Cytokine therapies for managing HIV and other viral infections

Abstract

Disclosed herein are methods of treating HIV comprising administering an effective amount of IL-15 and / or IL-15Ralpha optionally in combination with IL-12 or nucleic acids or vectors encoding the same to a human patient in need thereof. In certain embodiments, the IL-15 and / or IL-15Ralpha optionally in combination with IL-12 or nucleic acids or vector encoding the same are administered in combination with a multi-drug anti-retroviral therapy (ART).

Description

[0001] CYTOKINE THERAPIES FOR MANAGING HIV AND OTHER VIRAL INFECTIONS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 559,255 filed February 29, 2024. The entirety of this application is hereby incorporated by reference for all purposes.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under AH48377 and OD010947 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM

[0007] The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 24095PCT.xml. The XML file is 13,414 bytes, was created on February 25, 2025, and is being submitted electronically via the USPTO Patent Center filing system.

[0008] BACKGROUND

[0009] There are millions of humans living with HIV / AIDS. Combination antiretroviral therapy (ART) treatment regimens have successfully prolonged the lives of infected individuals. Stopping combination antiretroviral therapy (ART) leads to the HIV re-emergence. It is reported that HIV reservoirs residing in immune privileged areas are able to shield the virus from the immune system. See Churchill et al., Nat Rev Microbiol, 2016, 14:55-60. Thus, there is a need to identify improved methods of treating HIV.

[0010] Lin et al. report the effect of Interleukin (IL)- 12 and IL- 15 on activated natural killer and antibody-dependent cellular cytotoxicity in HIV Infection. Journal of Clinical Immunology, 1998, 18(5):335-345.

[0011] Guo report immunobiology of the IL-15-IL-15Ralpha complex as an antitumor and antiviral agent. Cytokine Growth Factor Rev, 2017, 38:10-21. Harwood et al. report therapeutic potential of IL-15 and N-803 in HIV / SIV infection. Viruses, 2021, 13, 1750.

[0012] Harwood et al. report transient T cell expansion, activation, and proliferation in therapeutically vaccinated simian immunodeficiency virus-positive macaques treated with N-803. J Virol, 2022, 96(23):e0142422.

[0013] Li et al. report IL- 15 enhances HIV-1 infection by promoting survival and proliferation of CCR5+CD4+ T cells. JCI Insight, 2023, 8(7):el66292.

[0014] See also US Patent Nos. 8,507,222, 9,255,141, 10,265,382, 10,335,460, and 10,808,022. References cited herein are not an admission of prior art.

[0015] SUMMARY

[0016] Disclosed herein are methods of treating HIV comprising administering an effective amount of IL-15 and / or IL-15Ralpha, or nucleic acid or vector encoding the same, optionally in combination with IL-12, or nucleic acid or vector encoding the same, to a subject, e.g., human patient, in need thereof. In certain embodiments, the IL- 15 and / or IL-15Ralpha, or nucleic acid or vector encoding the same, optionally in combination with IL- 12, or nucleic acid or vector encoding the same, are administered in combination with a multi-drug anti-retroviral therapy (ART).

[0017] In certain embodiments, IL-15, IL-15Ralpha, and / or IL-12 are administered in combination with and prior or during or at the initiation of the first administration of a multi-drug anti-retroviral therapy (ART). In certain embodiments, IL-15 and IL-12 are administered in combination with and prior or during or at the initiation of the first administration of a multi-drug anti-retroviral therapy (ART). In certain embodiments, IL-15Ralpha and IL-12 are administered in combination with and prior or during or at the initiation of the first administration of a multi-drug anti-retroviral therapy (ART). In certain embodiments, IL- 15 is administered in combination with and prior or during or at the initiation of the first administration of a multi -drug anti-retroviral therapy (ART). In certain embodiments, IL-15Ralpha is administered in combination with and prior or during or at the initiation of the first administration of a multi-drug anti-retroviral therapy (ART). In certain embodiments, IL- 12 is administered in combination with and prior or during or at the initiation of the first administration of a multi-drug anti-retroviral therapy (ART).

[0018] In certain embodiments, this disclosure relates to pharmaceutical compositions comprising IL- 15 and IL-15Ralpha, or nucleic acid or vector encoding the same, optionally in combination with IL-12, or nucleic acid, or vector encoding the same, and a pharmaceutically acceptable excipient. In certain embodiments, this disclosure relates to pharmaceutical compositions comprising agent(s) or combinations as disclosed herein.

[0019] In certain embodiments, this disclosure relates to uses of IL-15 and IL-15Ralpha, or nucleic acid or vector encoding the same, optionally in combination with IL- 12, or nucleic acid, or vector encoding the same, for the production of a medicament.

[0020] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0021] Figure 1 shows a scheme of a study used to test the effects of IL-12 and IL-15 / IL-15Ra as a mono or combination therapy in SIV infected, ART-treated rhesus macaques.

[0022] Figures 2A-2D show data indicating IL-15 / IL-15Ra treatment increased effector memory CD4 T cells during the second phase of cytokine treatment and sustained the elevation until at the time of ATI in blood.

[0023] Figure 2A shows data on temporal dynamics of total CD4 T cells during post cytokine treatment in SIV infected and ART treated animals.

[0024] Figure 2B shows data on temporal dynamics of proliferating CD4 T cell levels during post cytokine treatment.

[0025] Figure 2C shows data on temporal dynamics of frequency of central memory CD4 T cells.

[0026] Figure 2D shows data on temporal dynamics of frequency effector memory CD4 T cell during post cytokine treatment in SIV infected and ART treated animals.

[0027] Figure 3 shows data indicating IL-15 / IL-15Ra and IL- 12 plus IL-15 / IL-15R treated group animals induced CD8+ T cells in the B cell follicles of the LN during post-ATI. Images were obtained from about 10 different regions of a LN section per animal (N=3 for IL- 12, IL-15 / IL- 15R, IL-12 plus IL-15+IL-15R treated and ART-only control group). Row A shows data on quantity of total SIV Gag-CM9 tetramer-specific CD8+ T cells using in situ Gag-CM9 tetramer staining at the week 4 ATI time point in total lymph node area, B cell follicular area and T cell zone area. Row B shows data on the absolute counts of Tetramer+ GrzB+ cells. Row C shows data on the percentage of Tetramer+ GrzB+ cells. DETAILED DISCUSSION

[0028] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims or as amended during prosecution.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0030] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0031] As used herein, the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - a percentage of the specified value. In embodiments, about includes the specified value. In certain embodiments, the term “about” can include a 5 % or 10 % difference.

[0032] As used herein, "subject" refers to any animal, preferably a human patient, livestock, or domestic pet.

[0033] As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also contemplate treatment that merely reduces symptoms, and / or delays disease progression.

[0034] As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

[0035] As used herein, "administering" refers to any variety of administration, e.g., oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release biocompatible material or device to a subject. Administration is contemplated to include parenteral and transmucosal routes (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0036] As used herein, the term "combination with" when used to describe administration of an agent with an additional treatment means such that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof, such that multiple agents are pharmacologically available at some overlapping time, e.g. considering the half-life of each agent.

[0037] The term "effective amount" or "therapeutically effective amount" refers to that amount of an agent, peptide, vector, or pharmaceutical composition described herein that is sufficient to effect the intended application including, but not limited to, disease treatment, as illustrated below. The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells. The specific dose will vary depending on, for example, the particular agents chosen, the dosing regimen to be followed, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0038] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term “comprising” in reference to an oligonucleotide or peptide having a nucleotide or peptide sequence refers an oligonucleotide or peptide refers to an oligonucleotide or peptide that may contain additional 5’ (5’ terminal end) or 3’ (3’ terminal end) nucleotides orN- or C-terminal amino acids, i.e., the term is intended to include the oligonucleotide sequence or peptide sequence within a larger nucleic acid or peptide.

[0039] "Consisting essentially of or "consists of or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel character! stic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. The term “consisting of’ in reference to an oligonucleotide or peptide having a nucleotide or peptide sequence refers an oligonucleotide or peptide having the exact number of nucleotides or amino acids in the sequence and not more or having not more than a range of nucleotide expressly specified in the claim. For example, “5’ sequence consisting of’ is limited only to the 5’ end, i.e., the 3’ end may contain additional nucleotides. Similarly, a “3’ sequence consisting of’ is limited only to the 3’ end, and the 5’ end may contain additional nucleotides. In certain embodiments, the C-terminus may have 5, 10, 20, or 50 additional amino acids. In certain embodiments, the N-terminus may have 5, 10, 20, or 50 additional amino acids.

[0040] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p- acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art to provide a bioconjugate.

[0041] “IL-15” or “Interleukin- 15” and the like refers to a cytokine / protein, which in humans is involved in inflammatory and protective immune responses to microbials and reported to interact with immune cells of both the innate and adaptive immune systems. As documented by UniPro P40933, a consensus human sequence is

[0042] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVI SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLII LANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 1). Bold is the signal / pro-peptide sequence Many variants are known.

[0043] The natural receptor of IL- 15 in humans, i.e. IL- 15 receptor (IL- 15R) includes IL- 15Ralpha (IL-15a or CD25), CD122, and CD132 subunits. IL15 can be expressed in association with its high affinity IL-15Ra on the surface of IL 15 -producing cells.

