Cell-surface anchored cytokine therapy

Abstract

This disclosure provides compositions and methods for treating subjects having cancer. It is based, in part, on an improved ability to modulate and orchestrate the activity of different cytokines, and thereby mount an effective immune response at a primary tumor site (i.e., a primary site of injection, that may also be but need not be the primary site of cancer development in the subject) but also at distal tumor sites (i.e., metastatic lesions). Provided herein are methods that employ localized administration regimens for cytokine fusion proteins that target the universal leukocyte receptor CD45. The cytokine fusion proteins comprise the cytokine of interest, namely IL-12 or IL-15, fused to a CD45-specific binding moiety. The Examples demonstrate that such immunocytokine fusions can be used to achieve a highly efficacious and non-toxic cytokine therapy that elicits complete responses at injected lesions as well as distal un-injected lesions.

Description

[0001] CELL-SURFACE ANCHORED CYTOKINE THERAPY

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 589,629, filed October 11, 2023, the entire contents of which are incorporated by reference herein.

[0004] FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under EB031082 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The content of the electronic sequence listing (M065670536WO00-SEQ-MAT.xml; Size: 26,217 bytes; and Date of Creation: August 7, 2024) is incorporated by reference herein in its entirety.

[0008] BACKGROUND OF INVENTION

[0009] Cytokines are a class of small proteins that serve as modulators of immune signaling cascades. Because of their endogenous role in lymphocyte migration, activation, and proliferation, cytokines have been recognized as promising cancer immunotherapy candidates. Interleukin-2 (IL-2) was the first efficacious immunotherapy for advanced cancer, demonstrating a 17% objective response rate (ORR) as a monotherapy in metastatic melanoma. Despite this initial clinical validation, dose-limiting toxicities and severe immune-related adverse events have significantly hindered the utilization of cytokines for oncology. This can be attributed, at least in part, to systemic lymphocyte activation from the high administered cytokine doses necessary to achieve meaningful tumor penetration.

[0010] SUMMARY OF INVENTION

[0011] This disclosure provides compositions and methods for treating subjects having cancer. It is based, in part, on an improved ability to modulate and orchestrate the activity of different cytokines, and thereby mount an effective immune response at a primary tumor site (i.e., a primary site of injection, that may also be but need not be the primary site of cancer development in the subject) but also at distal tumor sites (i.e., metastatic lesions). Provided herein are methods that employ localized administration regimens for cytokine fusion proteins that target the universal leukocyte receptor CD45. The cytokine fusion proteins comprise the cytokine of interest, namely IL- 12 or IL- 15, fused to a CD45-specific binding moiety. The Examples demonstrate that such immunocytokine fusions can be used to achieve a highly efficacious and non-toxic cytokine therapy that elicits complete responses at injected lesions as well as distal un-injected lesions.

[0012] Certain methods provided herein aim to target IL- 12 intratumorally and to minimally target IL- 15 to the tumor draining lymph nodes (TDLN), for example by peritumoral and / or intranodal administration. This can be accomplished by delivering the IL- 12 containing immunocytokine fusion intratumorally and delivering the IL- 15 containing immunocytokine peritumorally and / or intranodally. Alternatively, this can be accomplished by administering both immunocytokine fusions intratumorally but at dose ratios that cause the IL- 12 immunocytokine fusion to be retained within the tumor and the IL- 15 immunocytokine fusion to travel to the TDLN.

[0013] Certain methods provided herein aim to stimulate an IL- 12 modulated immune response within the tumor initially and then stimulate an IL- 15 modulated immune response within the tumor, in the peritumoral region, and / or in the TDLN. This can be accomplished by spatial and / or temporal separation of the administration of the IL- 12 containing immunocytokine fusion and the IL- 15 containing immunocytokine fusion.

[0014] Accordingly, this disclosure provides, in part, a method for treating a subject having a solid tumor cancer comprising

[0015] (i) administering intratumorally to a subject having a solid tumor cancer, an effective amount of a first immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing, in a cell-free preparation(s), and

[0016] (ii) administering to the subject an effective amount of a second immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 15, a functional fragment of full-length IL- 15, IL-15 / IL-15Ra (IL- 15 superagonist) or a functional fragment thereof, or a functional variant of any of the foregoing. In some embodiments, the second immunocytokine fusion is administered peritumorally. In some embodiments, the second immunocytokine fusion is administered intranodally to tumor draining lymph nodes (TDLN). In some embodiments, wherein the second immunocytokine fusion is delivered to tumor draining lymph nodes (TDLN). In some embodiments, the second immunocytokine fusion is administered intratumorally. In some embodiments, the second immunocytokine fusion is administered after administration of the first immunocytokine fusion. In some embodiments, the second immunocytokine fusion is administered about 4, about 6, about 8, about 12, about 18, or about 24 hours after administration of the first immunocytokine fusion. In some embodiments, the second immunocytokine fusion is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after administration of the first immunocytokine fusion. In some embodiments, the first and second immunocytokine fusions are administered in a dose ratio of about 1:2, about 1:4, about 1:6, about 1:8, about 1:10, about 1:12, about 1:15, or about 1:20.

[0017] This disclosure further provides, in part, a method for treating a subject having a solid tumor cancer comprising administering, in a cell-free preparation(s), to a subject having a solid tumor cancer,

[0018] (i) an effective amount of a first immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing, and

[0019] (ii) an effective amount of a second immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 15, a functional fragment of full-length IL- 15, IL-15 / IL-15Ra (IL- 15 superagonist) or a functional fragment thereof, or a functional variant of any of the foregoing, wherein the second immunocytokine fusion is administered about 1 hour to about 21 days after administration of the first immunocytokine fusion.

[0020] In some embodiments, the first immunocytokine fusion is administered intratumorally. In some embodiments, the second immunocytokine fusion is administered intratumorally. In some embodiments, the second immunocytokine fusion is administered peritumorally. In some embodiments, the second immunocytokine fusion is administered intranodally to tumor draining lymph nodes (TDLN). In some embodiments, the second immunocytokine fusion is delivered to tumor draining lymph nodes (TDLN). In some embodiments, the second immunocytokine fusion is administered about 4, about 6, about 8, about 12, about 18, or about 24 hours after administration of the first immunocytokine fusion. In some embodiments, the second immunocytokine fusion is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after administration of the first immunocytokine fusion. In some embodiments, the first and second immunocytokine fusions are administered in a dose ratio of about 1:2, about 1:4, about 1:6, about 1:8, about 1:10, about 1:12, about 1:15, or about 1:20.

[0021] This disclosure further provides, in part, a method for treating a subject having a solid tumor cancer comprising

[0022] (i) administering intratumorally to a subject having a solid tumor cancer, an effective amount of a first immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing, in a cell-free preparation(s), and

[0023] (ii) administering to the subject an effective amount of a second immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 15, a functional fragment of full-length IL- 15, IL-15 / IL-15Ra (IL- 15 superagonist) or a functional fragment thereof, or a functional variant of any of the foregoing, wherein the first and second immunocytokine fusions are administered in a dose ratio of about 1:2 to about 1:20.

[0024] In some embodiments, the first and second immunocytokine fusions are administered in a dose ratio of about 1:2, about 1:4, about 1:6, about 1:8, about 1:10, about 1:12, about 1:15, or about 1:20. In some embodiments, the second immunocytokine fusion is administered intratumorally. In some embodiments, the second immunocytokine fusion is administered peritumorally. In some embodiments, the second immunocytokine fusion is administered intranodally to tumor draining lymph nodes (TDLN). In some embodiments, the second immunocytokine fusion is delivered to tumor draining lymph nodes (TDLN). In some embodiments, the second immunocytokine fusion is administered about 4, about 6, about 8, about 12, about 18, or about 24 hours after administration of the first immunocytokine fusion. In some embodiments, the second immunocytokine fusion is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after administration of the first immunocytokine fusion. This disclosure further provides, in part, a method for treating a subject having a solid tumor cancer comprising administering to a subject having a solid tumor cancer an effective amount of an immunocytokine fusion comprising a CD45 targeting moiety conjugated to full- length IL- 15, IL-15 / IL-15Ra (IL- 15 superagonist) or a functional fragment thereof, or a functional variant of any of the foregoing, in a cell-free preparation(s), wherein the subject was administered full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing, either in free form or in a fusion protein comprising an immune cell targeting protein.

[0025] In some embodiments, the immunocytokine fusion is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after administration of the free or fusion form of full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing. In some embodiments, the immunocytokine fusion is administered in a dose that is 2- fold to 20-fold greater than the dose of the free or fusion form of full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing. In some embodiments, the immunocytokine fusion is administered intratumorally. In some embodiments, the immunocytokine fusion is administered peritumorally and / or intranodally. In some embodiments, the free or fusion form of full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing was administered intratumorally. In some embodiments, the immune cell targeting protein is a CD45 targeting protein.

[0026] Various embodiments apply equally to the various methods and compositions provided herein and these are recited once for brevity with the intended understanding that they apply throughout. These are recited below.

[0027] In some embodiments, the CD45 targeting moiety is a CD45-specific antibody or antigen-binding antibody fragment. In some embodiments, the CD45 targeting moiety is CD45- specific antibody BC8, 4B2, GAP8.3, or 9.4, or a humanized version thereof, or an CD45- binding fragment thereof. In some embodiments, the CD45 targeting moiety is an Fab fragment, an F(ab’)2 fragment, or a single chain Fv, or antibodies BC8, 4B2, GAP8.3, or 9.4, or a humanized version thereof. In some embodiments, the solid tumor cancer is a colorectal cancer or a melanoma. In some embodiments, the solid tumor cancer has metastasized in the subject. In some embodiments, the solid tumor cancer has metastasized and the first and second immunocytokine fusion is administered at or near a primary solid tumor site.

