigens, to produce an expanded cell population comprising antigen-activated T cells enriched for the one or more marker antigens; e. contacting the expanded cell population comprising the antigen-activated T cells of step (d) with a T cell activating agent; f. administering to a subject an amount of antigen-activated T cells enriched for activation marker antigens from the expanded cell population comprising antigen-activated T cells enriched for one or more marker antigens of step (e); and g. administering a therapeutically effective amount of a third αβhIL2 mutein to the subject Including, The first, second and third αβhIL2 muteins are the same; the first and third αβhIL2 muteins are the same; the first and second αβhIL2 muteins are the same, or the first, second and third αβhIL2 muteins are each different αβhIL2 muteins.

[0055] The disclosure provides for the practice of any of the foregoing methods, wherein the tissue sample is selected from the group consisting of blood and solid tumor tissue.

[0056] The present disclosure provides for the practice of any of the aforementioned methods, wherein the subject is treated with a lymphocyte-depleting regimen prior to administration of the amount of antigen-activated T cells to the subject. The present disclosure provides for the practice of any of the aforementioned methods, wherein the αβhIL2 mutein is an IL2 mutein having at least 90% sequence identity to wt-hIL2 (SEQ ID NO:4), and wherein the αβhIL2 mutein comprises an amino acid substitution at position 18, 22, or 126 numbered according to wt-hIL2 (SEQ ID NO:4). The present disclosure provides for the practice of any of the aforementioned methods, wherein the αβhIL2 mutein comprises an amino acid substitution at one or more positions selected from R18, Q22, and / or Q126 numbered according to mature wild-type human IL2 (SEQ ID NO:4). The present disclosure provides for the practice of any of the aforementioned methods, wherein the αβhIL2 mutein comprises amino acid substitutions at positions R18E, Q22K, and Q126K numbered according to mature wild-type human IL2 (SEQ ID NO:4). In some embodiments, the αβhIL2 muteins are L18R, L18G, L18M, L18F, L18E, L18H, L18W, L18K, L18Q, L18S, L18V, L18I, L18Y, L18H, L18D, L18N, L18T, Q22F, Q22E, Q22G, Q22A, Q22L, Q22M, Q22F, Q22W , Q22K, Q22S, Q22V, Q22I, Q22Y, Q22H, Q22R, Q22N, Q22D, Q22T, Q22F, Q126H, Q126M, Q126K, Q126C, Q126D, Q126E, Q126G, Q126I, Q126R, Q126S, and Q126T. In some embodiments, the αβhIL2 mutein is an IL2 mutein having at least 90% sequence identity with wt-hIL2 (SEQ ID NO:4), including an αβhIL2 mutein that comprises three amino acid substitutions at positions 18, 22, and 126 numbered according to wt-hIL2 (SEQ ID NO:4).In some embodiments, the αβhIL2 mutein comprises amino acid substitutions at positions 18, 22 and 126, wherein (a) the amino acid substitution at position 18 of the αβhIL2 mutein is selected from the group consisting of L18R, L18G, L18M, L18F, L18E, L18H, L18W, L18K, L18Q, L18S, L18V, L18I, L18Y, L18H, L18D, L18N and L18T; and (b) the amino acid substitution at position 22 of the αβhIL2 mutein is selected from the group consisting of Q22F, Q22G, Q22H, Q22I ... and (c) the amino acid substitution at position 126 of the αβhIL2 mutein is selected from the group consisting of Q126H, Q126M, Q126K, Q126C, Q126D, Q126E, Q126G, Q126I, Q126R, Q126S, or Q126T.In some embodiments, the αβhIL2 mutein comprises the following sets of mutations: L18R, Q22E, and Q126K; L18R, Q22E, and Q126H; L18R, Q22E, and Q126M; L18R, Q22E, and Q126T; L18R; Q22E; V91K; V91R; Q126H; L18R, and Q126H; Q22E, and Q126H; L18G, Q22E, and Q126H; L18A, Q22E, and Q126H;L18M, Q22E and Q126H;L18F, Q22E and Q126H;L18W, Q22E and Q126H;L18K, Q22E and Q126H;L18Q, Q22E and Q126H;L18E, Q22E and Q126H;L18S, Q22E and Q126H;L18V, Q22E and Q126H;L18I, Q22E and Q126H;L18Y, Q22E and Q126H;L18H , Q22E and Q126H; L18N, Q22E and Q126H; L18D, Q22E and Q126H; L18T, Q22E and Q126H; L18R, Q22G and Q126H; L18R, Q22A and Q126H; L18R, Q22L and Q126H; L18R, Q22M and Q126H; L18R, Q22F and Q126H; L18R, Q22W and Q126H; L18R, Q22K and Q1 and L18R, Q22T and Q126H. In some embodiments, the αβhIL2 mutein comprises a set of mutations selected from the group consisting of: L18R, Q22S and Q126H; L18R, Q22V and Q126H; L18R, Q22I and Q126H; L18R, Q22Y and Q126H; L18R, Q22H and Q126H; L18R, Q22R and Q126H; L18R, Q22N and Q126H; L18R, Q22D and Q126H; and L18R, Q22T and Q126H. In some embodiments, the αβhIL2 mutein comprises a deletion of one, two, three, four, five, six, seven, eight, or nine N-terminal amino acids. In some embodiments, the αβhIL2 mutein comprises a deletion of one, two, or three N-terminal amino acids. In some embodiments, the αβhIL2 mutein comprises a deletion of the N-terminal alanine amino acid (des-Ala1). In some embodiments, the αβhIL2 mutein is modified to extend its duration of action in vivo.

[0057] The disclosure provides for the practice of any of the above methods, wherein the one or more marker antigens are selected from one or more antigens selected from a cell type antigen and an activation antigen. In some embodiments, the one or more antigens are selected from CD3, CD4, CD8, CD11a, CD11b, CD11c, CD14, CD16, CD19, CD25, CD27, CD28, CD38, CD45RA, CD45RO, CD58, CD61, CD62L, CD66b, CD69, CD103, CD122, CD127, CD197, CD279, D62L, CD69, FoxP3, PD-1, D62L, CCR4, CCR5, CCR6 (CD196), CCR7, CCR10, CXCR3, CTLA4, PD1, PDL1, TCRγδ, TCR Vα24, TCR Vβ1, HLA-DR, Ki67, T-bet, GATA-3, PU.1, RORγt, AHR, FOXO4, and FOXP3.

[0058] The present disclosure provides for the practice of any of the foregoing methods, wherein contacting the isolated cell population with the αβhIL2 mutein is performed in combination with one or more additional T cell activation agents. In some embodiments, the T cell activation agent is selected from a cytokine, a growth factor, or an antibody against a T cell activation antigen (e.g., an anti-CD3 antibody, an anti-CD137 antibody). Examples of T cell activation agents include CD3 / CD28 beads.

[0059] The disclosure provides for the practice of any of the above methods, wherein the isolated T cell population is contacted with a recombinant vector comprising a nucleic acid sequence encoding an engineered receptor, and the engineered receptor is selectively activated in response to administration of a cognate ligand that binds to the extracellular domain of the engineered receptor, resulting in intracellular signaling in T cells expressing the engineered receptor.

[0060] The present disclosure provides for the practice of any of the foregoing methods, wherein the method is practiced in combination with administering to the subject an ancillary agent. In some embodiments, the ancillary agent is selected from the group consisting of a chemotherapeutic agent, an antibody, an immune checkpoint modulator, and a physical method. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the adjunctive agent is selected from the group consisting of [fam]-trastuzumab deruxtecan, enfortumab vedotin, polatuzumab vedotin, cemiplimab, moxetumomab pasudotox, mogamulizumab, tildrakizumab, ibalizumab, durvalumab, inotuzumab ozogamicin, avelumab, atezolizumab, olaratumumab, ixekizumab, aratumumab, elotuzumab, necitumumab, dinutuximab, nivolumab, blinatumomab, pembrolizumab, ramucirumab, siltuximab, obinutuzumab, ado-trastuzumab emtansine, pertuzumab, brentuximab The antibody is selected from the group consisting of vedotin, ipilimumab, ofatumumab, certolizumab pegol, catumaxomab, panitumumab, bevacizumab, cetuximab, tositumomab-I131, ibritumomab tiuxetan, gemtuzumab ozogamicin, trastuzumab, infliximab, rituximab, and edrecolomab.

[0061] The present disclosure relates to a neoplastic disease, disorder, or condition, including but not limited to, adenoma, fibroma, hemangioma, hyperplasia, atypia, metaplasia, dysplasia, carcinoma, leukemia, breast cancer, sarcoma, leukemia, lymphoma, genitourinary cancer, ovarian cancer, urethral cancer, bladder cancer, prostate cancer, gastrointestinal cancer, colon cancer, esophageal cancer, stomach cancer, lung cancer; myeloma; pancreatic cancer; liver cancer; kidney cancer; endocrine cancer; skin cancer; glioma, neuroblastoma, astrocytoma, myelodysplastic disorders; cervical intraepithelial carcinoma; intestinal polyposis; oral leukoplakia; histiocytosis, hyperproliferative scars including keloid scars, cancers of the respiratory system, digestive system, genitourinary system, testicular cancer, breast cancer, prostate cancer, cancers of the endocrine system, melanoma, adenocarcinoma, myeloproliferative neoplasms, myeloid and lymphoid disorders with eosinophilia, myeloproliferative / myelodysplastic neoplasms, myelodysplastic syndromes, acute myeloid leukemia and related precursor neoplasms, and acute leukemia of ambiguous lineage, The practice of any of the foregoing methods is provided wherein the patient is selected from the group consisting of promyelocytic leukemia (APML), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), precursor lymphoid neoplasms, mature B-cell neoplasms, mature T-cell neoplasms, Hodgkin's lymphoma, and immunodeficiency-associated lymphoproliferative disorders, lymphoblastic leukemia (ALL), including B-cell ALL and T-cell ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM), erythroblastic leukemia and acute megakaryoblastic leukemia, malignant lymphomas, including, but not limited to, non-Hodgkin's lymphoma and variants thereof, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), and Hodgkin's disease.

[0062] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) isolating a tissue sample from a subject, wherein the tissue sample comprises a population of TILs; (b) contacting the isolated tissue sample of step (a) ex vivo with an amount of an αβhIL2 mutein at a concentration sufficient to induce proliferation and activation of TILs to produce an expanded cell population comprising activated TILs; (c) contacting the expanded cell population with a recombinant vector comprising a nucleic acid sequence encoding an engineered receptor that is selectively activated in response to administration of a cognate ligand that binds to the extracellular domain of the engineered receptor, resulting in intracellular signaling in T cells expressing the engineered receptor; (d) administering to a subject the expanded cell population comprising the activated TILs from step (b); and (e) administering to the subject a therapeutically effective amount of a cognate ligand for the engineered receptor. Includes.

[0063] In some aspects, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising: (a) isolating a tissue sample from a subject, wherein the tissue sample comprises a population of T cells; (b) applying an ex vivo cell selection process to the isolated tissue sample of step (a) to generate a population of T cells enriched for one or more marker antigens; (c) contacting the population of T cells from step (b) ex vivo with an amount of an αβhIL2 mutein at a concentration sufficient to induce T cell proliferation and activation; (d) contacting the population of T cells from step (c) with a recombinant vector comprising a nucleic acid sequence encoding an engineered receptor that is selectively activated in response to administration of a cognate ligand that binds to the extracellular domain of the engineered receptor, resulting in intracellular signaling in T cells expressing the engineered receptor; (e) administering the expanded cell population from step (d) to a subject; and (e) administering to the subject a therapeutically effective amount of a cognate ligand for the engineered receptor. Includes.

[0064] In some embodiments of the method, the subject is treated with a lymphocyte depletion regimen before administering the cell population to the subject. In some embodiments of the method, the cell population is contacted with a T cell activating agent before administering the cell population to the subject. In some embodiments of the method, the subject is pretreated in vivo with a therapeutically effective amount of an αβhIL2 mutein before administering the cell population engineered to express an engineered receptor. In some embodiments of the method, the cell population is engineered to express an engineered receptor, the engineered receptor is hCD122 comprising at least one amino acid substitution at a position selected from positions 133 or 134, numbered according to SEQ ID NO:2.

[0065] In some embodiments of the practice of the above methods, where the cell population is engineered to express an engineered receptor, the engineered receptor is hCD122 that contains amino acid substitutions at positions 133 and 134. In some embodiments, the engineered receptor is hCD122 that contains amino acid substitutions H133D and Y134F.

[0066] In some embodiments of the practice of the above methods, in which a cell population is engineered to express an engineered receptor, the cognate ligand is a hIL2 variant that selectively binds to hCD122 containing at least one amino acid substitution at a position selected from positions 133 or 134 numbered by SEQ ID NO:2. In some embodiments, the cognate ligand is a hIL2 variant comprising one or more amino acid substitutions at positions 15, 16, 19, 20, 22, 23, 51, or 81, as numbered according to wt hIL2 (SEQ ID NO:4), wherein the amino acid substitution at position 15 is selected from E15S, E15T, E15Q, or E15H; the amino acid substitution at position 16 is H16Q; the amino acid substitution at position 19 is selected from L19V or L19I; the amino acid substitution at position 20 is selected from D20T, D20S, D20L, or D20M; the amino acid substitution at position 22 is selected from Q22K, Q22N; the amino acid substitution at position 23 is selected from M23L, M23S, M23V, M23A, or M23T; and the amino acid substitution at position 81 is selected from R81D and R81Y. In some embodiments, the cognate ligand is a hIL2 variant comprising an amino acid substitution at position 15 selected from E15S, E15T, E15Q, or E15H; the amino acid substitution at position 16 is H16Q; the amino acid substitution at position 19 is selected from L19V or L19I; the amino acid substitution at position 20 is selected from D20T, D20S, D20L, or D20M; the amino acid substitution at position 22 is selected from Q22KQ22N; and the amino acid substitution at position 23 is selected from M23L, M23S, M23V, M23A, or M23T. In some embodiments, the cognate ligand is a hIL2 variant comprising the amino acid substitutions E15S, H16Q, L19V, D20L; Q22K, and M23A, and optionally further comprising a deletion of the N-terminal alanine residue. In some embodiments, the cognate ligand is modified to extend its duration of action in vivo. In some embodiments, the modification to extend the in vivo duration of action is PEGylation, hi some embodiments, the cognate ligand is a hIL2 mutein that has been modified by the N-terminal addition of a 40 kDa branched PEG molecule.

[0067] The present disclosure provides a cell product enriched for tumor antigen-experienced T cells, the cell product comprising: (a) isolating a tissue sample from a subject, wherein the tissue sample comprises a population of TILs; and (b) contacting the tissue sample of step (a) ex vivo with an amount of αβhIL2 mutein at a concentration sufficient to induce proliferation and activation of TILs to produce an expanded cell population comprising activated TILs. It is prepared by a process comprising:

[0068] The present disclosure provides a cell product enriched for tumor antigen-experienced T cells, the cell product comprising: (a) isolating a tissue sample from a subject, wherein the tissue sample comprises a population of antigen-activated T cells; (b) applying an ex vivo cell selection process to the isolated tissue sample of step (a) to generate a subpopulation of antigen-activated T cells enriched for antigen-activated T cells bearing one or more marker antigens; (c) expanding the subpopulation of antigen-activated T cells enriched for one or more marker antigens produced from step (b) by contacting the subpopulation of antigen-activated T cells enriched for one or more marker antigens with an amount of a second αβhIL2 mutein sufficient to induce proliferation of the antigen-activated T cells ex vivo to produce an expanded cell population comprising antigen-activated T cells enriched for one or more marker antigens; and (d) contacting the expanded cell population containing the antigen-activated T cells of step (c) with a T cell activating agent. It is prepared by a process comprising:

[0069] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. On the contrary, dimensions of various features have been arbitrarily expanded or reduced for the sake of clarity. [Brief explanation of the drawings]

[0070] [Figure 1] A graphical representation of the data is provided showing the percent of CD8+ T cells expressing IFNg (y-axis) in response to the indicated test agents. [Figure 2] A graphical representation of the data is provided showing the percent of CD8+ T cells expressing IFNg (y-axis) in response to the indicated test agents. [Figure 3] Figure 1 provides a graphic representation of the levels of in vivo STAT5 phosphorylation in non-human primate CD8+ T cells expressing various levels of CD25 and / or CD122 in response to increasing doses of PEGylated αβhIL2 mutein. The levels of pSTAT5, as determined by mean fluorescence intensity (MFI), in CD8+ T cells are presented on the y-axis. The figure legend indicates the different cell populations and symbols for the different dose levels and corresponding graphical symbols. The dose of PEGylated αβhIL2 mutein (in nanograms per milliliter) is presented on the x-axis. These data demonstrate that PEGylated abhIL2 mutein selectively activates T cells expressing CD25, and that such activation is dependent on the presence of CD122. [Figure 4]

[0033] Figure 1 provides a graphical representation of STAT5 phosphorylation levels in CD25 and CD25 CD8 T cells in non-human primates in response to two different doses (250 μg / kg and 20 μg / kg) of PEGylated αβhIL2 mutein. The percentage of STAT5-positive cells is presented on the y-axis. The figure legend indicates the symbols and corresponding graphical symbols for the different cell populations and different dose levels. The time course of the experiment in days is presented on the x-axis. [Figure 5] This figure provides a graphical representation of data generated in non-human primates treated with PEGylated αβhIL2 mutein, demonstrating that PEGylated αβhIL2 mutein induces selective proliferation of CD25+ CD8+ T cells in response to two dose levels (250 μg / kg and 20 μg / kg). The percentage of KI67+ CD8+ T cells is shown on the y-axis. The figure legend indicates the symbols and corresponding graphical symbols for the different cell populations and different dose levels. [Figure 6]1 provides a graphical representation of data generated in non-human primates treated with PEGylated non-α-hIL2 muteins. The percentage of KI67+ CD8+ T cells is presented on the y-axis. The figure legend indicates the different cell populations and the corresponding graphical symbols. The various evaluation time points are presented on the x-axis. [Figure 7]

[0023] Figure 1 provides a graphical representation of data generated in non-human primates treated with PEGylated αβhIL2 muteins. The levels of IL2 mutein species (in nanograms / ml) observed in the serum of the primates are presented on the y-axis. The time course of the study is presented on the x-axis. The figure legend indicates the different treatment conditions and the corresponding graphical symbols. [Figure 8] This figure provides a graphical representation of a time course study performed in non-human primates treated with PEGylated non-α-hIL2 muteins versus PEGylated αβhIL2. The y-axis provides the levels of pSTAT5+ in CD8+CD25+ cells. The figure legend indicates the different treatment conditions and the corresponding graphical symbols. The time course of the study is presented on the x-axis. [Figure 9] Figure 9 illustrates the antitumor efficacy of PEGylated αβ(REH)mIL2 in treating MC38 tumors in mice. Figure 9, panel A, provides a diagram of the study design showing the time of tumor implantation and the timeline (in days) and time points of administration of various PEGylated IL2 species. Figure 9, panel B, provides a graphical representation of estimated tumor volume (y-axis) versus time (x-axis) over the course of the study (29 days after implantation of MC38 tumor cells). The figure legend indicates the different treatment conditions and corresponding graphical symbols. CR is an abbreviation for complete response. Figure 9, panel C, provides a graphical representation of MC38 tumor weight (y-axis) in response to various treatments (x-axis). The legend indicates the different treatment conditions and corresponding graphical symbols used in panels B and C. [Figure 10]This figure summarizes the results of the evaluation of multiple parameters in response to various IL2 molecules. Figure 10, panel A, provides a diagram of the study design, showing the time of tumor implantation and the timeline (in days) and time points of administration of various PEGylated IL2 species. TILs were isolated from tumors on day 18, sorted for CD25 expression, and exposed to MC38 tumor cells ex vivo. Figure 10, panel B, provides the results of FACS sorting, with the percentage of CD8+ cells shown on the y-axis, while the percentage of CD25+ cells is shown on the x-axis. Rectangles indicate cells representing CD25+ TILs. In Figure 10, panels C, D, and E, the y-axis provides the levels of IFNγ (panel C), GM-CSF (panel D), and TNFα (panel E) in picograms / ml (pg / ml) in CD25+ CD8+ and CD25- CD8+ T cells isolated from tumors of MC38-injected mice. The figure legend indicates the different treatment conditions used and the corresponding graphical symbols. [Figure 11]Figure 11 provides data assessing the toxicity parameters of IL2 muteins in non-human primates. In Figure 11, panels A–F provide microscopic images of lung tissue from non-human primates treated with a PBS control (Panel A), wt-hIL2 (Panel B), a single dose of non-α-IL-2-PEG (Panel C); two doses of non-α-IL-2-PEG (Panel D); a 20 μg / kg dose of αβhIL2-PEG mutein (low dose or “LD”; see legend in Panel I) in Panel E; and a 250 μg / kg dose of αβhIL2-PEG mutein (high dose or “HD”; see legend in Panel I) in Panel F. Figure 11, panel G, provides data regarding the percentage of CD25+ CD8+ T cells that are phospho-STAT5 positive (y-axis) and the time course of the experiment (x-axis). The figure legend indicates the different treatment conditions and the corresponding graphical symbols. Figure 11, panel H, provides data regarding the concentration of FoxP3+ cells / millimeter squared observed in the lungs of animals treated with each of the test agents. The figure legend indicates the different treatment conditions and corresponding graphic symbols for CD25. Figure 11, panel H, provides data regarding the relative lung weights of animals normalized to untreated control animals (y-axis) in response to various test agents. The figure legend indicates the different test agents and corresponding graphic symbols. As previously noted, LD = low dose and HD = high dose. [Figure 12] Figure 12 of the accompanying drawings provides a graphical representation of pSTAT5 levels as measured in NKL cells treated with 293T transfection supernatants containing the indicated IL2 muteins (and controls) as described for various human IL2 muteins. The vertical axis represents IL2 activity levels as determined by the maximal induction level of phospho-STAT5, and each bar shows the activity level of a particular IL2 peptide assessed in association with a construct identified by the three-letter abbreviation corresponding to the amino acids at positions 18, 22, and 126 of the hIL2 muteins as numbered according to wild-type hIL2, except for the V91K mutein, which has a valine to lysine substitution at position 91. [Figure 13]Figure 13 of the accompanying drawings provides a comparison of pSTAT5 activity in CD25-positive (CD25+) and CD25-negative (CD25-) YT cells treated with 293T transfection supernatants containing the indicated human IL2 muteins (and controls). The vertical axis is a measure of selectivity calculated as the ratio of the level of pSTAT5 activity observed in CD25-positive YT cells divided by the level of pSTAT5 activity measured in CD25-negative YT cells, and each bar represents the activity level of a particular IL2 peptide assessed as identified by the three-letter abbreviations corresponding to the amino acids at positions 18, 22, and 126 of the hIL2 muteins numbered according to wild-type hIL2, with the exception of the V91K mutein, which has a valine to lysine substitution at position 91. [Figure 14] 1 provides tabular data illustrating that hIL2 muteins exhibited preferential pSTAT5 signaling activity compared to wild-type hIL2 on CD25-positive YT CD25 cells compared to CD25-negative YT cells at various dilutions. [Figure 15A] Figure 1 provides data on cell proliferation of 3F8 cells contacted with hIL2 muteins. The figure legend indicates the different test agents and the corresponding graphical symbols. Luminescence as a measure of cell proliferation is provided on the y-axis. Protein concentration (picomolar) is provided on the x-axis. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 16A] Figure 1 provides data on interferon gamma production from 3F8 cells contacted with hIL2 muteins. Interferon gamma expression is provided on the y-axis. Protein concentration (picomolar) is provided on the x-axis. The figure legend indicates the different test agents and the corresponding graphic symbols. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. DETAILED DESCRIPTION OF THE INVENTION

[0071] Detailed Description Abbreviation To facilitate understanding of this disclosure, certain terms and phrases are defined below, as well as throughout the specification. The definitions provided herein are non-limiting and should be read in light of the knowledge known to those of ordinary skill in the art.

[0072] Before the present methods and compositions are described, it is to be understood that this invention is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing aspects only, and is not intended to be limiting.

[0073] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of the range is also specifically disclosed. Each smaller range from any stated or intervening value in a stated range to any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded within the range, and each range in which one, neither, or both limits are included within the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. When a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included within the invention.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All patents, patent applications, and publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0075] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, such as polypeptides known to those skilled in the art.

[0076] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0077] Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric. Standard abbreviations are used, including: bp = base pairs; kb = kilobase; pl = picoliter; s or sec = seconds; min = minutes; h or hr = hours; AA or aa = amino acid; kb = kilobase; nt = nucleotide; pg = picogram; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromolar; mM = Millimolar concentration; M = molar concentration; kDa = kilodaltons; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; QD = once daily; BID = twice daily; QW = weekly; QM = monthly; HPLC = high performance liquid chromatography; BW = body weight; U = unit; ns = not statistically significant; PBS = phosphate buffered saline; PCR = polymerase chain reaction; HSA = human serum albumin; MSA = mouse serum albumin; DMEM = Dulbecco's modified Eagle's medium; EDTA = ethylenediaminetetraacetic acid.

[0078] It will be appreciated that throughout this disclosure, amino acids will be referred to by either single-letter or three-letter codes. For the convenience of the reader, the single-letter and three-letter amino acid codes are provided in Table 1 below.

[0079] Table 1: Amino acid abbreviations TIFF2024529340000002.tif98128

[0080] Standard methods in molecular biology are described in the scientific literature (see, e.g., Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugate and protein expression (Vol. 3), and bioinformatics (Vol. 4)). The scientific literature describes methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and protein glycosylation (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wiley and Sons, Inc., NY).

[0081] Unless otherwise indicated, the following terms are intended to have the meanings indicated below. Other terms are defined elsewhere throughout the specification.

[0082] ActivateAs used herein, the term "activate" refers to a receptor or receptor complex and is used to reflect the biological effect of binding of an agonist ligand to the receptor. An activator is a molecule that increases, activates, promotes, or enhances activation, e.g., sensitizes or upregulates a gene, protein, ligand, receptor, or cell. For example, binding of an IL2 agonist to intermediate or high affinity IL2 "activates" receptor signaling to produce one or more intracellular biological effects (e.g., phosphorylation of STAT5). Evaluable parameters for measuring T cell activation are well known in the art. In some embodiments, the level of T cell activation in response to administration of a test agent can be determined by the described flow cytometry method, which is determined by the level of STAT5 phosphorylation by methods well known in the art. STAT5 phosphorylation can be measured using flow cytometry techniques described in the art using commercially available kits such as the phospho-STAT5(Tyr694) kit (commercially available from Perkin-Elmer / cisbio Waltham MA as part number 64AT5PEG), substantially according to the manufacturer's instructions.

[0083] activityAs used herein, the term "activity" is used in reference to a molecule to describe a property of the molecule in relation to a test system (e.g., an assay) or a biological or chemical property of the molecule (e.g., the degree of binding of the molecule to another molecule) or a physical property of a material or cell (e.g., alteration of cell membrane potential). Examples of such biological properties include, but are not limited to, the catalytic activity of a biological agent, the ability to stimulate intracellular signaling, induce gene expression, induce or maintain cell proliferation, or modulate immune activity such as an inflammatory response. "Activity" is typically expressed as the level of biological activity per unit of test agent, such as [catalytic activity] / [mg protein], [immune activity] / [mg protein], International Units of activity (IU), [STAT5 phosphorylation] / [mg protein], [T-cell proliferation] / [mg protein], plaque-forming units (pfu), etc. As used herein, the term "proliferative activity" refers to activity that promotes cell growth and replication, including dysregulation of cell division, such as that observed in neoplastic diseases, inflammatory diseases, fibrosis, metaplasia, cell transformation, metastasis, and angiogenesis.

[0084] Administer / AdministerThe terms "administration" and "administering" are used interchangeably herein to refer to the act of contacting a subject, including contacting the subject's cells, tissues, organs, or biological fluids with an agent (e.g., an αβhIL2 mutein or a pharmaceutical formulation thereof) in vitro, in vivo, and / or ex vivo. Administration of an agent may be accomplished by any of a variety of art-recognized methods, including, but not limited to, topical administration, intravascular injection (including intravenous or intra-arterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, intranodal injection, transdermal, transmucosal, iontophoretic delivery, intralymphatic injection (Senti and Kundig (2009) Current Opinions in Allergy and Clinical Immunology 9(6):537-543), intragastric injection, intraprostatic injection, intravesical instillation (e.g., bladder), respiratory inhalers including nebulizers, intraocular injection, intraperitoneal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intracerebroventricular injection (ICVI), etc. The term "administration" includes contact of an agent with a cell, tissue, or organ as well as contact of an agent with a fluid in contact with a cell. The term "administration" includes ex vivo contacting of cells (or cell populations) that may be isolated from a subject and contacted with an agent, where the cells (or cell populations) are administered to the same subject from which the cells were obtained (autologous cell transfer) or to a subject different from the subject from which the cells were obtained (allogeneic cell transfer).