[0044] “IL- 15Ralpha” or “IL-15Ra” and the like refers to protein in the subunit. As documented by UniPro Q1326, a human consensus sequence is

[0045] MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRER YICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTP QPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTA KNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVE MEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 2). Variants are known. Non-limiting examples of GenBank Accession Nos. for the amino acid sequence of various species of native mammalian interleukin- 15 include NP 000576 (human, immature form), CAA62616 (human, immature form), NP_001009207 (Felis catus, immature form), AAB94536 (rattus, immature form), AAB41697 (rattus, immature form), NP_032383 (Mus musculus, immature form), AAR19080 (canine), AAB60398 (macaca mulatta, immature form), AAI00964 (human, immature form), AAH23698 (mus musculus, immature form), and AAH18149 (human).

[0046] In certain embodiments, the ratio of IL-15 to IL-15Ralpha is between 1 / 10 to 2 / 5. In certain embodiments, the ratio of IL-15 to IL-15Ralpha is about 1 / 5.

[0047] In certain embodiments, amino acid sequence of the immature / precursor form of native human IL- 15, which comprises the long signal peptide (bold) and the mature human native IL- 15 (without the signal sequence), is provided:

[0048] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVI SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLII LANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 1), signal sequence MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA (SEQ ID NO: 7)

[0049] It is contemplated that IL- 15 is used therapeutically without the signal sequence, as provided below: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 3).

[0050] In some embodiments, native IL- 15 is the immature or precursor form of a naturally occurring mammalian IL-15. In other embodiments, native IL- 15 is the mature form of a naturally occurring mammalian IL-15. In a specific embodiment, native IL- 15 is the precursor form of naturally occurring human IL-15. In another embodiment, native IL-15 is the mature form of naturally occurring human IL-15. In one embodiment, the native IL-15 protein / polypeptide is isolated or purified.

[0051] As used herein, the terms “native IL- 15” and “native interleukin- 15” in the context of nucleic acids refer to any naturally occurring nucleic acid sequences encoding mammalian interleukin-15, including the immature or precursor and mature forms. Non-limiting examples of GenBank Accession Nos. for the nucleotide sequence of various species of native mammalian IL- 15 include NM_000585 (human), NM_008357 (Mus musculus), and RNU69272 (rattus norvegicus).

[0052] In a specific embodiment, the nucleic acid is an isolated or purified nucleic acid. In some embodiments, nucleic acids encode the immature or precursor form of a naturally occurring mammalian IL-15. In other embodiments, nucleic acids encode the mature form of a naturally occurring mammalian IL-15. In a specific embodiment, nucleic acids encoding native IL- 15 encode the precursor form of naturally occurring human IL-15. In another embodiment, nucleic acids encoding native IL- 15 encode the mature of naturally occurring human IL-15.

[0053] As used herein, the terms “IL- 15 derivative” and “interleukin- 15 derivative” in the context of proteins or polypeptides refer to: (a) a polypeptide that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to a native mammalian IL-15 polypeptide; (b) a polypeptide encoded by a nucleic acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical a nucleic acid sequence encoding a native mammalian IL-15 polypeptide; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and / or substitutions) relative to a native mammalian IL-15 polypeptide; (d) a polypeptide encoded by nucleic acids can hybridize under high, moderate or typical stringency hybridization conditions to nucleic acids encoding a native mammalian IL- 15 polypeptide; (e) a polypeptide encoded by a nucleic acid sequence that can hybridize under high, moderate or typical stringency hybridization conditions to a nucleic acid sequence encoding a fragment of a native mammalian IL- 15 polypeptide of at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 100 contiguous amino acids, or at least 150 contiguous amino acids; or (f) a fragment of a native mammalian IL- 15 polypeptide. IL-15 derivatives also include a polypeptide that comprises the amino acid sequence of a naturally occurring mature form of a mammalian IL- 15 polypeptide and a heterologous signal peptide amino acid sequence. In a specific embodiment, an IL-15 derivative is a derivative of a native human IL- 15 polypeptide. In another embodiment, an IL- 15 derivative is a derivative of an immature or precursor form of naturally occurring human IL- 15 polypeptide. In another embodiment, an IL- 15 derivative is a derivative of a mature form of naturally occurring human IL- 15 polypeptide. In one embodiment, an IL- 15 derivative is isolated or purified.

[0054] In a preferred embodiment, IL-15 derivatives retain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of native mammalian IL- 15 polypeptide to bind IL-15Ra polypeptide, as measured by assays well known in the art, e.g., ELISA, coimmunoprecipitation. In another preferred embodiment, IL-15 derivatives retain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of native mammalian IL- 15 polypeptide to induce IL-15-mediated signal transduction, as measured by assays well-known in the art, e.g., electromobility shift assays, ELISAs and other immunoassays.

[0055] IL- 15 derivative nucleic acid sequences include codon-optimized nucleic acid sequences that encode native mammalian IL- 15 polypeptide, including mature and immature forms of IL- 15 polypeptide. In other embodiments, IL- 15 derivative nucleic acids include nucleic acids that encode mammalian IL- 15 RNA transcripts containing mutations that eliminate potential splice sites and instability elements (e.g., A / T or A / U rich elements) without affecting the amino acid sequence to increase the stability of the mammalian IL- 15 RNA transcripts.

[0056] As used herein, the terms “IL-15” and “interleukin- 15” refer to a native IL-15, an IL-15 derivative, or a native IL- 15 and an IL- 15 derivative.

[0057] As used herein, the terms “native IL-15Ra” and “native interleukin- 15 receptor alpha” in the context of proteins or polypeptides refer to any naturally occurring mammalian interleukin- 15 receptor alpha (“IL-15Ra”) amino acid sequence, including immature or precursor and mature forms and naturally occurring isoforms. The amino acid sequence of the immature form of the native full length human IL-15Ra, which comprises the signal peptide (bold) and the mature human native IL-15Ra, is provided:

[0058] MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSR

[0059] ERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGV TPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQT TAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLAS VEMEAMEALPVTWGTSSRDEDLENCSHHL. (SEQ ID NO: 2), signal sequence

[0060] MAPRRARGCRTLGLPALLLLLLLRPPATRG (SEQ ID NO 8).

[0061] It is contemplated that IL-15a is used therapeutically without the signal sequence, as provided below:

[0062] ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH

[0063] WTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIV PGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAI STSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL. (SEQ ID NO: 4).

[0064] The amino acid sequence of the immature form of the native soluble human IL-15Ra, which comprises the signal peptide (bold) and the mature human native IL-15Ra, is provided:

[0065] MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSR

[0066] ERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGV TPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQT TAKNWELTASASHQPPGVYPQGHSDTT (SEQ ID NO: 5).

[0067] It is contemplated that IL- 15a is used therapeutically without the signal sequence, as provided below:

[0068] ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH

[0069] WTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIV

[0070] PGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT (SEQ ID NO: 6).

[0071] In some embodiments, native IL-15Ra is the immature form of a naturally occurring mammalian IL-15Ra polypeptide. In other embodiments, native IL-15Ra is the mature form of a naturally occurring mammalian IL-15Ra polypeptide. In certain embodiments, native IL-15Ra is a soluble form of a naturally occurring mammalian IL-15Ra polypeptide. In other embodiments, native IL-15Ra is the full-length form of a naturally occurring mammalian IL-15Ra polypeptide. In a specific embodiment, native IL-15Ra is the immature form of a naturally occurring human IL-15Ra polypeptide. In another embodiment, native IL-15Ra is the mature form of a naturally occurring human IL-15Ra polypeptide. In certain embodiments, native IL-15Rais the soluble form of a naturally occurring human IL-15Ra polypeptide. In other embodiments, native IL-15Ra is the full-length form of a naturally occurring human IL-15Ra polypeptide. In one embodiment, a native IL-15Ra protein or polypeptide is isolated or purified.

[0072] As used herein, the terms “IL-15Ra derivative” and “interleukin- 15 receptor alpha derivative” in the context of a protein or polypeptide refer to: (a) a polypeptide that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to a native mammalian IL- 15 polypeptide; (b) a polypeptide encoded by a nucleic acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical a nucleic acid sequence encoding a native mammalian IL-15Ra polypeptide; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and / or substitutions) relative to a native mammalian IL-15Ra polypeptide; (d) a polypeptide encoded by a nucleic acid sequence that can hybridize under high, moderate or typical stringency hybridization conditions to a nucleic acid sequence encoding a native mammalian IL-15Ra polypeptide; (e) a polypeptide encoded by a nucleic acid sequence that can hybridize under high, moderate or typical stringency hybridization conditions to nucleic acid sequences encoding a fragment of a native mammalian IL- 15 polypeptide of at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 100 contiguous amino acids, or at least 150 contiguous amino acids; or (f) a fragment of a native mammalian IL-15Ra polypeptide. IL-15Ra derivatives also include a polypeptide that comprises the amino acid sequence of a naturally occurring mature form of mammalian IL-15Ra polypeptide and a heterologous signal peptide amino acid sequence. In a specific embodiment, an IL-15Ra derivative is a derivative of a native human IL-15Ra polypeptide. In another embodiment, an IL-15Ra derivative is a derivative of an immature form of naturally occurring human IL-15 polypeptide. In another embodiment, an IL-15Ra derivative is a derivative of a mature form of naturally occurring human IL- 15 polypeptide. In one embodiment, an IL-15Ra derivative is the soluble form of a native mammalian IL-15Ra polypeptide. In a specific embodiment, an IL-15Ra derivative is purified or isolated. In a preferred embodiment, IL-15Ra derivatives retain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a native mammalian IL-15Ra polypeptide to bind an IL-15 polypeptide, as measured by assays well known in the art, e.g., ELISA, co-immunoprecipitation. In another preferred embodiment, IL-15Ra derivatives retain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a native mammalian IL-15Ra polypeptide to induce IL- 15 -mediated signal transduction, as measured by assays well-known in the art, e.g., electromobility shift assays, ELIS As and other immunoassays.