[0028] This disclosure further provides, in part, a immunomodulatory composition comprising a first and a second immunocytokine fusion, each immunocytokine fusion comprising an immunomodulatory cytokine conjugated to a CD45 targeting moiety, in a cell-free preparation, wherein the first immunocytokine fusion comprises a CD45 targeting moiety conjugated to full- length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing, wherein the second immunocytokine fusion comprises a CD45 targeting moiety conjugated to full-length IL- 15, IL-15 / IL-15Ra (IL-15 superagonist) or a functional fragment thereof, or a functional variant of any of the foregoing, and wherein the first and second immunocytokine fusions are present in a dose ratio in the range of 1:2 to 1:20.

[0029] These and other aspects and embodiments are described in greater detail herein.

[0030] BRIEF DESCRIPTION OF DRAWINGS

[0031] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying Figures, which are schematic and are not intended to be drawn to scale. In the Figures:

[0032] FIGs. 1A-1F show that CD45 immunocytokines were preferentially retained on the cell surface over wildtype cytokines, eliciting sustained downstream pSTAT5 signaling in both cis and trans. FIG. 1A is an exemplary schematic of IL- 15 immunocytokine. FIG. IB is an equilibrium binding curve of aCD45-IL15 and control IgG-IL15 to plate-bound mouse CD45 (n= 3). FIG. 1C is a graph depicting proliferation after CTLL-2 cells were incubated with control IgG-IL15 or aCD45-IL15 for 48 hours at the stated concentrations. Proliferation was read-out by Cell-Titer Gio. FIGs. 1D-1F are a flow cytometric analyses of primary CD8 T cells isolated from mouse splenocytes activated for 48 hours with aCD3 / 28 antibodies and then rested for 48 hours. In FIG. ID is an analysis of control IgG-IL15 or test aCD45-IL15 labeled with AF488, were spiked in at various time points at 10 pg / mL and surface signal was detected with an aAF488 quenching antibody. Cells were analyzed by flow cytometry. In FIG. IE primary CD8 T cells were starved of IL-2 for 24 hours prior to a 1-hour Ab-IL-15 pulse at 10 pg / lxlO6T cells. Twenty-four hours after washing, cells were fixed, permeabilized, and stained for flow cytometry analysis of pSTAT5, CD25, and Ki67. In FIG. IF primary CD8 T cells were labeled with CFSE and incubated at 10 pg / lM cells for 1 hour (“preloaded”), washed, and then serially diluted into non-labeled “bystander” cells for 1 hour. Cells were then fixed, permeabilized, and stained for flow cytometry analysis of pSTAT5. “aCD45” intends “aCD45” throughout.

[0033] FIGs. 2A-2H show that CD45-IL15 demonstrated altered biodistribution through cell-surface retention at the tumor and TDLN while sparing systemic exposure. FIG. 2A is a study diagram. FIGs. 2B-2E show flow cytometric analyses of AF647-labeled aCD45-IL15 or IgG-IL15 dosed (10 pg) intratumorally 24 hours prior to analysis (n = 4 mice / group). FIG. 2B shows AF647 labeling on tumor immune cells. FIG. 2C shows AF647 MFI on tumor immune cells. FIG. 2D shows representative AF647 signal within the tumor. FIG. 2E shows AF647 labeling on TDLN immune cells. FIG. 2F shows Aeso measurements of serum from treated mice. Concentrations were calculated with a standard curve, nd = not detected. FIG. 2G shows representative TDLN images. FIG. 2H shows a representative tumor image. “IM” intends “1 million” throughout.

[0034] FIGs. 3A-3F show that «CD45-cytokine therapy eradicated single MC38 and

[0035] B16F10 tumors. FIG. 3A shows probability of survival for mice (n = 5 / group) that were dosed with Ipg of Ab-IL12 and lOpg of Ab-IL15 as described. FIG. 3B shows probability of survival for mice (n=10-19 / group) that were dosed with Ipg of Ab-IL12 and lOpg of Ab-IL15 as described. FIG. 3C shows percent rejecting secondary rechallenge for mice cured of B 16F10 tumors and rechallenged with 0.1M B16F10 cells 100 days after the initial challenge. FIG. 3D shows probability of survival in depletion experiments with each curve showing a specific antibody or knockout cell depletion. Antibodies were given i.p. FIG. 3E shows probability of survival for mice bearing B16F10 tumors that were dosed in the same paradigm as FIG. 3B with aCD45-IL12 or aCD45-IL15 monotherapy. FIG. 3F shows tumor area for mice bearing B16F10 tumors that were dosed in the same paradigm as FIG. 3B with varying delivery routes: intratumoral (i.t.), peritumoral (p.t.), or the aCD45-IL12 dose i.t. and the aCD45-IL15 dose p.t. Arrows indicate treatment. Control fusions are referred to as IgG-Cyt and test fusions are referred as aCD45-Cyt, where Cyt refers to the administration of the IL- 12 and IL- 15 fusions as provided in FIG. 3 A.

[0036] FIGs. 4A-4D show that «CD45-Cyt therapy drove abscopal responses. FIG. 4A shows probability of survival for mice bearing contralateral MC38 tumors that were treated with Ipg control or test IL-12 fusion and lOpg control or test IL-15 fusion as shown (n = 9-10 mice / group). FIG. 4B shows individual tumor curves from a representative experiment from FIG. 4A. FIG. 4C shows analysis of mice bearing contralateral MC38 tumors as described in FIG. 4A but using lOpg of AF647 labeled control or test IL-15 fusions and analyzed for biodistribution 24 hours later. FIG. 4D shows probability of survival for mice bearing contralateral B16F10 tumors that were treated with Ipg IL- 12 fusion (control or test) and lOpg IL- 15 fusion (control or test) as shown (n=5 mice / group).

[0037] FIGs. 5A-5J show that «CD45-cytokine therapy reprogrammed the TDLN leading to an optimal tumor-specific CD8 T cell effector subset, which then infiltrated the tumor. FIG. 5A shows the number of CD8+ cells in the CD45+ cell population in TDLN following PBS, IgG-Cyt, or aCD45-Cyt therapy. FIG. 5B shows the increased percentage of PD1+ cells in the CD45+ cell population of the TDLN after PBS, IgG-Cyt, or aCD45-Cyt therapy. FIG. 5C shows the number of CD25+ cells in the PD1+ CD8+ T cell population in TDLN after PBS, IgG-Cyt, or aCD45-Cyt therapy. FIG. 5D shows the number of GzmB+ cells in the PD1+ CD8+ T cell population in TDLN after PBS, IgG-Cyt, or aCD45-Cyt therapy. FIG. 5E shows the number of IFNg+ cells in the PD1+ CD8+ T cell population in TDLN after PBS, IgG-Cyt, or aCD45-Cyt therapy. FIG. 5F shows the number of TCF-TIM3+ or TCF+TIM3- cells in the PD1+ CD8+ T cell population in TDLN after PBS, IgG-Cyt, or aCD45-Cyt therapy. FIG. 5G shows the percentage of CD8+ T cells in the CD45+ cell population of the tumor following PBS, IgG-Cyt, or aCD45-Cyt therapy. FIG. 5H shows the percentage of CD25+ cells in the PD1+ CD8+ cell population of the tumor following PBS, IgG-Cyt, or aCD45-Cyt therapy. FIG. 51 shows the percentage of IFNg+ cells in the PD1+ CD8+ cell population of the tumor following PBS, IgG-Cyt, or aCD45-Cyt therapy. FIG. 5J shows the percentage of TNF+ cells in the PD1+ CD8+ cell population of the tumor following PBS, IgG-Cyt, or aCD45-Cyt therapy. DETAILED DESCRIPTION OF INVENTION

[0038] This disclosure provides improved methods for treating subjects having solid tumor cancers using immunocytokine fusion proteins that comprise a cytokine, namely in the form of IL- 12 or IL- 15, fused to a CD45 targeting moiety. When these fusions are used in a manner that targets IL- 12 predominantly to the tumor microenvironment and targets IL- 15 to tumor draining lymph nodes (TDLN), significant therapeutic benefit is surprising achieved including reduction or elimination of the tumor at the primary site of injection and reduction or elimination of distal tumors, suggesting their utility in the treatment of subjects presenting with a single localized tumor as well as subjects presenting with metastatic spread.

[0039] Immunocytokine fusions

[0040] An immunocytokine fusion (or immunocytokine fusion protein or immunocytokine fusion compound, as the terms are used interchangeably herein) as described herein comprises a cytokine (or a cytokine molecule, as the terms are used interchangeably herein) coupled to a CD45 targeting moiety (or CD45- specific binding moiety or a CD45 binding moiety, as the terms are used interchangeably herein), such as but not limited to an antibody an antibody fragment or a variant thereof, capable of binding to CD45. The cytokine and the CD45 targeting moiety may be covalently or non-covalently linked to each other (e.g., by chemical coupling, genetic fusion resulting from recombinant techniques, noncovalent association or otherwise), and such linkage may be referred to as an association, coupling, conjugation, and the like, as these terms are used interchangeably herein in this regard).