[0085] Adverse events As used herein, the term "adverse event" refers to any undesirable experience associated with the use of a therapeutic or prophylactic agent in a subject. Adverse events do not necessarily have to be caused by the administration of a therapeutic or prophylactic agent (e.g., an IL2 mutein) and may arise from unrelated circumstances. Adverse events are typically classified as mild, moderate, or severe. As used herein, the classification of adverse events used herein follows the Common Terminology Criteria for Adverse Events v5.0 (CTCAE), published by the U.S. Department of Health and Human Services, the National Institutes of Health, and the National Cancer Institute, dated November 27, 2017.

[0086] affinity As used herein, the term "affinity" refers to the degree of specific binding between a first molecule (e.g., a ligand) and a second molecule (e.g., a receptor), and is determined by the dissociation constant (K off ) and the association constant (K on ) is the ratio of K d The binding kinetics is measured by the binding kinetics expressed as

[0087] AgonistsAs used herein, the term "agonist" refers to a first agent that specifically binds to a second agent (a "target") and interacts with the target, causing or promoting increased activation of the target. In some cases, an agonist is an activator of a receptor protein that modulates cell activation, enhances activation, sensitizes cells to activation by a second agent, or upregulates the expression of one or more genes, proteins, ligands, receptors, biological pathways that can lead to cell proliferation or pathways that result in cell death, such as cell cycle arrest or apoptosis. In some embodiments, an agonist is an agent that binds to a receptor, changes the state of the receptor, and results in a biological response. The response mimics the effect of the receptor's endogenous activating molecule. The term "agonist" includes partial agonists, full agonists, and superagonists. Agonists may be described as "full agonists" when they produce substantially the complete biological response induced by the receptor under study (i.e., the response associated with the natural ligand / receptor binding interaction), or as partial agonists. In contrast to agonists, antagonists can specifically bind to receptors but do not result in the signal cascade typically initiated by the receptor, and can modify the activity of agonists at that receptor. Inverse agonists are agents that produce pharmacological responses opposite to those of agonists. A "superagonist" is a type of agonist that can produce a maximum response greater than that of the endogenous agonist at the target receptor, and therefore has greater than 100% activity of the natural ligand. A superagonist is typically a synthetic molecule that exhibits a response in an evaluable quantitative or qualitative parameter of greater than 110%, alternatively greater than 120%, alternatively greater than 130%, alternatively greater than 140%, alternatively greater than 150%, alternatively greater than 160%, or alternatively greater than 170% of the naturally occurring form of the molecule when evaluated at similar concentrations in an equivalent assay.Assessment of the agonist activity of the αβhIL2 muteins is performed with reference to the WHO international standard (NIBSC code: 86 / 500) wild-type mature human IL2 assessed at similar concentrations in an equivalent assay.

[0088] Antagonist As used herein, the term "antagonist" or "inhibitor" refers to a molecule that opposes the action of an agonist. An antagonist prevents, reduces, inhibits, or neutralizes the activity of an agonist, and an antagonist can also prevent, inhibit, or reduce the constitutive activity of a target, such as a target receptor, even in the absence of a specified agonist. An inhibitor is, for example, a molecule that reduces, blocks, prevents, delays activation, inactivates, desensitizes, or downregulates a gene, protein, ligand, receptor, biological pathway, or cell.

[0089] antibody As used herein, the term "antibody" refers collectively to: (a) glycosylated and non-glycosylated immunoglobulins (including, but not limited to, mammalian immunoglobulin classes IgG1, IgG2, IgG3, and IgG4) that specifically bind to a target molecule, and (b) IgG(1-4) delta C, which compete with the immunoglobulin from which it was derived for binding to the target molecule. H 2, F(ab')2, Fab, ScFv, V H , V L

[0033] The term "antibody" refers to immunoglobulin derivatives, including, but not limited to, tetrabodies, triabodies, diabodies, dsFv, F(ab')3, scFv-Fc, and (scFv)2. The term "antibody" is not limited to any particular synthetic means, and includes naturally occurring antibodies isolable from natural sources as well as engineered antibodies.

[0090] CD25As used herein, the terms "CD25," "IL2 receptor alpha," "IL2Rα," "IL2Ra," "low-affinity IL2 receptor," and "p55" are used interchangeably to refer to a 55 kD polypeptide constitutively expressed in Treg cells and inducibly expressed on other T cells in response to activation. The nucleic acid and protein sequences for human CD25 (hCD25) can be found under GenBank accession numbers NM_000417 and NP_0004Q8, respectively. Human CD25 is expressed as a 272-amino acid preprotein containing a 21-amino acid signal sequence, which is post-translationally removed to yield the 251-amino acid mature protein. Amino acids 22-240 (amino acids 1-219 of the mature protein) correspond to the extracellular domain. Amino acids 241-259 (amino acids 220-238 of the mature protein) correspond to the transmembrane domain. Amino acids 260-272 (amino acids 239-251 of the mature protein) correspond to the intracellular domain. The amino acid sequence of the mature form of hCD25 (not including the preprotein signal sequence) is: TIFF2024529340000003.tif32133.

[0091] CD122As used herein, the terms "CD122," "interleukin-2 receptor beta," "IL2Rb," "IL2Rβ," "IL15Rβ," and "p70-75" are used interchangeably to refer to the human CD122 transmembrane protein. Human CD122 (hCD122) is expressed as a 551-amino acid protein, the first 26 amino acids of which contain a signal sequence that is post-translationally cleaved to yield the 525-amino acid mature protein. Amino acids 27-240 (amino acids 1-214 of the mature protein) correspond to the extracellular domain, amino acids 241-265 (amino acids 225-239 of the mature protein) correspond to the transmembrane domain, and amino acids 266-551 (amino acids 240-525 of the mature protein) correspond to the intracellular domain. As used herein, the term CD122 includes naturally occurring variants of the CD122 protein, including S57F and D365E (as numbered according to the mature hCD122 protein). hCD122 is referenced in the UniProtKB database as entry P14784. The nucleic acid and protein sequences of human CD122 can be found under GenBank accession numbers NM_000878 and NP_000869, respectively. The amino acid sequence of the mature hCD122 protein without the signal sequence is: The file is TIFF2024529340000004.tif66134.

[0092] CD132As used herein, the terms "CD132," "IL2 receptor gamma," "IL2Rg," and "IL2Rγ" refer to the type 1 cytokine receptor, shared by the receptor complexes for IL-4, IL-7, IL-9, IL-15, and IL21, hence the term "common" gamma chain. Human CD132 (hCD132) is expressed as a 369-amino acid preprotein, including a 22-amino acid N-terminal signal sequence. Amino acids 23-262 (amino acids 1-240 of the mature protein) correspond to the extracellular domain, amino acids 263-283 (amino acids 241-262 of the mature protein) correspond to the 21-amino acid transmembrane domain, and amino acids 284-369 (amino acids 262-347 of the mature protein) correspond to the intracellular domain. hCD132 is referenced in the UniProtKB database as entry P31785. The nucleic acid and protein sequences of human CD132 can be found under GenBank accession numbers NM_000206 and NP_000197, respectively. The amino acid sequence of the mature hCD132 protein is: The file is TIFF2024529340000005.tif44134.

[0093] Cell Selection ProcessThe terms "cell selection process" and "ex vivo cell selection process" are used interchangeably to describe any of a variety of techniques for isolating specific cell subpopulations from a mixed cell population based on the expression or presence of one or more specific "marker" molecules, such as surface-expressed proteins or intracellular molecules, in or on the cells. A variety of methods for isolating specific subpopulations characterized by the presence of one or more such markers may be used in the practice of the present disclosure, and markers for specific cell types and subtypes may be used to isolate specific cell types in a mixed cell population and are well known in the art. One example of a cell selection process in such methods is affinity / immunoaffinity separation, in which cells are incubated with a molecule that specifically binds to such markers, and unbound cell types are washed away, leaving cells expressing the marker of interest (positive selection), or unwanted cells are retained and the cells of interest are recovered from the wash (negative selection). In some embodiments, the mixed cell population is contacted with an amount of magnetic beads conjugated to one or more binding molecules that selectively bind to markers present on the cells. Cells expressing such markers can be removed from a cell population by using a magnet to attract magnetic beads to which cells expressing markers bound by conjugated antibodies adhere. This process is described in more detail in Molday et al., U.S. Pat. No. 4,452,773. A variety of such antibody-coated magnetic beads are commercially available under the tradenames Dynabeads® or MACS® beads. In some embodiments, flow cytometry, particularly preparative-scale (FACS) sorting, optionally combined with a MEMS chip (WO 2010 / 033140), facilitates the isolation of T cell subpopulations with high levels of purity. Additionally, automated systems are available that provide for the isolation of specific T cell types, such as the CliniMACS Prodigy system commercially available from Miltenyi Biotech.

[0094] It should be noted that current surface marker-based cell separation protocols generally do not provide for the preparation of a 100% pure cell population expressing a marker of interest, but rather provide a sample enriched for cells expressing a particular marker of interest. While it is not necessary to provide a pure cell population expressing one or more markers of interest, in some embodiments, it is desirable to provide a cell population that is substantially composed of a particular cell type. In some embodiments, a population that is substantially composed of a particular cell type is composed of >50%, alternatively >60%, alternatively >70%, alternatively >80%, or alternatively >90% of a particular cell type. To provide additional levels of cell purity, samples can be subjected to multiple rounds of selection to attempt to prepare cells that are almost entirely composed of cells expressing one or more markers of interest. However, it has been observed that 100% purity in a cell population is not required to prepare an effective cell product, and the rarity of tumor antigen-experienced TILs in a sample may be lost in such a multi-step isolation process.

[0095] Examples of surface markers that can be used to identify and isolate specific T cell or TIL types in a mixed population include one or more cell surface markers selected from the group consisting of CD3, CD4, CD8, CD11a, CD11b, CD11c, CD14, CD16, CD19, CD25, CD27, CD28, CD38, CD45RA, CD45RO, CD58, CD61, CD62L, CD66b, CD69, CD103, CD122, CD127, CD197, CD279, D62L, CD69, FoxP3, PD-1, D62L, CCR4, CCR5, CCR6 (CD196), CCR7, CCR10, CXCR3, CTLA4, PD1, PDL1, TCRγδ, TCR Vα24, TCR Vβ11, and HLA-DR. In some embodiments, T cell subtypes are identified by the expression of one or more surface markers, and populations of T cells expressing one or more surface markers are isolated by positive or negative selection techniques. Examples of T cell subpopulations that can be isolated following such a cell selection process expressing multiple markers include CD4+ T cells, CD8+ T cells, CD25+ T cells, CD28 + T cells, CD62L + , CCR7 + T cells, CD27 + T cells, CD127 + T cells, CD45RA + T cells, CD45RO + Examples of T cell subpopulations expressing multiple markers that can be isolated following such a cell selection process include CD25+ CD8+ T cells, CD25- CD8+ T cells, CD28 + T cells, CD62L + , CCR7 + T cells, CD27 + T cells, CD127 + T cells, CD45RA + T cells, CD25+ CD8+ PD1+ T cells and CD62+ CD45RO +In addition to cell surface markers, intracellular markers such as Ki67, T-bet, GATA-3, PU.1, RORγt, AHR, FOXO4, and FOXP3 can also be used to select specific cell types, including, but not limited to, T cells.

[0096] Equivalent As used herein, the term "equivalent" is used to describe the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, if a first measurement of an evaluable quantitative parameter (e.g., CTLL-2 proliferation or IL2 activity level as determined by phospho-STAT5 assay) and a second measurement of an evaluable parameter do not deviate from a range that a person skilled in the art would recognize as not producing a statistically significant difference in the effect between the two results under the circumstances, the two measurements are considered "equivalent." In some cases, measurements can be considered "equivalent" if one measurement deviates from the other measurement by less than 30%, alternatively less than 25%, alternatively less than 20%, alternatively less than 15%, alternatively less than 10%, alternatively less than 7%, alternatively less than 5%, alternatively less than 4%, alternatively less than 3%, alternatively less than 2%, or even less than 1%. In certain embodiments, if one measurement deviates from a reference standard by less than 15%, alternatively less than 10%, or alternatively less than 5%, it is equivalent to the reference standard.

[0097] Originates from As used herein, the term "derived" in the context of the amino acid sequence of a mutein refers to the parent version of the protein from which the mutein is derived. For example, an IL2 mutein is said to be "derived" from a reference wild-type IL2 polypeptide to indicate that the polypeptide or nucleic acid has a sequence based on the sequence of the reference polypeptide. The term "derived" when applied to a mutein is not meant to be limiting with respect to the source or manner in which the mutein is derived.

[0098] Effective concentration (EC)As used herein, the term "effective concentration" or its abbreviation "EC" are used interchangeably to refer to the concentration of an agent (e.g., a hIL2 mutein) in an amount sufficient to elicit a response in a given parameter in a test system. The abbreviation "E" refers to the magnitude of a given biological effect observed in a test system when the test system is exposed to a test agent. The abbreviation "EC" is used when the magnitude of the response is expressed as a factor of the concentration ("C") of the test agent. In the context of a biological system, the term Emax refers to the maximum magnitude of a given biological effect observed in response to a saturating concentration of an activating test agent. When the abbreviation EC is provided with a subscript (e.g., EC 40 , E.C. 50 (e.g., where the subscript refers to the percent of Emax of the biological parameter observed at that concentration. For example, a concentration of such a test agent sufficient to result in the induction of a measurable biological parameter in a test system that is 30% of the maximum level of such measurable biological parameter in response to the test agent is the "EC" of the test agent with respect to such biological parameter. 30 Similarly, it is called "EC 100 The term "effective concentration" is used to mean the effective concentration of an agent that results in a maximal (100%) response of a measurable parameter in response to such an agent. 50 The term "EC" (commonly used in the field of pharmacodynamics) refers to the concentration of an agent sufficient to result in a half-maximal (50%) change in a measurable parameter. The term "saturation concentration" refers to the maximum possible amount of a test agent that can be dissolved in a standard volume of a particular solvent (e.g., water) under standard conditions of temperature and pressure. In pharmacodynamics, the saturation concentration of a drug is typically used to mean a drug concentration sufficient for all available receptors to be occupied by the drug, and is referred to as the EC 50is the drug concentration for giving a half-maximal effect. The EC for a particular effective concentration of a test agent may be abbreviated for a particular parameter and test system. For example, the concentration of an IL2 mutein for inducing 50% of the maximal level of STAT5 phosphorylation in CD25+ T cells may be referred to as "EC" depending on the context. 50 pSTAT5-CD25+ " or similar terms. Emax is a factor of the parameter being measured (e.g., pSTAT5 induction, proliferation), the test agent (e.g., a particular IL2 mutein such as "REH" below), and the test system (e.g., CD25+ human T cells, human CD25- cells, primary human T cells), and therefore the concentration of test agent (e.g., EC 20 , E.C. 50 Determination of the activity of a molecule (e.g., activity of a specific antibody or antibody against a specific antigen) can be empirically determined for a particular test system. In some cases, there is a generally accepted standardized measure of bioactivity that has been established for the molecule. For example, with respect to the potency of hIL2, the standard methodology for assessing hIL2 potency in International Units (IU) is measured in the murine cytotoxic T cell line CTLL-2 according to standard procedures as fully described by Wadhwa, et al. (2013) "The 2nd International standard for Interleukin-2 (IL2) Report of a collaborative study" Journal of Immunological Methods 397:1-7.

[0099] EC proliferation : "An effective concentration sufficient to induce proliferation of CD3-activated primary human T cells" (referred to herein as "EC PROThe term "IL2 mutein concentration" (abbreviated as "IL2 mutein concentration") refers to the effective concentration of an IL2 mutein sufficient to induce proliferation of CD3-activated primary human T cells as determined according to the teachings of standard protocols in the art, for example, using a carboxyfluorescein diacetate succinimidyl diester (CFSE) dilution assay or by thymidine incorporation. Alternatively, assessment of proliferation of primary human T cells can be measured using a bioluminescence assay that generates a luminescent signal proportional to the amount of ATP present, which is directly proportional to the number of cells present in culture, as described in Crouch, et al. (1993) "The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity," J. Immunol. Methods 160: 81-8, or a standard commercially available assay system such as the CellTiter-Glo® 2.0 Cell Viability Assay or the CellTiter-Glo® 3D Cell Viability Kit, commercially available from Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, catalog numbers G9241 and G9681, respectively, substantially according to the instructions provided by the manufacturer. Abbreviations: EC PRO When used with a subscript, this is provided to indicate the concentration of a test agent sufficient to induce the indicated percent of maximal proliferation of primary human T cells in response to the test agent as measured by a given test protocol. Illustratively, the abbreviation EC 30 PRO can be used with respect to a hIL2 mutein to indicate the concentration associated with 30% of the maximal level of proliferation of CD3-activated primary human T cells in response to that IL2 mutein as measured by the CellTiter-Glo® 2.0 cell viability assay.

[0100] EC activation : "An effective concentration sufficient to induce T cell activation" (referred to herein as "EC ACTThe term EC (abbreviated as "EC") refers to the effective concentration of an IL2 mutein sufficient to induce activation and / or differentiation of human T cells. ACT When used with a subscript, this is provided to indicate the concentration of a test agent sufficient to induce the indicated percent of maximal STAT5 phosphorylation in T cells in response to application of the test agent, as measured according to the test protocol. Illustratively, the abbreviation EC 30 PRO can be used with respect to hIL2 muteins to indicate the concentration associated with 30% of the maximal level of STAT5 phosphorylation in T cells responding with such IL2 muteins. A variety of techniques are available to those skilled in the art for assessing STAT5 phosphorylation, including flow cytometry as described in Horta, et al. (2019) Oncoimmunology 8(6): e1238538, as well as commercially available kits, such as the PathScan® Phospho-Stat5 (Tyr694) Sandwich ELISA Kit available from Cell Signaling Technology, Inc. (Danvers MA) under catalog number #7113; the STAT5A ELISA Kit available from LifeSpan BioSciences (Seattle WA) under catalog number LS-F38421-1; the Human STAT5A ELISA Kit available from Novus Biologicals (Centennial CO) under catalog number NVP2-80280; or the Phospho-STAT5 (Tyr694) Kit (available from Perkin-Elmer / cisbio Waltham MA under product number 64AT5PEG), substantially following the manufacturer's instructions. Abbreviations: EC ACT When used with a subscript, this is provided to indicate the concentration of test agent sufficient to produce the indicated subscript percent of maximal STAT5 phosphorylation in T cells in response to application of the test agent, as measured according to the STAT5 protocol. Illustratively, the abbreviation EC 30 PROcan be used, with respect to a hIL2 ortholog, to indicate the concentration associated with 30% of the maximal level of STAT5 phosphorylation in a T cell response to such a hIL2 ortholog as measured using a phospho-STAT5(Tyr694) kit.

[0101] It is concentrated As used herein, the term "enriched" refers to a sample in which a species (e.g., molecule or cell) of interest is present at a concentration: (a) greater than the concentration of the species in a starting sample, e.g., a biological sample (e.g., a sample in which the molecule naturally occurs or in which the molecule occurs after administration) (e.g., at least 3-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 50-fold greater, alternatively at least 100-fold greater, or alternatively at least 1000-fold greater); or (b) a sample that has been non-naturally engineered such that the species (e.g., molecule or cell) is present at a concentration greater than the concentration of the species in the starting sample, e.g., a biological sample (e.g., a sample in which the molecule naturally occurs or in which the molecule occurs after administration); or (b) a sample that has been non-naturally engineered such that the species is present at a concentration greater than the environment in which the molecule was made (e.g., in recombinantly modified bacteria or mammalian cells).

[0102] Extracellular domain As used herein, the term "extracellular domain" or its abbreviation "ECD" refers to the portion of a cell surface protein (e.g., a cell surface receptor) that is on the outside plasma membrane of the cell. The term "ECD" can include the extracytoplasmic portion of a transmembrane protein or the extracytoplasmic portion of a cell surface (or membrane-associated protein).

[0103] identityThe term "identity" as used herein in connection with polypeptide or DNA sequences refers to the subunit sequence identity between two molecules. If a subunit position in both molecules is occupied by the same monomer subunit (i.e., the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity between two amino acid sequences or two nucleotide sequences is a direct function of the number of identical positions. Generally, these sequences are aligned to obtain the highest level of match. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the BLAST 2.0 algorithm described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul, et al. (1977) Nucleic Acids Res. 25: 3389-3402. Software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information (NCBI). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score "T" when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the total alignment score can be increased. For nucleotide sequences, the total score is calculated using the parameters "M" (reward score for a pair of matching residues; always >0) and "N" (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the total score.Extension of word hits in each direction is stopped when (a) the total alignment score drops by an amount X from its maximum achieved value; the accumulation of alignments of one or more negatively scoring residues causes the total score to fall below zero; or (b) the end of either sequence is reached. The BLAST algorithm parameters "W," "T," and "X" determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) works similarly, but uses as defaults a word size ("W") of 28, an expectation ("E") of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, (1989) PNAS (USA) 89:10915-10919).

[0104] IL2 As used herein, the term "interleukin-2" or "IL2" refers to a naturally occurring IL2 polypeptide having IL2 activity. In some embodiments, IL2 refers to mature wild-type human IL2. Mature wild-type human IL2 (hIL2) exists as a 133 amino acid mature polypeptide (minus a signal peptide consisting of an additional 20 N-terminal amino acids) as described in Fujita, et. al, PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of a naturally occurring variant of mature wild-type human IL2 (hIL2) is: TIFF2024529340000006.tif26128. As used herein, the numbering of residues in hIL2 muteins is based on the hIL2 sequence UniProt ID P60568, excluding the signal peptide, which is the same as that of SEQ ID NO:4.

[0105] IL2 activityThe term "IL2 activity" refers to one or more biological effects on a cell in response to contacting the cell with an effective amount of an IL2 polypeptide. As previously noted, IL2 is a pleiotropic cytokine that results in one or more biological effects on a variety of cell types. An example of IL2 activity can be measured in a cell proliferation assay using CTLL-2 murine cytotoxic T cells. See Gearing, AJH and CB Bird (1987) in Lymphokines and Interferons, A Practical Approach. Clemens, MJ et al. (eds): IRL Press. 295. The specific activity of recombinant human IL2 (rhIL2) is approximately 2.1 x 10 4 IU / μg, which is calibrated against the WHO International Standard for Recombinant Human IL2 (NIBSC code: 86 / 500).

[0106] in sufficient quantities to bring about change As used herein, the phrase "in an amount sufficient to cause a change" refers to an amount of a test agent sufficient to provide a detectable difference between an indicator measured before (e.g., baseline level) and after application of the test agent to a system, e.g., the level of biological function assessed in a cell-based assay in response to administration of an amount of the test agent. While "in an amount sufficient to cause a change" can be sufficient to be a therapeutically effective amount, "in an amount sufficient to cause a change" can be more or less than a therapeutically effective amount.

[0107] Needs treatment The term "in need of treatment" as used herein refers to a judgment made by a physician or other caregiver regarding a subject that the subject needs or would potentially benefit from treatment. This judgment is made based on a variety of factors within the physician's or caregiver's expertise.

[0108] Needs preventionThe term "in need of prevention" as used herein refers to a judgment made by a physician or other caregiver regarding a subject that the subject is in need of or would potentially benefit from preventative care. This judgment is made based on a variety of factors within the physician's or caregiver's expertise.

[0109] inhibitors As used herein, the term "inhibitor" refers to a molecule that reduces, blocks, prevents, delays the activation of, inactivates, desensitizes, or downregulates, for example, a gene, protein, ligand, receptor, or cell. An inhibitor can also be defined as a molecule that reduces, blocks, or inactivates the constitutive activity of a cell or organism.

[0110] Isolated As used herein, the term "isolated" is used in reference to a polypeptide of interest that is in an environment different from that in which it would naturally occur if it were naturally occurring. "Isolated" is meant to include a polypeptide that is in a sample in which the polypeptide of interest has been substantially enriched and / or in which the polypeptide of interest has been partially or substantially purified. When the polypeptide is non-naturally occurring, "isolated" indicates that the polypeptide has been separated from the environment in which it was created, either by synthetic or recombinant means.

[0111] LigandAs used herein, the term "ligand" refers to a molecule that exhibits specific binding to a receptor, resulting in a change in the biological activity of the receptor such that the molecule binds to the molecule and causes a change in the activity of the receptor. In one aspect, the term "ligand" refers to a molecule, or complex thereof, that can act as an agonist or antagonist of the receptor. As used herein, the term "ligand" encompasses natural and synthetic ligands. "Ligand" also encompasses small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies. A complex of a ligand and receptor is termed a "ligand-receptor complex."

[0112] metastasis As used herein, the term "metastasis" refers to the spread of cancer cells from a primary tumor to surrounding tissues and distant organs.

[0113] Modified IL2 muteinAs used herein, the term "modified IL2 mutein" is used to refer to an IL2 mutein that has and comprises one or more additional modifications (i.e., modifications other than the core amino acid sequence of the IL2 mutein), such as PEGylation, glycosylation (N-linked and O-linked), acylation, or polysialylation, or modification by conjugation (as a chemical conjugation or fusion protein) to other polypeptide carrier molecules, including, but not limited to, albumin fusion polypeptides and / or Fc-fusion proteins, including serum albumin (e.g., human serum albumin (HSA) or bovine serum albumin (BSA)), or modification with a targeting moiety, such as an IL2 orthogonal polypeptide fusion protein, a target IL2 mutein polypeptide, e.g., an IgG, including ScFv-IL2 mutein polypeptide fusion protein, and a VHH-IL2 mutein polypeptide fusion protein. Modified IL2 muteins can be prepared to enhance one or more properties, for example, to modulate immunogenicity; to increase water solubility, bioavailability, serum half-life, and / or therapeutic half-life; and / or to modulate biological activity. Certain modifications can also be useful, for example, to generate antibodies for use in detection assays (e.g., epitope tags) and to provide ease of protein purification.

[0114] Modulate As used herein, the terms "modulate," "modulation," and the like refer to the ability of a test agent to affect a response, either positively or negatively, directly or indirectly, in a biological system or a system that includes a biochemical pathway.

[0115] MuteinAs used herein, the term "mutein" refers to a polypeptide that contains one or more modifications to its primary structure (e.g., insertions, deletions, substitutions, and modifications at one or more amino acid sites) compared to the primary structure of the parent polypeptide from which it is derived. In some cases, the parent polypeptide from which a mutein is derived is a wild-type polypeptide. Typical terminology refers to a mutein relative to the parent molecule from which it is derived. In the absence of any specific indication that the mutein is derived from another mutein, it is assumed that the term mutein is used in reference to the wild-type form of the protein. For example, a "human IL2 mutein" refers to a polypeptide that contains one or more modifications to its primary structure compared to the amino acid sequence of wild-type human IL2.

[0116] N-terminus As used herein in the context of a polypeptide's structure, the terms "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxyl terminus") refer to the extreme amino and carboxyl termini of a polypeptide, respectively, whereas the terms "N-terminally" and "C-terminally" refer to the relative positions toward the N-terminus and C-terminus, respectively, in the amino acid sequence of a polypeptide and can include residues at the N-terminus and C-terminus, respectively. The terms "directly N-terminally" or "directly C-terminally" are used to refer to the position of a first amino acid residue relative to a second amino acid residue, where the first and second amino acid residues are covalently linked to provide a contiguous amino acid sequence.

[0117] Neoplastic diseaseAs used herein, the term "neoplastic disease" refers to a disorder or condition in a subject that results from excessive cell proliferation or unregulated (or dysregulated) cell replication. The term neoplastic disease refers to a disorder that results from the presence of a neoplasm in a subject. Neoplasms can be classified as (1) benign, (2) pre-malignant (or "pre-cancerous"); and (3) malignant (or "cancerous"). The term "neoplastic disease" includes neoplasia-related diseases, disorders, and conditions, which refer to conditions that are directly or indirectly associated with neoplastic disease, including, for example, angiogenesis and pre-cancerous conditions such as dysplasia.