[0073] As used herein, the terms “IL-15Ra derivative” and “interleukin- 15 receptor alpha derivative” in the context of nucleic acids refer to: (a) a nucleic acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the naturally occurring nucleic acid sequence encoding a mammalian IL-15Ra polypeptide; (b) a nucleic acid sequence encoding a polypeptide that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical the amino acid sequence of a native mammalian IL-15Ra polypeptide; (c) a nucleic acid sequence that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid mutations (i.e., additions, deletions and / or substitutions) relative to the naturally occurring nucleic acid sequence encoding a mammalian IL-15Ra polypeptide; (d) a nucleic acid sequence that hybridizes under high, moderate or typical stringency hybridization conditions to a naturally occurring nucleic acid sequence encoding a mammalian IL-15Ra polypeptide; (e) a nucleic acid sequence that hybridizes under high, moderate or typical stringency hybridization conditions to a fragment of a naturally occurring nucleic acid sequence encoding a mammalian IL-15Ra polypeptide; and (f) a nucleic acid sequence encoding a fragment of a naturally occurring nucleic acid sequence encoding a mammalian IL- 15Ra polypeptide. In a specific embodiment, an IL-15Ra derivative in the context of nucleic acids is a derivative of a naturally occurring nucleic acid sequence encoding a human IL-15Ra polypeptide. In another embodiment, an IL-15Ra derivative in the context of nucleic acids is a derivative of a naturally occurring nucleic acid sequence encoding an immature form of a human IL- 15Ra polypeptide. In another embodiment, an IL- 15Ra derivative in the context of nucleic acids is a derivative of a naturally occurring nucleic acid sequence encoding a mature form of a human IL-15Ra polypeptide. In one embodiment, an IL-15Ra derivative refers to a nucleic acid sequence encoding a mammalian IL-15Ra polypeptide that is soluble. IL-15Ra derivative nucleic acid sequences include codon-optimized nucleic acid sequences that encode native IL-15Ra polypeptide, including mature and immature forms of IL- 15Ra polypeptide. In other embodiments, IL-15Ra derivative nucleic acids include nucleic acids that encode IL-15RaRNA transcripts containing mutations that eliminate potential splice sites and instability elements (e.g., A / T or A / U rich elements) without affecting the amino acid sequence to increase the stability of the IL-15Ra RNA transcripts.

[0074] As used herein, the terms “IL-15Ra” and “interleukin-15 receptor alpha” refer to a native IL-15Ra, an IL-15Ra derivative, or a native IL-15Ra and an IL-15Ra derivative.

[0075] As used herein, the term “IL-15 / IL-15Ra complex” refers to a complex comprising IL-15 and IL-15Ra covalently or noncovalently bound to each other. In a preferred embodiment, the IL- 15Ra has a relatively high affinity for IL- 15, e.g., Kd of 10 to 50 pM as measured by a technique known in the art, e.g., KinEx™ A assay, plasma surface resonance (e.g., BIAcore™ assay). In another preferred embodiment, the IL-15 / IL-15Ra complex induces IL-15-mediated signal transduction, as measured by assays well-known in the art, e.g., electromobility shift assays, ELISAs and other immunoassays. In some embodiments, the IL- 15 / IL- 15Ra complex retains the ability to specifically bind to the y chain.

[0076] In certain embodiments, IL- 15 and IL-15Ralpha are used in combination with IL- 12.

[0077] As used herein, the terms “IL-15” and “interleukin- 15” refer to a native IL-15, an IL-15 derivative, or a native IL- 15 and an IL- 15 derivative.

[0078] “IL-12” or “Interleukin- 12” and the like refers to a heterodimer of IL-12a with IL-12b to form the IL-12 cytokine. As documented by UniPro P29459, the IL-12a consensus human sequence is

[0079] MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQK ARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKT SFMMALCL S SI YEDLKM YQ VEFKTMN AKLLMDPKRQIFLDQNMLAVIDELMQ ALNFNS ETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 9). Bold is the signal / pro-peptide sequence. Many variants are known. IL-12 is reported to be naturally produced by antigen-presenting cells (APCs) such as B-cells and dendritic cells (DCs) as well as macrophages and granulocytes.

[0080] It is contemplated that IL-12a is used therapeutically without the signal sequence, as provided below: RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKD KTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEF KTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCIL LHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 10).

[0081] As documented by UniPro P29460, the IL- 12b consensus human sequence is

[0082] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTP EEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGA ATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDII KPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTD KTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 11). Bold is the signal sequence. Many variants are known.

[0083] It is contemplated that IL- 12b is used therapeutically without the signal sequence, as provided below:

[0084] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKN YSGRFTCWWLTTISTDLTF SVKS SRGS SDPQGVTCGAATLS AERVRGDNKEYEYS VECQE D S ACPAAEE SLPIE VMVD AVHKLKYENYT S SFFIRDIIKPDPPKNLQLKPLKNSRQ VE VS W EYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYS SSWSEWASVPCS (SEQ ID NO: 12).

[0085] In some embodiments, native IL- 12 is the immature or precursor form of a naturally occurring mammalian IL-12. In other embodiments, native IL-12 is the mature form of a naturally occurring mammalian IL-12. In a specific embodiment, native IL-12 is the precursor form of naturally occurring human IL-12. In another embodiment, native IL-12 is the mature form of naturally occurring human IL-12. In one embodiment, the native IL-12 protein / polypeptide is isolated or purified.

[0086] As used herein, the terms “native IL- 12” and “native interleukin- 12” in the context of nucleic acids refer to any naturally occurring nucleic acid sequences encoding mammalian IL-12a and IL-12b, including the immature or precursor and mature forms.

[0087] In a specific embodiment, the nucleic acid is an isolated or purified nucleic acid. In some embodiments, nucleic acids encode the immature or precursor form of naturally occurring mammalian IL-12a and IL-12b polypeptides. In other embodiments, nucleic acids encode the mature form of naturally occurring mammalian IL-12a and IL-12b polypeptides. In a specific embodiment, nucleic acids encoding native IL- 12 encode the precursor form of naturally occurring human IL- 12a and IL- 12b polypeptides. In another embodiment, nucleic acids encoding native IL- 12 encode the mature of naturally occurring human IL- 12a and IL- 12b polypeptides.

[0088] As used herein, the terms “IL- 12 derivative” and “interleukin- 12 derivative” in the context of proteins or polypeptides refer to: (a) polypeptides that are at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to native mammalian IL-12a and IL- 12b polypeptides; (b) polypeptides encoded by nucleic acid sequences that are at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to nucleic acid sequences encoding native mammalian IL- 12a and IL- 12b polypeptides; (c) polypeptides that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and / or substitutions) relative to native mammalian IL-12a and IL- 12b polypeptides; (d) polypeptides encoded by nucleic acids can hybridize under high, moderate or typical stringency hybridization conditions to nucleic acids encoding native mammalian IL-12a and IL-12b polypeptides; (e) polypeptides encoded by nucleic acid sequences that can hybridize under high, moderate or typical stringency hybridization conditions to nucleic acid sequences encoding a fragment of a native mammalian IL-12a and IL-12b polypeptides of at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 100 contiguous amino acids, or at least 150 contiguous amino acids; or (f) a fragment of native mammalian IL-12a and IL-12b polypeptides. IL-12 derivatives also include polypeptides that comprises the amino acid sequence of a naturally occurring mature form of a mammalian IL- 12a and IL- 12b polypeptides and a heterologous signal peptide amino acid sequence. In a specific embodiment, an IL-12a and IL-12b derivatives are derivatives of native human IL- 12a and IL- 12b polypeptides. In another embodiment, IL- 12a and IL- 12b derivatives are derivatives of an immature or precursor form of naturally occurring human IL-12a and IL-12b polypeptides. In another embodiment, IL-12a and IL-12b derivatives are derivatives of a mature form of naturally occurring human IL-12a and IL-12b polypeptides. In one embodiment, IL-12a and IL-12b polypeptides are isolated or purified.

[0089] In a preferred embodiment, IL-12a and IL-12b polypeptides retain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of native mammalian IL-12a and IL-12b polypeptides to bind IL-12 receptors as measured by assays well known in the art, e.g., ELISA, co-immunoprecipitation. In another preferred embodiment, IL-12a and IL-12b derivatives retain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of native mammalian IL-12a and IL-12b polypeptides to induce IL-12 receptors-mediated signal transduction, as measured by assays well-known in the art, e.g., electromobility shift assays, ELISAs and other immunoassays.

[0090] IL-12a and IL-12b derivative nucleic acid sequences include codon-optimized nucleic acid sequences that encode native mammalian IL- 12a and IL- 12b polypeptides, including mature and immature forms of IL-12a and IL-12b polypeptides. In other embodiments, IL-12a and IL-12b nucleic acids include nucleic acids that encode mammalian IL- 12a and IL- 12b RNA transcripts containing mutations that eliminate potential splice sites and instability elements (e.g., A / T or A / U rich elements) without affecting the amino acid sequence to increase the stability of the mammalian IL-12a and IL-12b RNA transcripts.

