WO2022010928A1

WO2022010928A1 - Method for enhancing humoral immunity

Info

Publication number: WO2022010928A1
Application number: PCT/US2021/040558
Authority: WO
Inventors: Takahiro Miyazaki; Murali Krishna Addepalli
Original assignee: Nektar Therapeutics (India) Pvt. Ltd.; Nektar Therapeutics
Priority date: 2020-07-06
Filing date: 2021-07-06
Publication date: 2022-01-13

Abstract

The present disclosure provides, among other things, methods, compositions, and kits for promoting or enhancing humoral immunity in a subject. More particularly, provided is a method for enhancing humoral immunity in a subject by administering to the subject a long acting IL-2Rαβ-biased agonist in combination with a vaccine, such as, for example, a preventative vaccine for protection against an infectious disease.

Description

METHOD FOR ENHANCING HUMORAL IMMUNITY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. 119(a) to Indian

Patent Application No. 202041028745, filed July 6, 2020, which is incorporated by reference herein in its entirety.

FIELD

[0002] The instant application relates to (among other things) a method for promoting or enhancing humoral immunity in a subject. More particularly, the instant application relates to, among other things, the use of a long acting interleukin-2 receptor (IL-2R)a.p-biased agonist for enhancing humoral immunity (e.g., by directly or indirectly activating B cells) when administered to a subject in combination with a vaccine, such as, for example, a vaccine that confers antibody-mediated protection, and related combinations and methods.

BACKGROUND

[0003] The development and introduction of human vaccines has had a tremendous impact on global health by dramatically reducing the mortality and morbidity caused by infectious diseases. Vaccines have prevented disease, complications and the death of millions of infants and children by protecting against many deadly infectious diseases (Bloom et al, World Economics 6:15 (2005). The World Health Organization (WHO) currently recommends routine immunization against multiple diseases including bacillus calmette-guerin (BCG), hepatitis B, polio, diphtheria, tetanus, pertussis, haemophilus influenzae type B, pneumococcus, rotavirus, measles, rubella, and human papilloma virus. Additional vaccines are recommended for populations at high risk or regions with special needs.

[0004] Despite the successes attributed to vaccination, current vaccination efforts face a number of obstacles. Millions of people are still estimated to die annually from vaccine- preventable illnesses, and several childhood illnesses have begun to re-emerge. Infectious disease-related mortality can in some cases be explained by lack of efficacious vaccines, i.e., where vaccinology has failed due to factors such as antigenic drift, and the existence of more difficult target diseases. There exists a need not only for new prophylactic as well as new therapeutic vaccines, but also for improvements to conventional vaccines and vaccination regimens, which could have a tremendous impact on patient populations, including, for example, immune-compromised patients. Thus, the present disclosure seeks to address this and other needs.

SUMMARY

[0005] The present disclosure provides a new and surprisingly beneficial immunomodulation approach for enhancing antibody-mediated protection conferred by a vaccine. Thus, in a first aspect, amongst others, provided herein is a method comprising administering to a subject a vaccine and a humoral immunoenhancing amount of a long acting IL-2R.p-biased agonist to generate or enhance humoral immunity in the subject.

[0006] In one or more embodiments, administration of the long acting IL-2R.p-biased agonist is effective to enhance the humoral immune response of the subject when compared to the humoral immune response of the subject upon administration of the vaccine in the absence of the long acting IL-2R.p-biased agonist.

[0007] In yet another aspect, provided herein is a method for modulating (i.e., enhancing) a humoral immune response to vaccination by administering to a subject that has been vaccinated or is to be vaccinated, a humoral immunoenhancing amount a long acting IL-2Rj3-biased agonist.

[0008] In yet a further aspect, provided herein is a method for providing protection against infection or an infectious disease by administering to a subject a vaccine directed to a pathogen for which protection is antibody -mediated, the improvement comprising further administering a humoral immunoenhancing amount of a long acting IL-2R.p-biased agonist to generate or enhance humoral immunity in the subject.