[0118] nucleic acid The terms "nucleic acid," "nucleic acid molecule," "polynucleotide," and the like are used interchangeably herein to refer to polymeric forms of any length of nucleotides, be they deoxyribonucleotides or ribonucleotides or analogs thereof. Non-limiting examples of polynucleotides include linear or circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, and the like.

[0119] Functionally linkedThe term "operably linked" is used herein to refer to the relationship between nucleic acid sequences encoding different functions that, when combined into a single nucleic acid sequence, provide a nucleic acid capable of causing the transcription and / or translation of the particular nucleic acid sequence in a cell when introduced into a cell. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, certain genetic elements, such as enhancers, need not be contiguous with the sequences in which they provide their effect.

[0120] Parent Polypeptide As used herein, the terms "parent polypeptide" or "parent protein" are used interchangeably to refer to a naturally occurring polypeptide that has been subsequently modified to generate a variant or mutein. A parent polypeptide may be a wild-type (or native) polypeptide. A parent polypeptide may refer to the polypeptide itself or a composition comprising the parent polypeptide (e.g., glycosylated, PEGylated, a fusion protein comprising the parent polypeptide).

[0121] Partial agonistAs used herein, the term "partial agonist" refers to a molecule that specifically binds to and activates a given receptor, but only partially activates the receptor compared to a full agonist. A partial agonist may exhibit both agonist and antagonist effects. For example, when both a full agonist and a partial agonist are present, the partial agonist acts as a competitive antagonist by competing with the full agonist to bind to the receptor, resulting in a net decrease in receptor activation compared to the contact of the receptor with the full agonist in the absence of the partial agonist. Clinically, partial agonists can be used to activate receptors in the presence of insufficient endogenous ligands to provide desired submaximal responses, or they can reduce receptor overstimulation in the presence of excessive endogenous ligands. The maximum response (Emax) produced by a partial agonist is called its intrinsic activity and can be expressed in percentage terms, where a full agonist would produce a 100% response. An IL2 partial agonist may have more than 10%, alternatively more than 20%, alternatively more than 30%, alternatively more than 40%, alternatively more than 50%, alternatively more than 60%, or alternatively more than 70% of the activity of WHO International Standard (NIBSC code: 86 / 500) wild-type mature human IL2 when assessed at similar concentrations in an equivalent assay.

[0122] PolypeptidesAs used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified polypeptide backbones. These terms include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without an N-terminal methionine residue; fusion proteins with immunologically tagged proteins; fusion proteins of immunologically active proteins (e.g., antigenic diphtheria or tetanus toxin fragments); and the like.

[0123] Prevent As used herein, the terms "prevent," "preventing," "prevention," and the like, generally in relation to a subject predisposed to having a particular disease, disorder, or condition due to genetic, experiential, or environmental factors, refer to a course of action initiated in a subject prior to the onset of a disease, disorder, condition, or symptoms thereof, so as to temporarily or permanently prevent, suppress, inhibit, or reduce the risk of the subject developing, or delay the onset of, the disease, disorder, condition, or other (e.g., as determined by the absence of clinical symptoms). In certain instances, the terms "prevent," "preventing," and "prevention" are also used to refer to slowing the progression of a disease, disorder, or condition from its current state to a more deleterious state.

[0124] As used herein, the term "proliferation" refers to an increase in cell division, which may be symmetric or asymmetric cell division. In certain embodiments, "proliferation" refers to the symmetric or asymmetric division of T cells. "Increased proliferation" occurs when there is an increase in the number of cells in a treated sample compared to cells in an untreated sample.

[0125] receptorAs used herein, the term "receptor" refers to a polypeptide having a domain that specifically binds to a ligand, where binding of the ligand results in a change in at least one biological property of the polypeptide. In some embodiments, the receptor is a "soluble" receptor that is not associated with a cell surface. In some embodiments, the receptor is a cell surface receptor comprising an extracellular domain (ECD) and a membrane-associated domain that acts to anchor the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane polypeptide comprising an intracellular domain (ICD) and an extracellular domain (ECD) connected by a transmembrane domain, typically referred to as the transmembrane domain (TM). Binding of a ligand to the receptor results in a conformational change in the receptor, resulting in a measurable biological effect. In some cases where the receptor is a transmembrane polypeptide comprising an ECD, a TM, and an ICD, binding of a ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to binding of the ligand to the ECD. In some embodiments, the receptor is a component of a multi-component complex that promotes intracellular signal transduction. For example, a ligand may bind to a cell surface molecule that promotes the formation of heteromultimers, including heterodimeric (e.g., the intermediate affinity CD122 / CD132 IL2 receptor), heterotrimeric (e.g., the high affinity CD25 / CD122 / CD132 hIL2 receptor), or homomultimeric (e.g., homodimeric, homotrimeric, homotetrameric) complexes that are not alone associated with any intracellular signaling but that result in intracellular signaling upon ligand binding.

[0126] RecombinationAs used herein, the term "recombinant" is used as an adjective to refer to a polypeptide, nucleic acid, or cell that has been modified using recombinant DNA technology. A recombinant protein is a protein produced using recombinant DNA technology and may be referred to as such using the abbreviation "r" (e.g., rhIL2) to indicate the method by which the protein is produced. Similarly, if a cell has been modified using recombinant DNA technology by incorporating (e.g., transfecting, transducing, or infecting) an exogenous nucleic acid (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids, etc.), the cell is referred to as a "recombinant cell." Techniques and protocols for recombinant DNA technology are well known in the art; for example, they can be found in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0127] response For example, the term "response" of a cell, tissue, organ, or organism encompasses a quantitative or qualitative change in an assessable biochemical or physiological parameter (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, energy consumption rate, level or state of differentiation), where the change is correlated with activation, stimulation, or treatment, or with internal mechanisms such as genetic programming. In certain contexts, terms such as "activation," "stimulation," and the like refer to cellular activation regulated by external or environmental factors as well as internal mechanisms. In contrast, terms such as "inhibition," "downregulation," and the like refer to an adverse effect.

[0128] SelectiveAs used herein, the term "selective" refers to the property of an agent to preferentially bind to and / or activate a particular cell type based on certain properties of such cell populations. In some embodiments, the present disclosure provides IL2 muteins that are CD25-selective, in that such muteins exhibit preferential activation of cells expressing CD25 and / or CD25 / CD122 receptors relative to cells expressing CD132 receptors. Selectivity is typically assessed by activity measured as an assay characteristic of activity induced in response to ligand / receptor binding. In some embodiments, selective IL2 muteins exhibit significantly reduced binding. In some embodiments, selectivity is measured by activation of cells expressing CD25 (e.g., YTCD25 or YTCD25 cells) relative to activation of cells displaying significantly low (preferably undetectable) levels of CD25 (e.g., YTCD25 or YTCD25 cells). In some embodiments, selectivity is measured by activation of T cells expressing CD25 (e.g., Tregs) compared to T cells expressing low levels of CD25 (e.g., unstimulated CD8+ or CD4+ T cells). In some embodiments, an IL2 mutein of the present disclosure has at least a 3-fold, alternatively at least a 5-fold, alternatively at least a 10-fold, alternatively at least a 20-fold, alternatively at least a 30-fold, alternatively at least a 40-fold, alternatively at least a 50-fold, alternatively at least a 100-fold, alternatively at least a 200-fold difference in EC50 on CD25+ cells compared to CD25- cells when measured in the same assay.

[0129] Significantly reduced bindingAs used herein, the term "exhibiting significantly reduced binding" is used in reference to the binding affinity of a variant (e.g., an ortholog) of a ligand to an altered form of a receptor (e.g., orthogonal CD122) compared to the binding of the variant ligand to the native form of the receptor. In some embodiments, a ligand (e.g., an ortholog) exhibits significantly reduced binding to the native form of the ligand if the orthogonal ligand binds to the native form of the receptor with less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the affinity of the native ligand. Similarly, an orthogonal receptor exhibits significantly reduced binding to a native form of a ligand if the native form of the ligand binds to the orthogonal form of the receptor with less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the affinity of the native receptor.

[0130] Specific binding As used herein, the term "specifically bind" refers to the degree of selectivity or affinity with which one molecule binds to another. In the context of a binding pair (e.g., ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pair), a first molecule of the binding pair is said to specifically bind to a second molecule of the binding pair if the first molecule does not bind in significant amounts to other components present in the sample. A first molecule of a binding pair is said to specifically bind to a second molecule of the binding pair if the affinity of the first molecule for the second molecule is at least 2-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 20-fold greater, or alternatively at least 100-fold greater than the affinity of the first molecule for other components present in the sample. Specific binding may be assessed using techniques known in the art.

[0131] subjectThe terms "recipient," "individual," "subject," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. A "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and exhibition, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments, the mammal is a human.

[0132] In effect As used herein, the term "substantially" refers to an amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the reference amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length. In one aspect, "substantially the same" refers to an amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length that produces approximately the same effect, e.g., physiological effect, as the reference amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length.

[0133] suffering As used herein, the term "suffering" refers to a determination made by a physician regarding a subject that the subject requires or will benefit from treatment based on available information accepted in the art for identifying a disease, disorder, or condition, including, but not limited to, X-rays, CT scans, conventional clinical diagnostic tests (e.g., blood counts), genomic data, protein expression data, and immunohistochemistry. The term suffering is typically used in conjunction with specific medical conditions, such as "suffering from a neoplastic disease," which refers to a subject who has been diagnosed with the presence of a neoplasm.

[0134] Virtually pureAs used herein, the term "substantially pure" indicates that a component (e.g., a polypeptide) constitutes more than about 50% of the total content of the composition, typically more than about 60% of the total polypeptide content. More typically, "substantially pure" refers to a composition in which at least 75%, at least 85%, at least 90% or more of the total composition is the component of interest. In some cases, the polypeptide constitutes more than about 90% or more than about 95% of the total content of the composition.

[0135] T cells As used herein, the term "T-cell" or "T cell" is used in its conventional sense to refer to lymphocytes that differentiate within the thymus and have specific cell surface antigen receptors, including those that control the initiation or suppression of cell-mediated and humoral immunity as well as others that lyse antigen-bearing cells. In some embodiments, T cells are naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, e.g., T H 1. T H 2. T H 9. T H 11. T H 22, T FH ;regulatory T cells, e.g. T R 1. Tregs, inducible Tregs; memory T cells, such as central memory T cells, effector memory T cells, NKT cells, tumor-infiltrating lymphocytes (TILs), and engineered variants of such T cells, including, but not limited to, CAR-T cells, recombinantly modified TILs, and TCR-engineered cells.

[0136] T cell activation agentAs used herein, the term "T cell activating agent" refers to a molecule that results in the activation and proliferation of T cells, regardless of whether such T cells express intermediate- or high-affinity IL2 receptors. Examples of T cell activating agents include cytokines, including wild-type hIL2, growth factors, and antibodies against T cell activation antigens (e.g., anti-CD3 antibodies, anti-CD137 antibodies). T cell activating agents can be used in the rapid expansion phase of isolated TIL cell populations. In some embodiments, the initial phase of activation is performed in the presence of an αβhIL2 mutein, which provides enrichment of the cell population for antigen-experienced T cells, and the enriched cell population can then be expanded using a T cell activating agent that generally broadly activates T cells in the population, regardless of whether they express high- or intermediate-affinity receptors.

[0137] Therapeutically effective doseThe phrase "therapeutically effective amount" is used herein in connection with the administration of an active substance to a subject, either alone or as part of a pharmaceutical composition or treatment regimen, in a single dose or as part of a series of doses, in an amount that can have any detectable positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition when administered to a subject.The therapeutically effective amount can be confirmed by measuring the relevant physiological effect, which can be adjusted in relation to the dosing regimen and according to diagnostic analysis such as the subject's condition.The parameters for evaluation to determine the therapeutically effective amount of an active substance are determined by a physician using art-recognized diagnostic criteria, including but not limited to, age, weight, sex, general health, ECOG score, observable physiological parameters, blood levels, blood pressure, evidence such as electrocardiogram, computed tomography, X-ray, etc. Alternatively or additionally, other parameters typically assessed in a clinical setting, such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptom, aspect, or characteristic of a disease, disorder, or condition, biomarkers (e.g., inflammatory cytokines, IFN-γ, granzymes, etc.), reduction in serum tumor markers, improvement in Response Evaluation Criteria in Solid Tumors (RECIST), improvement in immune-related response criteria (irRC), increased survival, increased progression-free survival, increased time to progression, increased time to treatment failure, increased event-free survival, increased time to next treatment, improved response rate, improved duration of response, reduction in tumor burden, complete response, partial response, stable disease, etc., may be monitored to determine whether a therapeutically effective amount of an agent has been administered to a subject, and these parameters are relied upon by clinicians in the art to assess improvement in a subject's condition in response to administration of an agent. The terms "complete response (CR)," "partial response (PR)," "stable disease (SD)," and "progressive disease (PD)" when used herein in relation to target lesions, and the terms "complete response (CR)," "incomplete response / stable disease (SD)," and "progressive disease (PD)" in relation to non-target lesions, are understood to be as defined by RECIST criteria.As used herein, the terms "immune-related complete response (irCR)," "immune-related partial response (irPR)," "immune-related progressive disease (irPD)," and "immune-related stable disease (irSD)" are as defined according to the immune-related response criteria (irRC). As used herein, the term "immune-related response criteria (irRC)" refers to a system for evaluating response to immunotherapy as described in Wolchok, et al. (2009) Guidelines for the Evaluation of Immune Therapy Activity in Solid Tumors: Immune-Related Response Criteria, Clinical Cancer Research 15(23): 7412-7420. A therapeutically effective amount may be adjusted over the course of a subject's treatment in relation to the dosing regimen and / or assessment of the subject's condition and variations in the aforementioned factors. In one aspect, a therapeutically effective amount is an amount of an agent that, when used alone or in combination with another agent, does not result in irreversible serious adverse events during administration to a mammalian subject.

[0138] tissue samplesAs used herein, the term "tissue sample" refers to a quantity of tissue obtained from a subject's tissue from a neoplastic disease. For example, a tissue sample can be a quantity of a neoplasm obtained by physical destruction of the neoplasm, such as by surgical (including catheter) resection and biopsy (including needle biopsy), and other similar procedures that contact the neoplasm. A "tissue sample" can also be a quantity of a peripheral organ, a specific organ involved in the humoral or innate immune response, such as a lymph node (particularly a draining lymph node associated with the neoplasm), spleen, or bone marrow. A tissue sample can also be a quantity of a body fluid, such as blood (including not only whole blood but also blood components such as plasma or serum), mucus secretion, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), eye fluids (e.g., vitreous humor, aqueous humor), and lymph. TILs have been isolated from subjects suffering from neoplastic disease not only directly through surgical resection of the neoplastic mass, but also from various body fluids (such as blood) and other organs that may communicate with the tumor via the circulatory or lymphatic system, such as lymph nodes (particularly draining lymph nodes), as well as from blood and blood products. PBMCs containing TIL cells can be obtained from blood units or apheresis fractions collected from subjects suffering from neoplastic disease using a number of techniques known to those skilled in the art. In some embodiments, after isolating PBMCs from peripheral blood as described above, the cell population may be sorted (as described herein) to isolate specific subpopulations of T cells, such as cytotoxic CD8+ T cells, and CD4+ helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after activation, expansion, and / or genetic modification.

[0139] Transmembrane domainThe term "transmembrane domain" or "TM" refers to a domain of a transmembrane polypeptide (e.g., a transmembrane polypeptide such as CD122 or CD132 or CAR) that is embedded in the cell membrane and is peptide-bound to the extracellular domain (ECD) and intracellular domain (ICD) of the transmembrane polypeptide when the transmembrane polypeptide is associated with the cell membrane. The transmembrane domain can be homologous (naturally associated) or heterologous (not naturally associated) to one or both of the extracellular domain and / or intracellular domain. In some embodiments, the transmembrane domain is the transmembrane domain natively associated with the ECD domain of the cognate receptor from which the orthogonal receptor is derived. In some embodiments, the transmembrane domain is the transmembrane domain natively associated with the ICD domain of the cognate receptor from which the orthogonal receptor is derived. In some embodiments, the transmembrane domain is the transmembrane domain natively associated with a growth signaling domain. In some embodiments, the transmembrane domain is the transmembrane domain natively associated with a different protein. Alternatively, the transmembrane domain of a receptor can be an artificial amino acid sequence that spans the plasma membrane. In some embodiments, in which the receptor is a chimeric receptor comprising an intracellular domain derived from a first parent receptor and a second extracellular domain derived from a second, different parent receptor, the transmembrane domain of the chimeric receptor is the transmembrane domain normally associated with either the ICD or ECD of the parent receptor from which the chimeric receptor is derived.

[0140] TreatThe terms "treat," "treating," "treatment," and the like refer to a course of action initiated with respect to a subject after a disease, disorder, or condition, or a symptom thereof, has been diagnosed, observed, or otherwise, in a subject, to eliminate, reduce, inhibit, alleviate, or ameliorate, either temporarily or permanently, at least one underlying cause of, or at least one symptom associated with, the disease, disorder, or condition afflicting the subject. Treatment includes a course of action taken with respect to a subject suffering from a disease, which course of action results in the inhibition of the disease in the subject (e.g., halting the progression of the disease, disorder, or condition) or the amelioration of one or more symptoms associated with the presence of the disease.

[0141] Treg cells or regulatory T cells As used herein, the term "regulatory T cells" or "Treg cells" refers to CD4 T cells that are capable of suppressing the responses of other T cells, including, but not limited to, effector T cells (Teff). + This refers to a type of T cell. Treg cells are characterized by expression of CD4, the IL2 receptor α subunit (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)). "Conventional CD4 + T cells, which are CD4 T cells other than regulatory T cells. + T cells are meant.

[0142] Wild type By "wild-type" or "WT" or "native" herein is meant an amino acid or nucleotide sequence found in nature and unmodified by the hand of man.

[0143] The use of TIL therapy has been established for the treatment of cancer, and is often used to treat melanoma. In the basic implementation of TIL therapy, a quantity of cells is isolated from a tumor sample in a subject, lymphocytes are isolated from the sample, the isolated lymphocytes are expanded ex vivo, and the cell population is reinfused into the subject. The basic premise of TIL therapy is that a fraction of lymphocytes (TILs) isolated from the tumor sample are tumor antigen-specific lymphocytes capable of significant anti-tumor efficacy; in other words, the subject is able to generate lymphocytes capable of significant anti-tumor effects. It has been observed that these desirable tumor antigen-specific TILs represent a very small proportion of the total lymphocytes in the tumor sample, and in some cases represent less than about 5% of the total amount of T cells isolated from tumor tissue. A further challenge is that tumor antigen-specific TILs in the tumor are "exhausted" and no longer exert anti-tumor effects.

[0144] Conventional TIL therapy protocols attempt to overcome these limitations by recovering a population of T cells containing these tumor antigen-specific TILs from a tissue sample (often a tumor sample), exposing the isolated cell population (optionally selected for the presence of cell surface markers associated with antigen-experienced T cells, such as CD8, CD25, CD137, and / or PD1), expanding (growing) and activating the isolated T cell population containing the tumor antigen-specific TILs ex vivo, and reinfusing the subject with the expanded cell population (TIL cell product) containing a larger number of reactivated tumor antigen-specific TILs. Reinfusion of such TIL cell products prepared using established protocols has been observed to result in beneficial antitumor effects in a significant proportion of treated subjects.

[0145] In TIL protocols, activation and expansion of isolated lymphocytes is achieved by contacting the isolated lymphocyte cell population with hIL2 that has substantially the activity of wild-type hIL2. As previously mentioned, hIL2 is a pleiotropic cytokine that induces T cell activation and proliferation. In current clinical practice, the hIL2 typically employed is aldesleukin, des-Ala1, a C125S hIL2 mutein that is the active pharmaceutical ingredient in Proleukin®, the form of hIL2 approved by the U.S. FDA for human use.

[0146] To further promote the proliferation of isolated lymphocytes, hIL2 is used in combination with anti-CD3 and / or anti-CD28 antibodies to mimic T cell activation from antigen-presenting cells. Typically, magnetic beads conjugated with CD3 and CD28 antibodies are used, and the magnetic beads can be magnetically removed from the mixture. Such CD3 / CD28 antibody conjugated beads are well known in the art and are commercially available from various sources (e.g., Dynabeads®, available from ThermoFisher Scientific under catalog number 11131D; TransAct™ CD3 / 28 beads, available from Miltenyi Biotech). It is suggested that displaying anti-CD3 / anti-CD28 antibodies on beads is preferable because the beads mimic the size and three-dimensional presentation of antigen-presenting cells.

[0147] The problem with using conventional expansion protocols employing CD3 / CD28 beads in combination with hIL2 (e.g., aldesleukin) with substantially wild-type activity is that essentially all lymphocytes in the isolated population expand indiscriminately at roughly the same rate. As a result, the proportion of the most desirable tumor antigen-specific T cells in the expanded cell population remains extremely small.

[0148] To maximize the amount of these tumor antigen-experienced T cells in the population that should be reinfused into the subject to maximize the likelihood of therapeutic efficacy, a very large number of cells (e.g., 1 x 10 10 Typically, TIL therapy involves immunodepletion of the subject prior to reinfusion of the activated cell product (a cell product containing more than 1000 T cells). The administration of such a large amount of mixed-cell-type activated lymphocytes creates certain problems for patients. First, significant toxicity, such as autoimmune or autoimmune-like reactions, is observed with the administration of large amounts of activated lymphocytes (Yang, J.; Toxicities associated with adoptive T-cell transfer for Cancer (2015) Cancer J. 21:506-9; Yeh, et al. (2009) Ophthalmology 116:981-989). Additionally, typical implementation of TIL therapy involves immunodepletion of the subject prior to reinfusion of the activated cell product (Rohann, et al. (2018) Journal for ImmunoTherapy of Cancer 6:102). Although TIL therapy combined with lymphocyte depletion has been correlated with improved clinical outcomes for subjects compared with TIL therapy alone, lymphocyte depletion is associated with significant clinical toxicity (Yang, J., supra). Furthermore, supportive hIL2 therapy following administration of cell products, typically with aldesleukin in clinical practice, is also associated with significant clinical toxicity.

[0149] To mitigate these well-established problems with conventional TIL therapy, various ex vivo approaches have been employed to enrich cell populations for T cells that have experienced the desired antigen. Conventional TIL production involves two phases: (1) an early growth phase, in which isolated tissue is mechanically or enzymatically digested and cultured in the presence of human interleukin-2 (hIL2) for approximately 7–21 days (usually about 14 days), and (2) a "rapid expansion" phase, in which TILs from the early growth phase are stimulated and expanded to large numbers by contact with soluble anti-CD3 antibodies, irradiated (autologous or allogeneic) feeder cells, and IL2 for approximately 14 days, typically resulting in an approximately 1000-fold expansion.

[0150] Procedures for isolating specific cell populations expressing certain cell surface molecules are well known in the art, including bead separation and fluorescence-activated cell sorting (FACS) procedures. Cell sorting procedures have been employed to first enrich cell populations for desired T cell subpopulations before expansion. For example, enrichment for CD8+ cells (Dudley et al. (2010) Clin Cancer Research 16:6122-6131) has been reported to improve clinical responses. PD1 is highly expressed on the surface of tumor-reactive TILs (Inozume, et al. (2010) J Immunotherapy 33:956-64), and enrichment of cell populations for PD1+ cells has been reported to be associated with improved clinical outcomes. Additionally, selection based on the expression of CD137 / 4-1BB as an activation marker for CD8+ T cells could be used to select tumor-reactive TILs from melanoma samples. Ye, et al. (2014) CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumors. Clin Cancer Research 20:44-55. Administration of cell products generated using these sorting protocols has been reported to provide enhanced antitumor responses.

[0151] Nevertheless, improvements in TIL therapy resulting from the selection of desired cell populations have not been achieved, as conventional TIL expansion and treatment protocols typically employ aldesleukin, which has activity similar to wild-type human IL2 (wt-hIL2). This use of wt-hIL2 creates multiple problems.

[0152] First, as previously noted, wt-hIL2 is a pluripotent cytokine that broadly activates T cells in isolated populations and does not selectively activate or stimulate the proliferation of desired tumor antigen-activated T cells. As a result, the proportion of cells that are desired antigen-experienced T cells in the cell product for reinfusion into the subject remains suboptimal.

[0153] Second, the reliance on wt-hIL2 to "support" the continued activation and proliferation of TIL cell products following administration of the TIL cell product to a subject does not selectively support the proliferation and persistence of antigen-activated T cells, and high-dose IL2 therapy in particular results in significant (potentially life-threatening) toxicities, which are well documented in the art.

[0154] Third, the inability of wt-hIL2 to selectively expand T cells that have experienced the desired antigen in a cell population isolated from a subject results in the generation of a TIL cell product that contains a majority of non-tumor antigen-specific T cells. To provide a therapeutically sufficient number of TILs activated by the desired antigen, TIL cell products prepared using conventional methods result in the need to administer TIL cell products containing a very large number of cells. The large number of cells in conventionally prepared TIL cell products often requires lymphodepletion of the subject to enable successful engraftment of the large number of cells in the conventionally prepared TIL cell product. As mentioned above, the use of such lymphodepleting treatment regimens alone often has significant toxicity, requiring treatment in a hospital setting, and leaves the subject susceptible to infection from other sources.

[0155] The following is a discussion of a series of experiments that were performed to demonstrate the utility of αβhIL2 muteins in practicing the methods of the present disclosure. Details regarding experimental specifications are provided in the Examples.

[0156] The present disclosure provides αβhIL2 mutein compositions and methods of use thereof for preferentially activating tumor antigen-experienced T cells in a mixed cell population. As demonstrated by the experimental data provided herein, αβhIL2 muteins selectively activate tumor-specific, antigen-experienced T cells in a mixed cell population.

[0157] To perform these extensive in vitro characterization and in vivo studies demonstrating the utility of the disclosed αβhIL2 muteins in the effective treatment of neoplastic disease in mammalian subjects, exemplary αβhIL2 muteins containing amino acid substitutions at positions L18, Q22, and Q126 were used. As previously noted, modifications of hIL2 at positions L18, Q22, and Q126 substitutions provide hIL2 muteins with altered affinity for hCD132, yet still typically exhibit binding to hCD25 and hCD122 equivalent to wild-type hIL2. Two representative αβhIL2 muteins modified at positions L18, Q22, and Q126 were prepared: (1) desAla1-hREH (referred to as "REH" or "hREH") with the amino modifications des-Ala1, L18R, Q22E, and Q126, and (2) desAla1-hREK (referred to as "REK" or "hREK") with the amino modifications des-Ala1, L18R, Q22E, and Q126K. A surrogate mouse IL2 (mIL2) mutein (mREH) containing amino acid substitutions L32R, Q36E, and Q141H ("mREH") numbered according to mature mouse IL2 (UniProt P04351; SEQ ID NO:5) corresponds to the αβhIL2 mutein containing the amino acid substitutions L18R, Q22E, and Q126K of REK. Samples of the aforementioned IL2 mutein polypeptides have been recombinantly produced in Escherichia coli (E. coli) using conventional recombinant DNA techniques and isolated in substantially pure form by conventional procedures including dialysis, ion exchange chromatography, and size exclusion chromatography. By deleting the alanine typically present at position 1 of the hIL2 molecule, the N-terminal methionine is more efficiently removed by the bacterial producer cells.This is due to the proline at the position following the N-terminal methionine rather than alanine, resulting in the expression and recovery of a substantially more homogeneous hIL2 product, which offers both economic and technical advantages, such as increased process efficiency, lower costs, and simplified purification and refolding to produce a substantially pure, homogeneous protein product that results in a more consistent reagent when additional agents, such as carriers or targeting molecules, are conjugated to the N-terminus of the hIL2 polypeptide. As shown in previous reports and confirmed by this study, the removal of alanine at position 1 does not substantially alter the biological activity of the resulting hIL2 polypeptide. αβhIL2 mutein test agents were prepared substantially according to the teachings of the Examples.

[0158] To demonstrate the effectiveness of αβIL2 muteins for preferentially expanding tumor antigen-experienced T cells in isolated mixed cell populations, a series of experiments was performed in a mouse model demonstrating that a population of immune cells extracted from tumor tissue, when cultured ex vivo in the presence of αβIL2 muteins, preferentially expanded tumor antigen-experienced lymphocytes in the mixed cell population. Parameters involved in the mouse model are provided in the Examples.