[0041] The CD45 targeting moiety is capable of binding to CD45 and thus to an immune cell that expresses CD45, thereby targeting its associated cytokine, to the immune cell. The immune cell may be a lymphocyte, such as a naturally occurring lymphocyte in the subject being treated, including T cells. The targeting moiety is therefore capable of binding to CD45-expressing cells in a tumor and in lymph nodes such as tumor draining lymph nodes (TDLN), and in doing so it presents its associated cytokine to immune cells in these regions. The immunocytokine fusion is therefore directed to a cell of interest and it enhances cell surface availability of the cytokine. The CD45 target is typically abundantly present on the surface of an immune cell, and it may outnumber the number of receptors for the cytokine on the immune cell surface.

[0042] Cells of interest targeted by the immunocytokine fusions include, inter alia, immune cells, especially lymphocytes, and preferably T-cells (e.g., total CD3+ T cells, or CD4+ T cells, or CD8+ T cells), and can include other cell types. In some embodiments, the fusions can activate a subset of CD8+ T cells. The immunocytokine fusions are administered in a cell-free preparation (i.e., a preparation that lacks cells), and accordingly the cells of interest to which such fusions bind are present in vivo (i.e., in the subject being treated).

[0043] In some embodiments, the CD45 targeting moiety functions to deliver and retain its conjugated cytokine to and in an area of interest such as a tumor. The CD45 targeting moiety, and the immunocytokine fusion, need not be and typically is not internalized by the immune cell in order to effect its function. The CD45 targeting moiety may however cause an increase in binding and availability of its associated cytokine and resultant signaling into and activation of immune cells at the site of administration, including the cells to which the immunocytokine fusion is bound. In some embodiments, the immunocytokine fusion can cause localized and prolonged stimulation of proliferation and activation of immune cells, thus inducing the controlled expansion and activation of an immune response. The CD45 targeting moiety does not substantially interfere with the signaling function of its associated cytokine. Similarly, the activity of the cytokine is not substantially impacted by its presentation as a fusion.

[0044] The CD45 targeting moiety may be but is not limited to an antibody (e.g., a full length immunoglobulin (IgG)), or an antibody fragment, or an antibody variant such as but not limited to an Fab’ fragment, an F(ab’)2 fragment, an Fab fragment, an F(ab)2 fragment, an Fv fragment, a single chain variable fragment (scFv fragment), and a camelid-derived single domain antibody (sdAb, also referred to as a VHH or a nanobody). The targeting moiety may also be nonantibody in nature and may include non-antibody scaffolds and antibody mimetics (e.g., lipocalins (e.g., anticalins), affibodies, fibronectin (e.g., monobodies or Adnectins), knottins, ankyrin repeats (e.g., DARPins), and A domains (e.g., avimers)). The immunocytokine fusion may comprise any of these antibody or non-antibody scaffolds or antibody mimetics provided such antibodies, fragments, scaffolds, and mimetics recognize and bind to CD45 and can be conjugated to IL- 12 or IL- 15, as provided herein.

[0045] Cytokines

[0046] Some aspects of the present disclosure provide methods of using immunocytokine fusions comprising a cytokine (e.g., IL-12 or IL-15) conjugated to a CD45 targeting moiety that targets immune cells that express CD45. Cytokines typically have short serum half-lives and limited bioavailability, and as a result they have been used in high doses in order to achieve therapeutic efficacy. Such high doses however have been associated with dose-limiting toxicities. The immunocytokines fusions of this disclosure and their methods of use can offer several advantages over unconjugated (or unfused or free form) cytokine therapy, including reduced side effects, e.g., a lower systemic toxicity, while retaining desirable immuno stimulatory activity.

[0047] The present disclosure provides, inter alia, immunocytokine fusions engineered to comprise interleukin- 12 (IL-12) or interleukin- 15 (IL-15), including functional variants thereof. The cytokine (or interleukin) may be used in its full-length form, or as a fragment of the full- length form, or as a subunit of the full-length form, or as a superagonist form such as when complexed or fused with its receptor (an example of which is the IL-15 / IL-15Ra superagonist), or as a functional variant of the any of the foregoing.

[0048] Functional variants refer to a variant of a cytokine having one or more mutations yet retaining activity of the “parent” cytokine. In some embodiments, the functional variant may be at least 90% identical in amino acid sequence to its parent, including at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to its parent. The activity of the functional variant may be at least 70% of the activity of its parent, including at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of its parent.

[0049] Interleukin-12 (IL-12)

[0050] Interleukin- 12 (IL- 12), in its full-length form, is a heterodimeric cytokine composed of p35 and p40 subunits which are encoded by 2 separate genes, IL-12A and IL-12B, respectively. IL- 12 is involved in the differentiation of naive T cells into Thl cells. It is regarded as a T cellstimulating factor, which can stimulate the growth and function of T cells. It stimulates the production of interferon-gamma (IFN-y) and tumor necrosis factor-alpha (TNF-a) from T cells and natural killer (NK) cells and reduces IL-4 mediated suppression of IFN- y. IL- 12 plays a role in the activities of NK cells and T lymphocytes. It mediates enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes.

[0051] IL- 12 binds to the IL- 12 receptor, which is a heterodimeric receptor comprising IL- 12Rpi and IL-12RP2. IL-12RP2 is considered to play a key role in IL- 12 function, since it is found on activated T cells and is stimulated by cytokines that promote Thl cell development and inhibited by those that promote Th2 cells development. Upon binding, IL-12R-P2 becomes tyrosine phosphorylated and provides binding sites for kinases, Tyk2 and Jak2, which in turn activate transcription factor proteins such as STAT4 that are implicated in IL- 12 signaling in T cells and NK cells. IL- 12 may function by reversing or tempering the anti-inflammatory environment and / or by stimulating a pro-inflammatory environment in solid tumors.

[0052] Certain immunocytokine fusions of this disclosure comprise IL- 12 fused to the CD45 targeting moiety. The fusion may comprise full-length IL- 12 or a functional fragment thereof, or an IL- 12 subunit, or an IL- 12 functional variant. IL- 12 may be provided in a dimeric form or as an individual subunit or a single chain protein having the p35 (I1-12A) and p40 (IL-12B) subunits linked to each other directly or through a linker, for example. See W02019 / 010219 for linkers useful to creating single chain IL- 12 and for examples of single chain IL- 12. The term IL- 12 may be used herein in describing various aspects and embodiments but it is to be understood that such teachings apply equally to full-length IL- 12, functional fragments of full- length IL- 12, p35 IL- 12 subunit, p40 IL- 12 subunit, and functional variants of any of the foregoing. In some embodiments, IL- 12 is human IL- 12.

[0053] In some embodiments, the fusion comprises the p35 (IL-12A) subunit of wild-type, human IL- 12 having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the fusion comprises the p40 (IL-12B) subunit of wild-type, human IL- 12 having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the fusion comprises the p35 and p40 subunits presented in heterodimeric form, such subunits having amino acid sequence of SEQ ID NOs: 1 and 2, respectively. In some embodiments, the fusion comprises the p35 and p40 subunits covalently conjugated to each other (or fused to each other) resulting in a single chain molecule comprising amino acid sequences of SEQ ID NOs: 1 and 2.

[0054] Variants of IL-12 are known in the art. For example, IL-12 variants that lack a protease site (e.g., in the D3 domain) and therefore have improved stability are described in US 9,029,330 (Webster et al.). Variants of the p40 subunit of IL-12 also having improved stability are provided in US 7872107 (Webster et al.). Single chain IL-12 is described in Agarwal et al. Intratumourally injected alum-tethered cytokines elicit potent and safer local and systemic anticancer immunity. Nat. Biomed. Eng. 6, 129-143 (2022), as are methods for making such single chain IL- 12 variants. The teachings in these references relating to IL- 12 variants are incorporated by reference herein. This disclosure contemplates use of any of the IL- 12 variants provided in such references. In other embodiments, the fusion comprises a functional variant of wild type, human IL- 12, e.g., having one or more amino acid mutations, two or more amino acid mutations, three or more amino acid mutations, or four, five, or six or more amino acid mutations. Such mutations may be substitutions such as conservative substitutions, deletions or insertions. A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, the fusion comprises an IL-12 variant having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the combination of SEQ ID NOs: 1 and 2 and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the activity of wild type, human IL- 12 having the amino acid sequences of SEQ ID NOs:l and 2. Similar functional variants of IL-12A and IL-12B subunits and single chain IL- 12 (as described above) are also contemplated by this disclosure.

[0055] Immunocytokine fusions comprising IL- 12 may be referred to herein as IL- 12 containing immunocytokine fusions or IL- 12 containing fusions or IL- 12 fusions.

[0056] Interleukin-15 (IL-15)

[0057] The IL- 15 cytokine regulates the activation and proliferation of T cells and natural killer (NK) cells. For example, IL-15 provides signals that maintain memory T cells in the absence of antigen and is implicated in NK cell development. The IL- 15 receptor, when bound to IL- 15, activates JAK kinase, STAT3, STAT5 and STAT6 transcription factors to elicit downstream signaling events.

[0058] Certain immunocytokine fusions of this disclosure comprise IL- 15 fused to the CD45 targeting moiety. The fusion may comprise full-length IL- 15 or a functional fragment thereof, or IL-15 complexed or fused to its receptor IL-15Ralpha (referred to as an IL-15 superagonist), or a functional variant of any of the foregoing. The term IL- 15 may be used herein in describing various aspects and embodiments but it is to be understood that such teachings apply equally to full-length IL- 15, functional fragments of full-length IL- 15, IL- 15 complexed or fused to its receptor IL-15Ralpha (referred to as an IL- 15 superagonist), and functional variants of any of the foregoing. In some embodiments, IL- 15 is human IL- 15.