[0091] Percent identity can be determined using any method known to one of skill in the art. In a specific embodiment, the percent identity is determined using the “Best Fit” or “Gap” program of the Sequence Analysis Software Package (Version 10; Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, Madison, Wis.). Information regarding hybridization conditions (e.g., high, moderate, and typical stringency conditions) have been described, see, e.g., U.S. Patent Application Publication No. US 2005 / 0048549 (e.g., paragraphs 72-73).

[0092] As used herein, the terms “purified” and “isolated” in the context of a compound or agent (including, e.g., proteinaceous agents such as antibodies) that is chemically synthesized refers to a compound or agent that is substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, the compound or agent is 60%, 65%, 75%, 80%, 85%, 90%, 95%, or 99% free (by dry weight) of other, different compounds or agents.

[0093] As used herein, the terms “purified” and “isolated” when used in the context of a compound or agent (including proteinaceous agents such as antibodies and polypeptides) that can be obtained from a natural source, e.g., cells, refers to a compound or agent which is substantially free of contaminating materials from the natural source, e.g., soil particles, minerals, chemicals from the environment, and / or cellular materials from the natural source, such as but not limited to cell debris, cell wall materials, membranes, organelles, the bulk of the nucleic acids, carbohydrates, proteins, and / or lipids present in cells. The phrase “substantially free of natural source materials” refers to preparations of a compound or agent that has been separated from the material (e.g., cellular components of the cells) from which it is isolated. Thus, a compound or agent that is isolated includes preparations of a compound or agent having less than about 30%, 20%, 10%, 5%, 2%, or 1% (by dry weight) of cellular materials and / or contaminating materials.

[0094] An “isolated” nucleic acid sequence or nucleotide sequence is one which is separated from other nucleic acid molecules which are present in a natural source of the nucleic acid sequence or nucleotide sequence. Moreover, an “isolated”, nucleic acid sequence or nucleotide sequence, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors when chemically synthesized. In certain embodiments, an “isolated” nucleic acid sequence or nucleotide sequence is a nucleic acid sequence or nucleotide sequence that is recombinantly expressed in a heterologous cell.

[0095] In some embodiments, the terms “nucleic acid”, “nucleotide” and “polynucleotide” refer to deoxyribonucleotides, deoxyribonucleic acids, ribonucleotides, and ribonucleic acids, and polymeric forms thereof, and include either single- or double-stranded forms. In certain embodiments, such terms include known analogues of natural nucleotides, for example, peptide nucleic acids (“PNA”s), that have similar binding properties as the reference nucleic acid. In some embodiments, such terms refer to deoxyribonucleic acids (e.g., cDNA or DNA). In other embodiments, such terms refer to ribonucleic acid (e.g., mRNA or RNA).

[0096] In certain embodiments, the bioconjugate is an antibody heavy chain constant region conjugated to a protein disclosed herein. As used herein, the term "conjugated” when referring to two moieties means the two moieties are bonded, wherein the bond or bonds connecting the two moieties may be covalent or non-covalent. In embodiments, the two moieties are covalently bonded to each other (e.g. directly or through a covalently bonded intermediary). In embodiments, the two moieties are non-covalently bonded (e.g. through ionic bond(s), van der waal’s bond(s) / interactions, hydrogen bond(s), polar bond(s), or combinations or mixtures thereof).

[0097] As used herein, the terms “bioconjugate” and “bioconjugate linker” refers to the resulting association between atoms or molecules of “bioconjugate reactive groups” or “bioconjugate reactive moieties”. The association can be direct or indirect. For example, a conjugate between a first bioconjugate reactive group (e g., -NH2, -C(O)OH, -N-hydroxysuccinimide, or -maleimide) and a second bioconjugate reactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine, amine sidechain containing amino acid, or carboxylate) provided herein can be direct, e.g., by covalent bond or linker (e.g. a first linker of second linker), or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). In embodiments, bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e. the association of two bioconjugate reactive groups) including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels- Alder addition). In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., haloacetyl moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., pyridyl moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., -N-hydroxysuccinimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. an amine). In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., -sulfo-N-hydroxysuccinimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. an amine).

[0098] The bioconjugate reactive groups can be chosen such that they do not participate in, or interfere with, the chemical stability of the conjugate described herein. Alternatively, a reactive functional group can be protected from participating in the crosslinking reaction by the presence of a protecting group. In embodiments, the bioconjugate comprises a molecular entity derived from the reaction of an unsaturated bond, such as a maleimide, and a sulfhydryl group.

[0099] A "heterologous" nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or a peptide sequence that does not naturally occur, e.g., because the whole sequence contains a segment from other plants, bacteria, viruses, other organisms, or joinder of two sequences that occur the same organism but are joined together in a manner that does not naturally occur in the same organism or any natural state. The term "recombinant" when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques provided that the entire nucleic acid sequence does not occurring in nature, i.e., there is at least one mutation in the overall sequence such that the entire sequence is not naturally occurring even though separately segments may occur in nature. The segments may be joined in an altered arrangement such that the entire nucleic acid sequence from start to finish does not naturally occur. The term "recombinant" when made in reference to a protein or a peptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.

[0100] Proteins disclosed herein are contemplated to include functional variants, allelic variants, or active fragments. Variants may include 1 or 2 amino acid substitutions or conserved substitutions. Variants may include 3 or 4 amino acid substitutions or conserved substitutions. Variants may include 5 or 6 or more amino acid substitutions or conserved substitutions. Variants include those with not more than 1% or 2% of the amino acids are substituted. Variants include those with not more than 3% or 4% of the amino acids are substituted. Variants include proteins with greater than 80%, 89%, 90%, 95%, 98%, or 99% identity or similarity.

[0101] Variants can be tested by mutating the vector to produce appropriate codon alternatives for polypeptide translation. Active variants and fragments can be identified with a high probability using computer modeling. Shihab et al. report an online genome tolerance browser. BMC Bioinformatics. 2017, 18(l):20. Ng et al. report methods of predicting the effects of amino acid substitutions on protein function. Annu Rev Genomics Hum Genet. 2006, 7:61-80. Teng et al. Approaches and resources for prediction of the effects of non-synonymous single nucleotide polymorphism on protein function and interactions. Curr Pharm Biotechnol, 2008, 9(2): 123-33.

[0102] Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, RaptorX, ESyPred3D, HHpred, Homology Modeling Professional for HyperChem, DNAStar, SPARKS-X, EVfold, Phyre, and Phyre2 software. See also Saldano et al. Evolutionary Conserved Positions Define Protein Conformational Diversity, PLoS Comput Biol. 2016, 12(3): e 1004775; Marks et al. Protein structure from sequence variation, Nat Biotechnol. 2012, 30(11): 1072-80; Mackenzie et al. Curr Opin Struct Biol. 2017, 44: 161-167 Mackenzie et al. Proc Natl Acad Sci U S A. 113(47):E7438-E7447 (2016); Joseph et al. J R Soc Interface. 2014, 11(95):20131147, Wei et al. Int. J. Mol. Sci. 2016, 17(12), 2118. Variants can be tested in functional assays. Certain variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on).

[0103] The term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0104] The terms "vector" or " expression vector " refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free expression system. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. In certain embodiments, this disclosure contemplates a vector encoding a peptide disclosed herein in operable combination with a heterologous promoter.

[0105] In certain embodiments, the recombinant vector optionally comprises a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selected from gag, pol, and env.

[0106] In certain embodiments, the recombinant vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin / CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), FLAG tag coding region, c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col El origin of replication, fl origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides.

[0107] A “selectable marker” is a nucleic acid introduced into a recombinant vector that encodes a polypeptide that confers a trait suitable for artificial selection or identification (report gene), e.g., beta-lactamase confers antibiotic resistance, which allows an organism expressing beta-lactamase to survive in the presence antibiotic in a growth medium. Another example is thymidine kinase, which makes the host sensitive to ganciclovir selection. It may be a screenable marker that allows one to distinguish between wanted and unwanted cells based on the presence or absence of an expected color. For example, the lac-z-gene produces a beta-galactosidase enzyme which confers a blue color in the presence of X-gal (5-bromo-4-chloro-3-indolyl-P-D-galactoside). If recombinant insertion inactivates the lac-z-gene, then the resulting colonies are colorless. There may be one or more selectable markers, e.g., an enzyme that can complement to the inability of an expression organism to synthesize a particular compound required for its growth (auxotrophic) and one able to convert a compound to another that is toxic for growth. URA3, an orotidine-5' phosphate decarboxylase, is necessary for uracil biosynthesis and can complement ura3 mutants that are auxotrophic for uracil. URA3 also converts 5-fluoroorotic acid into the toxic compound 5 -fluorouracil. Additional contemplated selectable markers include any genes that impart antibacterial resistance or express a fluorescent protein. Examples include, but are not limited to, the following genes: ampr, camr, tetr, blasticidinr, neor, hygr, abxr, neomycin phosphotransferase type II gene (nptll), p-glucuronidase (gus), green fluorescent protein (gfp), egfp, yfp, mCherry, p- galactosidase (lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat), alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphos resistance gene (bar), phosphomannose isomerase (pmi), xylose isomerase (xylA), arabitol dehydrogenase (atlD), UDP- glucose:galactose-l-phosphate uridyltransferase (galT), feedback-insensitive a subunit of anthranilate synthase (OASA1D), 2-deoxy glucose (2-DOGR), benzyladenine-N-3 -glucuronide, E. coli threonine deaminase, glutamate 1 -semialdehyde aminotransferase (GSA-AT), D-amino acidoxidase (DAAO), salt-tolerance gene (rstB), ferredoxin-like protein (pflp), trehalose-6-P synthase gene (AtTPSl), lysine racemase (lyr), dihydrodipicolinate synthase (dapA), tryptophan synthase beta 1 (AtTSBl), dehalogenase (dhlA), mannose-6-phosphate reductase gene (M6PR), hygromycin phosphotransferase (HPT), and D-serine ammonialyase (dsdA).