[0159] To model the activity of human αβIL2 muteins in a mouse environment, mouse αβIL2 muteins were prepared and evaluated to demonstrate comparable activity. Data presented elsewhere herein demonstrate that human αβIL2 muteins containing the amino acid substitutions L18R, Q22E, and Q126K ("REK") have significant antitumor efficacy in human tumor models.

[0160] To demonstrate that the murine REH αβIL2 mutein is a suitable surrogate for human REK for use in mouse studies, the ability of REH and REK to provide signaling through the IL2 receptor, as assessed by STAT5 phosphorylation, was evaluated in YT CD25 (CD25 positive) and YT (CD25 negative) cells. Briefly, 293T cells were transfected with the IL2 mutein construct, and after 2–3 days, the supernatant containing soluble REK and REH IL2 muteins was removed. After 20 min of stimulation, the supernatant was added to YT and YT CD25 cells. YT cells are an NK lymphoma cell line that does not endogenously express detectable levels of CD25. The IL2 response of YT cells endogenously expressing CD25 and derived YT cells ("YT CD25") was compared. Untransfected cells, cells transfected with an empty expression cassette, and wild-type human IL2 were included in this study as controls. pSTAT5 levels were measured by flow cytometry. IL2 concentrations in the supernatants were measured by MSD assay. The mean fluorescence intensity data generated from this experiment are provided in Tables 2 and 3 below.

[0161] Table 2. MFI of pSTAT5 in treated YT CD25+ cells TIFF2024529340000007.tif98157

[0162] Table 3. MFI of P-STAT5 in treated YT (CD25-negative) cells TIFF2024529340000008.tif101157

[0163] As demonstrated by the data presented above, REH and REK provide selective activation of CD25-positive T cells relative to CD25-negative T cells, as demonstrated by enhanced pSTAT5 production in immune cells expressing the high-affinity trimeric CD25 / CD122 / CD132 hIL2 receptor (YT CD25 cells) compared with pSTAT5 in immune cells expressing the intermediate-affinity dimeric CD122 / CD132 hIL2 receptor, YT cells.

[0164] To further validate mREH as a valid alternative to hREK, a study was conducted to evaluate the relative potency of human wild-type IL-2 (huIL-2), REH, and REK by comparing the EC50 of each molecule for phospho-STAT5 (pSTAT5) induction in primary human CD8+ T cells activated by anti-CD3 / anti-CD28 stimulation and primary human NK cells. Both cell types were isolated from fresh donor peripheral blood mononuclear cells (PBMCs). Because REH is a murine IL-2 mutein, it was tested on an equivalent cell population freshly isolated from mouse spleen. The results of this study are provided in Table 4 below.

[0165] Table 4. Potency of human and mouse IL2 muteins in the pSTAT5 assay TIFF2024529340000009.tif36160

[0166] The data provided in Table 4 demonstrate that REH represents a reasonable surrogate for REK for use in in vivo efficacy models, as it has similar target specificity for mouse cells as REK exhibits for human cells.

[0167] Having established the REH mIL2 mutein as a suitable murine surrogate for the REK αβhIL2 mutein, studies were conducted in a murine setting to evaluate the efficacy of REH in selectively expanding tumor antigen-specific T cells from murine MC38 tumors. The MC38 tumor cell line is derived from murine colon adenocarcinoma cells and forms neoplastic lesions when implanted in mice. A detailed protocol for this experiment is provided in the accompanying Examples. Briefly, MC38 tumor cells were implanted subcutaneously into a series of female C57 / B16 mice. 14 days after tumor cell implantation, the mice were sacrificed and tumors were harvested. The isolated tumors were enzymatically digested, and CD4+ and CD8+ T cells were isolated, resulting in approximately 4×10 T cells from 55 tumors. 7The resulting CD4+ and CD8+ T cells were cultured in the presence of wild-type mouse IL2 (wt-mIL2) or REH (a murine surrogate of the REK human αβ-biased hIL2 mutein). Wild-type IL2 was included as a control. Approximately 5–7 days later, cells were harvested and plated with tumor target cells. MC38 tumor cells were used as the target cell line expressing the cognate tumor antigen, while lineage-matched C57BL / 6 and B16 cells were used as negative controls. Cells were incubated with the target cells overnight; after 16 h, the protein transport inhibitor monensin was added for 4–5 h. After this incubation, cells were stained for flow cytometry analysis. To demonstrate that the expansion of activated cells using the αβ-biased mIL2 mutein REH differed from that using wt-mIL2, live CD8+ T cells were isolated by FACS and then analyzed for IFN-gamma expression.

[0168] The results of these experiments are provided in Figures 1 and 2 of the accompanying drawings. As shown in the figures, wt-mIL2 increased the proliferation of T cells responding to both MC38 cells and lineage-matched B16 cells, resulting in nonselective expansion of T cells. In contrast, culture of antigen-activated T cells in REH αβIL2 mutein expanded T cells responding to MC38 cells, but significantly less expansion to lineage-matched B16 cells. This data demonstrates that TIL expansion in the presence of αβIL2 mutein results in the selective expansion of antigen-activated TILs that specifically bind to antigens presented on tumor cells in the subject from which the TIL cells were isolated. Consequently, the use of αβIL2 mutein for ex vivo expansion of TILs in the preparation of a TIL cell product results in a TIL cell product that is specifically enriched for tumor-specific antigen-activated TILs. Because the nature of the immune response in humans is to generate a polyclonal immune response, isolated TILs provide multiple T cell clones that react with tumor antigens, and the ability of αβIL2 muteins to selectively activate antigen-activated TILs provides a TIL cell product containing multiple tumor antigen-specific T cell clones.The methods and compositions of the present invention facilitate the generation of a population enriched for multiple tumor antigen-specific T cell clones, thereby enabling the provision of an enhanced polyclonal immune response for the treatment of neoplastic diseases.

[0169] The foregoing discussion demonstrates the ability of the disclosed compositions and methods to be useful ex vivo for preparing TIL cell products enriched for tumor antigen-specific activated T cells. The disclosed compositions and methods further provide methods for activating, maintaining, and expanding antigen-activated TILs in vivo.

[0170] In a preferred embodiment, the TIL cell product used in combination with the αβhIL2 mutein is prepared by the above-described method to provide a TIL cell product that is substantially enriched in tumor antigen-specific activated TILs, although the αβIL2 mutein may be administered to a subject in combination with a TIL cell product prepared using conventional TIL preparation protocols, such as the selected TIL method or young TIL method described herein.

[0171] As noted, administration of wt-hIL2 to a subject in combination with TIL therapy does not provide selective support for the proliferation and persistence of antigen-activated T cells and results in significant (potentially life-threatening) toxicity, especially with high-dose IL2 therapy. A series of experiments was performed demonstrating that (1) the αβhIL2 muteins of the present disclosure selectively activate antigen-activated CD8+ T cells in vivo, and (2) the αβhIL2 muteins do not result in the systemic toxicity associated with administration of wt-hIL2, the standard of care in the art.

[0172] As described herein, a series of αβhIL2 muteins were prepared. A representative αβhIL2 mutein containing a deletion of the N-terminal alanine residue and the amino acid substitutions L18R, Q22E, and Q126K ("hIL2-REK") and its murine surrogate mREH (described above) were selected for evaluation in primate and mouse systems.

[0173] The ability of αβ-hIL2 muteins to activate and proliferate TCR-activated T cells was evaluated in vitro and in vivo. The in vivo half-life of wild-type IL2 molecules (including most IL2 muteins) is short in mammalian subjects, typically on the order of minutes. To improve their pharmacokinetic properties, IL2 molecules are often modified to provide a prolonged half-life in vivo. Various methodologies for extending the in vivo half-life of IL2 molecules are applicable to αβ-IL-2 muteins and are described in more detail below. To demonstrate the selectivity of αβ-IL-2 mutein hREK and its murine surrogate mREH, a series of in vivo experiments was performed in which αβ-IL-2 human and murine muteins were modified by N-terminal covalent attachment of 40 kDa branched (2 × 20 kDa) polyethylene glycol ("PEG") moieties to the IL2 muteins using conventional aldehyde chemistry.

[0174] αβ-IL-2-PEG was evaluated in non-human primates. As a comparator and to demonstrate that retention of CD25 binding in the hIL2 mutein is a factor in the expansion of antigen-activated cells, primates were also dosed with a similarly PEGylated version of the neo-2 / 15 non-α-IL2 mutein ("non-α-hIL2-PEG") described in Silva, et al. (2019) Nature 565:186-19. As illustrated by the data presented in Figure 3, αβ-IL-2-PEG inhibited STAT5 phosphorylation and CD25 hi CD122 + CD8 + Preferentially induced in T cells, CD25 + CD122 - or CD25 - CD8 +The data presented in Figure 4 of the accompanying drawings demonstrate that the ability of the PEGylated αβhIL2 mutein to selectively activate CD25+CD8+ T cells was maintained over a wide dose range. As shown in the figure, the PEGylated αβhIL2 analog provided sustained high levels of STAT5 phosphorylation in CD25+CD8+ T cells at all doses evaluated. In contrast, the PEGylated αβhIL2 mutein significantly reduced CD25+CD8+ T cell activation at both doses. neg This resulted in significantly lower levels of STAT5 phosphorylation in CD8+ T cells. These data demonstrate that the PEGylated αβhIL2 mutein provides sustained activation of CD8+ T cells and that such activation is regulated by the expression of CD25 on these CD8+ T cells.

[0175] The data presented in Figure 5 of the accompanying drawings demonstrate that the αβhIL2 mutein, αβ-IL-2-PEG, induced proliferation of CD25+ CD8+ T cells immediately within one day of injection. As shown in the figure, at the 56-hour time point, the PEGylated αβhIL2 mutein at both the 250 μg / kg and 20 μg / kg doses resulted in a significant increase in the percentage of KI-67+ CD8+ T cells in the samples. In contrast, at the 56-hour time point, the PEGylated αβhIL2 mutein at both the 250 μg / kg and 20 μg / kg doses resulted in only a slight increase in the percentage of KI67+ CD8+ T cells in the samples. These data demonstrate that the PEGylated αβhIL2 mutein induces proliferation of CD8+ T cells and that such proliferation is regulated by the expression of CD25 on such CD8+ T cells.

[0176] In contrast, as shown in Figure 6, non-α-IL-2-PEG inhibited CD25 + CD8 + T cells and CD25 - CD8 + T cell proliferation equally, indicating that such agents induce the proliferation of both CD25 + CD8 +These data demonstrate that PEGylated non-αhIL2 muteins are unable to selectively stimulate T cell proliferation. neg and CD25 pos These data demonstrate that the PEGylated non-αhIL2 mutein results in activation of CD8+ T cells regardless of CD25 status and does not provide the selective activation of CD25+ CD8+ T cells observed with PEGylated αβhIL2, as shown in Figure 5.

[0177] To demonstrate the long-term in vivo half-life of the PEGylated αβhIL2 mutein, serum samples were obtained from the non-human primate model described above, and IL-2 levels in the samples were determined. The results of the study are presented in Figure 7 of the accompanying drawings. The data presented in Figure 7 show that the PEGylated αβhIL2 mutein provides sustained serum levels greater than about 10 ng / ml over a 168-hour period in response to a single subcutaneous administration of the PEGylated αβhIL2 mutein at both the 250 μg / kg and 20 μg / kg doses.

[0178] To assess the duration of action of the PEGylated αβhIL2 mutein, samples obtained above from primates treated with the PEGylated non-α-hIL2 mutein and PEGylated αβhIL2 were evaluated for the percentage of activated CD25+ CD8+ cells (represented by STAT5 activity, y-axis) over time (x-axis). The data show that the percentage of STAT5+ CD8+ CD25+ T cells following subcutaneous administration of either 250 μg / kg or 20 μg / kg doses of the PEGylated αβhIL2 mutein provided significant levels of sustained activation of CD25+ CD8+ cells over the course of the study (168 hours or 7 days). In contrast, the percentage of STAT5+ CD8+ CD25+ T cells declined rapidly following administration of the PEGylated non-α-hIL2 mutein. Taken together, this data demonstrates that not only does the PEGylated αβhIL2 mutein have a long in vivo lifespan, but that over this period the agent is maintained at levels that provide a significant activation effect on CD8+ CD25+ T cells.

[0179] To demonstrate that αβhIL2 muteins have in vivo antitumor activity and that administration of this activity correlates with the observed increase in CD8+CD25+ TILs in response to αβhIL2 muteins, a series of studies were conducted in mice using MC38 mouse tumors and murine IL2 muteins. Initially, the efficacy of different IL2 muteins was evaluated in a murine MC38 tumor model, substantially according to the MC38 model and examples described above. The study design and dosing schedule are described in panel A of Figure 9 of the accompanying drawings. Mice were treated with 10 μg of murine PEGylated αβ(REH)mIL2 in different dosing regimens: a dose of 2.5 mg of PEGylated wild-type murine IL2 and a PEGylated murine version of the neo2 / 15 molecule at 3 mg. As shown in Figure 9, panel B, mice treated with the highest non-lethal dose regimen of wt-mIL2-PEG reduced the growth of syngeneic MC-38 mouse colon cancer, but did not result in any complete responses (CRs). As shown in Figure 9, panels B and C, non-α-IL2-PEG was less effective than mIL-2-PEG and did not induce complete responses in mice. In contrast, αβ-IL2-PEG induced complete responses in more than 50% of treated mice. Furthermore, administration of αβIL2 muteins, either as monotherapy or in combination with other complementary agents, is useful for the treatment of neoplastic diseases, providing specific support for TIL cell production and enhancing the persistence of antigen-activated T cells.

[0180] T cell responses against tumor-derived neoantigens are thought to promote antitumor responses in patients. Rizvi, et al. (2015) Science 348:124-128. The αβ-IL2-PEG mutein was designed to preferentially target antigen-activated CD25+ T cells. Studies in mice were conducted to demonstrate that the αβ-IL2 mutein selectively activates tumor antigen-specific CD8+ tumor-infiltrating T cells (TILs). The study design and results are presented in Figure 10 of the accompanying drawings. Briefly, mice were injected with MC38 tumor cells and treated with various test agents (PBS, αβ-mIL2-PEG mutein, PEGylated wild-type murine IL2 (mIL2 PEG), and PEGylated non-α-IL2) according to the schedule shown in Figure 10, panel A. On day 18 of the study, tumors from each treatment group were harvested, lymphocytes (TILs) were isolated from the tumors corresponding to each treatment group, and the isolated cell populations were further sorted for CD25 expression into two subpopulations: CD8+ CD25+ and CD8+ CD25- T cells. Each subpopulation was exposed to MC38 tumor cells ex vivo, and the levels of IFNg, GM-CSF, and TNFa in each population in response to re-exposure to MC38 tumor cells were analyzed. The results of this study are presented in Figure 10, panels C, D, and E. As shown in the figure, CD25- CD25+ T cells isolated from tumors in mice exposed to TILs did not secrete IFNg in response to tumor challenge, whereas CD25+ TILs from αβ-IL2-PEG-treated mice secreted high levels of IFNg, GM-CSF, and TNFα. In these studies, mIL2-PEG exhibited reduced cytokine secretion compared to αβ-IL2-PEG, whereas non-α-IL2-PEG did not support antigen-reactive T cells. It should be noted that the tumor cell-specific activity of TILs associated with various treatment agents correlated with their respective efficacy observed in the MC-38 tumor efficacy model data presented in Figure 9.

[0181] Aside from the significant toxicity of high-dose (HD-) hIL2 therapy used in conjunction with TIL therapy described above, wt-hIL2 is nonselective, and as a result, relying on wt-hIL2 to "support" the continued activation and proliferation of the TIL cell product after administration to a subject does not provide residual enrichment for tumor antigen-activated cells. As described in more detail below, administration of an αβIL2 mutein in combination with reinfusion of the TIL cell product provides specific support for antigen-activated T cells, further reducing or avoiding the toxicity associated with administration of wt-hIL2, particularly HD-hIL2 therapy.

[0182] wt-hIL2 activates the high-affinity trimeric IL2 receptor (IL2Rαβ / γ) or intermediate-affinity receptor present on antigen-activated T cells and regulatory T cells (Tregs), as well as the intermediate-affinity dimeric receptor (IL2Rβ / γ) expressed on naive and resting T cells and NK cells. The αβIL2 mutein preferentially activates cells expressing the high-affinity trimeric receptor (e.g., tumor-specific CD25+ CD8+ T cells) over cells expressing the intermediate-affinity receptor (e.g., NK cells).

[0183] Acute toxicity in hIL2-treated patients includes vascular leak syndrome (VLS), which results in edema in peripheral tissues and lungs and limits blood oxygenation. Patients receiving high-dose IL2 therapy (HD-IL2) may begin to experience significant toxicity after 2 days and may require supportive care. Dutcher, et al. (2014) Journal for immunotherapy of cancer 2:26. Two common alternative hypotheses are that VLS is due to CD25+ endothelial cells (Krieg, et al. (2010) PNAS(USA) 107:11906-11911) or CD25 -These parameters are mediated by either NK cell and granulocyte extravasation (Peace and Cheever (1989) J Exp Med 169:161-173). To assess these parameters, non-human primates were exposed for 56 hours to three different IL2 agonists: (a) hIL2 with wild-type hIL2 activity, traditionally used in clinics, referred to as "wild-type hIL2" (Proleukin®, Prometheus Laboratories); (b) a PEGylated representative αβ-hIL2 mutein containing the amino acid substitutions L18R, Q22E, and Q126K ("αβ-hIL2-PEG"); and (c) a PEGylated version of the neo-2 / 15 non-α-IL2 mutein ("non-α-hIL2-PEG") described in Silva, et al. (2019) Nature 565:186-19. Using conventional aldehyde chemistry, PEGylated versions of αβ-hIL2 and non-α-IL2 were modified by N-terminal covalent attachment of a 40 kDa, two-arm branched PEG (NOF # SunBright GL2-400AL3). Non-α-hIL2-PEG was administered intravenously at a dose of 50 micrograms / kilogram for three doses on days 1, 8, and 15 of the study. αβ-hIL2-PEG was administered subcutaneously at a dose of 250 micrograms / kilogram for three doses on days 1, 8, and 15 of the study. Eight doses of Proleukin® were administered intravenously at a dose of 37 micrograms / kilogram three times daily for eight days. Acute toxicity was assessed 56 hours after completion of treatment. Toxicity was assessed by immunohistochemistry for immune cell composition in the lungs. Chronic toxicity following weekly administration of three doses of each PEGylated agent. Meanwhile, wt hIL2 was administered three times daily for eight doses to mimic conventional clinical HD-hIL2 therapy.

[0184] Animals treated with wt-IL2 or PEGylated non-α-IL2 showed significant infiltration of CD11b+ neutrophils into the lungs, corresponding to histological findings of pulmonary edema. In particular, non-α-IL2-PEG induced a strong subendothelial infiltration of CD11b+ granulocytes. Treatment with wt-IL2 or non-α-IL2-PEG resulted in not only increased numbers of CD3+ T cells and NK cells (granzyme B+ CD3- cells), but also IFNγ expression in NK cells and their proliferation in the lungs. In contrast, αβ-IL2-PEG did not increase CD11b+ cells, and there was no significant change in the cellular infiltrate in response to treatment with αβ-hIL2-PEG.

[0185] Further results of this study are presented in Figure 11 of the accompanying drawings. Figure 11, panels A–F, show lung histology in response to various PEGylated hIL2 muteins. On day 3 of IL-2 treatment (Figure 2), lung thickening (arrows) and cellular infiltration are observed in response to aldesleukin (Panel B) and non-α-IL-2-PEG (Panel C, single dose; Panel D, two doses), but not in the control (Panel A) or αβ-IL-2-PEG (Panels E and F). While HD-IL-2 (every 8 h) or two subsequent doses of non-α-IL-2 PEG continuously induced phospho-STAT5, the duration of phospho-STAT5 was limited to 48 h after the single dose of non-α-IL-2 PEG (Panel G), and non-α-IL-2-PEG Tregs infiltrated the lungs on day 3 (Panel H). Weekly chronic dosing of non-α-IL-2-PEG in Part B of the study induced similar CD11b+ infiltrates (not shown) and increased relative lung weight compared to control or αβ-IL-2-PEG. Figure 11, Panel I.

[0186] The above data generated in non-human primates demonstrates that the αβIL2 muteins of the present disclosure provide significantly reduced toxicity compared to wild-type hIL2 therapy or non-α-hIL2 muteins, and that long-term exposure to the αβhIL2 muteins of the present disclosure does not result in the systemic toxicity associated with wt-hIL2 therapy or non-α-hIL2 muteins.

[0187] αβ-IL2s muteins, which have substantially reduced binding to the dimeric intermediate-affinity CC122 / CD132 (IL2Rβ / γ) IL2 receptor, have an improved safety profile compared to wild-type hIL2 or IL2 muteins (referred to as "non-α-IL2 muteins") that have been modified to provide reduced binding to the CD25 component of the high-affinity trimeric IL2 receptor. Because they have substantially reduced binding to the dimeric IL2Rβ / γ receptor complex, they do not substantially activate or proliferate cells expressing the dimeric IL2Rβ / γ receptor, thereby avoiding direct NK cell activation and vascular leak toxicity. αβ-IL-2 also has an improved safety profile compared to wild-type and non-α-IL-2 because it does not activate cells expressing the dimeric IL-2Rβ / γ, thereby avoiding direct NK cell activation and vascular leak toxicity.

[0188] The present disclosure provides the use of αβhIL2 muteins in the implementation of TIL therapy during both or either the ex vivo cell expansion phase and the support of the TIL cell product. TIL cell products enriched for tumor antigen-specific T cell clones, generated using the compositions and methods of the present disclosure, are useful for the ex vivo preparation and in vivo support of polyclonal anti-tumor immune responses in subjects. The ex vivo preparation of TIL cell products using αβhIL2 muteins provides a method for preparing TIL cell products substantially enriched in tumor antigen-specific activated T cells. The αβhIL2 muteins of the present disclosure provide selective in vivo support of tumor antigen-specific activated T cell clones that promote polyclonal anti-tumor immune responses useful in the treatment of neoplastic diseases. The use of αβhIL2 muteins in the ex vivo preparation of TIL cell products provides cell products significantly enriched in antigen-activated T cells, avoiding the need for lymphodepletion of the subject prior to administration of the TIL cell product or allowing the use of a less aggressive form of lymphodepletion of the subject prior to administration of the TIL cell product.

[0189] Although ex vivo use of the αβhIL2 mutein produces a TIL cell product that is substantially enriched in activated tumor antigen-specific T cells, the αβhIL2 mutein may also be used in combination TIL therapy in which the TIL cell product is prepared using conventional TIL preparation protocols. In one aspect, the present disclosure provides a method of treating a subject by administering to the subject a therapeutically effective amount of an αβhIL2 mutein in combination with administration of a TIL cell product, wherein the TILs in the TIL cell product have been expanded using conventional methodologies employing wt-hIL2 or using the αβhIL2 mutein.

[0190] The compositions and methods of the present disclosure relate to IL2 muteins. Unless otherwise specified, the following terminology and conventions are used in reference to such IL2 muteins.

[0191] In some embodiments, an αβhIL2 mutein useful in practicing the present disclosure is an IL2 mutein having 85% or more sequence identity, alternatively 90% or more sequence identity, alternatively 91% or more sequence identity, alternatively 92% or more sequence identity, alternatively 93% or more sequence identity, alternatively 94% or more sequence identity, alternatively 95% or more sequence identity, alternatively 96% or more sequence identity, alternatively 97% or more sequence identity, alternatively 98% or more sequence identity, 90% or more sequence identity to wt-hIL2 (SEQ ID NO:4), wherein the αβhIL2 mutein contains one or more amino acid substitutions at positions 18, 22, and 126 numbered according to wt-hIL2 (SEQ ID NO:4).

[0192] In some embodiments, αβhIL2 muteins useful in practicing the present disclosure comprise deletions of one, two, three, four, five, six, seven, eight, nine, or more N-terminal amino acid residues. In some embodiments, αβhIL2 muteins useful in practicing the present disclosure comprise deletions of one, two, three, four, five, six, seven, eight, or nine N-terminal amino acid residues. In some embodiments, αβhIL2 muteins useful in practicing the present disclosure comprise deletions of one, two, three, four, or five N-terminal amino acid residues. In some embodiments, αβhIL2 muteins useful in practicing the present disclosure comprise deletions of one, two, or three N-terminal amino acid residues. In some embodiments, αβhIL2 muteins useful in practicing the present disclosure comprise deletions of an N-terminal alanine amino acid residue (abbreviated as des-Ala1).

[0193] As used herein, the terms "alpha / beta-biased IL2 mutein" and "α / β-biased IL2 mutein" and "αβIL2 mutein" are used interchangeably herein to refer to an IL2 polypeptide that contains one or more structural alterations (e.g., primary structure alterations including one or more amino acid substitutions, modifications, or deletions) that have significantly reduced binding affinity for the CD132 subunit of the IL2 receptor, but substantially retain wild-type binding affinity for the CD25 and CD122 subunits of the IL2 receptor. As used herein, the terms "alpha / beta-biased hIL2 mutein" and "α / β-biased hIL2 mutein" and "αβ hIL2 mutein" are used interchangeably herein to refer to a hIL2 polypeptide that contains one or more structural alterations (e.g., primary structure alterations including one or more amino acid substitutions, modifications, or deletions) that have significantly reduced binding affinity for the hCD132 subunit of the hIL2 receptor, but retain binding affinity equivalent to wild-type hIL2 for the hCD25 and hCD122 subunits of the hIL2 receptor.

[0194] The binding affinity of an hIL2 mutein may be assessed for one or more subunits of the IL2 receptor (e.g., CD25, CD122, and / or CD132) and may be determined by techniques known in the art. As used herein, when referring to the binding affinity of an IL2 mutein for an IL2 receptor subunit, the binding affinity is determined by surface plasmon resonance ("SPR"). In assessing the binding affinity of an IL2 mutein for an IL2 receptor subunit, one member of the binding pair may be immobilized, and the other element of the binding pair may be provided in the mobile phase. In some embodiments, a "chip" for immobilizing a protein of interest is conjugated with a substance required for derivatization of the protein to be immobilized, such as an anti-His tag antibody, protein A, or biotin. As a result, in order to assess binding, it is often necessary to modify the protein to provide for binding to the chip surface and the conjugated substance. For example, an IL-2 mutein can be modified by incorporating a polyhistidine sequence for retention on a chip conjugated with an anti-his tag antibody (e.g., an anti-histidine CM5 chip commercially available from Cytiva, Marlborough MA). Alternatively, the IL2 receptor component can be immobilized on the chip, and the test agent IL2 mutein can be provided in the mobile phase. In either situation, it should be noted that modification of some proteins for immobilization on a coated SPR chip may interfere with the binding properties of one or both components of the binding pair to be assessed by SPR. In such cases, it may be necessary to switch the mobile and binding elements of the binding pair or to use a chip with a binder that promotes uninterfering conjugation of the protein to be assessed.In some embodiments, when SPR is used to evaluate the binding affinity of an α / β-biased hIL2 mutein to a hIL2 receptor subunit, the α / β-biased hIL2 mutein may be derivatized by the C-terminal addition of a poly-His sequence (e.g., 6xHis6 or 8xHis8) immobilized on an SPR chip, and the hIL2 receptor subunit to which the binding affinity of the α / β-biased hIL2 mutein is to be evaluated is provided in the mobile phase. Means for incorporating a poly-His sequence at the C-terminus of an α / β-biased hIL2 mutein produced by recombinant DNA techniques are well known to those skilled in the art of biotechnology. In some embodiments, the binding affinity of an α / β-biased hIL2 mutein to a hIL2 receptor subunit is evaluated using SPR substantially according to the teachings of Example 7 herein.

[0195] In some embodiments, αβhIL2 muteins useful in the methods of the present disclosure contain a substitution, deletion, or insertion within the wild-type hIL-2 (wt hIL2) amino acid sequence that modulates binding of the hIL2 mutein to the extracellular domain of hCD132. The following nomenclature is used herein to refer to the substitution, deletion, or insertion: Residues may be named herein by their amino acid position in IL-2 followed by their single-letter or three-letter amino acid code; for example, "Cys125" or "C125" refers to the cysteine ​​residue at position 125 of SEQ ID NO:4. Substitutions are named herein by their single-letter amino acid code followed by the amino acid position in IL-2 that it substitutes; for example, "K35A" refers to the substitution of an alanine (A) residue for a lysine (K) residue at position 35 of SEQ ID NO:5. Deletions are indicated by "des" followed by the amino acid residue and its position in SEQ ID NO:4. For example, the term "des-Ala1" or "desA1" refers to the deletion of an alanine at position 1 of the polypeptide of SEQ ID NO:4.