[0009] In one or more further embodiments, the vaccine is selected from a live attenuated pathogen, a whole inactivated organism, an inactivated bacterial toxin, a recombinant vaccine, a subunit vaccine, a conjugate vaccine, and a viral vaccine. [0010] In some more particular embodiments, the vaccine is selected from a vaccine against diphtheria, hepatitis A, hepatitis B, herpes zoster, human papillomavirus, haemophilus influenza type a and/or b, chickenpox, measles, meningococcus, adenovirus, anthrax, cholera, diphtheria, Japanese ecephalitis, mumps, pertussis, pneumococcus, polio, rabies, rotavirus, rubella, shingles, whooping cough, smallpox, tetanus, tuberculosis, typhoid fever, varicella, and yellow fever.

[0011] By way of clarity, with regard to the sequence of administering, the vaccine and the long acting IL-2R.p-biased agonist may be administered concurrently or sequentially, in any order, and via the same and/or different routes of administration. Moreover, treatment may comprise a single cycle of administration (i.e., vaccination accompanied by administration of the long acting IL-2R.p-biased agonist, or may comprise multiple cycles (i.e., further administrations of a vaccine (i.e., “booster”) and/or the long acting IL-2R.p-biased agonist).

[0012] In one or more embodiments related to any one or more of the foregoing aspects or embodiments, the vaccine is administered to the subject separately from the long acting IL- 2Rj3-biased agonist. In yet one or more alternative embodiments, the vaccine is administered to the subject together with the long acting IL-2R.p-biased agonist.

[0013] In yet one or more further embodiments, the vaccine is administered to the subject prior to administering the long acting IL-2R]3-biased agonist. For example, in one or more embodiments, the vaccine and the long acting IL-2R]3-biased agonist are both administered on day 1 of treatment. In one or more alternative embodiments, the vaccine is administered on day 1 of treatment and the long acting IL-2R.p-biased agonist is administered on any one of days 1 to 7 of treatment. For example, the long acting IL-2R.p-biased agonist is administered on any one of days 1, 2, 3, 4, 5, 6, or 7 of treatment, or even thereafter.

[0014] In yet one or more further embodiments, the vaccine is administered to the subject subsequent to administering the long acting IL-2R.p-biased agonist. For example, in one or more embodiments, the vaccine and the long acting IL-2R]3-biased agonist are both administered on day 1 of treatment. In one or more alternative embodiments, the long acting IL-2R.p-biased agonist is administered on day 1 of treatment and the vaccine is administered on any one of days 1 to 7 of treatment. For example, the vaccine is administered on any one of days 1, 2, 3, 4, 5, 6, or 7 of treatment, or even thereafter.

[0015] In one or more particular embodiments, the long acting IL-2R.p-biased agonist and the vaccine are administered on the same day.

[0016] In some embodiments, the subject is a human subject.

[0017] In yet one or more embodiments, the long acting IL-2Rj3-biased agonist is administered at a dose in a range of from about 0.0005 to 0.3 mg/kg. In some embodiments, the long acting IL-2R.p-biased agonist is administered at a dosage amount of about 0.001 to about 0.01 mg/kg. In yet some further embodiments, the long acting IL-2R.p-biased agonist is administered at a dose ranging from about 0.003 to about 0.009 mg/kg. In some particular embodiments, the long acting IL-2R.p-biased agonist is administered at a dose of about 0.003 mg/kg. In yet some other embodiments, the long acting IL-2R.p-biased agonist is administered at a dose of about 0.006 mg/kg.

[0018] In one or more embodiments, the vaccine is a preventative vaccine for fighting an infection or an infectious disease.

[0019] In some further embodiments, the vaccine is a non-cancer vaccine. In some alternative embodiments, the vaccine is one that is not directed to a coronavirus; in some further more particular embodiments, the vaccine is not a SARS-CoV-2 vaccine or is not for prevention of COVID-19.

[0020] In yet some additional embodiments, the vaccine is a traditional vaccine.

[0021] In some embodiments related to any one or more of the aspects or embodiments provided herein, the long acting IL-2R.p-biased agonist comprises aldesleukin (des-alanyl-1, serine- 125 human interleukin-2) releasably covalently attached to polyethylene glycol. In yet some additional embodiments, the long acting IL-2R.p-biased agonist comprises aldesleukin releasably covalently attached to from 4, 5 and 6 polyethylene glycol polymers. In yet some further embodiments, the long acting IL-2R.p-biased agonist comprises aldesleukin releasably covalently attached to an average of about 6 polyethylene glycol polymers. In one or more additional embodiments, the polyethylene glycol polymers that are releasably covalently attached to aldesleukin are branched. In some further embodiments, the long acting IL-2R.p- biased agonist is multi(2,7-(bis-methoxyPEG-carboxyamide)(9H-fluorene-9-yl)methyl N- carbamate)interleukin-2, more particularly, (2,7-(bis-methoxyPEGio_kD-carboxyamide)(9H- fluorene-9-yl)methyl N-carbamate)_6avginterleukin-2.