[0059] In some embodiments, the fusion comprises a full length, wild-type, human IL- 15 having the amino acid sequence of SEQ ID NO: 3, or a functional fragment thereof. Examples of functional IL-15 variants are provided in US Publication No. US 20160184399, US Patent No. US 11365231 (Wong et al.), and US 2021 / 0325028 (Yueng et al.), and the specific teachings therein relating to IL- 15 variants are incorporated by reference herein and such variants are contemplated for use in the methods and compositions provided herein.

[0060] In some embodiments, the fusion comprises a cytokine is bound to its receptor. For example, the fusion may comprise IL- 15 bound to its receptor or an IL- 15 binding domain thereof. IL-15 receptor alpha has an amino acid sequence of SEQ ID NO:4. The IL-15 - IL- 15Ra complex is referred to herein as IL- 15 superagonist. A "superagonist" form of a cytokine may have increased activity, e.g., by at least 10%, 20%, 30%, compared to the cytokine when not covalently conjugated or complexed to its receptor. The IL- 15 superagonist may comprise an extracellular domain of an IL-15R alpha. IL-15 / IL-15Ra superagonists are described in WO 2010 / 059253, and these specific teachings are incorporated by reference herein.

[0061] In one embodiment, the IL- 15 receptor fragment comprises, or consists of, the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., at least 95% identical thereto, or having at least one, two, three, four, five amino acid alterations, but not more than ten, fifteen, twenty alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a fragment thereof, and having IL- 15 binding activity.

[0062] The wild-type IL-15 receptor alpha sequence is provided as SEQ ID NO: 4. IL- 15 receptor alpha contains an extracellular domain, a 23 amino acid transmembrane segment, and a 39 amino acid cytoplasmic tail. The sushi domain has been described in the literature including, e.g., Bergamaschi et al. (2008), JBC VOL. 283, NO. 7, pp. 4189-4199; Wei et al. (2001), Journal of Immunology 167:277-282; Schluns et al. (2004) PNAS Vol 110 (15) 5616- 5621; US 2016 / 0184399 (the specific contents of each of which relating to the sushi domain is incorporated by reference herein). Minimal sushi domain, wild-type: CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSL KC (SEQ ID NO: 6)

[0063] Extended sushi domain, wild-type:

[0064] ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIR (SEQ ID NO: 5)

[0065] In some embodiments, the fusion comprises a complex of the IL- 15 and an IL-

[0066] 15 receptor fragment, e.g., a sushi domain (e.g., as described herein). In some embodiments, the sushi domain is human or non-human. In one embodiment, the sushi domain includes a human sequence, or a substantially identical thereto (e.g., at least 95% identical thereto). In embodiments, the extracellular domain of IL- 15 receptor alpha comprises a domain referred to as herein as "the sushi domain," which binds IL-15. In embodiments, the sushi domain is provided as a 62-amino acid referred to herein as a "minimal domain" (e.g., having the amino acid sequence of SEQ ID NO: 6), or a 65-amino acid extended domain (e.g., having the amino acid sequence of SEQ ID NO: 5), or comprises a 77-amino acid domain comprising, e.g., consisting of, amino acids 31-107 or SEQ ID NO: 7. These sequences are known in the art and provided for example in PCT Publication No. WO 2019 / 010219.

[0067] In some embodiments, the IL- 15 receptor fragment has a wild type sequence. In other embodiments, the IL- 15 receptor fragment has a mutated sequence, e.g., a substitution at position 77, e.g., an L 771 substitution (with the numbering referring to the wild-type IL-15Ra of SEQ ID NO: 8). In some embodiments, the sushi domain comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, or SEQ ID NO: 10, or an amino acid sequence substantially identical thereto (e.g., at least 95% identical thereto, or having at least one, two, three, four, five amino acid alterations, but not more than ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, or SEQ ID NO: 10, and having IL- 15 binding activity.

[0068] In some embodiments, an IL- 15 receptor alpha extracellular domain consists of 62-171 amino acids of SEQ ID NO: 7 or an amino acid sequence substantially identical thereto (e.g., at least 95% identical thereto 96%, 97%, 98%, or 99% identity thereto or having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 alterations (e.g., substitutions) relative thereto, and having IL- 15 binding activity). In some embodiments, an IL-15 receptor alpha extracellular domain consists of 65- 171 amino acids of SEQ ID NO: 7 or an amino acid sequence substantially identical thereto (e.g., at least 95% identical thereto 96%, 97%, 98%, or 99% identity thereto or having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 alterations (e.g., substitutions) relative thereto, and having IL- 15 binding activity). In some embodiments, an IL- 15 receptor alpha extracellular domain consists of 62-171, 62-160, 62-150, 62-140, 62-130, 62-120, 62-110, 62-100, 62-90, 62-80, 62- 70, 65-171, 65-160, 65-150, 65-140, 65-130, 65-120, 65-110, 65-100, 65-90, 65-80, 65-70 or 65-77 amino acids of SEQ ID NO: 7 or a sequence having at least 95% identity thereto 96%, 97%, 98%, or 99% or having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 alterations (e.g., substitutions) relative thereto, and having IL- 15 binding activity. In some embodiments, an IL- 15 receptor alpha extracellular domain consists of 62-171, 62-160, 62-150, 62-140, 62-130, 62- 120, 62-110, 62-100, 62-90, 62-80, 62-70, 65-171, 65-160, 65-150, 65-140, 65-130, 65-120, 65- 110, 65-100, 65-90, 65-80, 65-70 or 65-77 amino acids of SEQ ID NO: 7.

[0069] Wild-type IL-15 receptor alpha sequence is deposited as Genbank Acc. No. AAI21141.1) (SEQ ID NO: 11). Wild-type IL-15 receptor alpha extracellular domain is a portion of the sequence deposited under accession number Q13261: SEQ ID NO: 7.

[0070] In other embodiments, the fusion comprises a functional variant of human IL-15, e.g., having one or more amino acid amino acid mutations, two or more amino acid mutations, three or more amino acid mutations, or four, five, or six or more amino acid mutations. Such mutations may be substitutions such as conservative substitutions, deletions or insertions. In some embodiments, the fusion comprises a functional variant of IL- 15 having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the activity of wild type human IL-15 having the amino acid sequence of SEQ ID NOG. Similar functional variants of IL-15 fragments and IL- 15 superagonists are also contemplated by this disclosure.

[0071] Immunocytokine fusions comprising IL- 15 may be referred to herein as IL- 15 containing immunocytokine fusions or IL- 15 containing fusions or IL- 15 fusions.

[0072] CD45 Targeting Moieties

[0073] Immuno stimulatory fusions disclosed herein include a CD45 targeting moiety. The CD45 targeting moiety can be chosen from an antibody or an antibody fragment (e.g., an antigen binding domain), an antibody variant, an antibody mimetic, or a ligand that binds to CD45 or a ligand fragment, or a combination thereof. The CD45 targeting moiety recognizes and binds specifically to CD45, and when used in vivo such CD45 is immune cell-bound CD45. In doing so, the CD45 targeting moiety targets (or delivers) the immunocytokine fusion (and the associated cytokine) to the CD45-expressing immune cell (e.g., a lymphocyte such as a T cell). CD45 is referred to as the leukocyte common antigen because it is expressed on leukocytes (i.e., white blood cells) but not erythrocytes (i.e., red blood cells). CD45, including human CD45, is a well-characterized antigen.

[0074] The CD45 targeting moiety may be chosen from an antibody (e.g., a full antibody (e.g., an antibody that includes at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains), or an antigen-binding antibody fragment (e.g., a Fab, F(ab')2, Fv, a single chain Fv (scFv), a single domain antibody, a diabody (dAb), or a nonantibody scaffold, or an antibody mimetic, or a moiety that binds to CD45.

[0075] The CD45 targeting moiety, in some embodiments, is one that binds human CD45. The CD45 targeting moiety may itself be of human origin or it may be of non-human origin yet still recognize and bind human CD45. In the case of antibodies or antibody fragments, the CD45 targeting moiety may be humanized. Techniques for humanizing an antibody are known in the art.

[0076] Examples of CD45 targeting moieties are known in the art. These include anti-CD45 human antibodies or humanized versions of animal anti-CD45 antibodies. Examples include but are not limited to BC8 (ACCT: HB-10507), 4B2, GAP8.3, and 9.4. These and other CD45 targeting moieties and CD45 specific antibodies may be found in PCT Publication No. W02019 / 010219, the specific teachings of which are incorporated by reference herein. Other anti-CD45 antibodies including anti-human CD45 antibodies whether such antibodies are of human origin or are humanized, are provided in US Patent Publication No. 20110076270 to Novartis, PCT Patent Publication No. W02003048327 to Abgenix, US Patent No. US 11692041 to UCB, US Patent Publication No. US 20170226209 to INSERM, US Patent No. US 9701756 to Miltenyi, PCT Patent Publication No. W02020092654 to Magenta, PCT Patent Publication No. W02022040088 to Synthekine, Inc. (providing examples of single domain antibodies that selectively bind to the extracellular domain of human CD45, as well as VHE, GAP8.3, 4B2 AND 9.4 anti-human CD45RO antibodies, the sequences of which are provided therein), PCT Patent Publication No. W02022040577 to City of Hope, PCT Patent Publication No. WO2022064191 to UCL (providing antibodies and fragments that specifically bind to human CD45 and comprise a light chain variable domain and a heavy chain variable domain, the sequences of which are provided therein), PCT Patent Publication No. WO2022063853, and US Patent Publication No. US 2021 / 0371524 (Palchaudhuri et al., describing antibodies such as Ab A, AbB, and AbC, the sequences of which are provided therein). The teachings in these references relating to anti-CD45 antibodies, CD45-binding fragments thereof, and functional variants thereof are incorporated by reference herein and this disclosure contemplates the use of such antibodies, fragments and variants in the compositions and methods provided herein.