[0196] Unless otherwise specified, when referring to amino acid substitutions in human IL2 muteins of the present disclosure, the amino acid positions are numbered according to hIL2 as used herein and refer to the identification of the particular amino acid position relative to the position where that amino acid normally occurs in the sequence of mature wild-type IL2. In some embodiments, the IL2 is hIL2 (SEQ ID NO:4). For example, with reference to hIL2, "R81" refers to the 81st amino acid (counting from the N-terminus) arginine present in the sequence of mature wild-type hIL2.

[0197] The present disclosure relates to the use of hIL2 muteins (herein referred to as "αβhIL2 muteins") that have reduced binding affinity for hCD132 while retaining at least substantially wild-type binding affinity for hCD25 and / or hCD122.

[0198] In some embodiments, αβhIL2 muteins useful in practicing the present disclosure provide modifications that modulate the affinity of the hIL2 mutein's binding not only to individual components of the hIL2 receptor (i.e., hCD25, hCD122, and hCD132), but also to combinations thereof, such as hCD122 / hCD132 (the "intermediate-affinity hIL2 receptor"), hCD25 (the "low-affinity IL2 receptor"), and hCD25 / hCD122 / hCD132 (the "high-affinity IL2 receptor"). In some embodiments, αβhIL2 muteins useful in practicing the present disclosure have reduced binding affinity for the intermediate-affinity hIL2 receptor compared to wt-hIL2.

[0199] In some embodiments, biased hIL2 muteins useful in the methods of the present disclosure contain substitutions, deletions, or insertions within the wild-type IL-2 amino acid sequence that reduce the affinity of the αβhIL2 mutein for the extracellular domain of hCD132 while retaining significant binding to hCD25.

[0200] In some embodiments, the αβhIL2 muteins useful in the methods of the present disclosure contain substitutions, deletions, or insertions within the wild-type IL-2 amino acid sequence that reduce the affinity of the αβhIL2 mutein for the extracellular domain of hCD132 while retaining significant binding to hCD122.

[0201] In some embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure comprise one or more amino acid substitutions selected from amino acid positions 18, 22, and 126 numbered according to mature wild-type hIL-2.

[0202] In some embodiments, αβhIL2 muteins useful in practicing the methods of the disclosure have reduced binding affinity to the extracellular domain of hCD132 (e.g., <50% of the affinity of wild-type hIL2, alternatively <45% of the affinity of wild-type hIL2, alternatively <40% of the affinity of wild-type hIL2, alternatively <35% of the affinity of wild-type hIL2, alternatively <25% of the affinity of wild-type hIL2, alternatively <20% of the affinity of wild-type hIL2, alternatively <15% of the affinity of wild-type hIL2, alternatively <10% of the affinity of wild-type IL2, or alternatively <5% of the affinity of wild-type IL2). In some embodiments, the hIL2 muteins exhibit reduced binding affinity for CD132 compared to wt hIL2 and exhibit increased binding affinity for CD122 in the presence of CD25, membrane-bound CD25, or sCD25 that is equal to or greater than wt hIL2. In some embodiments, the αβhIL2 muteins of the present disclosure comprise one or more amino acid substitutions that reduce binding to the CD132 receptor. In some embodiments, the one or more amino acid substitutions that reduce CD132 receptor binding affinity are selected from amino acids at the interface between hIL2 and hCD132. The crystal structure of hIL2 and its interface with hCD132 have been published, and other studies have been conducted that have identified positions in the hIL2 molecule that have been identified as interacting with hIL2 binding to CD132, including residues L18, Q22, Q126, T123, S127, I129, and S130. αβhIL2 muteins useful in practicing the methods of the present disclosure comprise amino acid substitutions or deletions and one or more of residues L18, Q22, Q126, T123, S127, I129, and S130.

[0203] Garcia et al. (International Application No. PCT / 2018 / 062122, published May 31, 2019, PCT International Publication No. WO2019 / 104092 A1, hereinafter "Garcia '092") describe, inter alia, certain IL2 muteins having modifications involving positions 18, 22, and 126 that are useful in practicing the methods described herein, which exhibit reduced binding to CD132 while retaining partial IL2 activity.

[0204] In some embodiments, αβhIL2 muteins useful in practicing the methods of the disclosure have reduced binding affinity to the extracellular domain of hCD132 (e.g., <50% of the affinity of wild-type hIL2, alternatively <45% of the affinity of wild-type hIL2, alternatively <40% of the affinity of wild-type hIL2, alternatively <35% of the affinity of wild-type hIL2, alternatively <25% of the affinity of wild-type hIL2, alternatively <20% of the affinity of wild-type hIL2, alternatively <15% of the affinity of wild-type hIL2, alternatively <10% of the affinity of wild-type IL2, or alternatively <5% of the affinity of wild-type IL2) while maintaining substantial affinity for the extracellular domain of the wild-type human CD122 receptor (e.g., 20% of the affinity of wild-type hIL2, alternatively >30%, alternatively >40%, alternatively The antibody retains a binding affinity of >50% of the affinity of wild-type hIL2, alternatively >60% of the affinity of wild-type hIL2, alternatively >65% of the affinity of wild-type hIL2, alternatively >70% of the affinity of wild-type hIL2, alternatively >75% of the affinity of wild-type hIL2, alternatively >80% of the affinity of wild-type hIL2, alternatively >85% of the affinity of wild-type hIL2, alternatively >90% of the affinity of wild-type IL2, alternatively >90% of the affinity of wild-type IL2, alternatively >95% of the affinity of wild-type IL2, alternatively >100% of the affinity of wild-type hIL2, alternatively >105% of the affinity of wild-type hIL2, alternatively >110% of the affinity of wild-type IL2, alternatively >115% of the affinity of wild-type hIL2, alternatively >125% of the affinity of wild-type IL2, or alternatively >150% of the affinity of wild-type hIL2.

[0205] In some embodiments, an αβhIL2 mutein useful in practicing the methods of the present disclosure has reduced binding affinity for the extracellular domain of the hCD132 receptor and further comprises one, two, three, four, five, six, seven, eight, nine, ten, or more mutations that increase affinity for the extracellular domain of the wild-type human CD122 receptor. In certain embodiments, a subject hIL-2 mutein useful in practicing the methods of the present disclosure comprises at least one mutation (e.g., deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) relative to wild-type IL-2 (e.g., SEQ ID NO:4) and binds to CD122 with greater affinity than wild-type IL-2. In certain embodiments, the IL-2 mutein binds to CD122 with at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more higher affinity than wild-type hIL-2. The binding affinity of the hIL-2 mutein can also be expressed as 1.2-fold, 1.4-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 100-fold, 200-fold, 250-fold or more higher affinity for the extracellular domain of hCD122 than wild-type hIL-2.

[0206] In some embodiments, the αβhIL2 mutein: (a) has a binding affinity to the extracellular domain of hCD132 that is greater than 10% but less than about 90%, alternatively greater than 10% but less than about 80%, alternatively greater than 10% but less than about 70%, alternatively greater than 10% but less than about 60%, alternatively greater than 10% but less than about 50%, alternatively greater than 10% but less than about 40%, or alternatively greater than 5% but less than about 40% of that of wild-type hIL2; and (b) has a binding affinity to the extracellular domain of hCD122 that is greater than 10% but less than about 90%, alternatively greater than 10% but less than about 80%, alternatively greater than 10% but less than about 70%, alternatively greater than 10% but less than about 60%, alternatively greater than 10% but less than about 50%, alternatively greater than 10% but less than about 40%, or alternatively greater than 5% but less than about 40% of that of wild-type hIL2. (c) has a binding affinity to the extracellular domain of hCD122 that is greater than 50%, alternatively greater than 60%, alternatively greater than 70%, alternatively greater than 80%, alternatively greater than 90%, alternatively greater than 100%, alternatively greater than 120%, alternatively greater than 150%, or alternatively greater than 200% compared to hIL2; and (c) has a binding affinity to the extracellular domain of hCD122 that is greater than 50%, alternatively greater than 60%, alternatively greater than 70%, alternatively greater than 80%, alternatively greater than 90%, alternatively greater than 100%, alternatively greater than 120%, or alternatively greater than 150% compared to wild-type hIL2.

[0207] In some embodiments, the αβhIL2 mutein: (a) has a binding affinity to the extracellular domain of hCD132 that is greater than 10% but less than about 90%, alternatively greater than 10% but less than about 80%, alternatively greater than 10% but less than about 70%, alternatively greater than 10% but less than about 60%, alternatively greater than 10% but less than about 50%, alternatively greater than 10% but less than about 40%, or alternatively greater than 5% but less than about 40% of that of wild-type hIL2; and (b) has a binding affinity to the extracellular domain of hCD122 that is greater than 50%, alternatively greater than 60%, alternatively greater than 70%, alternatively 80% of that of wild-type hIL2. (c) has a binding affinity for the extracellular domain of hCD122 that is greater than 50%, alternatively greater than 60%, alternatively greater than 70%, alternatively greater than 80%, alternatively greater than 90%, alternatively greater than 100%, alternatively greater than 120%, alternatively greater than 150%, or alternatively greater than 200% relative to wild-type hIL2; and (d) the amino acid sequence of the biased IL2 mutein is greater than 70%, alternatively greater than 80%, alternatively greater than 90%, alternatively greater than 95% identical to the amino acid sequence of wild-type hIL2.

[0208] In some embodiments, αβhIL2 muteins useful in practicing the methods of the disclosure have reduced binding affinity with the extracellular domain of hCD132 (e.g., <50% of the affinity of wild-type hIL2, alternatively <45% of the affinity of wild-type hIL2, alternatively <40% of the affinity of wild-type hIL2, alternatively <35% of the affinity of wild-type hIL2, alternatively <25% of the affinity of wild-type hIL2, alternatively <20% of the affinity of wild-type hIL2, alternatively <15% of the affinity of wild-type hIL2, alternatively <10% of the affinity of wild-type hIL2, or alternatively <5% of the affinity of wild-type hIL2), while retaining substantial affinity for the hCD25 / hCD122 receptor complex (e.g., >50% of the affinity of wild-type hIL2, alternatively <45% of the affinity of wild-type hIL2, alternatively <40% of the affinity of wild-type hIL2, alternatively <35% of the affinity of wild-type hIL2, alternatively <25% of the affinity of wild-type hIL2, alternatively <20% of the affinity of wild-type hIL2, alternatively <15% of the affinity of wild-type hIL2, alternatively <10% of the affinity of wild-type hIL2, or alternatively <5% of the affinity of wild-type hIL2). to >60% of the affinity of wild-type hIL2, alternatively >65% of the affinity of wild-type hIL2, alternatively >70% of the affinity of wild-type hIL2, alternatively >75% of the affinity of wild-type hIL2, alternatively >80% of the affinity of wild-type hIL2, alternatively >85% of the affinity of wild-type hIL2, alternatively >90% of the affinity of wild-type hIL2, alternatively >90% of the affinity of wild-type hIL2, alternatively >95% of the affinity of wild-type hIL2, alternatively >100% of the affinity of wild-type hIL2, alternatively >105% of the affinity of wild-type hIL2, alternatively >110% of the affinity of wild-type hIL2, alternatively >115% of the affinity of wild-type hIL2, alternatively >125% of the affinity of wild-type hIL2, or alternatively >150% of the affinity of wild-type hIL2. In certain embodiments, the αβhIL2 muteins of the present disclosure have reduced affinity for CD 132. In some embodiments, such αβhIL2 muteins incorporate modifications in the primary structure of wild-type IL2, incorporating one or more modifications at positions 18, 22, and 126 numbered according to wild-type hIL2.

[0209] In some embodiments, αβhIL2 muteins useful in practicing the methods of the disclosure have reduced binding affinity to CD132 while maintaining substantial affinity for hCD25 (e.g., >50% of the affinity of wild-type hIL2, alternatively >60% of the affinity of wild-type hIL2, alternatively >65% of the affinity of wild-type hIL2, alternatively >70% of the affinity of wild-type hIL2, alternatively >75% of the affinity of wild-type hIL2, alternatively >80% of the affinity of wild-type hIL2, alternatively >85% of the affinity of wild-type hIL2, alternatively >90% of the affinity of wild-type hIL2, alternatively >95% of the affinity of wild-type hIL2). Alternatively, the affinity of the wild-type hIL2 is >90%, alternatively >95% of the affinity of the wild-type hIL2, alternatively >100% of the affinity of the wild-type hIL2, alternatively >105% of the affinity of the wild-type hIL2, alternatively >110% of the affinity of the wild-type hIL2, alternatively >115% of the affinity of the wild-type hIL2, alternatively >125% of the affinity of the wild-type hIL2, alternatively >150% of the affinity of the wild-type hIL2, alternatively >200% of the affinity of the wild-type hIL2, alternatively >300% of the affinity of the wild-type hIL2, alternatively >400% of the affinity of the wild-type hIL2, alternatively >500% of the affinity of the wild-type hIL2.

[0210] In some embodiments, the αβhIL2 mutein: (a) has greater than 10% but less than about 90%, alternatively greater than 10% but less than about 80%, alternatively greater than 10% but less than about 70%, alternatively greater than 10% but less than about 60%, alternatively greater than 10% but less than about 50%, alternatively greater than 10% but less than about 40%, or alternatively greater than 5% but less than about 40% of the binding affinity of wild-type hIL2 to the extracellular domain of hCD132; (c) has a binding affinity to the extracellular domain of hCD25 that is greater than 50%, alternatively greater than 60%, alternatively greater than 70%, alternatively greater than 80%, alternatively greater than 90%, alternatively greater than 100%, alternatively greater than 120%, alternatively greater than 150%, or alternatively greater than 200% compared to hIL2; and (d) has a binding affinity to the extracellular domain of hCD25 that is greater than 50%, alternatively greater than 60%, alternatively greater than 70%, alternatively greater than 80%, alternatively greater than 90%, alternatively greater than 100%, alternatively greater than 120%, or alternatively greater than 150% compared to wild-type hIL2.

[0211] In some embodiments, the αβhIL2 mutein: (a) has a binding affinity to the extracellular domain of hCD132 that is greater than 10% but less than about 90%, alternatively greater than 10% but less than about 80%, alternatively greater than 10% but less than about 70%, alternatively greater than 10% but less than about 60%, alternatively greater than 10% but less than about 50%, alternatively greater than 10% but less than about 40%, or alternatively greater than 5% but less than about 40% of that relative to wild-type hIL2; (b) has a binding affinity to the extracellular domain of hCD122 that is greater than 50%, alternatively greater than 60%, alternatively greater than 70%, alternatively greater than 80%, %, alternatively greater than 90%, alternatively greater than 100%, alternatively greater than 120%, alternatively greater than 150%, or alternatively greater than 200%; (c) has a binding affinity for the extracellular domain of hCD25 that is greater than 50%, alternatively greater than 60%, alternatively greater than 70%, alternatively greater than 80%, alternatively greater than 90%, alternatively greater than 100%, alternatively greater than 120%, or alternatively greater than 150% relative to wild-type hIL2; and (d) the amino acid sequence of the biased IL2 mutein is greater than 70%, alternatively greater than 80%, alternatively greater than 90%, alternatively greater than 95% identical to the amino acid sequence of wild-type hIL2.

[0212] In certain embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure disrupt the association of CD122 with CD132 (i.e., formation of an intermediate-affinity IL2 receptor complex) such that this CD122 / CD132 interaction is reduced by about 2%, about 5%, about 10%, about 15%, about 20%, about 50%, about 75%, about 90%, about 95% or more compared to wild-type hIL-2. In some embodiments, the one or more mutations that reduce the binding affinity of the αβhIL2 mutein for CD132 are amino acid substitutions. In some embodiments, a subject αβhIL2 mutein consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to wild-type IL-2 (SEQ ID NO:4).

[0213] In certain embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure disrupt the association of the hCD25 / hCD122 complex with hCD132, such that the interaction between hCD25 / hCD122 and hCD132 (i.e., formation of a high-affinity IL2 receptor complex) is reduced by about 2%, about 5%, about 10%, about 15%, about 20%, about 50%, about 75%, about 90%, about 95% or more compared to wild-type hIL-2. In some embodiments, the one or more mutations that reduce the binding affinity of the αβhIL2 mutein for hCD132 are amino acid substitutions. In some embodiments, a subject αβhIL2 mutein consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to wild-type IL-2 (SEQ ID NO:4).

[0214] In some embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure are partial agonists with reduced ability to stimulate signaling in CD25neg cells compared to wild-type hIL-2. In some embodiments, αβhIL2 muteins stimulate pERK1 / ERK2 signaling in CD25neg cells at 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less of the level at which wild-type IL-2 stimulates pERK1 / ERK2 signaling in the same cells. In some embodiments, the CD25neg cells are T cells. In some embodiments, the CD25neg T cells are CD8+ T cells. In other embodiments, the CD8+ T cells are activated CD25neg CD8+ T cells. In some embodiments, the CD25 cells are natural killer (NK) cells. STAT5 and ERK1 / 2 signaling can be measured, for example, by phosphorylation of STAT5 and ERK1 / 2 using any suitable method known in the art.

[0215] In certain embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure have a reduced CD25neg In some embodiments, the CD25 neg The cells are natural killer (NK) cells.

[0216] In some embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure that are partial agonists have one or more reduced functions compared to wild-type IL-2.

[0217] In some embodiments, the αβhIL2 muteins useful in practicing the methods of the present disclosure are partial agonists, hi certain embodiments, the αβhIL2 muteins useful in practicing the methods of the present disclosure are partial agonists with reduced ability to stimulate one or more signaling pathways dependent on CD122 / CD132 heterodimerization.

[0218] In some embodiments, the αβhIL2 mutein has a reduced ability to stimulate phosphorylation in CD122+ cells compared to wild-type hIL-2. In some embodiments, the αβhIL2 mutein stimulates STAT5 phosphorylation in IL-2R+ cells at a level that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less of the level at which wild-type IL-2 stimulates STAT5 phosphorylation in the same cells.

[0219] In some embodiments, the αβhIL2 muteins useful in practicing the methods of the present disclosure are full agonists.

[0220] In some embodiments, the αβhIL2 muteins useful in practicing the methods of the present disclosure are superagonists.

[0221] In some embodiments, the one or more amino acid substitutions that provide a reduced binding affinity of the αβhIL2 mutein to the ECD of the hCD132 receptor subunit are selected from amino acids at the interface between hIL2 and the ECD of hCD132. The crystal structure of hIL2 and its interface with the ECD of hCD132 have been published, and other studies have been performed that have identified residues L18, Q22, Q126, T123, S127, I129, and S130 of wt-hIL2 as being involved in binding between wt-hIL2 and the ECD of hCD132. In some embodiments, the αβhIL2 mutein contains amino acid substitutions at positions 18, 22, and / or 126 numbered by wt-hIL2. As noted above, the numbering of residues in the αβhIL2 muteins of the present disclosure follows the numbering of residues in the mature (signal peptide-lacking) form of wild-type human IL2 (SEQ ID NO:4).

[0222] In some embodiments, the amino acid substitution at residue L18 of the αβhIL2 mutein is selected from the group consisting of L18R, L18G, L18M, L18F, L18E, L18H, L18W, L18K, L18Q, L18S, L18V, L18I, L18Y, L18H, L18D, L18N and L18T.

[0223] In some embodiments, the amino acid substitution at residue Q22 of the αβhIL2 mutein is selected from the group consisting of Q22F, Q22E, Q22G, Q22A, Q22L, Q22M, Q22F, Q22W, Q22K, Q22S, Q22V, Q22I, Q22Y, Q22H, Q22R, Q22N, Q22D, Q22T, and F.

[0224] In some embodiments, the amino acid substitution at residue Q126 of the αβhIL2 mutein is selected from the group consisting of Q126H, Q126M, Q126K, Q126C, Q126D, Q126E, Q126G, Q126I, Q126R, Q126S, or Q126T.

[0225] In some embodiments, the αβhIL2 mutein comprises a substitution at residue S130 selected from the group consisting of S130R and S130G.

[0226] In some embodiments, the αβhIL2 mutein has the following mutations at positions 18, 22, and 126: the leucine at position 18 (L18) is substituted with an amino acid selected from the group consisting of R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D and T; a substitution of the glutamine at position 22 (Q22) with an amino acid selected from the group consisting of E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, and F; and A glutamine at position 126 (Q126) is substituted with an amino acid selected from the group consisting of H, M, K, C, D, E, G, I, R, S, and T. It is a hIL2 mutein comprising:

[0227] In some embodiments, the αβhIL2 mutein has the following mutations at positions 1, 18, 22, and 126: Alanine (A1) at position 1 is deleted (des-Ala1), the leucine at position 18 (L18) is substituted with an amino acid selected from the group consisting of R, L, G, M, F, E, H, W, K, Q, S, V, I, Y, H, D and T; a substitution of the glutamine at position 22 (Q22) with an amino acid selected from the group consisting of E, G, A, L, M, F, W, K, S, V, I, Y, H, R, N, D, T, and F; and A glutamine at position 126 (Q126) is substituted with an amino acid selected from the group consisting of H, M, K, C, D, E, G, I, R, S, and T. It is a hIL2 mutein comprising:

[0228] In some embodiments, the αβhIL2 mutein comprises the following set of mutations: L18R, Q22E, and Q126H; L18R, Q22E, and Q126K; L18R, Q22E, and Q126M; L18R, Q22E Q126T; L18R; Q22E; V91K; V91R; Q126H; L18R, and Q126H; Q22E, and Q126H; L18G, Q22E, and Q126H; L18A, Q22E, and Q126H; L18M, Q22E, and Q126H; L18F, Q22E, and Q126H; L18W, Q22E, and Q126H; L18K, Q22E, and Q126H; L18 Q, Q22E and Q126H;L18E, Q22E and Q126H;L18S, Q22E and Q126H;L18V, Q22E and Q126H;L18I, Q22E and Q126H;L18Y, Q22E and Q126H;L18H, Q22E and Q126H;L18N, Q22E and Q126H;L18D, Q22E and Q126H;L18T, Q22E and Q126H;L18R, Q22G and Q126H;L18R, Q22A and Q126H;L18R, Q22L and Q126H;L18R, Q22M and Q126H;L18R, Q22F and Q126H;L18R, Q22W and Q126H;L18R, Q22K and Q126H;L18R, Q22S and Q126H;L18R, Q22V and Q126H;L18 L18R, Q22Y and Q126H; L18R, Q22H and Q126H; L18R, Q22R and Q126H; L18R, Q22N and Q126H; L18R, Q22D and Q126H; and L18R, Q22T and Q126H, and in each case may further comprise a deletion of the N-terminal alanine residue (des Ala1).

[0229] In some embodiments, the αβhIL2 mutein comprises the set of amino acid substitutions provided in Table 5 below at positions 18, 22, and 126 (numbered by hIL2) (each row corresponds to a set of amino acid substitutions). As a convenient nomenclature for these molecules, the molecules are referred to by the amino acids at positions 18, 22, and 126, such that a hIL2 mutein containing the substitutions L18W, Q22E, and Q126H is referred to as "WEH." These three-letter abbreviations are reflected in Figures 12 and 13 of the accompanying drawings.

[0230] Table 5. α / β-biased hIL2 substitutions at positions 18, 22, and 126 TIFF2024529340000010.tif196144TIFF2024529340000011.tif142144

[0231] When wild-type hIL2 is expressed endogenously in mammalian cells, it is expressed as a preprotein containing a signal peptide that is efficiently cleaved in mammalian cells to produce an alanine residue (Ala1) at the N-terminal amino acid of the mature hIL2 polypeptide. Expression of αβhIL2 muteins in mammalian cells is possible, but expression is typically more expensive than bacterial cell production, and expression in mammalian cells can also result in non-native glycosylation of the αβhIL2 mutein, depending on the cell line used. Consequently, production of αβhIL2 muteins in bacterial cells may be preferred in certain situations. However, direct expression of αβhIL2 muteins in bacterial cells (i.e., not as a fusion protein) results in the addition of an N-terminal methionine residue. If the Ala1 characteristic of the wild-type IL2 sequence is retained in the αβhIL2 mutein, this results in a proline at the +2 position relative to the N-terminal methionine. When a proline is present at the +2 position relative to the N-terminal methionine, the endogenous bacterial methionyl aminopeptidase (MAP) of the bacterial host cell does not efficiently cleave the N-terminal methionine. As a result, direct bacterial expression of an αβhIL2 mutein typically results in a mixture of αβhIL2 mutein species, with some species possessing the N-terminus and others lacking the N-terminal methionine. Such a mixture of IL2 species is difficult to resolve using typical manufacturing procedures, resulting in increased processing steps, product loss, and difficulties when attempting to conjugate molecules such as targeting molecules or carrier molecules such as PEG to the N-terminus of the αβhIL2 mutein. However, by deleting Ala1 from the αβhIL2 mutein, the residue at the +2 position relative to the N-terminal methionine is a threonine (T3), resulting in highly efficient cleavage of the N-terminal methionine, facilitating bacterial production of the IL2 mutein and providing a more uniform αβhIL2 mutein product. In some embodiments, the disclosure provides hIL2 muteins that include a deletion of alanine at position 1 (des-Ala1; des-A1, numbered according to hIL2).

[0232] A series of exemplary hIL2 muteins containing amino acid substitutions at positions 18, 22, and / or 126 linked to CD132, as listed in Table 5, were prepared and tested for IL2 activity and selectivity for CD25+ and CD25- T cells. The molecules were prepared and tested substantially according to the teachings of the Examples herein. Briefly, nucleic acid sequences encoding various human IL-2 muteins, human IL-2 REK, mouse IL-2-REH (L18R, Q22E, Q126H), CD25(22-240), and CD122(27-240), when expressed in Expi293 cells, were purified by Ni-Excel (Cytiva) affinity chromatography. The supernatant was supplemented with 5 mM imidazole, while washing and elution were performed in PBS supplemented with 30 and 250 mM imidazole, respectively. The affinity eluate was further purified by preparative size-exclusion chromatography (SEC) on a HiLoad 16 / 600 Superdex 200 pg column equilibrated with PBS buffer. Purity was established by reducing 4–20% Tris-glycine SDS-PAGE (Biorad), and SEC-MALS (Wyatt) was performed on a Superdex Increase 10 / 300 GL column equilibrated with PSB.

[0233] To demonstrate the activity of hIL2 muteins with reduced binding affinity for CD132 compared to wild-type hIL2 of the present disclosure and their preferential activation of CD25-expressing cells, a series of hIL2 muteins were prepared and evaluated for their ability to provide selective activation of YT cells, NK cells that express the intermediate-affinity dimeric form of the IL2 receptor, and a YT cell variant designated YT CD25, which is a YT cell engineered to express CD25 on its surface (iCD25+), resulting in human immune cells that express all three components of the high-affinity trimeric IL2 receptor.

[0234] The results of these experiments are provided in Figures 12, 13, and 14 of the accompanying drawings. As shown in Figure 12, hIL2 muteins containing amino acid substitutions at positions 18, 22, and / or 126 involved in hIL2 binding to hCD132 demonstrated significant increases in pSTAT5 signaling, demonstrating that the hIL2 muteins retain significant hIL2 activity compared to wild-type hIL2 in YT CD25 cells. As shown in Figure 2, the hIL2 muteins of the present disclosure demonstrated preferential pSTAT5 signaling activity compared to wild-type hIL2 on CD25-positive YT CD25 cells compared to CD25-negative YT cells. Data from dilutions of these molecules are provided in Figure 3 of the accompanying drawings.

[0235] Additional studies were performed to evaluate the disclosed αβhIL2 muteins for activity in 3F8 cells, CD4-positive human T cells. The 3F8 cell line was generated by activation of PBMCs obtained from a healthy human donor with the EBV-transformed B cell line JY. The CD4-positive T cell clone 3F8 expresses CD25 and CD122, proliferates in response to IL-2, and produces IFNγ. Additional representative αβhIL2 muteins, as detailed in Table 6 below, were evaluated for proliferation activity and IFNγ production in 3F8 cells according to the teachings of Example 8 herein. Data from this experiment are provided in Table 6 below and in Figures 15 (cell proliferation) and 16 (IFNγ production) of the accompanying drawings. IC values ​​were calculated for protein concentrations in the transfection supernatant. 50 Correct the following.