[0022] In some embodiments, the method or combination is effective to increase IgM antibody titers in a subject, that is to say, is effective to provide immunoenhancement of an IgM response in a subject.

[0023] In one or more further embodiments, the method or combination is useful to enhance the antibody response to a vaccine that mediates protection against disease through the induction of serum antibodies by prolonging the duration (persistence) of the antibody response at or above a threshold level effective to prevent or fight against infection.

[0024] In some further embodiments, the subject is elderly.

[0025] In some additional embodiments, the subject is immunocompromised.

[0026] In yet some further embodiments, the subject is hyporesponsive to traditional vaccination.

[0027] In yet a further aspect, provided is a kit comprising a humoral immunoenhancing amount of a long acting IL-2R.p-biased agonist and a vaccine, accompanied by instructions for use.

[0028] In one or more embodiments of the kit, the long acting IL-2R.p-biased agonist and the vaccine are comprised in a single composition for administration to the subject, where the single composition optionally comprises a pharmaceutically acceptable excipient.

[0029] In some alternative embodiments of the kit, the long acting IL-2R.p-biased agonist and the vaccine are provided in separate containers, and the kit comprises instructions for administering the vaccine and the long-acting IL-2R.p-biased agonist separately to the subject. [0030] In some embodiments of the kit, both the long-acting IL-2RP-biased agonist and the vaccine are in solid form. In one or more related embodiments, the long acting IL-2RP- biased agonist and the vaccine are in a solid form suitable for reconstitution in an aqueous diluent.

[0031] In yet one or more further embodiments, each of the long acting IL-2RP-biased agonist and the vaccine are comprised within separate compositions each comprising a pharmaceutically acceptable excipient.

[0032] Additional aspects and embodiments are set forth in the following description, including the examples and the claims, and the disclosure should not be considered to be limited in this regard.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG 1. is a graph of anti-tetanus toxoid IgM antibody titers from sera collected from mice from model vaccine treatment groups as described in Example 1. Study groups included (i) mice treated with tetanus toxoid (40 IU) and vehicle (open circles); (i) mice treated with tetanus toxoid (4 IU) and vehicle (open squares); (i) mice treated with tetanus toxoid (0.4 IU) and vehicle (open triangles); mice treated with tetanus toxoid (40 IU) and 0.4 mg/kg IL-2RP- biased agonist, (2,7-(bis-methoxyPEGio_kD-carboxyamide)(9H-fluorene-9-yl)methyl N- carbamate)_4-6interleukin-2 (also referred to herein more generally as RSLAIL-2) (closed circles); mice treated with tetanus toxoid (4 IU) and 0.4 mg/kg RSLAIL-2 (closed squares); and mice treated with tetanus toxoid (0.4 IU) and 0.4 mg/kg RSLAIL-2 (closed triangles). Treatment with the exemplary long acting IL-2RP-biased agonist, (2,7-(bis-methoxyPEGio_kD-carboxyamide)(9H- fluorene-9-yl)methyl N-carbamate)_4-6interleukin-2 (also referred to herein as “RSLAIL-2”) was found to significantly enhance IgM titers in tetanus toxoid-vaccinated mice over the course of treatment.

[0034] FIG. 2 is a graph demonstrating anti-tetanus toxoid IgG antibody titers from sera collected from mice from various model vaccine treatment groups as described in Example 1. Study groups included (i) mice treated with tetanus toxoid (40 IU) and vehicle (open circles); (i) mice treated with tetanus toxoid (4 IU) and vehicle (open squares); (i) mice treated with tetanus toxoid (0.4 IU) and vehicle (open triangles); mice treated with tetanus toxoid (40 IU) and 0.4 mg/kg RSLAIL-2 (closed circles); mice treated with tetanus toxoid (4 IU) and 0.4 mg/kg RSLAIL-2 (closed squares); and mice treated with tetanus toxoid (0.4 IU) and 0.4 mg/kg RSLAIL-2 (closed triangles).