[0077] The CD45 targeting moiety may be an antibody having amino acid changes in its Fc region to silence or reduce the effector function. Examples include antibodies L234A, L235A, and P329G (LALA-PG)). These amino acid changes may reduce or eliminate complement binding and fixation as well as Fc-y-dependent, antibody-dependent, cell-mediated cytotoxicity.

[0078] Immunocytokine Fusion Synthesis / Structure

[0079] An immunocytokine fusion may be synthesized through genetic engineering. This approach may involve removal of a stop codon from a cDNA sequence coding for one of the fusion protein components (e.g., IL-12 or IL-15), then appending the cDNA sequence of the second fusion protein components (e.g., CD45 specific antibody, fragment, or single chain) in frame through ligation or overlap extension PCR. If a full length or Ec containing antibody variant is used as the CD45 targeting moiety, the Ec receptor may be derived from an IgA (IgAl, IgA2), IgD, IgE, IgG (i.e., IgGl, IgG2, IgG3, IgG4), or IgM antibody. The resulting DNA sequence can be expressed as a single protein. The protein may be engineered to express the full sequence of both original proteins, or only a portion of one or both.

[0080] As an example, the fusion may comprise a CD45-binding fragment that is a Fab fragment, which optionally comprises a light chain and a heavy chain fragment optionally linked by a disulfide bond, and the cytokine (IL- 12 or IL- 15) is operably linked to the Fab fragment at a C-terminus of the light chain, an N-terminus of the light chain, a C-terminus of the heavy chain fragment, or an N-terminus of the heavy chain fragment.

[0081] In some embodiments, the cytokine (IL- 12 or IL- 15) is operably linked to a CD45- targeting moiety by a linker. The linker may be a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. In some embodiments, the linker is a peptide linker. The peptide linker can be 5-20, 8-18, 10-15, or about 8, 9, 10, 11, 12, 13, 14, 15-20, 20-25, or 25-30 amino acids long. In some embodiments the peptide linker can be 30 amino acids or longer; e.g., 30-35, 35-40, 40-50, or 50-60 amino acids long. In some embodiments, the peptide linker comprises Gly and Ser, e.g., a linker comprising the amino acid sequence (Gly3-Ser)n (SEQ ID NO: 19) or (Gly4-Ser)n (SEQ ID NO: 20), wherein n indicates the number of repeats of the motif, e.g., n= 1, 2, 3, 4 or 5 (e.g., a (Gly3- Ser)2 (SEQ ID NO: 21) or (Gly4Ser)2 (SEQ ID NO: 22), or a (Gly3-Ser)3 (SEQ ID NO: 23) or a (Gly 4Ser)3 (SEQ ID NO: 24) linker). In some embodiments, the peptide linker is a (GGGS)N (SEQ ID NO: 25) or a (GGGGS)N (SEQ ID NO:26) linker, wherein N indicates the number of repeats of the motif and is an integer selected from 1-10.

[0082] In embodiments, the linker comprises amino acids derived from an IgG hinge region, e.g., an IgGl, IgG2 or IgG4 hinge region. For example, the linker comprises a variant amino acid sequence from an IgG hinge, e.g., a variant having one or more cysteines replaced, e.g., with serines. In some embodiments, the linker comprises DKTHTCPPSCAPE (SEQ ID NO: 12), having one or both cysteines replaced with another amino acid, e.g., a serine. In some embodiments, the linker comprises amino acids DKTHTSPPSPAP (SEQ ID NO: 13), EPKSSDKTHTSPPSPAPE (SEQ ID NO: 14), or a derivative thereof. In embodiments, the linker comprises amino acids derived from an IgG2 hinge region, e.g., amino acids SVESPPSP (SEQ ID NO: 15), ERKSSVESPPSP (SEQ ID NO: 16), or a derivative thereof. In embodiments, the linker comprises amino acids derived from an IgG4 hinge region, e.g., amino acids PPSPSSP (SEQ ID NO: 17), ESKYGPPSPSSP (SEQ ID NO: 18), or a derivative thereof.

[0083] In other embodiments, the linker is a non-peptide, chemical linker. For example, the cytokine is covalently coupled to the CD45 targeting moiety by crosslinking. Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). In yet other embodiments, the cytokine is directly covalently coupled to the CD45 targeting moiety, without a linker. In yet other embodiments, the cytokine and the CD45 targeting moiety are not covalently linked, and may instead by non-covalently associated.

[0084] In other embodiments, the linker can be a protein or a fragment or derivative thereof, e.g., human albumin or an Fc domain, or a fragment or derivative thereof. In some embodiments, the CD45 targeting moiety is linked to the N-terminus and the cytokine is linked to the C-terminus.

[0085] In other embodiments, the linker non-covalently associates the CD45 targeting moiety to the cytokine. For example, the linker comprises a dimerization domain, e.g., a coiled coil or a leucine zipper.

[0086] Fusion based on other noncovalent interactions can also be used, e.g., using the high affinity of the IL-15 / sushi interaction to join two other proteins.

[0087] In the case of two-component fusions, the order of the components may be

[0088] N - cytokine - CD45 targeting moiety - C; or

[0089] N - CD45 targeting moiety - C; or

[0090] N - cytokine - linker - CD45 targeting moiety - C; or

[0091] N - CD45 targeting moiety - linker - cytokine - C.

[0092] Subjects

[0093] The term "subject" includes living organisms in which an immune response can be elicited (e.g., mammals, human). In some embodiments, the subjects may be humans and primates; domesticated or household animals such as cats and dogs; farm animals such as sheep, goats, cattle, horses and pigs; zoo animals; wild animals; and the like.

[0094] In some embodiments, the subject is a human subject who has been diagnosed with solid tumor cancer. In some embodiments, the subject has a solid tumor cancer that has metastasized to other organs.

[0095] Cancer

[0096] Cancer, as used herein, intends solid tumor cancers. Subjects being treated may have a localized cancer or metastatic cancer.

[0097] Solid tumor cancers include without limitation ovarian cancer, uterine cancer, breast cancer, testicular cancer, stomach cancer, cancer of the small intestine, colon cancer, rectal cancer, cancer of the anal region, renal-cell carcinoma, liver cancer, lung cancer including but not limited to non-small cell lung carcinoma, esophageal cancer, melanoma, Kaposi's sarcoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, brain stem glioma, pituitary adenoma, epidermoid cancer, carcinoma of the cervix squamous cell cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the vagina, sarcoma of soft tissue, cancer of the urethra, carcinoma of the vulva, cancer of the penis, cancer of the bladder, cancer of the ureter, carcinoma of the renal pelvis, spinal axis tumor, neoplasm of the central nervous system (CNS), primary CNS lymphoma, as well as metastatic lesions of said cancers, or combinations thereof.

[0098] The methods provided herein may also be used to treat subjects having lymphomas, including dosing the IL- 15 fusions and optionally the IL- 12 fusions in the affected lymph nodes, and making use of the differential temporal and / dose strategies described herein.

[0099] In some embodiments, the solid tumor cancer has metastasized in the subject.

[0100] Treatment

[0101] Various methods contemplated in this disclosure are intended to treat a subject having a solid tumor cancer. Treating a subject having a solid tumor cancer may be manifest as a solid tumor cancer that stops growing or begins to shrink. Thus, a reduction in tumor volume, as can be determined using standard imaging techniques such as ultrasound, MRI, CT or PET scanning, and the like, would indicate that the subject (and the tumor) have been treated, and consequently that the amount(s) of fusion(s) administered is effective. Treating a subject having a solid tumor cancer and metastatic lesions may manifest be manifest as one or more metastatic lesions that stop growing or that begin to shrink or that disappear altogether. Thus, a reduction in metastatic lesion volume or number would indicate that the subject (and the metastatic lesion(s)) have been treated, and consequently that the amount(s) of fusion(s) administered is effective, at least in the context of the metastatic lesions.

[0102] The IL- 12 containing fusion may be administered in an amount that is deemed effective when it is administered as a monotherapy. Such amounts can be determined in vivo (e.g., determining the amount that stimulates an immune response and / or tumor cell lysis in the tumor environment) or they may be determined using in vitro assays. Similarly, the IL-15 containing fusion may be administered in an amount that is deemed effective when it is administered as a monotherapy. Such amounts can be determined in vivo or by using in vitro assays. In still other embodiments, the IL-12 and / or the IL-15 containing fusions may be administered in amounts that are less than their respective effective amounts yet the combination of the two fusions results in therapeutic efficacy.

[0103] Cell-free preparation A cell-free preparation is a preparation that lacks cells (i.e., it is free of cells). Immunocytokine fusions may be administered in a cell-free preparation for in vivo use. Immunocytokine fusions therefore may be administered in a “free” or “soluble” form, intending that they are not bound to cells is required in an adoptive cell therapy approach.

[0104] Similarly the fusions are administered free of scaffolds such as polymeric scaffolds. The preparation to be administered may be regarded as scaffold- free or polymer- free or polymeric scaffold-free.