[0236] Table 6. Proliferation and IFNγ production of human CD4+ T cell clone 3F8 in response to hIL2 muteins TIFF2024529340000012.tif95142

[0237] The data in Table 6 above and Figures 15 and 16 demonstrate that the αβhIL2 muteins of the present disclosure, which have reduced binding affinity for CD132 compared to wild-type hIL2, are effective in stimulating the proliferation of and IFNγ production from CD25+ / CD122+ human immune cells.

[0238] In addition to those modifications incorporated into the αβhIL2 muteins that modulate the binding of hIL2 to hCD132, the αβhIL2 muteins of the present disclosure may optionally further contain one or more amino acid substitutions or deletions that confer additional beneficial properties to the αβhIL2 muteins, as described in more detail below.

[0239] In some embodiments, the αβhIL2 muteins contain one or more mutations at positions in the hIL-2 sequence that alter the orientation of positions that contact CD25 or other positions that contact CD25, resulting in the hIL2 mutein possessing increased affinity for CD25. In some embodiments, the αβhIL2 muteins of the present disclosure contain one or more of the substitutions V69A and Q74P, which have been described to increase the binding affinity of hIL2 for CD25.

[0240] In addition to modifications to the amino acid sequence of the αβhIL2 mutein that provide reduced binding to CD132 and, optionally, increased binding to CD25 and / or CD122, the αβhIL2 muteins of the present disclosure may further contain one or more conservative amino acid substitutions within the amino acid sequence of the αβhIL2 mutein that do not result in a substantial alteration of the activity profile of the αβhIL2 mutein. Such conservative substitutions include those described by Dayhoff in *The Atlas of Protein Sequence and Structure* 5 (1978) and by Argos in *EMBO J., 8:779-785 (1989). Conservative substitutions are generally made according to Table 7 below.

[0241] Table 7: Exemplary conservative amino acid substitutions TIFF2024529340000013.tif100142

[0242] In some embodiments, the αβhIL2 muteins of the present disclosure comprise one or more amino acid substitutions that increase hCD122 receptor binding (or binding to the ECD of hCD122). In some embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure that have reduced binding affinity for the CD132 receptor further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations that increase CD122 binding affinity. In certain embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure comprise at least one mutation (e.g., deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues) compared to wt hIL2, such that the hIL2 mutein binds to CD122 with higher affinity than wt hIL2. In certain embodiments, the hIL2 mutein binds to CD122 with an affinity that is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% greater than wild-type IL2. The binding affinity of the αβ hIL2 mutein may also be expressed as 1.2, 1.4, 1.5, 2, 5, 10, 15, 20, 25, 50, 100, 200, 250 or more times greater affinity for CD122 than wild-type hIL2.

[0243] In some embodiments, the αβhIL2 mutein contains one or more amino acid substitutions that increase hCD122 receptor binding affinity and are selected from amino acids at the interface between hIL2 and hCD122. Based on the crystal structure of hIL2 with its receptor, those positions that have been identified as interacting with hIL2 binding to hCD122 include, but are not limited to, Q74, L80, R81, L85, I86, I89V, and I92, numbered according to mature wt hIL2. In some embodiments, the αβhIL2 mutein contains one or more amino acid substitutions that enhance CD122 binding affinity, including, but not limited to, the group consisting of Q74N, Q74H, Q74S, L80F, L80V, R81D, R81T, L85V, I86V, I89V, and / or I92F, or a combination thereof. In some embodiments, the αβhIL2 mutein comprises the amino acid substitutions L80F, R81D, L85V, 186V, and 192F. In some embodiments, the αβhIL2 mutein comprises the amino acid substitutions N74Q, L80F, R81D, L85V, 186V, 189V, and 192F.

[0244] In one aspect, the present disclosure provides αβhIL2 muteins that exhibit significant or enhanced binding affinity for hCD25 and reduced binding affinity for the hCD132 (or extracellular domain of hCD132) receptor compared to wild-type human IL2 (hIL2). In some embodiments, the αβhIL2 muteins of the present disclosure contain one or more amino acid substitutions that increase hCD25 binding. In some embodiments, the one or more amino acid substitutions that increase hCD25 receptor binding affinity are selected from amino acids at the interface between hIL2 and hCD25. In some embodiments, the αβhIL2 muteins contain one or more mutations at positions in the IL2 sequence that alter the orientation of positions that contact CD25 or other positions that contact CD25, resulting in an αβhIL2 mutein that retains increased affinity for CD25. Based on the crystal structure of hIL2 with its receptor and other studies, those positions that have been identified as interacting with hIL2 binding to hCD25 include V69 and Q74, numbered according to mature wt hIL2. In some embodiments, the αβhIL2 muteins of the present disclosure contain one or more of the substitutions V69A and Q74P.

[0245] The αβhIL2 muteins of the present disclosure may contain modifications at the Thr3 (T3) position that eliminate the O-glycosylation site to facilitate the production of non-glycosylated hIL2 muteins when the IL2 muteins are expressed in eukaryotic expression systems, particularly mammalian host cells such as CHO or HEK cells. In one embodiment, the αβhIL2 muteins of the present disclosure contain an amino acid modification, deletion, or substitution at the Thr3 (T3) position to prevent O-glycosylation at T3. U.S. Patent No. 5,116,943; Weiger et al., (1989) Eur. J. Biochem., 180:295-300. In one embodiment, the modification at T3 is an amino acid substitution. In some embodiments, the αβhIL2 muteins of the present disclosure may contain an amino acid substitution in T3 selected from amino acid substitutions including T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P, which removes the glycosylation site at position 3 without eliminating biological activity. In one embodiment, the αβhIL2 muteins of the present disclosure contain the amino acid substitution T3A. In some embodiments, the T3 residue may be substituted with a cysteine ​​residue (T3S) to facilitate selective N-terminal modification of the sulfhydryl group of the cysteine, particularly PEGylation (see, e.g., U.S. Patent No. 5,206,344, issued April 27, 1993, to Katre et al.).

[0246] In some embodiments of the present disclosure, the αβhIL2 muteins of the present disclosure comprise amino acid substitutions to avoid vascular leak syndrome, a substantial negative dose-limiting side effect of the use of IL2 therapy in humans, without substantial loss of efficacy. See U.S. Patent No. 7,514,073 B2 to Epstein et al., issued April 7, 2009. In some embodiments, the αβhIL2 muteins of the present disclosure further comprise one or more amino acid substitutions selected from the group consisting of R38W, R38G, R39L, R39V, F42K, and H55Y.

[0247] In some embodiments of the present disclosure, the αβhIL2 muteins of the present disclosure may optionally include an amino acid substitution of methionine 104 with, in some cases, an alanine residue (M104A). Removal of methionine at position 104 provides αβhIL2 muteins with improved resistance to oxidation and loss of activity. U.S. Patent No. 4,752,585, issued June 21, 1988, to Koths et al.

[0248] The wild-type hIL2 sequence contains an unpaired cysteine ​​residue at position 125. Unpaired cysteines provide an opportunity for protein misfolding due to incorrect disulfide bridges between cysteine ​​sulfhydryl groups. This can be particularly problematic when αβhIL2 muteins are recombinantly expressed in bacteria and isolated from inclusion bodies. Consequently, the αβhIL2 muteins of the present disclosure can optionally contain an amino acid substitution at position 125. In some embodiments, the αβhIL2 muteins of the present disclosure can optionally contain a C125A or C125S amino acid substitution.

[0249] In some embodiments, an αβhIL2 mutein useful in practicing the methods of the disclosure comprises an amino acid substitution at position 91. In some embodiments, an αβhIL2 mutein comprises a substitution at position 91 selected from the substitutions V91K, V91R, V91K. In some embodiments, an αβhIL2 mutein useful in practicing the methods of the disclosure comprises an amino acid substitution at position 91 that can be presented as an Fc fusion as described in more detail in U.S. Pat. No. 9,580,486 B2 to Gavin et al., granted February 28, 2017, the teachings of which are incorporated herein by reference with respect to the construction of Fc fusions of IL2 muteins comprising a substitution at position 91.

[0250] It has been observed that deletion of amino acids at the N-terminus of the hIL2 molecule does not result in substantial loss of IL2 activity. The αβhIL2 muteins of the present disclosure may optionally contain N-terminal amino acid deletions (des-Ala1) at positions 1-9, alternatively at positions 1-8, alternatively at positions 1-7, alternatively at positions 1-6, alternatively at positions 1-5, alternatively at positions 1-4, alternatively at positions 1-3, alternatively at positions 1-2, or alternatively at position 1, while retaining the αβhIL2 mutein's hIL2 activity and reduced binding affinity for CD132. The αβhIL2 muteins may contain a deletion of an alanine (desAla1) at position 1 to facilitate recombinant production of substantially pure αβhIL2 muteins in bacterial expression systems. The αβhIL2 muteins may further contain a deletion at positions 1-3 to eliminate a glycosylation site at T3.

[0251] In some embodiments, the hIL2 mutein may be affinity matured to enhance its affinity for CD25 and / or CD122, resulting in modifications to the amino acid sequence of the hIL2 mutein. An "affinity matured" polypeptide is one that has one or more alterations to one or more residues that result in an improvement in the affinity of the polypeptide for its receptor (or vice versa), compared to a parent polypeptide that does not possess the alterations. Affinity maturation can be performed to increase the binding affinity of the IL2 mutein by at least about 10%, alternatively at least about 50%, alternatively at least about 100%, alternatively at least about 150%, or by 2-, 3-, 4-, or 5-fold, compared to the parent IL2 mutein polypeptide.

[0252] One problem associated with the use of wt-hIL2 in therapeutic applications in mammalian subjects is its relatively short lifespan in the circulation of the treated subject, often on the order of minutes or perhaps hours. In some embodiments of the present invention, the αβhIL2 mutein is modified to provide an extended duration of action (e.g., half-life) in the mammalian subject when administered to the mammalian subject. In some embodiments, the αβhIL2 mutein modified to provide an extended duration of action in a mammalian subject has a half-life in the mammal of greater than 4 hours, alternatively greater than 5 hours, alternatively greater than 6 hours, alternatively greater than 7 hours, alternatively greater than 8 hours, alternatively greater than 9 hours, alternatively greater than 10 hours, alternatively greater than 12 hours, alternatively greater than 18 hours, alternatively greater than 24 hours, alternatively greater than 2 days, alternatively greater than 3 days, alternatively greater than 4 days, alternatively greater than 5 days, alternatively greater than 6 days, alternatively greater than 7 days, alternatively greater than 10 days, alternatively greater than 14 days, alternatively greater than 21 days, or alternatively greater than 30 days.

[0253] Modifications of the αβhIL2 mutein to provide an extended duration of action in a mammalian subject include (but are not limited to) amino acid substitutions in the primary sequence of the αβhIL2 mutein, conjugation of the αβhIL2 mutein to one or more carrier molecules, providing the αβhIL2 mutein in the form of a fusion protein with an additional polypeptide sequence (e.g., an αβhIL2 mutein-Fc fusion), and PEGylated αβhIL2 mutein.

[0254] It should be noted that more than one modification that provides extended duration of action in a mammalian subject may be used for a given αβhIL2 mutein. For example, an αβhIL2 mutein of the present disclosure may contain both an amino acid substitution that provides extended duration of action and an amino acid substitution that provides for conjugation to a carrier molecule, such as a polyethylene glycol (PEG) molecule.

[0255] In some embodiments, in addition to the amino acid substitutions that result in reduced binding affinity to hCD132 while retaining significant binding affinity to hCD122 and / or hCD25, the primary sequence of the αβhIL2 mutein can be further modified by incorporation of one or more amino acid substitutions that provide a prolonged duration of action. See, e.g., Dakshinamurthi, et al. (2009) International Journal of Bioinformatics Research 1(2):4-13. Examples of such amino acid substitutions that provide a prolonged duration of action are one or more amino acid substitutions selected from the group consisting of one, two, or all three of V91R, K97E, and T113N. In some embodiments, in addition to the amino acid substitutions that result in reduced binding affinity to hCD132 while retaining significant binding affinity to hCD122 and / or hCD25, the αβhIL2 muteins useful in practicing the methods of the present disclosure comprise one or more amino acid substitutions selected from the group consisting of V91R, K97E, and T113N.

[0256] In some embodiments, the αβhIL2 muteins useful in the practice of the present disclosure, which have a prolonged duration of action in mammalian subjects, are achieved by covalently binding the αβhIL2 muteins to one or more carrier molecules. As used herein, the term "carrier molecule" refers to large, slowly metabolized macromolecules. Examples of such slowly metabolized macromolecular carriers include proteins; polysaccharides, such as sepharose, agarose, cellulose, or cellulose beads; polymeric amino acids, such as polyglutamic acid or polylysine; and amino acid copolymers. In certain embodiments, particularly when it is desirable to induce a host immune response, the αβhIL2 mutein may be conjugated to one or more immunogenic agents, such as inactivated virus particles; inactivated bacterial toxins, such as toxoids or leukotoxin molecules from diphtheria, tetanus, cholera; inactivated bacteria, dendritic cells, thyroglobulin; rotavirus VP6 polypeptide; influenza virus hemagglutinin, influenza virus nucleoprotein; keyhole limpet hemocyanin (KLH); and hepatitis B virus core protein and surface antigen S.

[0257] Examples of protein carrier molecules that can be covalently attached to αβhIL2 muteins to provide a prolonged duration of action in vivo include, but are not limited to, albumin, antibodies and antibody fragments, such as the Fc domain of an IgG molecule.

[0258] In some embodiments, the carrier molecule is an albumin molecule. It is known in the art that conjugation of proteins to albumin molecules facilitates prolonged in vivo exposure. In one embodiment of the present invention, the αβhIL2 mutein is conjugated to albumin via chemical linkage or expressed as a fusion protein with an albumin molecule, herein referred to as an "αβhIL2 mutein albumin fusion." The term "albumin," when used in connection with an αβhIL2 mutein albumin fusion, includes albumins such as human serum albumin (HSA), monkey (cyno) serum albumin, and bovine serum albumin (BSA). In some embodiments, the HSA comprises a C34S or K573P amino acid substitution compared to the wild-type HSA sequence. According to the present disclosure, albumin can be conjugated to the αβhIL2 mutein at the carboxyl terminus, the amino terminus, both the carboxyl terminus and the amino terminus, and internally (see, e.g., U.S. Pat. Nos. 5,876,969 and 7,056,701). Various forms of albumin, such as the albumin secretory presequence and variants thereof, fragments and variants thereof, and HSA variants, can be used in the HAS-αβhIL2 mutein conjugates contemplated by the present disclosure. Such forms generally retain one or more desired albumin activities. In additional aspects, the present disclosure involves fusion proteins comprising an αβhIL2 mutein fused directly or indirectly to albumin, albumin fragments, albumin variants, and the like, wherein the fusion protein has greater plasma stability than the unfused drug molecule and / or the fusion protein retains the therapeutic activity of the unfused drug molecule. As an alternative to chemical linkage between the αβhIL2 muteins, the αβhIL2 mutein-albumin conjugate can be provided as a fusion protein comprising an albumin polypeptide sequence and the αβhIL2 mutein recombinantly expressed as a single polypeptide chain in a host cell, optionally comprising a linker molecule between the albumin and the αβhIL2 mutein. Such fusion proteins can be readily prepared by those skilled in the art through recombinant techniques.Nucleic acid sequences encoding such fusion proteins can be ordered from any of a variety of sources. The nucleic acid sequence encoding the fusion protein is incorporated into an expression vector operably linked to one or more expression control elements, the vector is introduced into suitable host cells, and the fusion protein is isolated from the host cell culture by techniques well known in the art.

[0259] In some embodiments, an extended in vivo duration of action of the αβhIL2 mutein can be achieved by conjugation to an Fc domain derived from a mammalian (preferably human) immunoglobulin, such as an IgG1 or IgG4 molecule. The Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells lining blood vessels; upon binding, the Fc fusion molecule is protected from degradation and re-release into the circulation, allowing the molecule to remain in circulation longer. This Fc binding is thought to be the mechanism by which endogenous IgG retains its long plasma half-life. A wide variety of modifications have been introduced into naturally occurring Fc domains, including modified hinge regions, modifications that reduce Fc effector function, modifications that facilitate disulfide bonding between Fc subunits, and the incorporation of amino acid substitutions in Fc monomers that provide geometrically complementary structures that allow consistent 1:1 association of such modified Fc dimer subunits.

[0260] The Fc domain of an αβhIL2 mutein-Fc fusion can be a naturally occurring or synthetic polypeptide homologous to the IgG C-terminal domain produced by digestion of IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. αβhIL2 mutein fusions can include the entire Fc region or a smaller portion that retains the ability to extend the circulating half-life of the chimeric polypeptide of which it is a part. Typically, a nucleic acid sequence encoding an αβhIL2 mutein is provided in frame with one or both subunits of the Fc domain and expressed as a fusion protein as discussed above. The use of Fc fusions as carrier molecules for heterologous polypeptide sequences is well known in the art and is used in many approved pharmaceutical biological agents.

[0261] An example of a geometrically complementary Fc monomer subunit is the "knobs-into-holes" Fc modification, as described in Ridgeway et al. (1996) Protein Eng. 9 617-621 and U.S. Patent No. 5,731,168, issued March 24, 1998. In one embodiment, the "knobs-into-holes" modification comprises the amino acid substitutions T366W and, optionally, S354C in one antibody heavy chain and the amino acid substitutions T366S, L368A, Y407V, and, optionally, Y349C in the other antibody heavy chain. The knobs-into-holes format is often used to facilitate the expression of a first polypeptide (e.g., a hIL2 mutein) on a first Fc monomer bearing the "knobs" modification and a second polypeptide on a second Fc monomer bearing the "holes" modification, facilitating the expression of heterodimeric polypeptides or bispecific binding molecules. Engineered Fc domains useful in preparing extended αβhIL2 muteins can optionally be modified by the introduction of cysteine ​​residues at positions S354 and Y349, which result in stable disulfide bridges between the two antibody heavy chains in the Fe region (Carter, et al. (2001) Immunol Methods 248, 7-15). Engineered Fc domains useful in preparing extended αβhIL2 muteins can optionally contain mutations that inhibit complement binding and Fc receptor binding. Engineered Fc domains useful in preparing extended αβhIL2 muteins can optionally be designed to be lytic, i.e., capable of binding complement or lysing cells via another mechanism, such as antibody-dependent complement lysis (ADCC).

[0262] In some embodiments, extended duration of action of the αβhIL2 muteins in vivo may be achieved by conjugation to one or more polymeric carrier molecules, such as XTEN polymers or water-soluble polymers.

[0263] The αβhIL2 mutein can further comprise an XTEN polymer. The XTEN polymer can be conjugated to the αβhIL2 mutein (either chemically or as a fusion protein) to provide an extended lifespan similar to PEGylation and can be produced as a recombinant fusion protein in Escherichia coli (E. coli). Suitable XTEN polymers for use with the hIL2 muteins of the present disclosure are provided in Podust, et al. (2016) "Extension of in vivo half-life of biologically active molecules by XTEN protein polymers," J Controlled Release 240:52-66 and Haeckel et al. (2016) "XTEN as a Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic," PLOS ONE | DOI:10.1371 / journal.pone.0157193 June 13, 2016. The XTEN polymer fusion protein can incorporate a protease-sensitive cleavage site, such as an MMP-2 cleavage site, between the XTEN polypeptide and the hIL2 mutein.

[0264] In some embodiments, extended in vivo duration of action of the αβhIL2 mutein can be achieved by conjugation to one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of the present invention include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), polyolefin alcohols, polysaccharides, poly-alpha-hydroxy acids, polyvinyl alcohol (PVA), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof.

[0265] In some embodiments, an extended in vivo duration of action of the αβhIL2 mutein may be achieved by conjugation of the αβhIL2 mutein to one or more polyethylene glycol molecules ("PEGylation").

[0266] PEGs suitable for conjugation to αβhIL2 muteins are generally water soluble at room temperature and have the general formula R(O—CH2—CH2) n PEGs have the formula OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, R generally has 1 to 8 carbons. PEGs conjugated to polypeptide sequences can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-arm PEGs are contemplated by the present disclosure.

[0267] The molecular weight of PEG useful in the present disclosure is not limited to any particular range. The PEG component of the PEG-IL2 mutein can have a molecular mass of greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular mass is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa. A linear or branched PEG molecule having a molecular weight of about 2,000 to about 80,000 daltons, alternatively about 2,000 to about 70,000 daltons, alternatively about 5,000 to about 50,000 daltons, alternatively about 10,000 to about 50,000 daltons, alternatively about 20,000 to about 50,000 daltons, alternatively about 30,000 to about 50,000 daltons, alternatively about 20,000 to about 40,000 daltons, alternatively about 30,000 to about 40,000 daltons. In one embodiment of the invention, the PEG is a 40 kD branched PEG comprising two 20 kD arms.

[0268] The present disclosure also contemplates compositions of conjugates in which PEGs have different n values, and thus various different PEGs are present in specific ratios. For example, some compositions contain a mixture of conjugates where n=1, 2, 3, and 4. In some compositions, the percentage of conjugates where n=1 is 18-25%, the percentage of conjugates where n=2 is 50-66%, the percentage of conjugates where n=3 is 12-16%, and the percentage of conjugates where n=4 is up to 5%. Such compositions can be produced using reaction conditions and purification methods known in the art. Chromatography can be used to separate conjugate fractions, and then fractions containing conjugates with the desired PEG number can be identified and purified to remove unmodified protein sequences and conjugates with other PEG numbers.

[0269] PEG suitable for conjugation to polypeptide sequences is generally water soluble at room temperature and has the general formula R(O-CH-CH) n It has the formula OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, R generally has 1 to 8 carbons.

[0270] Two widely used first-generation activated monomethoxy PEGs (mPEGs) are succinimidyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15:100-114) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., U.S. Pat. No. 5,650,234 to Dolence et al.), which react preferentially with lysine residues to form carbamate linkages, but are also known to react with histidine and tyrosine residues. The use of PEG-aldehyde linkers targets a single site on the N-terminus of a polypeptide through reductive amination.

[0271] PEGylation frequently occurs at the α-amino group at the N-terminus of polypeptide, the epsilon amino group on the side chain of lysine residue, and the imidazole group on the side chain of histidine residue.Most recombinant polypeptides have a single alpha and multiple epsilon amino and imidazole groups, so depending on linker chemistry, many positional isomers can be generated.General PEGylation strategies known in the art can be applied herein.

[0272] PEG can be attached to the αβhIL2 muteins of the present disclosure via a terminal reactive group ("spacer") that mediates a bond between one or more free amino or carboxyl groups of the polypeptide sequence and polyethylene glycol. PEGs having a spacer that can be attached to a free amino group include N-hydroxysuccinylimide polyethylene glycol, which can be prepared by activating the succinate ester of polyethylene glycol with N-hydroxysuccinylimide.

[0273] The PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-arm PEGs are contemplated by the present disclosure.In a specific embodiment, PEG useful in the practice of the present invention includes 10 kDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601). Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 10 kDa linear PEG-NHS ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 20 kDa linear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF), 20 kDa linear PEG-NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), 20 kDa two-arm branched PEG-aldehyde, which is a 20 kDa compound containing two 10 kDa linear PEG molecules. PEG-aldehyde (e.g., Sunbright® GL2-200AL3, NOF), 20 kDa two-arm branched PEG-NHS ester comprising two 10 kDa linear PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), 40 kDa two-arm branched PEG-aldehyde comprising two 20 kDa linear PEG molecules, 40 kDa two-arm branched PEG-NHS ester (e.g., Sunbright® GL2-400AL3), 40 kDa two-arm branched PEG-NHS ester comprising two 20 kDa linear PEG molecules. Included are PEG-NHS ester (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), linear 30 kDa PEG-aldehyde (e.g., Sunbright® ME-300AL), and linear 30 kDa PEG-NHS ester.

[0274] As mentioned above, PEG can be attached to the αβhIL2 mutein directly or via a linker molecule. Suitable linkers include "flexible linkers," which are generally long enough to allow some movement between the modified polypeptide sequence and the linked components and molecules. Linker molecules are generally about 6-50 atoms in length. Linker molecules can also be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. Suitable linkers can be readily selected and can be of any suitable length, for example, 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, or more than 50 amino acids. Examples of flexible linkers include glycine polymers (Gly). n , glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Glycine and glycine-serine polymers are relatively structure-indefinite and therefore can serve as neutral tethers between components. Further examples of flexible linkers include glycine polymers (G), n , glycine-alanine polymers, alanine-serine polymers, and glycine-serine polymers. Glycine and glycine-serine polymers are relatively structure-indefinite and, therefore, may serve as neutral tethers between components. Multimers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) of these linker sequences may be linked together to provide flexible linkers that can be used to conjugate heterologous amino acid sequences to the polypeptides disclosed herein.

[0275] In one embodiment, the branched 40 kD PEG and linker conjugated to the N-terminal proline of the αβhIL2 mutein has the following structure: TIFF2024529340000014.tif14128

[0276] In a particular embodiment of the present disclosure, the αβhIL2 mutein is a hIL2 mutein comprising the amino acid substitutions L18R, Q22E and Q126K, numbered according to SEQ ID NO:4, a deletion of the N-terminal alanine residue (des-Ala1), and a branched 40kD PEG and linker conjugated to the N-terminus of the mutein, wherein the branched 40kD PEG and linker have the following structure: TIFF2024529340000015.tif14128

[0277] In one embodiment of the present disclosure, the αβIL2 mutein modified to provide an extended duration of action in vivo is a PEGylated αβIL2 mutein useful in the practice of the present disclosure, and has the following structure: [PEG]-[Linker]n-[desAla1-hIL2[L18R / Q22E / Q126K] where n=0 or 1, or

[0278] In one embodiment of the present disclosure, the αβIL2 mutein modified to provide an extended duration of action in vivo is a PEGylated αβIL2 mutein useful in the practice of the present disclosure, and has the following structure: TIFF2024529340000016.tif19138 where n=0 (absent) or 1 (present).

[0279] In one embodiment of the present disclosure, the αβIL2 mutein modified to provide an extended duration of action in vivo is a PEGylated αβIL2 mutein useful in the practice of the present disclosure, and has the following structure: TIFF2024529340000017.tif19138 where 40kD-PEG-linker (n=1) is a molecule of the following structure: TIFF2024529340000018.tif21128.

[0280] While the method or site of PEG attachment to the αβhIL2 mutein can vary, in certain embodiments, PEGylation does not alter or only minimally alters the activity of the αβhIL2 mutein. Site-specific PEGylation of the αβhIL2 mutein may be used to avoid interference with the binding characteristics of PEG to one or more IL2 receptor subunits. Site-specific PEGylation of the αβhIL2 mutein can be achieved by substituting one or more amino acids with naturally occurring amino acids (e.g., cysteine) whose side chains facilitate PEGylation, or by site-specific incorporation of unnatural amino acids with side chains that facilitate selective PEG conjugation. For example, the αβhIL2 mutein may contain a substitution of cysteine ​​with threonine at position 3 (3TC) to facilitate N-terminal PEGylation using specific chemistry. Incorporation of unnatural amino acids with side chains that facilitate selective PEG conjugation chemistry is as described by Ptacin et al. (PCT International Application No. PCT / US2018 / 045257, filed August 3, 2018, published February 7, 2019 as International Publication No. WO 2019 / 028419A1).

[0281] Conversely, site-specific conjugation of PEG to hIL2 muteins may be used to generate αβhIL2 muteins by incorporating unnatural amino acids bearing specific PEGylatable moieties at sequences or residues of hIL2 identified to interact with hCD132, including amino acids such as residues 18, 22, 109, 126, and 119 to 133. Site-specific PEGylation at one or more of these residues identified to be at the interface between IL2 and CD132 may be used to prepare αβhIL2 muteins with reduced binding to hCD132 that are useful in practicing the methods of the present disclosure.

[0282] In some embodiments, αβhIL2 muteins useful in the practice of the present disclosure that have an extended duration of action in mammalian subjects are achieved by covalently attaching the αβhIL2 mutein to a fatty acid molecule, as described in Resh (2016) Progress in Lipid Research 63: 120-131. Examples of fatty acids that can be conjugated include myristate, palmitate, and palmitoleic acid. Myristoyl is typically linked to the N-terminal glycine, although lysine can also be myristoylated. Palmitoylation is typically achieved by enzymatic modification of free cysteine-SH groups, such as with DHHC proteins that catalyze S-palmitoylation. Palmitoylation of serine and threonine residues is typically achieved enzymatically using the PORCN enzyme. In some embodiments, the αβhIL2 mutein is acetylated at the N-terminus by an N-terminal acetyltransferase and an enzymatic reaction with, for example, acetyl-CoA. Alternatively, or in addition to N-terminal acetylation, the αβhIL2 mutein is acetylated at one or more lysine residues, for example, by enzymatic reaction with lysine acetyltransferase. See, e.g., Choudhary et al. (2009) Science 325 (5942):834L2 ortho840.