DETAILED DESCRIPTION

TERMS / DEFINITIONS

[0035] In describing and claiming certain features of this disclosure, the following terminology will be used in accordance with the definitions described below unless indicated otherwise.

[0036] As used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0037] It is to be understood that wherever aspects are described herein with the language

"comprising," otherwise analogous aspects described in terms of "consisting of' and/or "consisting essentially of' are also provided.

[0038] Reference to a range or sub-range is meant to expressly recite sub-ranges formed by any two members of a disclosed range. For example, a range described as being from 20 to 50, or from 10 to 30, will thereby expressly include ranges from 10 to 20, 20 to 30, 10 to 50, 30 to 50, and so forth.

[0039] "Water soluble, non-peptidic polymer" refers to a polymer that is at least 35% (by weight) soluble in water at room temperature. Preferred water soluble, non-peptidic polymers are however preferably greater than 70% (by weight), and more preferably greater than 95% (by weight) soluble in water. Typically, an unfiltered aqueous preparation of a "water-soluble" polymer transmits at least 75% of the amount of light transmitted by the same solution after filtering. Preferably, such unfiltered aqueous preparation transmits at least 95% of the amount of light transmitted by the same solution after filtering. Most preferred are water-soluble polymers that are at least 95% (by weight) soluble in water or completely soluble in water. With respect to being "non-peptidic," a polymer is non-peptidic when it contains less than 35% (by weight) of amino acid residues.

[0040] The terms "monomer," "monomeric subunit" and "monomeric unit" are used interchangeably herein and refer to one of the basic structural units of a polymer. In the case of a homo-polymer, a single repeating structural unit forms the polymer. In the case of a co-polymer, two or more structural units are repeated — either in a pattern or randomly — to form the polymer. Preferred polymers used in connection with the present invention are homo-polymers. The water-soluble, non-peptidic polymer comprises one or more monomers serially attached to form a chain of monomers.

[0041] "PEG" or "polyethylene glycol," as used herein, is meant to encompass any water-soluble poly(ethylene oxide). Unless otherwise indicated, a "PEG polymer" or a polyethylene glycol is one in which substantially all (preferably all) monomeric subunits are ethylene oxide subunits, though, the polymer may contain distinct end capping moieties or functional groups, e.g., for conjugation. PEG polymers for use in the moieties described herein will typically comprise one of the two following structures: "-(CH₂CH₂0)_n-" or "-(CH₂CH₂0)_n-iCH₂CH₂-," depending upon whether or not the terminal oxygen(s) has been displaced, e.g., during a synthetic transformation. As stated above, for a PEG-based polymer or a PEG-based polymer conjugate, the variable (n) will typically fall within a range from about 3 to 4000, and the terminal groups and architecture of the overall PEG can vary.

[0042] "Branched," in reference to the geometry or overall structure of a polymer, optionally forming part of a polymer-conjugate, refers to a polymer having two or more polymer "arms" or “chains” extending from a branch point or central structural feature. In some embodiments, a branched polymer comprises two polymer arms or chains emanating from a central structural feature.

[0043] Molecular weight in the context of a water-soluble polymer, such as PEG, can be expressed as either a number average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques. Most commonly employed are gel permeation chromatography and gel filtration chromatography. Other methods for measuring molecular weight values can also be used, such as the use of end-group analysis or the measurement of colligative properties (e.g., freezing-point depression, boiling-point elevation, or osmotic pressure) to determine number average molecular weight or the use of light scattering techniques, ultracentrifugation, or viscometry to determine weight average molecular weight. Matrix assisted laser desorption/ionization time of flight may also be used. PEG polymers are typically polydisperse (i.e., number average molecular weight and weight average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.

[0044] A "physiologically cleavable" or "hydrolyzable" bond is a relatively labile bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to hydrolyze in water may depend not only on the general type of linkage connecting two atoms within a given molecule but also on the substituents attached to these atoms. Appropriate hydrolytically unstable or weak linkages may include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides, oligonucleotides, thioesters, and carbonates.

[0045] An "enzymatically degradable linkage" means a linkage that is subject to degradation by one or more enzymes.

[0046] A "stable" linkage or bond refers to a chemical bond that is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages may generally include but are not limited to the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, amines, and the like. Generally, a stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks. [0047] A covalent “releasable” linkage, for example, in the context of a polyethylene glycol that is covalently attached to an active moiety such as interleukin-2, is one that, under physiological conditions by any suitable release mechanism, releases or detaches a polyethylene glycol polymer moiety from the active moiety such as interleukin-2.