[0105] Immunocytokine Fusion Administration

[0106] Some methods provided herein effect separate administration of IL- 12 containing immunocytokine fusions and IL- 15 containing immunocytokine fusions. Separate administration in this context intends that the different immunocytokine fusions may be administered to different locations and / or at different times and / or in different amounts (or doses). While not intending to be bound by any particular theory or mechanism of action, certain methods aim to deliver and retain IL- 12 containing fusions in the tumor environment, thereby substantially limiting the immunostimulatory effect of IL- 12 to the tumor environment. Certain methods similarly aim to deliver IL- 15 containing fusions to proximal tumor-draining lymph nodes (TDLN), thereby targeting the effect of IL- 12 to TDLN. Different spatial and / or temporal administration strategies are described in greater detail below.

[0107] Differential spatial administrations strategies may involve administering the IL- 12 containing immunocytokine fusion intratumorally. Intratumoral injection can trigger tumor cell lysis, followed by release of tumor-derived antigens and subsequent activation of tumor- specific effector T cells, or the direct activation of tumor- specific effector cells. Intratumoral administration intends that the fusion is administered into a tumor. The site of injection is referred to herein as the “primary” site or “primary” tumor regardless of whether that is also the first location of the cancer in the subject. Typically the primary tumor / site is also the largest tumor mass in the subject and thus reasonably accessible by the end user. If a subject presents with multiple tumor masses, then injection may be made into one or more than one such mass. As demonstrated in the Examples, the effect of administration at and / or near one tumor lesion (i.e., the primary tumor) had beneficial impact on other tumor lesions (i.e., metastatic sites / tumors) in the body. Accordingly, the methods provided herein do not require administration at every tumor site and rather benefit from the anti-cancer immune response generated at or near that primary tumor site and transduced throughout the body.

[0108] Differential spatial administrations strategies also involve administering the IL- 15 containing immunocytokine fusion peritumorally and / or intranodally, both with the intended effect of delivering the IL- 15 fusion to TDLN. Peritumoral administration refers to administration in the tissue area proximal to but outside the tumor and having the same lymphatic drainage basin as the tumor. The primary tumor site to which the IL- 12 fusion is administered and the peritumoral region to which the IL- 15 containing fusion is administered both drain to the same lymph node(s). Depending on the tumor nature, size and other characteristics, the peritumoral region may be millimeters (e.g., tens or hundreds) or centimeters (e.g., 1 or 2) from the margins of the tumor. Medical practitioners, including oncology surgeons, may readily identify the peritumoral area of a given tumor.

[0109] Intranodal administration refers to administration to lymph nodes into which the primary tumor drains. Medical practitioners, including oncology surgeons, are able to readily identify the lymph nodes into which a tumor drains. The IL-15 fusions may be injected into one or more lymph nodes in such regions.

[0110] When the IL- 12 containing fusion is administered intratumorally and the IL- 15 containing fusion is administered peritumorally or intranodally, these injections may occur at substantially simultaneously (e.g., within 10, 20, 30, 40 or 50 minutes of each other) and they may occur in any order (e.g., the IL- 12 containing fusion may be administered first followed by the IL- 15 containing fusion, or vice versa).

[0111] Differential temporal administrations strategies involve first administering the IL- 12 containing immunocytokine fusion and then administering the IL- 15 containing immunocytokine fusion. In some embodiments, the IL- 15 containing immunocytokine fusion may be administered within 1 day after administration of the IL- 12 containing immunocytokine fusion. The IL-15 containing immunocytokine fusion may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after administration of the IL- 12 containing immunocytokine fusion. The IL- 15 containing immunocytokine fusion may be administered 1-6, 6-12, 12-18, or 18-24 hours after administration of the IL-12 containing immunocytokine fusion. In some embodiments, the IL- 15 containing immunocytokine fusion is administered days, weeks or months after administration of the IL- 12 containing immunocytokine fusion. The IL- 15 containing immunocytokine fusion may be administered 2, 3, 4, 5, or 6 days after administration of the IL- 12 containing immunocytokine fusion. The IL- 15 containing immunocytokine fusion administration may be even further delayed in other embodiments and therefore it may be administered 1, 2, 3, 4, 5, 6, 7, or 8 weeks after administration of the IL-12 containing immunocytokine fusion. The IL-15 containing immunocytokine fusion administration may be administered 1-4 weeks after administration of the IL- 12 containing immunocytokine fusion, or 1-3 weeks after administration of the IL- 12 containing immunocytokine fusion, or 1-2 weeks after administration of the IL- 12 containing immunocytokine fusion, or 7-10 days after administration of the IL-12 containing immunocytokine fusion.

[0112] When a differential temporal administration strategy is used, the IL- 12 containing immunocytokine fusion may be administered intratumorally and the IL- 15 containing immunocytokine fusion may be administered peritumorally and / or intranodally. Alternatively, a differential temporal administration strategy may involve administering the IL- 12 containing immunocytokine fusion and the IL- 15 containing immunocytokine fusion intratumorally, albeit at different times as described above.

[0113] This disclosure therefore contemplates both spatial and temporal separation of the IL- 12 containing immunocytokine fusion and the IL- 15 containing immunocytokine fusion.

[0114] In still other embodiments, the IL- 12 containing immunocytokine fusion is administered in a lower dose than the IL- 15 containing immunocytokine fusion. This embodiment is aimed at retaining the IL- 12 containing immunocytokine fusion at the tumor site while allowing the IL- 15 containing immunocytokine fusion to travel from the tumor site to the TDLN. Doses as used herein refer to the mass of the IL-12 cytokine and the mass of the IL-15 cytokine in the immunocytokine fusions. If the remainder of the fusion is identical between the IL- 12 and IL- 15 containing immunocytokine fusions, then the dose can be expressed as the mass of the IL- 12 containing fusion and the mass of the IL- 15 containing fusion. The difference between these doses may be expressed as a dose (or mass) ratio. Accordingly, the dose ratio of the IL- 12 containing fusion to the IL-15 containing fusion administered to a subject may be about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, or 1:500.

[0115] When the IL- 12 containing fusion is administered in a lower dose relative to the IL- 15 containing fusion, the fusions may both be administered intratumorally, or the IL- 12 containing fusion may be administered intratumorally and the IL- 15 containing fusion may be administered peritumorally and / or intranodally. Additionally or alternatively, the IL- 12 containing fusion may be administered ahead of the IL- 15 containing fusion, including using any of the administration schedules provided herein.

[0116] Accordingly, in some embodiments, the IL- 12 containing fusion may be administered intratumorally and the IL- 15 containing fusion may be administered peritumorally and / or intranodally more than 1 hour later, including but not limited to 6-12 hours, 12-24 hours, 1-7 days, 7-10 days, 1-2 weeks, or 1-3 weeks after the administration of the IL- 12 containing fusion. In any of these embodiments, the dose ratio of the IL-12 containing fusion to the IL-15 containing fusion may be in the range of 1:2 to 1:50, including 1:2 to 1:20, 1:2 to 1:10, or any of the dose ratio set forth herein.

[0117] Certain methods provided in this disclosure comprise administering an IL- 15 containing fusion to a subject having a solid tumor cancer that has already received (i.e., been administered) an IL-12 containing fusion or unconjugated IL-12. The IL-15 containing fusion may be administered peritumorally and / or intranodally to such subject. Additionally or alternatively, the IL-15 containing fusion may be administered within hours, weeks, or months after the IL-12 containing fusion (or IL- 12) was administered, including but not limited to 1-12 hours, 12-24 hours, 1-3 days, 7-10 days, 1-2 weeks, or 1-3 weeks after administration of the IL-12 containing fusion (or IL-12). The dosing of the IL-15 containing fusion may follow the dosing regimens and schedules set forth herein.

[0118] Compositions

[0119] Also provided in this disclosure are compositions comprising mixtures of the IL- 12 containing immunocytokine fusion and the IL- 15 containing immunocytokine fusion. The IL- 12 containing fusions and IL- 15 containing fusions may be present in the mixture in a range of dose (or mass) ratios. The dose ratios may range from 1:2 to 1:100, or from 1:2 to 1:50, or from 1:2 to 1:20, or from 1:2 to 1:10, or from 1:5 to 1:15, including about 1:10. As noted herein regarding dose ratios, these ratios refer to the dose (or mass) of the cytokine component in the fusion rather than the dose (or mass) of the entire fusion. However, if the remainder of the fusion is identical between the two, then these ratios will still apply.

[0120] These compositions may be administered intratumorally. They may also be administered only once to the subject. Therefore, unlike other methods provided herein that involve an intratumoral administration of at least the IL- 12 containing fusion and a peritumoral and / or intranodal administration of at least the IL- 15 containing fusion, these mixtures of the IL- 12 and IL- 15 containing fusions may be administered once, for example, intratumorally. In some instances, these intratumoral administrations may be followed with peritumoral and / or intranodal administration of IL- 15 containing fusions.

[0121] In some instances, the compositions are intended for in vivo use and therefore are regarded as being pharmaceutical compositions. Such pharmaceutical compositions may further comprise pharmaceutically acceptable carriers, excipients, and / or stabilizers.

[0122] The following examples are provided to illustrate specific instances of the practice of the present disclosure and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.