[0283] In some embodiments, the αβhIL2 mutein can comprise a functional domain of a chimeric polypeptide. The hIL2 mutein fusion proteins of the present disclosure can be readily produced by recombinant DNA methodology using techniques known in the art by constructing a recombinant vector containing a nucleic acid sequence comprising a nucleic acid sequence encoding the αβhIL2 mutein in frame with a nucleic acid sequence encoding a fusion partner at either the N- or C-terminus of the hIL2 mutein, which optionally further comprises a nucleic acid sequence encoding a linker or spacer polypeptide in frame.

[0284] In other embodiments, the αβhIL2 mutein can be modified to contain an additional polypeptide sequence that functions as an antigenic tag, such as a FLAG sequence. The FLAG sequence is recognized by a biotinylated, highly specific anti-FLAG antibody, as described herein (see, e.g., Blanar et al. (1992) Science 256:1014, and LeClair, et al. (1992) PNAS-USA 89:8145). In some embodiments, the hIL2 mutein polypeptide further comprises a C-terminal c-myc epitope tag.

[0285] In other embodiments, the αβhIL2 muteins can be modified to contain additional polypeptide sequences that facilitate isolation or purification. Non-limiting examples include molecules comprising binding molecules, such as biotin (biotin-avidin specific binding pair), antibodies, receptors, ligands, lectins, or solid supports, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes.

[0286] In some embodiments, the αβhIL2 muteins (including αβhIL2 mutein fusion proteins) of the present disclosure are expressed as fusion proteins with one or more transition metal chelating polypeptide sequences. The incorporation of such transition metal chelating domains facilitates purification by immobilized metal affinity chromatography (IMAC), as described in U.S. Pat. No. 4,569,794 to Smith et al., issued February 11, 1986. Examples of transition metal chelating polypeptides useful in the practice of the present invention are described in Smith et al. (supra) and U.S. Pat. No. 5,320,663 to Dobeli et al., issued May 10, 1995, the entire teachings of which are incorporated herein by reference. A particular transition metal chelating polypeptide useful in the practice of the present invention is a peptide containing three to six consecutive histidine residues, such as the hexa-histidine peptide (His)6, often referred to in the art as a "His-tag."

[0287] In some embodiments, the αβhIL2 mutein is conjugated to a molecule (a "targeting domain") that provides selective binding to a particular cell type or tissue that expresses a cell surface molecule that specifically binds to such targeting domain, optionally incorporating a linker molecule of 1 to 40 (alternatively 2 to 20, alternatively 5 to 20, alternatively 10 to 20) amino acids between the αβhIL2 mutein sequence and the sequence of the targeting domain of the fusion protein.

[0288] In other embodiments, chimeric polypeptides can be produced comprising an αβhIL2 mutein and an antibody or antigen-binding portion thereof. The antibody or antigen-binding component of the chimeric protein can serve as a targeting moiety. For example, this can be used to localize the chimeric protein to a specific cell subset or target molecule. Methods for producing cytokine-antibody chimeric polypeptides are described, for example, in U.S. Patent No. 6,617,135. In some embodiments, the targeting moiety is an antibody (including single-domain antibodies such as VHHs and scFvs) that specifically binds to at least one cell surface molecule associated with tumor cells (i.e., at least one tumor antigen), where the cell surface molecule associated with tumor cells is selected from the group consisting of GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3Rα2, CD19, mesothelin, Her2, EpCam, Muc1, ROR1, CD133, CEA, EGFRRVIII, PSCA, GPC3, Pan-ErbB, and FAP.

[0289] In other embodiments, the chimeric polypeptide comprises an αβhIL2 mutein and a heterologous polypeptide that functions to enhance expression or direct cellular localization of the αβhIL2 mutein, such as the Aga2p agglutinin subunit (see, e.g., Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0290] In some embodiments, the targeting moiety can be an antibody or antibody fragment. In particular, antibodies selective for binding to tumor cell-associated antigens are useful for targeted delivery of systemically administered αβhIL2 muteins to tumors, supporting antigen-specific TILs in the tumor.

[0291] In some embodiments, the αβhIL2 muteins may also be linked to additional therapeutic agents, including therapeutic compounds as described elsewhere in this disclosure, e.g., anti-inflammatory compounds or anti-neoplastic agents, therapeutic antibodies (e.g., Herceptin), immune checkpoint modulators, immune checkpoint inhibitors (e.g., anti-PD1 antibodies), cancer vaccines. Antimicrobial agents include aminoglycosides including gentamicin, antiviral compounds such as rifampicin, 3'-azido-3'-deoxythymidine (AZT) and acyclovir, antifungal agents such as azoles including fluconazole, plyre macrolides such as amphotericin B and candicidin, antiparasitic compounds such as antimonial agents, and the like. The hIL2 mutein can be conjugated to additional cytokines such as CSF, GSF, GMCSF, TNF, erythropoietin, immunomodulators or cytokines, such as interferons or interleukins, neuropeptides, reproductive hormones such as HGH, FSH or LH, thyroid hormones, neurotransmitters such as acetylcholine, hormone receptors such as estrogen receptors. Also included are nonsteroidal anti-inflammatory drugs such as indomethacin, acetylsalicylic acid, ibuprofen, sulindac, piroxicam and naproxen, and anesthetics or analgesics. Also included are radioisotopes, such as those useful for imaging and treatment.

[0292] The αβhIL2 muteins of the present disclosure can be chemically conjugated to such carrier molecules using well-known chemical conjugation methods. Bifunctional cross-linking reagents, such as homofunctional and heterofunctional cross-linking reagents, well known in the art, can be used for this purpose. The type of cross-linking reagent to be used depends on the nature of the molecule to be coupled to the αβhIL2 mutein and can be easily identified by one skilled in the art. Alternatively or additionally, the αβhIL2 mutein and / or the molecule intended to be conjugated can be chemically derivatized so that the two can be conjugated in separate reactions, which is also well known in the art.

[0293] Generally, in the practice of adoptive cell therapy, during the ex vivo phase, a sample of tissue (e.g., neoplasm) containing T cells (e.g., TILs) is obtained and subjected to a two-step process: an initial expansion step and a rapid expansion (REP) step.

[0294] The expansion process begins with the excision of a sample of the neoplasm, which is cut into small pieces (a few millimeters) or enzymatically digested into a single-cell suspension. The fragments or digests are then incubated in the presence of αβhIL2 at a concentration sufficient to induce proliferation (e.g., EC 300 of the αβhIL2 mutein). 10 PRO or above, alternatively EC 20 PRO or above, alternatively EC 30 PRO or above, alternatively EC 40 PRO or above, EC 50 PRO or above, alternatively EC 60 PRO The culture is cultured for a period of about 7-21 days, alternatively about 12-18 days, alternatively about 12-16 days, about 12 days, about 13 days, about 14 days, or longer, for a period of about 7-21 days, alternatively about 12-18 days, alternatively about 12-16 days, about 12 days, about 13 days, about 14 days. The culture contains approximately 5 x 10 7Up to 100 TILs are maintained. During the expansion of the digest, tumor cells typically disappear from the culture. The use of tumor fragments or digests during the expansion phase typically does not affect the success rate of expansion and / or clinical response.

[0295] In some embodiments, the expansion step can optionally provide a selection step to further enrich for tumor-specific cell populations. Once the culture is composed largely of CD3+ T cells, its specificity is tested by quantitation of interferon-γ (IFN-γ) during a short culture period in the presence of an autologous or HLA-matched tumor cell line. The selected cell population is then further expanded for an additional period of time, as described above, substantially following the procedure described immediately above.

[0296] During the ex vivo phase, activated tumor-reactive T cells can be further selected, sorted, and enriched (e.g., by FACS) based on one or more additional cell surface markers. Improvements in TIL therapy have been reported by selecting cells that express PD1, resulting in T cells bearing CD8, CD25, and PD1 (i.e., CD8+CD25+PD1+ T cells) in some embodiments. Enrichment / selection of CD8+PD1+ T cells: In some embodiments, the preselection procedure involves enriching the cell population for PD-1+CD8+ T cells. Salas-Benito, et al. (2018) J Immunol Sci. (2018);2(1): 55-59 reported that preselection of PD-1+ tumor-infiltrating CD8+ T cells before ex vivo expansion improves the efficacy of TIL adoptive T cell therapy. PD-1 + CD8 T cells can be easily and rapidly isolated using FACS or magnetic techniques. The use of pre-enriched tumor-specific T cells simplifies the TIL production process and may also aid in the generation of T cell products with high antitumor activity. PD-1 expression may enable the isolation of rare tumor-specific TILs, facilitating the application of TIL therapy to solid tumors.

[0297] In the REP step, cells obtained from the expansion step are stimulated to grow into a large number (typically 1 x 10 10 ~2×10 11 The cells obtained from the expansion step are further expanded to 100-200 fold excess irradiated feeder cells (of autologous or allogeneic origin) at a concentration sufficient to induce proliferation (e.g., EC 10 PRO or above, alternatively EC 20 PRO or above, alternatively EC 30 PRO or above, alternatively EC 40 PRO or above, EC 50 PRO or above, alternatively EC 60 PRO The irradiated feeder cells are mixed for a period of about 7-21 days, alternatively about 12-18 days, alternatively about 12-16 days, about 12 days, about 13 days, or about 14 days (or longer). The irradiated feeder cells release growth factors into the culture medium and will be suitable for large-scale TIL expansion (usually greater than 1000-fold). During the final stage of REP, a bioreactor (such as a WAVE or Xuri, or a gas-permeable G Rex bottle) is typically used to facilitate high cell density culture. The REP process can optionally be performed in the presence of an activating compound, such as a CD3 antibody.

[0298] In some embodiments, as described in Forget, et al (2017) Frontiers in Immunology 8:908 and Idorn, et al (2016) Methods Mol Biol. 1428:261-76, TILs can optionally be engineered during the ex vivo stage using techniques well known in the art, such as CRISPR-cas9 or expression vectors (e.g., lentiviral expression vectors or mRNA) to express additional proteins that support anti-tumor effects (e.g., CXCR2 receptor).

[0299] In the in vivo phase of adoptive T cell therapy, the expanded T cells obtained from the ex vivo phase are readministered to the subject in the presence of the biased IL2 muteins of the present disclosure at a concentration sufficient to expand the activated cell population, optionally in combination with one or more adjuvant agents.

[0300] In some embodiments, αβhIL2 muteins useful in performing the in vivo steps of the disclosed methods provide modifications that alter the binding of the IL2 mutein to other proteins, particularly CD25, CD122, and CD132, and combinations of such proteins, such as CD122 / CD132 (the "intermediate-affinity IL2 receptor"), CD25 (the "low-affinity IL2 receptor"), and CD25 / CD122 / CD132 (the "high-affinity IL2 receptor"). The present disclosure provides methods and compositions for the treatment and / or prevention of neoplastic diseases, disorders, or conditions by administration of a therapeutically effective amount of a human IL-2 mutein that has reduced binding affinity for CD132 but still retains significant binding affinity for CD122 and / or CD25 equivalent to that of wild-type human IL-2.

[0301] In some embodiments, IL2 muteins useful in practicing the methods of the disclosure have substantial affinity for the extracellular domain of the wild-type human CD122 receptor (e.g., 20% of the affinity of wild-type hIL2, alternatively >30% of the affinity of wild-type hIL2, alternatively >40%, alternatively >50% of the affinity of wild-type hIL2, alternatively >60% of the affinity of wild-type hIL2, alternatively >65% of the affinity of wild-type hIL2, alternatively >70% of the affinity of wild-type hIL2, alternatively >75% of the affinity of wild-type hIL2, alternatively >80% of the affinity of wild-type hIL2, alternatively >85% of the affinity of wild-type hIL2, alternatively >90% of the affinity of wild-type IL2, alternatively >90% of the affinity of wild-type IL2, alternatively >95% of the affinity of wild-type IL2, alternatively >100% of the affinity of wild-type IL2). %, alternatively >105% of the affinity of wild-type hIL2, alternatively >110% of the affinity of wild-type IL2, alternatively >115% of the affinity of wild-type hIL2, alternatively >125% of the affinity of wild-type IL2, or alternatively >150% of the affinity of wild-type hIL2), while possessing reduced binding affinity to the extracellular domain of hCD132 (e.g., <50% of the affinity of wild-type hIL2, alternatively <45% of the affinity of wild-type hIL2, alternatively <40% of the affinity of wild-type hIL2, alternatively <35% of the affinity of wild-type hIL2, alternatively <25% of the affinity of wild-type hIL2, alternatively <20% of the affinity of wild-type hIL2, alternatively <15% of the affinity of wild-type hIL2, alternatively <10% of the affinity of wild-type IL2, or alternatively <5% of the affinity of wild-type IL2).

[0302] In some embodiments, αβhIL2 muteins useful in practicing the methods of the disclosure have reduced binding affinity for the extracellular domain of the hCD132 receptor and further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations that increase affinity for the extracellular domain of the wild-type human CD122 receptor. In certain embodiments, αβhIL2 muteins useful in practicing the methods of the disclosure comprise at least one mutation (e.g., deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues) compared to wild-type IL-2 (e.g., SEQ ID NO:4) and bind to CD122 with greater affinity than wild-type IL-2. In certain embodiments, the αβhIL2 mutein binds to CD122 with an affinity that is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% greater than wild-type IL-2. The binding affinity of the αβhIL2 mutein can also be expressed as 1.2, 1.4, 1.5, 2, 5, 10, 15, 20, 25, 50, 100, 200, 250 or more times greater affinity for the extracellular domain of hCD122 than wild-type hIL-2.

[0303] In some embodiments, αβhIL2 muteins useful in practicing the methods of the disclosure have substantial affinity (e.g., >50% of the affinity of wild-type hIL2, alternatively >60% of the affinity of wild-type hIL2, alternatively >65% of the affinity of wild-type hIL2, alternatively >70% of the affinity of wild-type hIL2, alternatively >75% of the affinity of wild-type hIL2, alternatively >80% of the affinity of wild-type hIL2, alternatively >85% of the affinity of wild-type hIL2, alternatively >90% of the affinity of wild-type hIL2, alternatively >90% of the affinity of wild-type hIL2, alternatively >95% of the affinity of wild-type hIL2, alternatively >100% of the affinity of wild-type hIL2, alternatively >105% of the affinity of wild-type hIL2) at the hCD25 / hCD122 receptor complex. , alternatively >110% of the affinity of wild-type hIL2, alternatively >115% of the affinity of wild-type hIL2, alternatively >125% of the affinity of wild-type hIL2, or alternatively >150% of the affinity of wild-type hIL2, while possessing reduced binding affinity to the extracellular domain of hCD132 (e.g., <50% of the affinity of wild-type hIL2, alternatively <45% of the affinity of wild-type hIL2, alternatively <40% of the affinity of wild-type hIL2, alternatively <35% of the affinity of wild-type hIL2, alternatively <25% of the affinity of wild-type hIL2, alternatively <20% of the affinity of wild-type hIL2, alternatively <15% of the affinity of wild-type hIL2, alternatively <10% of the affinity of wild-type hIL2, or alternatively <5% of the affinity of wild-type hIL2). In certain embodiments, the αβhIL2 muteins of the present disclosure possess reduced affinity for CD 132. In some embodiments, such IL2 muteins incorporate modifications in the primary structure of wild-type IL2, incorporating one or more modifications at positions 18, 22, and 126, numbered according to wild-type hIL-2.

[0304] In some embodiments, αβhIL2 muteins useful in practicing the methods of the disclosure have substantial affinity for hCD25 (e.g., >50% of the affinity of wild-type hIL2, alternatively >60% of the affinity of wild-type hIL2, alternatively >65% of the affinity of wild-type hIL2, alternatively >70% of the affinity of wild-type hIL2, alternatively >75% of the affinity of wild-type hIL2, alternatively >80% of the affinity of wild-type hIL2, alternatively >85% of the affinity of wild-type hIL2, alternatively >90% of the affinity of wild-type hIL2, alternatively >90% of the affinity of wild-type hIL2, alternatively >90% of the affinity of wild-type hIL2). Alternatively, the antibody may possess a reduced binding affinity to CD132 while retaining a binding affinity of >95% of the affinity of wild-type hIL2, alternatively >100% of the affinity of wild-type hIL2, alternatively >105% of the affinity of wild-type hIL2, alternatively >110% of the affinity of wild-type hIL2, alternatively >115% of the affinity of wild-type hIL2, alternatively >125% of the affinity of wild-type hIL2, alternatively >150% of the affinity of wild-type hIL2, alternatively >200% of the affinity of wild-type hIL2, alternatively >300% of the affinity of wild-type hIL2, alternatively >400% of the affinity of wild-type hIL2, alternatively >500% of the affinity of wild-type IL2.

[0305] In some embodiments, IL2 muteins useful in practicing the methods of the present disclosure exhibit significant or enhanced binding affinity to hCD25 and reduced binding affinity to the extracellular domain of the hCD132 receptor compared to wild-type human IL-2 (hIL-2).

[0306] In some embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure contain one or more amino acid substitutions that reduce CD132 receptor binding affinity selected from amino acid positions 18, 22 and 126, numbered according to mature wild-type hIL-2.

[0307] In some embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure that are partial agonists have one or more reduced functions compared to wild-type IL-2.

[0308] In certain embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure disrupt the association of CD122 with CD132 such that the CD122 / CD132 interaction is reduced by about 2%, about 5%, about 10%, about 15%, about 20%, about 50%, about 75%, about 90%, about 95% or more compared to wild-type hIL-2. In some embodiments, the one or more mutations that reduce the binding affinity of the IL-2 mutein for CD132 are amino acid substitutions. In some embodiments, the subject hIL-2 muteins consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to wild-type IL-2 (SEQ ID NO:4).

[0309] In some embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure are partial agonists. In certain embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure are partial agonists that have a reduced ability to stimulate one or more signaling pathways that depend on CD122 / CD132 heterodimerization. In some embodiments, αβhIL2 muteins have a reduced ability to stimulate phosphorylation in CD122+ cells compared to wild-type hIL-2. In some embodiments, the IL-2 mutein stimulates STAT5 phosphorylation in IL-2RP+ cells at 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less of the level at which wild-type IL-2 stimulates STAT5 phosphorylation in the same cells.

[0310] In some embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure are partial agonists with reduced ability to stimulate signaling in CD122+ cells compared to wild-type hIL-2. In some embodiments, αβhIL2 muteins stimulate pERK1 / ERK2 signaling in CD122+ cells at 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less of the level at which wild-type IL-2 stimulates pERK1 / ERK2 signaling in the same cells. In some embodiments, the CD122+ cells are T cells. In certain embodiments, the CD122+ T cells are CD8+ T cells. In some embodiments, the CD122+ CD8+ T cells are CD122+ CD8+ T cells isolated from a subject. In other embodiments, the CD8+ T cells are activated CD122+ CD8+ T cells. In other embodiments, the CD122+ cells are natural killer (NK) cells. STAT5 and ERK1 / 2 signaling can be measured, for example, by phosphorylation of STAT5 and ERK1 / 2 using any suitable method known in the art. For example, STAT5 and ERK1 / 2 phosphorylation can be measured in T cells using antibodies specific to the phosphorylated forms of these molecules.

[0311] In certain embodiments, the αβhIL2 muteins useful in practicing the methods of the present disclosure are partial agonists with reduced ability to induce lymphocyte proliferation compared to wild-type hIL-2. In some embodiments, the lymphocytes are T cells. In certain embodiments, the lymphocytes are primary CD8+ T cells. In other embodiments, the lymphocytes are activated CD8+ T cells. Cell proliferation can be measured using any suitable method known in the art. For example, lymphocyte proliferation can be measured using a carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay or by thymidine incorporation. In some embodiments, the αβhIL2 muteins of the present disclosure induce lymphocyte proliferation at 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less of the level at which wild-type hIL-2 induces lymphocyte proliferation.

[0312] In some embodiments, αβhIL2 muteins useful in practicing the methods of the present disclosure are partial agonists with reduced ability to activate CD25 expression in lymphocytes compared to wild-type IL-2. In some embodiments, the αβhIL2 muteins activate CD25 expression in lymphocytes at 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less of the level at which wild-type IL-2 activates CD25 expression in the same cells. In some embodiments, the lymphocytes are CD8+ T cells. In some embodiments, the CD8+ T cells are freshly isolated CD8+ T cells. In other embodiments, the CD8+ T cells are activated CD8+ T cells.

[0313] In some embodiments, the αβhIL2 muteins useful in practicing the methods of the present disclosure are full agonists.

[0314] In some embodiments, the αβhIL2 muteins useful in practicing the methods of the present disclosure are superagonists.

[0315] In some aspects, the present disclosure provides methods and compositions for the treatment and / or prevention of neoplastic diseases, disorders or conditions by administering a population of CD8+ CD25+ enriched T cells in combination with a therapeutically effective amount of an αβhIL2 mutein, optionally in combination with one or more adjunctive agents, including, but not limited to, one or more of a chemotherapeutic agent, an immune checkpoint modulator, radiation therapy and / or physical interventional treatment modalities such as surgery.

[0316] In some aspects, the present disclosure provides methods and compositions for the treatment and / or prevention of neoplastic diseases, disorders, or conditions by administering a population of CD8+ CD25+ enriched T cells in combination with a therapeutically effective amount of an αβhIL2 mutein that has reduced binding affinity for CD132 but still retains significant binding affinity for CD122 and / or CD25 equivalent to the activity of wild-type human IL-2, wherein the serum concentration of the αβhIL2 mutein is increased over a period of time (e.g., at least 24 hours, alternatively at least 48 hours, alternatively at least 72 hours, alternatively at least 96 hours, alternatively at least 12 hours). and / or at or above an effective concentration of IL2 mutein sufficient to promote proliferation of CD3-activated primary human T cells for the αβhIL2 mutein (e.g., EC 0, EC 10, EC 20, EC 30, EC 40, EC 50, EC 60, EC 70, EC 80, EC 90, EC 100, EC 110, EC 120, EC 130, EC 140, EC 150, EC 200, EC 250, EC 300, EC 400, EC 500, EC 600, EC 700, EC 800, EC 900, EC 1000, EC 1500, EC 1600, EC 1700, EC 1800, EC 1900, EC 2000, EC 2100, EC 2200, EC 2300, EC 2400, EC 2500, EC 2600, EC 2700, EC 2800, EC 3000, EC 3100, EC 3200, EC 3300, EC 3400, EC 3500, EC 3600, EC 3700, EC 3800, EC 3900, EC 4000, EC 4100, EC 4200, EC 4300, EC 4400, EC 4500, EC 4600, EC 4700, EC 4800, EC 4900, EC 5000, EC 5100, EC 52 10 PRO or above, alternatively EC 20 PRO or above, alternatively EC 30 PRO or above, alternatively EC 40PRO or above, EC 50 PRO or above, alternatively EC 60 PRO and at or above an effective concentration (e.g., EC 70 ACT or above, alternatively EC 60 ACT or above, alternatively EC 50 ACT or above, alternatively EC 50 ACT or above, EC 40 ACT or above, alternatively EC 40 ACT The present invention provides methods and compositions for administering the compound of formula I to human subjects, wherein the compound is maintained at a serum concentration (at or above) of 100 mg / kg / day.

[0317] In some embodiments, the present disclosure provides methods of using populations of CD8+ CD25+ enriched T cells in combination with one or more αβhIL2 muteins for the treatment of neoplastic disease.

[0318] During the in vivo phase, the αβhIL2 mutein may be administered in combination with one or more adjunctive agents, as described below. In some embodiments, administration of the αβhIL2 mutein to the subject occurs prior to administration of the enriched adoptive T cell population.

[0319] In some embodiments of the disclosed methods, the subject is optionally subjected to a lymphocyte-depleting non-myeloablative chemotherapy regimen (NMA chemotherapy) prior to the in vivo phase and re-administration of the expanded cell population. Several studies have been conducted evaluating the role of preconditioning lymphocyte-depleting regimens. Lymphocyte-depleting regimens induce short but profound lymphopenia and neutropenia, with complete bone marrow recovery within 7-10 days and without the need for hematopoietic stem cell support. In one embodiment, the NMA comprises the following regimen: cyclophosphamide at a dose of approximately 60 mg / kg for approximately 2 days, followed by approximately 25 mg / m for 5 days. 2 Intravenous administration of fludarabine at a dose of

[0320] In some embodiments, the lymphocyte depletion regimen includes administration of cyclophosphamide and fludarabine. Lymphocyte depletion regimens are commonly used in combination with adoptive cell therapy protocols and agents, and dosage ranges for administration of lymphocyte depleting agents are well known in the art. In one embodiment of the implementation of the aforementioned method, the subject is treated with a lymphocyte depletion regimen including cyclophosphamide in combination with fludarabine. In some embodiments, the lymphocyte depletion regimen involves administering cyclophosphamide in combination with fludarabine for 1, 2, 3, 4, or 5 days prior to administration of the adoptively transferred cells. In some embodiments, the dose of cyclophosphamide used in the lymphocyte depletion regimen is about 100, 200, 300, 400, 500, 600 mg / m for 1, 2, 3, 4, or 5 days prior to administration of the adoptively transferred cells. 2 In some embodiments, the lymphocyte depletion regimen is 300 mg / m for 3 days to the subject. 2 / day cyclophosphamide and 30 mg / m 2 In some embodiments, the dose of fludarabine used in the lymphocyte depletion regimen is about 10, 20, 30, 40, 50, 60 mg / m for 1, 2, 3, 4, or 5 days prior to administration of the GPC CAR T cells. 2 In one embodiment, the dose is about 500 mg / m for 3 days prior to administration of the adoptively transferred cells. 2 / day~about 600mg / m2 / day cyclophosphamide dose and approximately 30 mg / m 2 In one embodiment, the dose of fludarabine is about 300 mg / m for 3 days prior to administration of the adoptively transferred cells. 2 / day~about 600mg / m 2 / day cyclophosphamide dose and approximately 30 mg / m 2 / day fludarabine dose.

[0321] In some embodiments of the disclosed methods, the subject is optionally or additionally lymphodepleted with total body ionizing irradiation (TBI) at a dose of about 1 Gray to about 80 Gray, optionally about 1 Gray to about 20 Gray, and optionally about 2 Gray to about 15 Gray. Murine models have shown that prior lymphodepletion with total body irradiation (TBI) improves response rates to TIL therapy. These models have shown that depletion of endogenous lymphocytes creates physical space, resulting in lower competition for the homeostatic cytokines IL-7 and IL-15, and eliminating immunosuppressive lymphocyte and myeloid populations.

[0322] In radiotherapy, the radiation dose applied varies depending on the type and stage of the cancer being treated. Higher radiation doses are typically administered for solid epithelial tumors, while lower doses of about 0.5 Gray to about 4 Gray, preferably about 1 to 2 Gray, may be sufficient for non-solid tumors such as lymphomas as part of a maintenance protocol.

[0323] In an alternative embodiment to administering an abhIL2 mutein to a subject to provide in vivo support for tumor antigen-specific activated T cells prepared according to the methods of the present disclosure, an enriched cell population comprising tumor antigen-experienced activated T cells may be selectively activated through the use of an engineered receptor-ligand pair that provides for the selective proliferation and activation of cells expressing the engineered receptor in the subject in response to administration of the cognate ligand for the engineered receptor.

[0324] In some embodiments, in response to binding of a cognate ligand to the extracellular domain (ECD) of the engineered receptor, the intracellular domain (ICD) of the engineered receptor initiates intracellular signaling in TILs, resulting in activation and / or proliferation of the engineered cell. In some embodiments, the engineered receptor comprises an ICD that activates JAK / STAT in T cells, such that contact of a T cell expressing the engineered receptor with its cognate ligand results in JAK / STAT signaling in the cell, resulting in activation and / or proliferation of the T cell expressing the engineered receptor.