[0108] As used herein, a recombinant viral vector, and the like, is a virus, the genome of which has been altered by the hand of man, e.g., not capable of replicating or attenuated / weakened, i.e., the altered virus is still capable of replicating its genome, so as not to pose a threat to a subject. Such alterations include, but are not limited to, insertion mutations, including insertion of one or more nucleotides, deletion mutations, including deletion of one or more nucleotides, substitution mutations, including substitution of one or more nucleotides, and / or insertions of heterologous nucleic acid sequences into the genome, e.g., a selectable marker. Any virus can be used to construct recombinant virus vectors of this disclosure, so long as the resulting recombinant virus vector has the desirable characteristics. Viruses used to construct recombinant virus vectors of this disclosure can be eukaryotic viruses or prokaryotic viruses. Moreover, elements from viruses or bacteria used to construct recombinant virus vectors of this disclosure can be from eukaryotic cells, eukaryotic viruses, prokaryotic viruses, bacteria, or any combination thereof. Examples of such elements include, but are not limited to, ORF sequences, gene sequences, promoter sequences, enhancer sequences, repressor sequences, cleavage sequences, or any useful fragments thereof. Examples of viruses useful for constructing recombinant viral vectors include, but are not limited to, poxviruses, iridoviruses, adenoviruses, adeno-associated viruses, Simian Virus 40 (SV40), Epstein-Barr virus, herpesvirus, JC virus, bacteriophage T7, bacteriophage, T3 and bacteriophage SP6.

[0109] Methods of treating HIV

[0110] In certain embodiments, this disclosure relates to methods of treating HIV comprising administering an effective amount of IL-15 and / or IL- 15 alpha, or nucleic acid or vector encoding the same, optionally in combination with IL-12, or nucleic acid or vector encoding the same, to a human patient in need thereof.

[0111] In certain embodiments, IL-15, IL-15Ralpha, and / or IL-12 are administered in combination with and prior or during or at the initiation of the first administration of a multi-drug anti-retroviral therapy (ART). In certain embodiments, IL-15 and IL-12 are administered in combination with and prior or during or at the initiation of the first administration of a multi-drug anti-retroviral therapy (ART). In certain embodiments, IL-15Ralpha and IL-12 are administered in combination with and prior or during or at the initiation of the first administration of a multi-drug anti-retroviral therapy (ART). In certain embodiments, IL- 15 is administered in combination with and prior or during or at the initiation of the first administration of a multi -drug anti-retroviral therapy (ART). In certain embodiments, IL-15Ralpha is administered in combination with and prior or during or at the initiation of the first administration of a multi-drug anti-retroviral therapy (ART). In certain embodiments, IL-12 is administered in combination with and prior or during or at the initiation of the first administration of a multi-drug anti-retroviral therapy (ART).

[0112] In certain embodiments, the method of treating HIV by administering an effective amount of IL- 15 and IL- 15 alpha, or RNA, DNA or vector encoding the same, optionally in combination with IL- 12, or RNA, DNA or vector encoding the same, to a human patient in need thereof are also administered in combination with a multi-drug anti-retroviral therapy (ART). In certain embodiments, the multi-drug anti-retroviral therapy comprises a nucleoside reverse transcriptase inhibitor (NRTI), abacavir, emtricitabine, lamivudine, tenofovir disoproxil fumarate, zidovudine, non-nucleoside reverse transcriptase inhibitor (NNRTI), doravirine, efavirenz, etravirine, nevirapine, rilpivirine, protease inhibitor (PI), atazanavir, darunavir, fosamprenavir, ritonavir, tipranavir, fusion inhibitor, enfuvirtide, CCR5 antagonist, maraviroc, capsid Inhibitor, lenacapavir, attachment inhibitor, fostemsavir, post-attachment inhibitor, ibalizumab, ibalizumab-uiyk, integrase strand transfer inhibitor (INSTI), cabotegravir, dolutegravir, raltegravir, or combinations thereof.

[0113] In certain embodiments, the combination is abacavir and lamivudine; or abacavir, dolutegravir, and lamivudine; or abacavir, lamivudine, and zidovudine; or atazanavir and cobicistat; or bictegravir, emtricitabine, and tenofovir alafenamide; or cabotegravir and rilpivirine; or darunavir and cobicistat; or darunavir, cobicistat, emtricitabine, and tenofovir alafenamide; or dolutegravir and lamivudine; or dolutegravir and rilpivirine; or doravirine, lamivudine, and tenofovir disoproxil fumarate; or efavirenz, emtricitabine, and tenofovir disoproxil fumarate; or efavirenz, lamivudine, and tenofovir disoproxil fumarate; or elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide; or elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate; or emtricitabine, rilpivirine, and tenofovir alafenamide; or emtricitabine, rilpivirine, and tenofovir disoproxil fumarate; or emtricitabine and tenofovir alafenamide; or emtricitabine and tenofovir disoproxil fumarate; or lamivudine and tenofovir disoproxil fumarate; or lamivudine and zidovudine; or lopinavir and ritonavir.

[0114] In certain embodiments, the ratio of IL-15 to IL-15 alpha is between 1 / 10 to 2 / 5. In certain embodiments, the ratio of IL-15 to IL-15 alpha is about 1 / 5.

[0115] Pharmaceutical compositions

[0116] In certain embodiment, this disclosure contemplates pharmaceutical compositions comprising compounds disclosed herein and pharmaceutically acceptable excipient. In certain embodiments, this disclosure contemplates the production of a medicament comprising agents disclosed herein and uses for methods disclosed herein.

[0117] Pharmaceutical compositions typically comprise an effective amount of agents and a suitable pharmaceutical acceptable carrier. The preparations can be prepared in a manner known per se, which usually involves mixing the agents according to the disclosure with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active agents, when necessary under aseptic conditions. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences.

[0118] In certain embodiment, the pharmaceutical composition comprises IL- 15 and / or IL- 15 alpha and optionally in combination with IL- 12 or nucleic acid, RNA, DNA, or vector encoding the same and a pharmaceutically acceptable excipient.

[0119] In certain embodiment, the pharmaceutical composition is in the form of a pH buffered aqueous saline solution optionally comprising a saccharide or polysaccharide.

[0120] In certain embodiment, the pharmaceutical composition is in the form of tablet, pill, capsule, gel, gel capsule, micronized particles, or lipid particles.

[0121] In certain embodiment, the pharmaceutically acceptable excipient is selected from lactose, sucrose, mannitol, triethyl citrate, dextrose, cellulose, methyl cellulose, ethyl cellulose, hydroxyl propyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, croscarmellose sodium, polyvinyl N-pyrrolidone, crospovidone, ethyl cellulose, povidone, methyl and ethyl acrylate copolymer, polyethylene glycol, fatty acid esters of sorbitol, lauryl sulfate, gelatin, glycerin, glyceryl monooleate, silicon dioxide, titanium dioxide, talc, com starch, carnauba wax, stearic acid, sorbic acid, magnesium stearate, calcium stearate, castor oil, mineral oil, calcium phosphate, starch, carboxymethyl ether of starch, iron oxide, triacetin, acacia gum, esters, or salts thereof.

[0122] In certain embodiments, the disclosure relates to pharmaceutical compositions comprising IL-15 and / or IL-15Ralpha and optionally IL-12 and a pharmaceutically acceptable excipient. In certain embodiments, the composition is a pill or in a capsule or the composition is an aqueous buffer, e.g., a pH between 6 and 8. In certain embodiments, the pharmaceutically acceptable excipient is selected from a filler, glidant, binder, disintegrant, lubricant, and saccharide. Optionally, the pharmaceutical composition further comprises a second clotting agent such as aminocaproic acid (s-aminocaproic acid), tranexamic acid, fibrinogen, and vitamin K.

[0123] Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable (such as olive oil, sesame oil) and injectable organic esters such as ethyl oleate.

[0124] Prevention of the action of microorganisms may be controlled by addition of any of various antibacterial and antifungal agents, example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0125] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the agents may be admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or: (a) fdlers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, as for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium agents, (g) wetting agents, as for example cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.

[0126] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the agents, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, com germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.

[0127] In certain embodiments, production processes are contemplated which two components, agents disclosed herein and a pharmaceutical carrier, are provided already in a combined dry form ready to be reconstituted together. In other embodiments, it is contemplated that agents disclosed herein and a pharmaceutical carrier are admixed to provide a pharmaceutical composition.