[0123] EXAMPLES

[0124] Materials

[0125] Gene blocks (gBlock, IDT) encoding for the light and heavy chain variable regions of anti-CD45 (clone: 30-F11) (Orozco et al. Therapy of Myeloid Leukemia using Novel Bispecific Fusion Proteins Targeting CD45 and 90Y-DOTA. Mol. Cancer Ther. 19, 2575-2584 (2020)) or untargeted isotype control (anti-FITC, clone: 4-4-20) were cloned into a mouse kappa light chain and IgG2c backbone with LALA-PG mutations (Lo et al. Effector- attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice. J. Biol. Chem. 292, 3900-3908 (2017)), respectively, in the mammalian expression vector gWiz (Genlantis). Murine IL-15 / IL-15Rasushi, as described previously (Agarwal et al. Intratumourally injected alum-tethered cytokines elicit potent and safer local and systemic anticancer immunity. Nat. Biomed. Eng. 6, 129-143 (2022)), was then cloned at the C terminus of the heavy chain; murine single chain IL- 12 (scIL-12), as described previously (Agarwal et al. Intratumourally injected alum-tethered cytokines elicit potent and safer local and systemic anticancer immunity. Nat. Biomed. Eng. 6, 129-143 (2022), was cloned at the N terminus of the heavy chain. A gBlock encoding for extracellular domain of mouse CD45RO (used for ELIS As; sequence obtained from Uniprot) was cloned into gWiz with a His6 tag. Plasmid sequences confirmed by Sanger sequencing (Quintara Biosciences) were transformed into Stellar Competent Cells (Takara Bio) and purified using the NucleoBond Xtra Midi endotoxin-free midi-prep kit (Takara Bio). For immunocytokines, equal mass of heavy and light chain plasmids were transfected into Expi293F cells (Gibco) per manufacturer’s instructions and harvested 6 days after transfection. All immunocytokines were purified using rProteinA Sepharose Fast Flow resin (Cytiva Life Sciences) and validated for size by SDS-PAGE. His-tagged CD45RO was purified by TALON affinity resin (Takara). Purified proteins were confirmed to be endotoxin-free (<0.1 EU per dose) using the Endosafe Nexgen-PTS system (Charles River). Purified proteins were flash- frozen in liquid nitrogen and stored at -80C until use.

[0126] Example 1:

[0127] We have shown that antibodies against the ubiquitous leukocyte surface receptor CD45 are able to bind to the surface of T cells and other lymphocytes without triggering internalization. We hypothesized that cell surface-displayed cytokines using CD45 targeting can exhibit new biological effects relative to the native forms of these cytokines. To test this, we generated aCD45 immunocytokine fusions with IL- 15 as a testbed pay load. Specifically, murine IL-15 / IL-15Ra was fused to the C terminus of the heavy chain of a murine IgG2c backbone with LALA-PG effector attenuating mutations (hereafter, aCD45-IL15; FIG. 1A). A non-targeting size-matched immunocytokine was also developed, referred to as IgG-IL15. We verified that recombinantly expressed aCD45-IL15 was able to bind plate-bound and cell-surface CD45 as well as maintain its cytokine bioactivity on reporter cells (FIGs. 1B-1C).

[0128] A fluorescence quenching assay and dye-labeled constructs were used to assess the binding and internalization behavior of aCD45 immunocytokines incubated with activated CD8 T cells isolated from murine splenocytes. Non-targeted cytokines were rapidly internalized, while aCD45-IL15 had a cell surface half-life of 30 hours, validating the ability of CD45 anchoring to maintain cytokines on the cell surface despite their rapid native internalization behavior (FIG. ID).

[0129] To evaluate whether this extended surface retention enabled enhanced cytokine activity, primary CD8 T cells were pulsed and washed with aCD45-IL15 or IgG-IL-15 fusions, and downstream signaling was evaluated after 24 hours. Prolonged cell surface residence time enabled T cells briefly pulsed with aCD45 -cytokine for 1 hour to exhibit robust pSTAT5, CD25, and Ki67 expression 24 hours later (FIG. IE). Non-CD45 targeted IL-15 (IgG-IL-15) performed similarly to media controls (FIG. IE). Further, when CD8+ T cells were pre-loaded with aCD45-IL15 by pulsing for 1 hour with the construct, then washed and mixed with non-loaded “bystander” CD8+T cells, the presence of just 1 pre-loaded T cell per 100 total cells led to pSTAT5 induction in a majority T cell culture 1 hour later (FIG. IF). Fluorescent labeling of the aCD45-IL-15 construct showed minimal transfer onto bystander cells at this ratio (data not shown). The ability to stimulate bystander cells suggests that cells decorated with aCD45-cytokines are able to present the payload effectively in trans. Non-targeted IL-15 (IgG-IL-15) led to robust pSTAT5 signal in the preloaded population, but failed to stimulate any bystander cells, likely due to the rapid internalization of the cytokine (FIG. IF).

[0130] Then, the in vivo biodistribution of these fusion proteins was assessed following intratumoral administration. For these experiments, test and control IL- 15 fusions (i.e., aCD45- IL-15 and IgG-IL-15) were labeled with AF647, and the cytokine fusions were injected into established MC38 flank tumors. Association of the constructs was then assessed on cells in the tumor, tumor-draining lymph nodes (TDLNs), and in the blood 24 hours later by flow cytometry and fluorescence measurements (FIG. 2A). Injection of 10 pg CD45-targeted IL15 (1.4 pg on a cytokine basis) led to robust association with essentially all CD45+cells in the tumor (FIG. 2B). By contrast, the control non-targeted cytokine (IgG-IL-15) only showed association with -40% of intratumoral CD45+cells at very dim levels (FIGs. 2B-2D). This dose also led to labeling of -75% of CD45+cells in the TDLN by aCD45-IL15 but no detectable free cytokine fusion in the blood (FIGs. 2E-2F). Control IgG-IL15 labeling only reached around -5% of TDLN CD45+cells and was readily detected in serum (FIGs. 2E-2F). Confocal microscopy confirmed these results, showing broad distribution of aCD45-IL15 throughout the tumor and the TDLN, with striking B cell colocalization (FIGs. 2G-2H). Thus, CD45 targeting increased localization of the cytokine on immune cells in both tumors and TDLNs, and lowered systemic exposure when doses were selected to not exceed the binding capacity of leukocyte surface CD45 in the tumor.

[0131] Next, the impact of this altered biodistribution on the therapeutic potential of a cytokine therapy was evaluated by employing two cytokines, IL-15 and IL-12. Syngeneic flank tumors were treated with a single intratumoral injection of aCD45-IL12 followed by one injection of aCD45-IL15 several days later. The relative doses were informed by biodistribution experiments and aimed to retain IL-12 at the tumor and IL-15 at the tumor and TDLN. Hereafter, this sequential combination is referred to as aCD45-Cyt or IgG-Cyt therapy for the CD45-targeted vs. non-CD45-targeted control treatments, respectively. Before efficacy experiments, it was confirmed that aCD45-IL12 is retained on the surface of primary lymphocytes and bioactive on reporter cells (not shown).

[0132] The aCD45-cytokine therapy and non-targeted control were tested in two common syngeneic tumor models: the MC38 colorectal carcinoma that is partially responsive to CBI, and B16F10 which is a very aggressive and poorly responsive melanoma model. Injection of control IgG-Cyt elicited an extension in survival in both syngeneic tumor models, however few (MC38) or no (B16F10) animals were long-term survivors (FIGs. 3A-3B). By contrast, 100% (MC38) or >90% (B16F10) of tumors treated with aCD45-Cyt therapy were rejected, and animals experienced no weight loss (FIGs. 3A-3B). On a cytokine mass basis, these doses did not surpass 2pg of total cytokine, highlighting the potency of this approach. Mice cured by aCD45- Cyt therapy displayed robust immune memory with >75% rejecting a secondary tumor rechallenge (FIG. 3C).

[0133] The mechanism of aCD45-Cyt was further assessed through antibody depletions and knockout mice. These data show that aCD45-Cyt therapy is dependent on CD8 T cells and batf3 dendritic cells, indicating a role of cross-presentation and de novo priming at the TDLN (FIG. 3D). Natural Killer (NK), neutrophils, and macrophages were dispensable for the observed antitumor efficacy (FIG. 3D). Use of both cytokines in the treatment was preferred, as aCD45-IL15 or aCD45-IL12 monotherapy elicited no long-term survivors (FIG. 3E).

[0134] The relevant contributions to efficacy from intratumoral versus TDLN leukocytes were analyzed by varying the route of administration of aCD45-Cyt therapy. While dosing both cytokines peritumorally impacted efficacy, it was found that peritumorally delivered aCD45- IL15, which should predominantly act on the TDLN while sparing the tumor, displayed comparable tumor control to the full intratumoral paradigm.

[0135] Abscopal immune responses to this localized therapy were also evaluated. Injection of MC38 tumors on opposite flanks of mice followed by treatment of only the right-flank tumor with aCD45-Cyt treatment strikingly led to 90% of mice rejecting both the treated and untreated tumors and becoming long-term survivors, while only 20% of animals receiving control IgG-Cyt therapy eliminated both tumors (FIGs. 4A-4B). Tumor control at the untreated lesion was not driven by leakage or diffusion out of the treated tumor and TDLN, as there was no significant accumulation of aCD45-IL15 at the contralateral tumor (FIG. 4C). In contrast, unanchored IL- 15 was readily detected at the distal lesion following intratumoral delivery on the opposite flank (FIG. 4C). A similar experiment was performed in the aggressive B16F10 model. While control IgG-Cyt treatment led to some delay of treated tumors, all untreated tumors grew progressively (FIG. 4D). By contrast, aCD45-Cyt therapy led to primary tumor regression and only 2 of 5 untreated tumors escaped.