[0048] Reference to a long acting IL-2Ra.p-biased agonist as described herein is meant to encompass pharmaceutically acceptable salt forms thereof.

[0049] A traditional (or conventional) vaccine is a vaccine that functions to promote antibody-production (humoral immunity), that is, a vaccine that confers antibody -mediated immunity against infection and/or disease to a subject.

[0050] "Substantially" or "essentially" means nearly totally or completely, for instance,

95% or greater of a given quantity.

[0051] Similarly, “about” or “approximately” as used herein means within plus or minus

5% of a given quantity.

[0052] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a component that may be included in the compositions described herein and causes no significant adverse toxicological effects to a subject.

[0053] The term "patient," or “subject” as used herein refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a compound or composition or combination as provided herein and includes both humans and animals. Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, caprinae, canines, felines, and the like), avians, and preferably are human. An elderly subject, in reference to a human subject, is a subject that is aged 65 or older.

[0054] An “immunocompromised” subject is a subject with a weakened or suppressed immune system; immunocompromised subjects have a reduced ability to fight infections and other diseases. [0055] "Administering" refers to the introduction of a therapeutic agent to a subject, including vaccines, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. A therapeutic agent can also be administered via a non- parenteral route, or orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering of one or more therapeutic agents can also be performed for each of the therapeutic agents, for example, once, a plurality of times, and/or over one or more extended periods.

[0056] A "therapeutically effective amount" or "therapeutically effective dosage" of a therapeutic agent is any amount of the agent that, when used alone or in combination with another therapeutic agent, (i) protects a subject against the onset of a disease such as an infectious disease, or (ii) promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in one or more suitable in vitro assays. For example, protection conferred by a vaccine can be measured by, for example, immune responses to the vaccine, prevention of infection, reduction in disease severity, decreased rate of hospitalization or shortened duration of hospitalization.

[0057] An enhancement in humoral immunity can be conferred, e.g., by any of a number of measures for evaluating antibody -mediated efficacy of a vaccine, for example, by providing an increase in the peak of vaccine-induced antibody titers, by providing an increase in vaccine- induced antibody titers at a given timepoint post-vaccination, by providing greater persistence of vaccine antibodies above a protective threshold, by prolonging the maintenance of immune memory cells capable of reactivation with subsequent microbial exposure, and the like.

Overview

[0058] In an effort to address at least some of the shortcomings associated with current vaccine strategies, such as for example, weak and/or ineffective humoral immune responses, provided herein is a method comprising administering to a subject a vaccine such as, for example, a traditional vaccine, and a humoral immunoenhancing amount of a long acting IL- 2Rj3-biased agonist. Thus, the present disclosure is based, at least in part, on the discovery of a particularly beneficial therapeutic combination comprising a vaccine and a long-acting IL-2R agonist, and more specifically, an IL-2R.p-biased agonist, for enhancing a humoral immune response in a subject to thereby generate or enhance humoral immunity, for example, by stimulating IgM production. This finding was somewhat unexpected, given some previous reports of IL-2 or IL-2 based proteins resulting in suppression of a humoral immune response following immunization. See, e.g., Kunzendorf, U., J Clin Invest. 1996; 97(5): 1204-1210, describing a chimeric protein comprising mouse IL-2 fused to mouse IgG2b Fc domains, i.e., an IL2-IgG2b fusion protein, which, when administered in vivo was found to suppress the humoral immune response (i.e., by virtue of a profound inhibition of IgM production) following immunization with sheep erythrocytes. Similarly, Gottlieb, D.J., etal, Clin Exp Immunol., 1992, Mar 87(3): 493 - 498, reported an abrogated humoral response in humans following IL-2 infusion. Due to complexity of IL-2 and its fine tuning of the balance of T effector cell and B cell helper aspects of the immune response, the foregoing finding was unexpected.