[0128] Providing a pharmaceutic composition is possible in a one-step process, simply by adding a suitable pharmaceutically acceptable diluent to the composition in a container. In certain embodiments, the container is preferably a syringe for administering the reconstituted pharmaceutical composition after contact with the diluent. In certain embodiments, the agents can be filled into a syringe, and the syringe can then be closed with the stopper. A diluent is used in an amount to achieve the desired end-concentration. The pharmaceutical composition may contain other useful component, such as ions, buffers, excipients, stabilizers, etc.

[0129] A container can be any container suitable for housing and storing pharmaceutically compositions such as syringes, vials, tubes, etc. The pharmaceutical composition may then preferably be applied via specific needles of the syringe or via suitable catheters. Atypical diluent comprises water for injection, and NaCl (preferably 50 to 150 mM, especially 110 mM), CaCh (preferably 10 to 80 mM, especially 40 mM), sodium acetate (preferably 0 to 50 mM, especially 20 mM) and mannitol (preferably up to 10% w / w, especially 2% w / w). Preferably, the diluent can also include a buffer or buffer system so as to buffer the pH of the reconstituted dry composition, preferably at a pH of 6.2 to 7.5, especially at pH of 6.9 to 7.1. In certain embodiments, the diluent is provided in a separate container. This can preferably be a syringe. The diluent in the syringe can then easily be applied to the container for reconstitution of the dry compositions. If the container is also a syringe, both syringes can be finished together in a pack. It is therefore contemplated to provide the dry compositions in a syringe, which is finished with a diluent syringe with a pharmaceutically acceptable diluent for reconstituting, said dry and stable composition.

[0130] In certain embodiments, this disclosure contemplates a kit comprising a pharmaceutical composition IL- 15 and / or IL-15Ralpha and optionally IL- 12 and a container with a suitable diluent. Further components of the kit may be instructions for use, administration means, such as syringes, catheters, brushes, etc. (if the compositions are not already provided in the administration means) or other components necessary for use in medical (surgical) practice, such as substitute needles or catheters, extra vials or further wound coverings. In certain embodiments, the kit comprises a syringe housing the dry and stable hemostatic composition and a syringe containing the diluent (or provided to take up the diluent from another diluent container).

[0131] In certain embodiments, a pharmaceutical composition comprising IL- 15 and / or IL- 15Ralpha and optionally IL-12 may also contain an adjuvant or be administered in combination with an adjuvant. Contemplated adjuvants suitable for inclusion in the pharmaceutical composition and delivery methods include cationic or polycationic compounds, liposomes, chitosan, alum solution, aluminium hydroxide, aluminium salts aluminium phosphate gel, aluminium hydroxide gel (alum), polyphosphazene, squalene, squalene water emulsion, CpG oligonucleotides (nucleic acids with unmethylated CpG motifs), 1 -al pha,25-dihydroxy -vitamin D3, calcium phosphate gel, dimethyl dioctadecyl ammonium bromide, dehydroepiandrosterone, dimyristoylphosphatidylcholine, myristoyl phosphatidylglycerol, deoxycholic acid sodium salt, imiquimod, interferon gamma, interleukin-1 beta, interleukin-2, interleukin-7, interleukin- 12, 7- allyl-8-oxoguanosine, acetylmuramyl-alanyl-isoglutamine, N-acetyl muramyl-L-threonyl-D- isoglutamine, NAc-Mur-L-Ala-D-Gln-OCFb, QS-21, Quil-A (Quil-A saponin), sorbitan trioleate, 2,6,10,15,19,23-hexamethyltetracosan, stearyl tyrosine, lipid A, 4 '-monophosphoryl lipid A, 3-0- desacyl-4'-monophosphoryl lipid A (MPL™), liposomes containing lipid A, or lipid A adsorbed on aluminium hydroxide. IL-15 / IL-15Ra Cytokine Therapy Enhances Control of Viral Rebound in SIV-Infected Macaques

[0132] Immunotherapeutic cytokines can enhance immune responses against chronic infections. Cytokines such as IL-15 and IL-12 expand CD8 T and NK cells and increase their cytotoxicity. Importantly IL- 15 can enhance follicular homing of CD8 T cells, and IL- 15 + IL- 12 enhances follicular homing of NK cells during chronic SIV infection. Here we tested the therapeutic effects of IL-15 and IL-12 when administered alone or in combination during chronic SIV infection in rhesus macaques (RMs).

[0133] Twenty -two RMs infected with SIVmac251 were initiated on antiretroviral therapy (ART) at 8 weeks post-SIV infection for 9-months. Three groups of animals received cytokine treatments - IL-15 / IL-15Ra (n=6), IL- 12 (n=5), IL-15 / IL-15Ra+IL-12 (n=6) - in two phases (5 doses once a week). The first phase was administered at 6 weeks post SIV (2 weeks prior to ART) and the second phase was during ART at 12 weeks pre- ATI (ART Treatment Interruption). ART alone (n=5) group served as the control. Animals were monitored longitudinally for immunological and virological parameters.

[0134] IL-15 / IL-15Ra and IL-15 / IL-15Ra+IL-12 therapies induced significant expansion of functional SIV-specific CD8 T cells with proliferative capacity (Ki-67) and follicular homing (CXCR5); and CD16+ NK cells in blood and LN. Importantly, IL-15 / IL-15Ra therapy resulted in the significant expansion of degranulating CD107a+CD8 T cells pre-ATI (P=0.03). In addition, the blood transcriptomic profile confirmed the induction of cytolytic molecules (granzyme-B, perforin), Jak / Stat signaling pathway in IL-15 / IL-15a group, while genes associated with cell cycle arrest, DNA damage (MDM2, DDIT4) were significantly reduced. Post-ATI, virus rebounded in all animals but IL-15 / IL-15Ra treated animals showed nearly 3-log lower viremia compared to ART only animals (p=0.004) with 83% of animals below 500 copies / ml at 20 weeks post ATI. SIV- specific CXCR5+ CD8 T cells in blood (p=0.04) and LN (p=0.02) and SIV-specific CD28+ cells in blood (p=0.002 and LN (p=0.03), were associated with control of viremia post ATI.

[0135] The IL-15 / IL-15Ra therapy at the initiation of ART and during ART markedly enhance the magnitude and function of SIV-specific CXCR5+CD8 T cells and CD16+NK cells and contributed to profound control of viremia post ATI. These studies define IL-15 / IL-15Ra as a potentially effective immune therapy for HIV cure strategy. IL-12 and IL-15 / IL-15Ra cytokine therapy for sustained HIV remission

[0136] The impact of IL-12 and IL-15 / IL-15Ra mono and combination therapies were assessed before and during the initiation of anti-retroviral therapy (ART). The magnitude and function of SIV-specific CD8 T and total NK cells for effective viral control following ART treatment interruption (ATI) was evaluated in rhesus macaques (RMs). RMs were intrarectally infected with a SIVmac251 monkey infectious dose. At week 6, once a clear viral set point was established, the RMs were randomized and distributed into 4 groups (at least 5 RM per group) as depicted in Fig. 1.

[0137] Animals received anti-retroviral therapy (ART; TFV, FTC, DTG) starting from week 8 post-infection. Animals in Group 1 did not receive any cytokine therapy and served as no cytokine / ART-alone controls. Groups 2-4 received different cytokine therapies via subcutaneous route weekly for 5 weeks, starting from week 6 post-infection. Group 2 animals received recombinant IL-12 (lOug / kg), Group 3 animals received IL-15 (20ug / kg) / IL- 15Ra (lOOug / kg) and Group 4 animals received IL- 12 plus IL-15 / IL-15Ra. Cytokine infusion was performed in 2 phases: 1) before initiation of ART at week 6 post-infection and 2) during suppressive ART at week 34 post-infection. The aim of phase 1 of cytokine infusion was to identify the effects of cytokines alone during the early stage of chronic infection on enhancing or restoring CD8 T cell and NK cell functions in the presence of viral antigen, followed by their combined effects with the initiation of ART 2 weeks post cytokine infusion. The second phase of cytokines was infused during the virally suppressed chronic infection with 26 weeks of ongoing ART. ART was interrupted at week 45. All animals were followed for induction of immune responses and viral load kinetics post first phase of cytokine treatment (prior to ART), post second phase of cytokine treatment (during ART), and after analytical treatment interruption (ATI).

[0138] The Effector Memory CD4 T cells were elevated in the IL-15 / IL-15Ra treated group at the time of ATI:

[0139] HIV primarily infects CD4 T cells, and monitoring CD4 T cell count is an important aspect of managing HIV infection. Initially, the temporal dynamics of CD4 T cell levels in different experimental groups were studied. The frequency of CD4 T cells was comparable in all groups before SIV infection. Similarly, after the cytokine treatment, CD4 T cells appeared to be similar in both control and cytokine-treated groups during the first phase and second phase (see Fig. 2A). In addition, the proliferating CD4 T cells also followed the similar trend. The ART-alone treated group and IL- 15 / IL- 15Ra treated group had slightly lower levels of Ki-67+ CD4 T cells during the first phase of cytokine treatment. All the groups demonstrated similar levels of Ki-67+ CD4 T cells during the second phase of cytokine treatment as well as at the time of ATI (see Fig. 2B). Central memory CD4 T cells showed a slightly higher trend in all cytokine-treated groups at the end of the first phase cytokine treatment compared to ART alone group. There was no noticeable difference observed in the central memory CD4 T cell levels during the second phase of cytokine treatment and at the time ATI between the experimental groups (see Fig. 2C). However, effector memory CD4 T cells were higher in IL-15 / IL-15Ra groups post the before SIV infection and during the first phase of cytokine treated groups. The effector memory CD4 T cells showed a slightly higher trend compared to the control group. During the second phase of cytokine treatment, effector memory CD4 T cells were elevated in the IL-15 / IL-15Ra treated group. The IL-12 and IL-12 plus IL-15 / IL-15Ra treated groups also showed a higher trend compared to the ART alone group. The effector memory CD4 T cells were maintained at a higher level in the IL-15 / IL-15Ra group until the time of ATI (see Fig. 2D). Overall, the IL-15 / IL-15R treated group showed higher EM CD4 T cells which may contribute to viral control during post-ATI time point.