[0325] The present disclosure further provides a method of treating a subject suffering from a neoplastic disease, the method comprising the steps of: a. isolating a tissue sample from a subject suffering from a neoplastic disease, wherein the tissue sample comprises a population of TILs; b. contacting the tissue sample of step (a) ex vivo with an amount of an αβhIL2 mutein at a concentration sufficient to induce proliferation and activation of TILs to produce an expanded cell population comprising activated TILs; and c. contacting the expanded cell population of step (b) with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding the engineered receptor operably linked to one or more expression control sequences active in the T cell receptor; d. administering to a subject a cell population comprising activated TILs recombinantly modified to express the engineered receptor prepared according to step (c); e. Administering to a subject a large amount of a cognate ligand that specifically binds to the extracellular domain of the engineered receptor, wherein binding of the ligand to the receptor results in intracellular signaling in TILs that express the receptor, and such intracellular signaling results in activation and proliferation of TILs that express the engineered receptor.

[0326] Because the antigen-experienced TILs expanded in response to administration of the αβhIL2 mutein do not bind to the same antigen on tumor cells but rather are polyclonal in nature, the aforementioned method provides a method of providing a polyclonal anti-tumor response in a subject that can be modulated in response to administration of a cognate ligand for the engineered receptor. Consequently, the aforementioned method provides a method of inducing a polyclonal anti-tumor immune response in a subject, wherein the polyclonal response can be modulated in response to administration to the subject of an effective amount of a ligand that specifically binds to engineered cells expressing the engineered receptor and activates intracellular signaling.

[0327] A variety of engineered receptor-ligand pairs that result in the activation and / or proliferation of T cells expressing the engineered receptor in response to contact with the cognate ligand are known in the art and may be used in the methods of the present disclosure.

[0328] In one aspect, the engineered receptor / ligand pair is the "orthogonal" IL2 receptor-ligand system described in U.S. Patent No. 10,869,887 to Garcia et al., published December 22, 2020, the entire teachings of which are incorporated by reference. Garcia et al. describe hCD122 receptor subunits that are modified at positions 133 and / or 134 of the ECD of hCD122. These modifications effectively abolish binding of wild-type hIL2 to the engineered receptor. However, Garcia et al. further engineered hIL2 variants that selectively bind to the ECD of the engineered hCD122 receptor, enabling the engineered hIL2 variants to selectively activate the JAK / STAT signaling cascade of the ICD of hCD122, resulting in the selective in vivo activation and / or proliferation of T cells expressing the engineered CD122 receptor in response to administration of the engineered hIL2 variant ligand to a subject. Engineering TILs to express the receptor described by Garcia et al. is described in Chartier-Courtaud et al., PCT International Application No. PCT / US20 / 065892, published June 25, 2020, as WO 2020 / 131547.

[0329] In one aspect, the present disclosure provides a method of treating a subject suffering from a neoplastic disease, the method comprising the steps of: a. isolating a tissue sample from a subject suffering from a neoplastic disease, wherein the tissue sample comprises a population of TILs; b. contacting the tissue sample of step (a) ex vivo with an amount of an αβhIL2 mutein at a concentration sufficient to induce proliferation and activation of TILs to produce an expanded cell population comprising activated TILs; and c. contacting the expanded cell population of step (b) with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding an engineered T cell receptor operably linked to one or more expression control sequences active in the T cell receptor; d. administering to a subject a cell population comprising activated TILs recombinantly modified to express the engineered receptor according to step (c), wherein the receptor is hCD122 comprising amino acid substitutions at positions H133 and Y134; e. Administering to a subject a large amount of a cognate ligand that specifically binds to the extracellular domain of the engineered receptor, wherein binding of the ligand to the receptor results in intracellular signaling in TILs that express the receptor, wherein the cognate ligand is a hIL2 mutein, and such intracellular signaling results in activation and proliferation of TILs that express the receptor.

[0330] In some embodiments, the engineered receptor is a human CD122 protein comprising the amino acid substitutions H133D and Y134F.In some embodiments, the expression vector is a lentiviral or retroviral vector. In one aspect, the engineered receptor is a human CD122 protein comprising amino acid substitutions at positions 133 and 134, and the engineered ligand is as described by Garcia et al., wherein the engineered ligand is a human IL2 mutein comprising: an amino acid substitution at position 15 selected from E15S, E15T, E15Q, or E15H; an amino acid substitution at position 16 of H16Q; an amino acid substitution at position 19 selected from L19V or L19I; an amino acid substitution at position 20 selected from D20T, D20S, D20L, or D20M; and an amino acid substitution at position 23 selected from M23L, M23S, M23V, M23A, or M23T; and optionally, an amino acid substitution at position 22 selected from Q22K, Q22N, R81D, R81Y, T51I, or a combination thereof. In some embodiments, the engineered ligand is a human IL2 mutein (designated SQVLKA; SEQ ID NO:6) containing the substitutions E15S, H16Q, L19V, D20L; Q22K, and M23A. In some embodiments, the engineered ligand is modified to provide an extended in vivo half-life, as described in more detail elsewhere herein. In one embodiment, the engineered ligand is a PEGylated version of SQVLKA (SEQ ID NO:7) containing a des-Ala1 deletion, with an N-terminal 40 kDa branched PEG added to P2 of the des-Ala1 SQVLKA ligand.

[0331] In one embodiment of the disclosure, the cognate ligand is a human IL2 variant of the following structure: [PEG]-[Linker]n-[desAla1-hIL2[E15S-H16Q-L19V-D20L-Q22K-M23A] where n=0 or 1, or

[0332] In another embodiment of the disclosure, the cognate ligand is a human IL2 variant of the following structure: TIFF2024529340000019.tif26135 where n=0 (absent) or 1 (present).

[0333] In one embodiment, the cognate ligand is a human IL2 variant of the following structure: TIFF2024529340000020.tif26135 where 40kD-PEG-linker (n=1) is a molecule of the following structure: TIFF2024529340000021.tif22128.

[0334] In some aspects, the present disclosure provides for the administration of a pharmaceutical formulation comprising a therapeutically effective amount of a cognate ligand to a subject in need of treatment. Administration to a subject can be accomplished as an intravenous bolus or by continuous infusion over a period of time. Alternative routes of administration include intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The cognate ligand can also be administered intratumorally, peritumorally, intralesionally, intranodal, or perilesional, or appropriately administered into the lymph, to exert local and systemic therapeutic effects.

[0335] A therapeutically effective amount of the N-terminal 40 kDa branched PEG-des-Ala1 SQVLKA ligand is about 0.5 mg to about 20 mg, alternatively about 1 mg to about 15 mg, and alternatively about 1.5 mg to about 12 mg administered subcutaneously weekly. In one embodiment, a therapeutically effective amount of the N-terminal 40 kDa branched PEG-des-Ala1 SQVLKA ligand for a human subject is about 1.5 mg to about 12 mg administered subcutaneously weekly.

[0336] An alternative ligand / receptor system useful in practicing the aforementioned methods is described in U.S. Patent Application Publication No. US2021 / 0205365A1 to Price et al., published July 8, 2021. Price et al. describe a chimeric growth factor receptor that is selectively activatable in T cells (e.g., NK cells, CARs, TILs) in response to administration of the approved small molecule thrombopoietin receptor agonist eltrombopag (sold as Promacta®, Novartis). In some embodiments of the aforementioned methods, the engineered receptor is the chimeric receptor of Price et al. and the activating ligand is eltrombopag.

[0337] To provide expression of the engineered receptor in T cells, the nucleic acid sequence encoding the engineered receptor is incorporated into a vector comprising a nucleic acid sequence operably linked to one or more expression control sequences functional in T cells. Viral vector systems useful in the practice of the present invention include, for example, naturally occurring or recombinant viral vector systems. Viral vectors can be derived from the genomes of human or bovine adenovirus, vaccinia virus, lentivirus, herpesvirus, adeno-associated virus, human immunodeficiency virus, Sindbis virus, and retrovirus (including but not limited to Rous sarcoma virus), and hepatitis B virus. Typically, a gene of interest is inserted into such a vector, allowing packaging of the gene construct (typically with viral genome sequences), followed by infection of susceptible host cells, resulting in expression of the gene of interest (e.g., the engineered receptor). When a viral vector system is used for transfection, retroviral or lentiviral expression vectors are preferred for transfecting T cells, because these systems enhance the efficiency of gene transfer into T cells, thereby reducing the culture time of large quantities of T cells for clinical application. In particular, gammaretroviruses are particularly preferred for genetic modification of clinical-grade T cells, and therapeutic efficacy has been demonstrated. Pule, et al. (2008) Nature Medicine 14(11):1264-1270. Similarly, self-inactivating lentiviral vectors are also useful, as their integration into resting T cells has been demonstrated. June, et al. (2009) Nat Rev Immunol 9(10):704-716. Garcia et al.'s method for transducing TILs with vectors encoding modified CD122 proteins is described in U.S. Patent Application No. US 2020 / 0347350A1 by Karyampudi et al., published November 5, 2020.

[0338] As an alternative to expressing an orthogonal receptor from a vector, the genome of a cell may be modified to express the orthogonal receptor using techniques known in the art. In some embodiments, the compositions and methods of the present disclosure include genetically modifying a human immune cell by using at least one endonuclease to facilitate the incorporation of an engineered hCD122 ECD modification into the genomic sequence of the human immune cell. A method for such modification of T cells is described in U.S. Patent Application Publication No. US 2013 / 015884A1 to Galetto et al., published November 28, 2013, and a method for TCR alpha-deficient T cells by expressing pT alpha, which results in the restoration of a functional CD3 complex, is described in U.S. Patent No. 10,426,795B2 to Galetto et al., published October 21, 2019.

[0339] The IL2 muteins of the present disclosure may be produced by conventional methodologies for constructing polypeptides, including recombinant or solid phase synthesis.

[0340] An αβhIL2 mutein can be generated by affinity maturation of a wild-type hIL2 peptide to enhance its affinity for CD25 and / or CD122 and reduce its binding affinity for CD132. An "affinity-matured" polypeptide is one that contains one or more alterations in one or more residues that result in an improved polypeptide for a given receptor component compared to the parent wild-type polypeptide. Affinity maturation can be performed to increase the binding affinity of the hIL2 mutein by at least about 10%, alternatively at least about 50%, alternatively at least about 100%, alternatively at least about 150%, or 1-5 times compared to the "parent" polypeptide. Instructions on affinity maturation of polypeptides are well known in the art. See, for example, Rao, et al. (2003) Protein Engineering vol. 16(12): 1081-1087; Levin and Weiss (2006) Molecular BioSystems 2: 49-57. Rao et al. applied affinity maturation techniques to hIL2 analogs with enhanced affinity for CD25.

[0341] In addition to producing mutant polypeptides through the expression of nucleic acid molecules modified by recombinant molecular biology techniques, the subject αβhIL2 muteins can also be chemically synthesized. Chemically synthesized polypeptides are routinely produced by those skilled in the art. Chemical synthesis involves the direct synthesis of peptides by chemical means from protein sequences encoding αβhIL2 muteins exhibiting the described properties. This method allows the incorporation of both natural and unnatural amino acids at positions that affect the interaction of IL2 with CD25, CD122, and CD132.

[0342] In some embodiments, the IL2 muteins of the present disclosure can be prepared by chemical synthesis. Chemical synthesis of IL2 muteins can proceed via liquid phase or solid phase. Solid phase peptide synthesis (SPPS) allows the incorporation of unnatural amino acids and / or peptide / protein backbone modifications. Various forms of SPPS are available for synthesizing the IL2 muteins of the present disclosure and are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero JA et al., (2005) Protein Pept Lett. 12:723-8). During chemical synthesis, alpha functional groups and any reactive side chains may be protected with acid-labile or base-labile groups that are stable under conditions for amide bond linkage but can be easily cleaved without harming the formed peptide chain.

[0343] In solid-phase synthesis, either the N-terminal or C-terminal amino acid can be coupled to a suitable support material. Suitable support materials are inert to the reagents and reaction conditions of the stepwise condensation and cleavage reactions of the synthetic process and are insoluble in the reaction medium used. Examples of commercially available support materials include styrene / divinylbenzene copolymers modified with reactive groups and / or polyethylene glycol; chloromethylated styrene / divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene / divinylbenzene copolymers, etc. The sequential coupling of protected amino acids can be carried out according to conventional methods in peptide synthesis, typically in an automated peptide synthesizer.

[0344] At the end of solid-phase synthesis, the peptide is cleaved from the support material with concomitant cleavage of the side chain protecting groups. The resulting peptide can be purified by a variety of chromatographic methods, including, but not limited to, hydrophobic adsorption chromatography, ion exchange chromatography, partition chromatography, high performance liquid chromatography (HPLC), and reverse-phase HPLC.

[0345] Recombinant production: Alternatively, the IL2 muteins of the present disclosure are produced by recombinant DNA technology. In a typical implementation of recombinant production of a polypeptide, a nucleic acid sequence encoding the desired polypeptide is incorporated into an expression vector appropriate for the host cell in which expression is to be achieved, and the nucleic acid sequence is operably linked to one or more expression control sequences encoded by the vector and functional in the target host cell. The recombinant protein can be recovered through disruption of the host cell or, if the polypeptide incorporates a secretory leader sequence (signal peptide), from the cell culture medium. The recombinant protein may also be purified and concentrated for further use, including integration. Processes for recombinant production of IL2 polypeptides are known in the art and are described in U.S. Patent No. 4,604,377, issued August 5, 1986, to Fernandes and Taforo, U.S. Patent No. 4,512,584, issued May 21, 1985, to Mark et al., and U.S. Patent No. 4,401,756, issued August 30, 1983, to Gillis, the entire teachings of which are incorporated herein by reference.

[0346] Construction of nucleic acid sequences encoding IL2 muteins In some embodiments, the IL2 mutein is produced by recombinant methods using a nucleic acid sequence encoding the IL2 mutein (or a fusion protein comprising the IL2 mutein). A nucleic acid sequence encoding the desired αβhIL2 mutein can be synthesized by chemical means using an oligonucleotide synthesizer.

[0347] Nucleic acid molecules are not limited to sequences that code for polypeptides; they can also include some or all of the non-coding sequences upstream or downstream of the coding sequence (for example, the coding sequence of IL-2).Those with ordinary skill in the field of molecular biology are familiar with routine procedures for isolating nucleic acid molecules.For example, they can be produced by treating ge...

Claims

1. A pharmaceutical composition comprising an αβhIL2 mutein for use in a method of treating a subject suffering from a neoplastic disease, wherein the method comprises: (a) isolating a tissue sample from the subject, the tissue sample comprising a population of TILs; (b) contacting the isolated tissue sample of step (a) ex vivo with an amount of the αβhIL2 mutein at a concentration sufficient to induce the proliferation and activation of the TILs to produce an expanded cell population comprising activated TILs; and (c) administering the expanded cell population comprising activated TILs from step (b) to the subject A pharmaceutical composition comprising the same.

2. The pharmaceutical composition according to claim 1, wherein the method further comprises administering to the subject a therapeutically effective amount of a second αβhIL2 mutein before step (a) or after step (c), and the first and second αβhIL2 muteins are the same or different.

3. The method comprises: (i) administering to the subject a therapeutically effective amount of an αβhIL2 mutein before step (a); and (ii) administering to the subject a therapeutically effective amount of an αβhIL2 mutein after step (c) further comprising the αβhIL2 muteins are each the same, or the αβhIL2 muteins administered in steps (i) and (ii) are the same, or the αβhIL2 mutein administered in step (i) and used in step (b) is the same, or the αβhIL2 muteins are each different αβhIL2 muteins, The pharmaceutical composition according to claim 1.

4. The pharmaceutical composition according to claim 1, wherein the TILs comprise antigen-activated T cells. Claim 5: (i) an αβhIL2 mutant or each αβhIL2 mutant is an IL2 mutant having at least 90% sequence identity with wt-hIL2 (SEQ ID NO: 4) and containing an amino acid substitution at position 18, 22 or 126 numbered by wt-hIL2 (SEQ ID NO: 4), and optionally, the amino acid substitution at position 18 is selected from the group consisting of L18R, L18G, L18M, L18F, L18E, L18H, L18W, L18K, L18Q, L18S, L18V, L18I, L18Y, L18H, L18D, L18N and L18T, the amino acid substitution at position 22 is selected from the group consisting of Q22E, Q22F, Q22G, Q22A, Q22L, Q22M, Q22F, Q22W, Q22K, Q22S, Q22V, Q22I, Q22Y, Q22H, Q22R, Q22N, Q22D, Q22T and Q22F, and the amino acid substitution at position 126 is selected from the group consisting of Q126K, Q126H, Q126M, Q126C, Q126D, Q126E, Q126G, Q126I, Q126R, Q126S and Q126T, or (ii) an αβhIL2 mutant or each αβhIL2 mutant is an IL2 mutant having at least 90% sequence identity with wt-hIL2 (SEQ ID NO: 4) and containing three amino acid substitutions at positions 18, 22 and 126 numbered by wt-hIL2 (SEQ ID NO: 4), and optionally, (a) the amino acid substitution at position 18 of the αβhIL2 mutant is selected from the group consisting of L18R, L18G, L18M, L18F, L18E, L18H, L18W, L18K, L18Q, L18S, L18V, L18I, L18Y, L18H, L18D, L18N and L18T; (b) the amino acid substitution at position 22 of the αβhIL2 mutant is selected from the group consisting of Q22E, Q22F, Q22G, Q22A, Q22L, Q22M, Q22F, Q22W, Q22K, Q22S, Q22V, Q22I, Q22Y, Q22H, Q22R, Q22N, Q22D, Q22T, and Q22F; and (c) the amino acid substitution at position 126 of the αβhIL2 mutant is selected from the group consisting of Q126K, Q126H, Q126M, Q126C, Q126D, Q126E, Q126G, Q126I, Q126R, Q126S, or Q126T, Optionally, the αβhIL2 mutant or each αβhIL2 mutant comprises a set of mutations selected from the group consisting of: L18R, Q22E, and Q126K; L18R, Q22E, and Q126H; L18R, Q22E and Q126M; L18R, Q22E, Q126T; L18R; Q22E; V91K; V91R; Q126H; L18R and Q126H; Q22E and Q126H; L18G, Q22E and Q126H; L18A, Q22E and Q126H; L18M, Q22E and Q126H; L18F, Q22E and Q126H; L18W, Q22E and Q126H; L18K, Q22E and Q126H; L18Q, Q22E and Q126H; L18E, Q22E and Q126H; L18S, Q22E and Q126H; L18V, Q22E and Q126H; L18I, Q22E and Q126H; L18Y, Q22E and Q126H; L18H, Q22E and Q126H; L18N, Q22E and Q126H; L18D, Q22E and Q126H; L18T, Q22E and Q126H; L18R, Q22G and Q126H; L18R, Q22A and Q126H; L18R, Q22L and Q126H; L18R, Q22M and Q126H; L18R, Q22F and Q126H; L18R, Q22W and Q126H; L18R, Q22K and Q126H; L18R, Q22S and Q126H; L18R, Q22V and Q126H; L18R, Q22I and Q126H; L18R, Q22Y and Q126H; L18R, Q22H and Q126H; L18R, Q22R and Q126H; L18R, Q22N and Q126H; L18R, Q22D and Q126H; and L18R, Q22T and Q126H, Optionally, the αβhIL2 mutant or each αβhIL2 mutant comprises a set of mutations selected from the group consisting of L18R, Q22E, and Q126K, and L18R, Q22E, and Q126H, Optionally, the αβhIL2 mutant or each αβhIL2 mutant comprises the set of mutations L18R, Q22E, and Q126K, The pharmaceutical composition according to claim 1. **Claim 6**: The pharmaceutical composition according to claim 5, wherein the αβhIL2 mutein or each αβhIL2 mutein contains a deletion of 1, 2, 3, 4, 5, 6, 7, 8, or 9 N-terminal amino acids, optionally, the αβhIL2 mutein or each αβhIL2 mutein contains a deletion of 1, 2, or 3 N-terminal amino acids, and optionally, the αβhIL2 mutein or each αβhIL2 mutein contains a deletion of the N-terminal alanine amino acid (des-Ala1). **Claim 7** The pharmaceutical composition according to claim 2 or 3, wherein the αβhIL2 mutein administered to the subject is modified to extend its in vivo duration of action. **Claim 8**: (i) The tissue sample is selected from the group consisting of blood and solid tumor tissue, and / or (ii) The subject is treated with a lymphodepletion regimen prior to administering the amount of antigen-activated T cells to the subject, and / or (iii) The step of contacting the isolated cell population with the αβhIL2 mutein is performed in combination with one or more additional T cell activating agents, optionally, the one or more additional T cell activating agents are cytokines, growth factors, or antibodies that bind to T cell activating antigens, and optionally, (a) The cytokine is selected from the group consisting of human interleukin-10 (hIL10), human interleukin-7 (hIL7), human interleukin-9 (hIL9), human interleukin-4 (hIL4), and human interleukin-15 (hIL15), or (b) The antibody that binds to the T cell activating antigen is selected from the group consisting of anti-CD3 antibody, anti-CD28 antibody, and anti-CD137 antibody, and optionally, the αβhIL2 mutein is used in combination with anti-CD3 antibody and anti-CD28 antibody. The pharmaceutical composition according to claim 1. **Claim 9**: The method is performed in combination with administration of an adjuvant agent to the subject, optionally, the adjuvant agent is selected from the group consisting of chemotherapeutic agents, antibodies, immune checkpoint modulators, and physical methods, Optionally, (a) The adjuvant agent is an immune checkpoint modulator, optionally, the immune checkpoint modulator is an anti-PD-1 antibody or an anti-PD-L1 antibody, or, (b) The auxiliary agent is an antibody selected from the group consisting of [fam]-trastuzumab deruxtecan, enfortumab vedotin, polatuzumab vedotin, semipramab, moxetumomab pasudotox, mogamulizumab, tiludakizumab, ibalizumab, dulvalumab, inotuzumab, ozogamicin, abemaciclib, atezolizumab, orlatumumab, ixekizumab, aratumumab, elotuzumab, necitumumab, dinutuximab, nivolumab, blinatumomab, pembrolizumab, ramucirumab, siltuximab, obinutuzumab, ado-trastuzumab emtansine, pertuzumab, brentuximab vedotin, ipilimumab, ofatumumab, certolizumab pegol, catumaxomab, panitumumab, bevacizumab, cetuximab, tositumomab-I131, ibritumomab tiuxetan, gemtuzumab ozogamicin, trastuzumab, infliximab, rituximab, and edrecolomab. The pharmaceutical composition according to claim 1.

10. The method is (c) contacting a recombinant vector containing a nucleic acid sequence encoding the engineered receptor with the expanded cell population from step (b), wherein the engineered receptor is selectively activated in response to administration of a cognate ligand that binds to the extracellular domain of the engineered receptor and results in intracellular signaling in T cells expressing the engineered receptor; (d) administering the expanded cell population containing the activated TILs from step (c) to the subject; and (e) administering a therapeutically effective amount of the cognate ligand for the engineered receptor to the subject The pharmaceutical composition according to claim 1, comprising

11. (i) the subject is treated with a lymphodepletion regimen before administering the cell population to the subject and / or (ii) the cell population is contacted with a T cell activating agent before administering the cell population to the subject and / or (iii) the subject is pre-treated in vivo with a therapeutically effective amount of αβhIL2 mutein before isolating the tissue sample and / or (iv) the vector is a lentiviral vector or a retroviral vector and / or (v) The engineered receptor is hCD122 that contains at least one amino acid substitution at a position selected from position 133 or 134 numbered by SEQ ID NO: 2, optionally, the engineered receptor is hCD122 that contains amino acid substitutions at positions 133 and 134, optionally, hCD122 contains the amino acid substitutions H133D and Y134F, and / or (vi) The cognate ligand is an hIL2 variant that selectively binds to hCD122 that contains at least one amino acid substitution at a position selected from position 133 or 134 numbered by SEQ ID NO: 2, Optionally, the hIL2 variant contains one or more amino acid substitutions at positions 15, 16, 19, 20, 22, 23, 51 or 81 numbered by wt hIL2 (SEQ ID NO: 4); the amino acid substitution at position 15 is selected from E15S, E15T, E15Q, or E15H; the amino acid substitution at position 16 is H16Q; the amino acid substitution at position 19 is selected from L19V or L19I; the amino acid substitution at position 20 is selected from D20L, D20T, D20S or D20M; the amino acid substitution at position 22 is selected from Q22K, Q22N; the amino acid substitution at position 23 is selected from M23A, M23L, M23S, M23V or M23T; and the amino acid substitution at position 81 is selected from R81D and R81Y, Optionally, the hIL2 variant contains an amino acid substitution at position 15 selected from E15S, E15T, E15Q, or E15H; the amino acid substitution at position 16 is H16Q; the amino acid substitution at position 19 is selected from L19V or L19I; the amino acid substitution at position 20 is selected from D20L, D20T, D20S or D20M; the amino acid substitution at position 22 is selected from Q22K, Q22N; the amino acid substitution at position 23 is selected from M23A, M23L, M23S, M23V, or M23T, optionally, the hIL2 variant contains the amino acid substitutions E15S, H16Q, L19V, D20L; Q22K and M23A, Optionally, the hIL2 variant further contains a deletion of the N-terminal alanine residue, and / or (vii)The cognate ligand has been modified to extend its in vivo duration of action, optionally the modification for extending the in vivo duration of action is PEGylation, and optionally the hIL2 mutein has been modified by N-terminal addition of a 40 kDa branched PEG molecule. The pharmaceutical composition according to claim 10.

12. The neoplastic disease is selected from the group consisting of adenoma, fibroma, hemangioma, hyperplasia, atypia, metaplasia, dysplasia, carcinoma, leukemia, breast cancer, sarcoma, leukemia, lymphoma, urogenital cancer, ovarian cancer, urethral cancer, bladder cancer, prostate cancer, gastrointestinal cancer, colon cancer, esophageal cancer, gastric cancer, lung cancer; myeloma; pancreatic cancer; liver cancer; kidney cancer; endocrine cancer; skin cancer; glioma, neuroblastoma, astrocytoma, myelodysplastic syndrome; cervical intraepithelial neoplasia; intestinal polyposis; oral leukoplakia; histiocytosis, hypertrophic scar including keloid scar, respiratory cancer, digestive cancer, urogenital cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, melanoma, adenocarcinoma, myeloproliferative neoplasm, myeloid and lymphoid disorders with eosinophilia, myeloproliferative / myelodysplastic neoplasm, myelodysplastic syndrome, acute myeloid leukemia and related progenitor cell neoplasms, and acute leukemia of undetermined lineage, promyelocytic leukemia (APML), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), precursor lymphoid neoplasms, mature B cell neoplasms, mature T cell neoplasms, Hodgkin lymphoma, and immunodeficiency-related lymphoproliferative disorders, lymphoblastic leukemia (ALL) including B cell ALL and T cell ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenström macroglobulinemia (WM), erythroleukemia and acute megakaryoblastic leukemia, malignant lymphoma including but not limited to non-Hodgkin lymphoma and its variants, peripheral T cell lymphoma, adult T cell leukemia / lymphoma (ATL), cutaneous T cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), and Hodgkin disease, the pharmaceutical composition according to claim 1.

13. An ex vivo method for preparing a population of antigen-activated T cells, comprising: contacting a tissue sample containing a population of TILs isolated from a subject with an amount of αβhIL2 mutein at a concentration sufficient to induce the proliferation and activation of the TILs to generate an expanded cell population containing activated TILs. Optionally, before said contacting, applying an ex vivo selection process to said isolated tissue sample to generate a subpopulation of antigen-activated T cells enriched for antigen-activated T cells having one or more marker antigens, An ex vivo method. [

14. ] A step of contacting a recombinant vector comprising a nucleic acid sequence encoding an engineered receptor with a cell product, wherein the engineered receptor is selectively activated in response to administration of a cognate ligand that binds to the extracellular domain of the engineered receptor, and which results in intracellular signaling in T cells expressing the engineered receptor. Optionally, (i) the vector is a lentiviral vector or a retroviral vector, and / or (ii) the engineered receptor is hCD122 comprising at least one amino acid substitution at a position selected from positions 133 or 134 numbered by SEQ ID NO: 2, optionally hCD122 comprising amino acid substitutions at positions 133 and 134, and optionally the hCD122 comprises the amino acid substitutions H133D and Y134F. The method according to claim 13. [

15. ] A cell product produced by the method according to claim 13 or 14. [

16. ] A cell product according to claim 15 for use in a method of treating a subject suffering from a neoplastic disease, the method comprising administering the cell product to the subject, and optionally the method further comprising administering to the subject a therapeutically effective amount of a second αβhIL2 mutein. [

17. ] Use of an αβhIL2 mutein ex vivo for activation and expansion of antigen-activated T cells.

Images