Vaccines

[0059] The treatment methods, uses, compositions and combinations provided herein encompass administration of a vaccine, e.g., a traditional vaccine that confers protective immunity against infection through the induction of antibodies. The compositions and methods provided herein find use in, among other things, both clinical and research applications. [0060] Illustrative vaccines for use in the methods, combinations, compositions, and uses provided include, but are not limited to, traditional vaccines, such as for example, live attenuated pathogens, whole inactivated organisms, inactivated bacterial toxins, subunit vaccines, conjugate vaccines, and viral vaccines. Live attenuated vaccines suitable for use include vaccines for, for example, measles, mumps, rubella, varicella, influenza, and rotavirus. Inactivated/killed vaccines for use in the methods and combinations described herein include vaccines for polio and hepatitis A. Suitable toxoid (inactivated toxin)-based vaccines include diphtheria and tetanus vaccines. Subunit/conjugate-based vaccines include hepatitis B, haemophilus influenza type b, pertussis, pneumococcal, and meningococcal vaccines. Additional vaccines suitable for use in the instant methods and related compositions are described in Kallerup, R. S., et al, Chapter 2, Classification of Vaccines, in Subunit Vaccine Delivery, Foged, C; Rades, Th., et al. (Eds), Springer Science + Business Media, NY 2015, see, e.g., Table 2.2, and in Amanna, I.J; Slifka, M.K., in Virology. 2011 Mar 15; 411(12): 206- 215, herein incorporated by reference. In some embodiments, the vaccine is preferably a non-cancer vaccine. In some additional related embodiments, the vaccine is not a vaccine that targets tumor-specific antigens. In yet some further embodiments, the vaccine is not a vaccine that activates cytotoxic T-cells. In some further embodiments, the vaccine is not a vaccine directed to a coronavirus, or is not a SARS- CoV-2 vaccine.

[0061] Illustrative vaccines whose effect may be enhanced by administration in combination with a long acting, IL-2R.p-biased agonist such as, for example, multi(2,7-(bis- methoxyPEG-carboxyamide)(9H-fluorene-9-yl)methyl N-carbamate)interleukin-2, i.e., for enhancing a humoral immune response, include but are not limited to, for example, the following vaccines: hepatitis A (e.g., HepA (Havrix, Vaqta), HepA-HepB (Twinrix)), hepatitis B ((e.g., HepB (Engerix-B, Recombivax HB, Heplisav-B), DTaP-HepB-IPV (Pediarix), HepA-HepB (Twinrix)), herpes zoster, human papillomavirus (e.g., HPV9 (Gardasil 9)), haemophilus influenza type b (e.g., Hib (ActHIB, PedvaxHIB, Hiberix), DTaP-IPV/Hib (Pentacel)), seasonal influenza (e.g., IIV (Afluria, Fluad, Flublok, Flucelvax, FluLaval, Fluarix, Fluvirin, Fluzone, Fluzone High Dose, Fluzone Intradermal, LAIV (FluMist)), measles (e.g., MMR (M-M-RII), MMRV (ProQuad)), meningococcal (e.g., MenACWY (Menactra, Menveo), MenB (Bexsero, Trumenba)), adenovirus, anthrax (e.g., AVA (BioThrax)), cholera, diphtheria (e.g., DTaP (Daptacel, Infanrix), Td (Tenivac, generic), DT, Tdap (Adacel, Boostrix), DTaP-IPV (Kinrix, Quadracel), DTaP-HepB-IPV (Pediarix), DTaP-IPV/Hib (Pentacel)), Japanese encephalitis (e.g., JE (Ixiaro)), mumps (e g., MMR (M-M-RII), MMRV (ProQuad), pertussis (e g., DTaP (Daptacel, Infanrix), Tdap (Adacel, Boostrix), DTaP-IPV (Kinrix, Quadracel), DTaP-HepB-IPV (Pediarix), DTaP-IPV/Hib (Pentacel)), pneumococcal (e.g., PCV123 (Prevnar 13), PPASV23 (Pneumovax 23)), polio (e.g., polio (Ipol), DTaP-IPV (Kinrix, Quadracel), DTaP-HepB-IPV (Pediarix), DTaP-IPV/Hib (Pentacel)), rabies (e.g., Imovax Rabies, RabAvert), rotavirus (e.g., RV1 (Rotarix), RV5 (RotaTeq)), rubella (e.g., MMR, MMRV), shingles (e.g., ZVL (Zostavax), RZV (Shingrix)), smallpox (e.g., Vaccinia (ACAM2000)), tetanus (e.g., DTaP (Daptacel, Infanrix), Td (Tenivac), DT, Tdap (Adacel, Boostrix), DTaP-IPV (Kinrix, Quadracel), DTaP- HepB-IPV (Pediarix), DTaP-IPV/Hib (Pentacel)), tuberculosis, typhoid fever (e.g., Typhoid Oral (Vivotif), Typhoid Polysaccharide (Typhim Vi)), varicella (e.g,. VAR (Varivax), MMRV (ProQuad)), and yellow fever (VF (YF-Vax)). For vaccines that are ineffective in mounting a rapid antibody response to vaccination, for example, by producing IgM antibodies, vaccination accompanied by administration of a long acting, IL-2RP-biased agonist as described herein may be particularly advantageous. For example, administration of the IL-2R.p-biased agonist may be particularly beneficial in instances in which vaccination may be insufficient to mount a protective immune response against a pathogen with a short incubation period following exposure and a rapid onset of symptoms.