[0140] Treatment with IL-15 / IL-15Ra increased the central memory CD8 T cells

[0141] To understand the influence of cytokines IL- 12 and IL-15 / IL-15Ra (either as monotherapy or as a combination therapy) on induction of CD8 T cell responses, the frequency of CD8 T cells and functional markers were monitored during phase I and phase II of cytokine therapy. The total number of CD8 T cells was increased using IL-12, IL-15 / IL-15Ra, when compared to the ART alone group, and the level was sustained slightly higher level in cytokine treated groups compared to ART alone group during phase I, and during ART treatment. During phase II cytokine treatment, no difference was observed in total CD8 levels between ART alone and cytokine-treated groups. However, total CD8 T cells maintained a slightly higher level in IL-15 / IL-15Ra, and the combined treatment group compared to the ART alone group at the time of ATI. Proliferating CD8 T cells showed a slight increase after the first week of cytokine treatment in IL-15 / IL-15Ra treated group during phase I cytokine treatment. During phase II cytokine treatment there was no difference observed in proliferating CD8 T cells between control and experimental groups. To understand the memory differentiation of total T cells, post cytokine therapy. The naive (CD28+CD95-), central memory (CM:CD28+CD95+), and effector memory (EM: CD28-CD95+) CD8 T cells were examined. The central memory CD8 T cells were increased after IL-15 / IL-15Ra treatment during the first phase of cytokine treatment, and not much difference was observed in other cytokine-treated groups. Similarly, the IL-15 / IL-15Ra group showed a slight increase in the CM CD8 T cells during the phase II cytokine treatment, which maintained a higher level until the ATI time point. In contrast, effector memory (EM) CD8 T cells were slightly decreased in the IL- 15 / IL-15Ra treated group and slightly increased in the IL-12 plus IL-15 / IL-15Ra, IL-12, and ART control during phase I cytokine treatment. During phase II cytokine treatment, IL- 12 plus IL-15 / IL- 15Ra combined cytokine-treated group showed a slight increase in the EM CD8 T cells, whereas not much difference was observed in other experimental and ART control groups. In summary, the IL-15 / IL-15Ra treated group had elevated CM CD8 T cells during phase I cytokine treatment and maintained a higher level during the ATI time points, which may contribute to viral control during post-ATI.

[0142] The magnitude of lymph node homing and co-stimulatory markers was elevated in both blood and lymph nodes after treatment with IL-15 / IL-15Ra and IL-15 / IL-15Ra plus IL-12 treated groups

[0143] Immunological studies of spontaneous HIV / SIV controllers have identified lymph node homing and germinal center homing virus-specific CD8+ T cells as a key immune mechanism of viral control. To understand the antigen-specific CD8 T cell response to mono and dual cytokine therapy, SIV-specific GagCM9+ tetramer-specific CD8 T cells were analyzed in blood and LN. The percentage of GagCM9+ CD8 T cells was not much different, which is comparable between groups during phase I and phase II cytokine treatment in blood. Proliferating GagCM9+ CD8 T cells were significantly increased in IL-15 / IL-15Ra treated group during phase I cytokine treatment, but there was not much difference in proliferating GagCM9+ CD8 T cells during phase

[0144] 11 cytokine treatment between experimental and ART alone groups in blood. Antigen-specific follicular homing CD8 T cells were increased after cytokine treatment in IL-15 / IL-15Ra and IL-

[0145] 12 plus IL-15 / IL-15Ra treated groups during phase I treatment and this was maintained during ART treatment compared to ART alone and IL- 12 groups. Furthermore, CXCR5+GagCM9+ CD8 T cells were enhanced during phase II cytokine treatment in IL-15 / IL-15Ra and combined treated groups, which was maintained at the time ATI in the blood. Antigen-specific co-stimulatory marker expression (CD28) was elevated during phase I cytokine treatment in IL-15 / IL-15Ra and IL- 12 plus IL-15 / IL-15R treated group. During phase II cytokine treatment, the level was comparable between the groups. However, the level was higher in IL-15 / IL-15Ra and dual cytokine-treated groups at the time of ATI in the blood. The lymph node compartment was evaluated for the influence of cytokines IL- 12 and IL-15 / IL-15Ra (either mono or as a combination therapy) to determine the effects on induction of antigen-specific CD8 T cells and functional markers. The percentage of GagCM9+ CD8 T cells was elevated in IL-12 and IL-15 / IL-15Ra treated groups during phase I cytokine treatment and there was not much difference observed during phase II cytokine treatment between groups in LN. In contrast, there was no difference observed in Ki67+GagCM9+ CD8 T cells during phase I and phase II cytokine treatment in LN. Antigenspecific CXCR5+ CD8+ T cells were expanded after IL-15 / IL-15Ra treated group during phase I cytokine treatment compared to ART alone group. The level was sustained higher during ART treatment and the level was slightly elevated in IL-15 / IL-15Ra and dual cytokine-treated groups during phase II cytokine treatment and at the time of ATI in LN. Antigen-specific co-stimulatory marker expression looks similar across the group during phase I and phase II cytokine treatment. In summary, the heightened presence of antigen-specific CD8 T cells in LN, CXCR5+ CD8 T cells in both blood and LN at the time of ATI could potentially contribute to the control of viremia after post- ATI in the IL-15 / IL-15Ra treated group.

[0146] The magnitude of NK cells was elevated in both blood and lymph nodes after treatment with IL-15 / IL-15Ra and IL-15 / IL-15Ra plus IL-12 treated groups

[0147] NK cells are a component of the innate immune system that plays a central role in host defense against viral infections. NK cells influence viral control during SIV / HIV infection. To study the therapeutic benefits of mono and dual cytokine therapy, the kinetics of NK cells and functional markers were assessed in blood and lymph nodes (LN). Total NK cells were increased after IL- 12 plus IL-15 / IL-15Ra treated animals compared to the ART alone and other experimental groups during phase I of cytokine treatment in blood. The cytokine-treated groups, especially IL- 15 / IL-15Ra and combined cytokine-treated groups maintained higher NK cells during ART treatment. Similarly, total NK cells were elevated in IL-15 / IL-15Ra and combined cytokine-treated groups and during the phase II cytokine treatment. Likewise, proliferating NK cells increased in the IL-15 / IL-15Ra treated group during phase I cytokine treatment. However, during phase IT cytokine treatment, not much difference was observed in proliferating NK cell levels in control and experimental groups. Among a subset of CD16+CD56- NK cells, a slight increase was observed in IL-15 / IL-15Ra treated and double cytokine treated groups during the phase I cytokine treatment, and the level was sustained higher during ART treatment. The level was further enhanced in the IL-15 / IL-15Ra treated group during phase II cytokine treatment. The CD16+CD56- NK cells maintained higher levels in the IL- 15 / IL-15Ra treated and double cytokine treated group at the time of ATI in the blood. In addition, total NK cells were expanded to higher levels in IL- 12 plus IL-15 / IL-15Ra treated group during the phase I cytokine treatment. During phase II cytokine treatment, a slight increase in total NK cells was observed in all cytokine-treated groups compared to ART alone control, and this maintenance continued until ATI in LN. Proliferating NK cells in LN were comparable between groups during phase I and phase II cytokine treatment in LN. CD16+CD56- NK cells were slightly increased after cytokine treatment in the IL- 15 and combined cytokine treated groups during phase I. Similarly, IL-15 / IL-15Ra and combined cytokine-treated groups showed expansion of CD16+CD56- NK cells during phase II cytokine treatment, and this expansion was maintained at the time of ATI in LN. Overall, elevated levels of total NK cells and CD16+CD56- NK cells were higher after cytokine treatment, and were maintained until ATI, which may contribute for post viremia control in IL-15 / IL-15Ra and combined cytokine treated groups.

[0148] IL-15 / IL-15Ra treatment near the start of ART and during ART enhances SIV viral control post-ATI

[0149] To understand the therapeutic benefit of the mono and dual cytokine therapy, the kinetics of viral RNA levels were assessed in the plasma until about 20 weeks post ATI (5 months). Following ATI, the plasma viremia increased rapidly in all animals by 4 weeks. Notably, all the cytokine treatment post-ATI resulted in a mixed virologic response. Some animals responded well and showed a decline in virus levels and some animals did not. During this phase, the ART alone group tended to have high viral load levels and did not show any signs of viral control. Interestingly, the IL-15 / IL-15Ra treated animals tend to control the reemerging viral levels very early post-ATI phase. These animals experienced delayed peaking and displayed blunt viral load at peak time points, which was at week 4 post-ATI, compared to the other experimental groups studied. The dual cytokine group also responded well; however, they did not show any reduction in peak viral levels post ATI. Despite the mixed virologic response, when the pre -ART set-point viral load were compared, a significant reduction in post-ATI viral load was observed in both the IL-15 / IL-15Ra group and in the combination group. Both the IL-15 / IL-15Ra and the combination of IL-15 / IL-15Ra plus IL-12 group showed increased expression of CXCR5+NK cells post-first phase of cytokine therapy. Hence, this suggests there is an association between these cells and the observed reduction in viral load. Viral blips were not observed during the second phase of cytokine therapy, i.e., for either mono or dual cytokines treatments.