[0136] The immune response to aCD45-Cyt therapy was profiled by analyzing T cells in the tumor and TDLN 2 days after completion of aCD45-Cyt therapy. Though a significant swelling of the CD8 compartment over PBS was seen by aCD45-Cyt therapy in the TDLN, there was no significant difference between the two cytokine therapies (FIG. 5A). aCD45-Cyt therapy led to robust phenotypic differences within the CD8 T cell compartment in the TDLN. For example, aCD45-Cyt therapy led to significant increases in PD1 expression by TDLN CD8+ cells (FIG. 5B), and this activated population was profiled further. Striking increases in the number of CD25+, GzmB+, and IFNg+ activated T cells were seen only in the aCD45-Cyt group (FIGs. 5C-5E). aCD45-Cyt therapy also drove strong differentiation of PD1+ TDLN CD8+ T cells as seen by a significant increase in TIM3+TCF1- cells (FIG. 5F). This ability to drive cytolytic effectors at the TDLN was unique to aCD45-Cyt therapy, as unanchored cytokine controls led to non-significant increases across these various markers (FIGs. 5A-5F). Both anchored and unanchored cytokine treatments led to significant increases in the number of stem-like PD1+TCF1+TIM3- CD8+ T cells in the lymph node (FIG. 5F). Analysis of intratumoral CD8+ T cells revealed similar phenotypic patterns as in the lymph node. aCD45-Cyt therapy did not drive increased CD8+ T cell infiltration over IgG-Cyt, but did lead to upregulation of CD25, IFNg, and TNF within activated PD1+ CD8+ T cells (FIGs. 5G-5J).

[0137] EQUIVALENTS

[0138] The foregoing written specification is considered to be sufficient to enable one ordinarily skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as mere illustrations of one or more aspects of the invention. Other functionally equivalent embodiments are considered within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.

[0139] Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0140] All references, patents and patent applications that are recited in this application are incorporated by reference herein in their entirety.

Claims

What is claimed is:CLAIMS1. A method for treating a subject having a solid tumor cancer comprising administering intratumorally to a subject having a solid tumor cancer, an effective amount of a first immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing, in a cell-free preparation(s), and administering to the subject an effective amount of a second immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 15, a functional fragment of full-length IL- 15, IL-15 / IL-15Ra (IL- 15 superagonist) or a functional fragment thereof, or a functional variant of any of the foregoing.

2. The method of claim 1, wherein the second immunocytokine fusion is administered peritumorally.

3. The method of claim 1, wherein the second immunocytokine fusion is administered intranodally to tumor draining lymph nodes (TDLN).

4. The method of claim 1, wherein the second immunocytokine fusion is delivered to tumor draining lymph nodes (TDLN).

5. The method of claim 1, wherein the second immunocytokine fusion is administered intratumorally.

6. The method of any one of claims 1-5, wherein the second immunocytokine fusion is administered after administration of the first immunocytokine fusion.

7. The method of claim 6, wherein the second immunocytokine fusion is administered about 4, about 6, about 8, about 12, about 18, or about 24 hours after administration of the first immunocytokine fusion.

8. The method of claim 6, wherein the second immunocytokine fusion is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after administration of the first immunocytokine fusion.

9. The method of any one of claims 1-8, wherein the first and second immunocytokine fusions are administered in a dose ratio of about 1:2, about 1:4, about 1:6, about 1:8, about 1:10, about 1:12, about 1:15, or about 1:20.

10. A method for treating a subject having a solid tumor cancer comprising administering, in a cell-free preparation(s), to a subject having a solid tumor cancer,(i) an effective amount of a first immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing, and(ii) an effective amount of a second immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 15, a functional fragment of full-length IL- 15, IL-15 / IL-15Ra (IL- 15 superagonist) or a functional fragment thereof, or a functional variant of any of the foregoing, wherein the second immunocytokine fusion is administered about 1 hour to about 21 days after administration of the first immunocytokine fusion.

11. The method of claim 10, wherein the first immunocytokine fusion is administered intratumorally.

12. The method of claim 10 or 11, wherein the second immunocytokine fusion is administered intratumorally.

13. The method of claim 10 or 11, wherein the second immunocytokine fusion is administered peritumorally.

14. The method of claim 10 or 11, wherein the second immunocytokine fusion is administered intranodally to tumor draining lymph nodes (TDLN).

15. The method of claim 10 or 11, wherein the second immunocytokine fusion is delivered to tumor draining lymph nodes (TDLN).

16. The method of any one of claims 10-15, wherein the second immunocytokine fusion is administered about 4, about 6, about 8, about 12, about 18, or about 24 hours after administration of the first immunocytokine fusion.

17. The method of any one of claims 10-15, wherein the second immunocytokine fusion is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after administration of the first immunocytokine fusion.

18. The method of any one of claims 10-17, wherein the first and second immunocytokine fusions are administered in a dose ratio of about 1:2, about 1:4, about 1:6, about 1:8, about 1:10, about 1:12, about 1:15, or about 1:20.

19. A method for treating a subject having a solid tumor cancer comprising administering intratumorally to a subject having a solid tumor cancer, an effective amount of a first immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing, in a cell-free preparation(s), and administering to the subject an effective amount of a second immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 15, a functional fragment of full-length IL- 15, IL-15 / IL-15Ra (IL- 15 superagonist) or a functional fragment thereof, or a functional variant of any of the foregoing, wherein the first and second immunocytokine fusions are administered in a dose ratio of about 1:2 to about 1:20.

20. The method of claim 19, wherein the first and second immunocytokine fusions are administered in a dose ratio of about 1:2, about 1:4, about 1:6, about 1:8, about 1:10, about 1:12, about 1:15, or about 1:20.

21. The method of claim 19 or 20, wherein the second immunocytokine fusion is administered intratumorally.

22. The method of claim 21, wherein the second immunocytokine fusion is administered peritumorally.

23. The method of claim 21, wherein the second immunocytokine fusion is administered intranodally to tumor draining lymph nodes (TDLN).

24. The method of claim 21, wherein the second immunocytokine fusion is delivered to tumor draining lymph nodes (TDLN).

25. The method of any one of claims 19-24, wherein the second immunocytokine fusion is administered about 4, about 6, about 8, about 12, about 18, or about 24 hours after administration of the first immunocytokine fusion.

26. The method of any one of claims 19-24, wherein the second immunocytokine fusion is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after administration of the first immunocytokine fusion.

27. A method for treating a subject having a solid tumor cancer comprising administering to a subject having a solid tumor cancer an effective amount of an immunocytokine fusion comprising a CD45 targeting moiety conjugated to full-length IL- 15, IL-15 / IL-15Ra (IL- 15 superagonist) or a functional fragment thereof, or a functional variant of any of the foregoing, in a cell-free preparation(s), wherein the subject was administered full-length IL- 12, a functional fragment of full- length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or afunctional variant of any of the foregoing, either in free form or in a fusion protein comprising an immune cell targeting protein.

28. The method of claim 27, wherein the immunocytokine fusion is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after administration of the free or fusion form of full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing.

29. The method of claim 27 or 28, wherein the immunocytokine fusion is administered in a dose that is 2-fold to 20-fold greater than the dose of the free or fusion form of full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing.

30. The method of any one of claims 27-29, wherein the immunocytokine fusion is administered intratumorally.

31. The method of any one of claims 27-29, wherein the immunocytokine fusion is administered peritumorally and / or intranodally.

32. The method of any one of claims 27-31, wherein the free or fusion form of full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing was administered intratumorally.

33. The method of any one of claims 27-32, wherein the immune cell targeting protein is a CD45 targeting protein.

34. The method of any one of the foregoing claims, wherein the CD45 targeting moiety is a CD45- specific antibody or antigen-binding antibody fragment.

35. The method of any one of the foregoing claims, wherein the CD45 targeting moiety is CD45-specific antibody BC8, 4B2, GAP8.3, or 9.4, or a humanized version thereof, or an CD45 -binding fragment thereof.

36. The method of any one of the foregoing claims, wherein the CD45 targeting moiety is an Fab fragment, an F(ab’)2 fragment, or a single chain Fv, or antibodies BC8, 4B2, GAP8.3, or 9.4, or a humanized version thereof.

37. The method of any one of the foregoing claims, wherein the solid tumor cancer is a colorectal cancer or a melanoma.

38. The method of any one of the foregoing claims, wherein the solid tumor cancer has metastasized in the subject.

39. The method of claim 38, wherein the solid tumor cancer has metastasized and the first and second immunocytokine fusion is administered at or near a primary solid tumor site.

40. An immunomodulatory composition comprising a first and a second immunocytokine fusion, each immunocytokine fusion comprising an immunomodulatory cytokine conjugated to a CD45 targeting moiety, in a cell-free preparation, wherein the first immunocytokine fusion comprises a CD45 targeting moiety conjugated to full-length IL- 12, a functional fragment of full-length IL- 12, a single chain IL- 12, a subunit of IL- 12, IL- 12 subunit A, IL- 12 subunit B, or a functional variant of any of the foregoing, wherein the second immunocytokine fusion comprises a CD45 targeting moiety conjugated to full-length IL-15, IL-15 / IL-15Ra (IL-15 superagonist) or a functional fragment thereof, or a functional variant of any of the foregoing, and wherein the first and second immunocytokine fusions are present in a dose ratio in the range of 1:2 to 1:20.

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