Long acting, IL-2R.p-Biased Agonist

[0062] The immunomodulating methods, formulations, combinations, compositions, kits and the like described herein encompass the administration of a long acting, IL-2R.p-biased agonist discovered to surprisingly enhance the antibody -mediated protection conferred by vaccination.

[0063] Non-limiting examples of long acting, IL-2R.p-biased agonists are described in

International Patent Publication Nos. WO 2012/065086 and in WO 2015/125159. An exemplary and preferred long acting, IL-2RP-biased agonist is RSLAIL-2 referenced in Examples land 2 in the present disclosure, where the releasable PEG is based upon a 2,7,9-substituted fluorene as shown below, with poly(ethylene glycol) chains extending from the 2- and 7- positions on the fluorene ring via amide linkages (fluorene-C(0)-NH~), and releasable covalent attachment to IL- 2 via attachment to a carbamate nitrogen atom attached via a methylene group (-CEL-) to the 9- position of the fluorene ring, also referred to herein as multi(2,7-(bis-methoxyPEG- carboxyamide)(9H-fluorene-9-yl)methyl N-carbamate)interleukin-2. In this regard, RSLAIL-2 is a composition comprising compounds encompassed by the following formula:

wherein IL-2 is an IL-2 (interleukin-2), and pharmaceutically acceptable salts thereof, where “n” is an integer from about 3 to about 4000. As indicated in the formula above, the IL-2 molecule preferably possesses 4, 5, or 6 branched polyethylene glycol moieties as shown above covalently attached thereto. Representative ranges for each “n” include, for example, an integer from about 40 to about 550, or an integer from about 60 to about 500, or an integer from about 113 to about 400, or from about 200-300. In one or more preferred embodiments, the value of “n” in each of the polyethylene glycol chains is substantially the same, such that the two PEG chains extending from the central fluorenyl core have substantially the same weight average molecular weight. In certain preferred embodiments, “n” in each of the polyethylene glycol chains is about 227 (i.e., where each polyethylene glycol chain extending from the central fluorenyl core has a weight average molecular weight of about 10,000 daltons, such that the weight average molecular weight of the overall branched PEG moiety is about 20,000 daltons), i.e., referred to herein (2,7- (bis-methoxyPEGio_kD-carboxyamide)(9H-fluorene-9-yl)methyl N-carbamate)_4-6interleukin-2,

[0064] In one or more embodiments, the composition contains no more than 10% (based on a molar amount), and preferably no more than 5% (based on a molar amount), of compounds encompassed by the following formula:

wherein IL-2 is an interleukin-2, (n) (referring to the number of polyethylene glycol moieties attached to IL-2) is an integer selected from the group consisting of 1, 2, 3, 7 and >7, and pharmaceutically acceptable salts thereof.

[0065] In some embodiments, RSLAIL-2 possesses on average about six polyethylene glycol moieties attached to IL-2, as depicted in the structure below.

[0066] To determine average degree of PEGylation for a composition such as the

RSLAIL-2 compositions described herein, typically the protein is quantified by a method such as an bicinchoninic acid (BCA) assay or by UV analysis, to determine moles of protein in the sample. The PEG moieties are then released by exposing the sample to conditions in which the PEG moieties are released, and the released PEG is then quantified (e.g., by BCA or UV) and correlated with moles protein to determine average degree of PEGylation.