[0150] IL-15 / IL-15R cytokine therapy maintains low viremia post-ART Interruption

[0151] ART treatment interruption was performed in study animals. Animals were analyzed for viral loads weekly for the first four weeks and were followed until week 20 post-ATI. Viremia for individual RMs was collected. Interestingly the IL-15 / IL-15Ra treated animals displayed blunt peak viremia. Five out of six animals did not peak above 10A4 viral RNA copies at the peak of ART (4 weeks post ATI). On the other hand, IL- 12 and dual cytokine treatment group animals tend to have higher viral levels. Starting at 4 weeks post-ATI, IL-15 / IL-15R treated RMs controlled viremia to significantly lower levels than the other groups. The IL-15 / IL-15Ra therapy-induced long-term control of plasma viremia (<1000 copies / mL) up to 20 weeks post-ATI, with significantly lower area under the curve (AUC) than control RMs. One out of 6 RMs from the IL- 15 / IL-15R and dual cytokine treatment group controlled the viral RNA levels below 60 copies throughout the follow-up period. Pre-ART versus post-ART viral load were compared. A significant reduction in post-ATI viral load was observed in both the IL-15 / IL-15Ra group and the dual cytokine-treated group. Collectively, these data demonstrated that the IL-15 / IL-15Ra treated group significantly lowered the viral burden at the time post-ATI.

[0152] IL-12 plus IL-15 / IL-15R treated group animals induced CD8+ T cells in the B cell follicles of the LN during post-ATI

[0153] To understand the influence of cytokine treatments on SIV-specific CD8+ T cells in the LNs, LN biopsies were collected 4 weeks after the ART treatment interruption. Phenotypic analysis of the LN CD8+ T cells using flow cytometry revealed higher proliferating CD8+ T cells in the IL-15 / IL-15Ra group, although not significantly different compared with dual or control groups. However, the frequency of SIV-specific CXCR5+ CD8 T cells is higher in IL-15 / TL-15Ra and IL- 15 / IL-15Ra plus IL-12 groups, i.e., post-second phase were sustained higher at the time of ATI and significant compared to the ART-only group. Similarly, the frequency of SIV-specific CD107a+ CD8+ T cells was also higher in the IL- 15 / IL-15Ra group compared with the ART-only group. The other groups also showed a marginal increase in the SIV-specific CD107a+ CD8 T cell responses but not significantly different compared with the control.

[0154] To gain insight into the localization of SIV-specific CD8+ T cells, in situ GagCM9 tetramer staining of LN sections were performed in Mamu-A*01+ animals in each treatment. GagCM9+ cells were present in both the T cell zone (TCZ) and BCF regions of the LN of all groups and expressed GrzB (Fig. 3). The density of these cells was marginally higher in TCZ compared with BCF, and the density of total GagCM9+ T cells correlated positively with the density of total GagCM9+ expressing GrzB. To get a better understanding of the localization of CD8+ T cells in all animals, the density of bulk cytolytic (GrzB+) CD8+ T cells was investigated. Frozen LN sections at 4 weeks post-ATI (the time when the viral load was significantly reduced in the IL- 15 / IL-15R groups) time points were stained for CD20 (B cells), CD3 (T cells), CD8, and GrzB (cytolytic protein). The magnitude and spatial distribution of CD3+CD8+GrzB+ T cells were analyzed in the TCZ, BCF, and germinal centers (Fig 3). The frequency of CD3+ CD8+GrzB+ T cells were markedly higher in the TCZ and BCF of IL-15 / IL-15R and IL-15 / IL-15R plus IL-12 animals compared with IL-12 or ART-only animals (Fig 3). Between the three regions, the density was highest in the TCZ and lowest in GC. While these responses were comparable between the groups, only the IL-15 / IL-15Ra and IL-15 / IL-15Ra plus IL- 12 groups showed significantly higher CD8+GrzB+ T cells in the GC compartment. Collectively, the data indicates that IL-15 / IL-15Ra plus IL- 12 induced significant localization of the CD8+ T cells having cytolytic potential in the lymphoid tissue compartments and combining IL-15 / IL-15Ra plus IL- 12 with ART therapy potentiated their localization in the germinal centers.

[0155] Functional homing and central memory SIV-specific CD8 T cells associated with peak SIV viral control at post-ATI

[0156] Consistent enhancement of antigen-specific follicular homing CXCR5+ CD8 T cells, central memory CD8 T cells, CD28+ CD8 T cells, and CD 16+ NK cells during post-ATI in the IL- 15 or IL- 15 plus IL- 12 treatment groups was observed. Subsequently, the frequency of these SIV Gag-specific CD8 T cell markers post-ATI were correlated with plasma viral RNA levels across all experimental groups. It was revealed that SIV-specific CXCR5+ CD8 T cells, CD28+ CD8 T cells, central memory CD8 T cells, and CD 16+ NK cells were inversely correlated with SIV plasma RNA levels in both blood and in the lymph node. These data suggest a role for anti-viral CD8 T cells in viral control following IL-15 / IL-15Ra plus IL-12 treatment.

Claims

CLAIMS1. A method of treating HIV comprising administering an effective amount of IL- 15 and IL- 15Ralpha in combination with IL- 12 or nucleic acid or vector encoding the same to a human patient in need thereof.

2. The method of claim 1, wherein the IL- 15, IL-15Ralpha, and IL- 12 are administered in combination with a multi-drug anti-retroviral therapy (ART).

3. The method of claim 2, wherein the IL-15, IL-15Ralpha, and IL-12 are administered in combination with and during the initiation of the first administration of a multi-drug anti-retroviral therapy (ART) in the subject.

4. The method of any of claims 1-3, wherein the multi-drug anti-retroviral therapy comprises a nucleoside reverse transcriptase inhibitor (NRTI), abacavir, emtricitabine, lamivudine, tenofovir disoproxil fumarate, zidovudine, non-nucleoside reverse transcriptase inhibitor (NNRTI), doravirine, efavirenz, etravirine, nevirapine, rilpivirine, protease inhibitor (PI), atazanavir, darunavir, fosamprenavir, ritonavir, tipranavir, fusion inhibitor, enfuvirtide, CCR5 antagonist, maraviroc, capsid inhibitor, lenacapavir, attachment inhibitor, fostemsavir, post-attachment inhibitor, ibalizumab, integrase strand transfer inhibitor (INSTI), cabotegravir, dolutegravir, raltegravir, or combinations thereof.

5. The method of claim 4, wherein the combination is abacavir and lamivudine; or abacavir, dolutegravir, and lamivudine; or abacavir, lamivudine, and zidovudine; or atazanavir and cobicistat; or bictegravir, emtricitabine, and tenofovir alafenamide; or cabotegravir and rilpivirine; or darunavir and cobicistat; or darunavir, cobicistat, emtricitabine, and tenofovir alafenamide; or dolutegravir and lamivudine; or dolutegravir and rilpivirine; or doravirine, lamivudine, and tenofovir disoproxil fumarate; or efavirenz, emtricitabine, and tenofovir disoproxil fumarate; or efavirenz, lamivudine, and tenofovir disoproxil fumarate; or elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide; or elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate; or emtricitabine, rilpivirine, and tenofovir alafenamide; or emtricitabine, rilpivirine, and tenofovir disoproxil fumarate; or emtricitabine and tenofovir alafenamide; or emtricitabine andtenofovir disoproxil fumarate; or lamivudine and tenofovir disoproxil fumarate; or lamivudine and zidovudine; or lopinavir and ritonavir.

6. The method of any of claims 1-5, wherein the ratio of IL- 15 to IL-15Ralpha is between 1 / 10 to 2 / 5.

7. The method of any of claims 1-5, wherein the ratio of IL-15 to IL-15Ralpha is about 1 / 5.

8. A pharmaceutical composition comprising IL-15 and IL-15Ralpha in combination with IL- 12 or nucleic acid or vector encoding the same and a pharmaceutically acceptable excipient.

9. The pharmaceutical composition of claim 8, in the form of a pH buffered aqueous saline solution optionally comprising a saccharide or polysaccharide.

10. The pharmaceutical composition of claim 8, in the form of tablet, pill, capsule, gel, gel capsule, micronized particles, or lipid particles.

11. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable excipient is selected from lactose, sucrose, mannitol, triethyl citrate, and dextrose.

12. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable excipient is selected from cellulose, methyl cellulose, ethyl cellulose, hydroxyl propyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, croscarmellose sodium, polyvinyl N- pyrrolidone, crospovidone, and ethyl cellulose.

13. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable excipient is selected from povidone, methyl and ethyl acrylate copolymer.

14. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable excipient is polyethylene glycol.

15. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable excipient is selected from sorbic acid, fatty acid esters of sorbitol, lauryl sulfate, gelatin, glycerin, and glyceryl monooleate.

16. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable excipient is selected from silicon dioxide, titanium dioxide, and iron oxide.

17. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable excipient is selected from stearic acid, magnesium stearate, and calcium stearate.

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