[0067] In some further embodiments, RSLAIL-2 is generally considered to be an inactive prodrug, i.e., inactive upon administration, and by virtue of slow release of the polyethylene glycol moieties in vivo following administration, providing active conjugated forms of interleukin-2 having fewer PEG moieties attached than in the conjugate composition that is initially administered. RSLAIL-2 may be considered to be a CD-122 (also known as IL-2RP) agonist, that is, a molecule capable of activating or stimulating CD-122 (IL-2Rp). Moreover, RSLAIL-2 may be considered to be a CD-122 agonist that selectively binds and activates IL- 2RPy over IL-2RaPY. See, for example Charych, D., et ah, PLOS One , 12(7): 0179431, July 5, 2017.

[0068] Additional exemplary compositions of RSLAIL-2 comprise compounds in accordance with the above formula wherein the overall polymer portion of the molecule has a weight average molecular weight in a range of from about 250 Daltons to about 90,000 daltons. Additional suitable ranges include weight average molecular weights in a range selected from about 1,000 daltons to about 60,000 daltons, in a range of from about 5,000 daltons to about 60,000 daltons, in a range of about 10,000 daltons to about 55,000 daltons, in a range of from about 15,000 daltons to about 50,000 daltons, and in a range of from about 20,000 daltons to about 50,000 daltons.

[0069] Additional illustrative weight-average molecular weights for the polyethylene glycol polymer portion include about 200 daltons, about 300 daltons, about 400 daltons, about 500 daltons, about 600 daltons, about 700 daltons, about 750 daltons, about 800 daltons, about 900 daltons, about 1,000 daltons, about 1,500 daltons, about 2,000 daltons, about 2,200 daltons, about 2,500 daltons, about 3,000 daltons, about 4,000 daltons, about 4,400 daltons, about 4,500 daltons, about 5,000 daltons, about 5,500 daltons, about 6,000 daltons, about 7,000 daltons, about 7,500 daltons, about 8,000 daltons, about 9,000 daltons, about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, about 20,000 daltons, about 22,500 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, and about 75,000 daltons. In some embodiments, the weight-average molecular weight of the polyethylene glycol polymer is about 20,000 daltons.

[0070] As described above, the long-acting, IL-2RP-biased agonist may be in the form of a pharmaceutically-acceptable salt. Typically, such salts are formed by reaction with a pharmaceutically-acceptable acid or an acid equivalent. The term "pharmaceutically-acceptable salt" in this respect, will generally refer to the relatively non-toxic, inorganic and organic acid addition salts. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a long-acting interleukin-2 as described herein with a suitable organic or inorganic acid, and isolating the salt thus formed.

Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, oxylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts ", J. Pharm. Sci. 66:1-19). Thus, salts as described may be derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; or prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

[0071] In reference to the foregoing IL-2RP-biased agonist compounds and compositions, the term "IL-2" as used herein, refers to a moiety having human IL-2 activity. The term, ‘residue’, in the context of residue of IL-2, means the portion of the IL-2 molecule that remains following covalent attachment to a polymer such as a polyethylene glycol, at one or more covalent attachment sites, as shown in the formulae above. It will be understood that when the unmodified IL-2 is attached to a polymer such as polyethylene glycol, the IL-2 is slightly altered due to the presence of one or more covalent bonds associated with linkage to the polymer(s). This slightly altered form of the IL-2 attached to another molecule is referred to a "residue" of the IL-2. [0072] For example, proteins having an amino acid sequence corresponding to any one of

SEQ ID NOs: 1 through 4 described in International Patent Publication No. WO 2012/065086 are exemplary IL-2 proteins, as are any proteins or polypeptides substantially homologous thereto. These sequences are also provided herein. The term substantially homologous means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. For the purposes herein, sequences having greater than 95 percent homology, equivalent biological activity (although not necessarily equivalent strength of biological activity), and equivalent expression characteristics are considered substantially homologous. For purposes of determining homology, truncation of the mature sequence should be disregarded. As used herein, the term "IL-2" includes such proteins modified deliberately, as for example, by site directed mutagenesis or accidentally through mutations. These terms also include analogs having from 1 to 6 additional glycosylation sites, analogs having at least one additional amino acid at the carboxy terminal end of the protein wherein the additional amino acid(s) includes at least one glycosylation site, and analogs having an amino acid sequence which includes at least one glycosylation site. The term includes both natural and recombinantly produced moieties. In addition, the IL-2 can be derived from human sources, animal sources, and plant sources. One exemplary and preferred IL-2 is recombinant IL-2 referred to as aldesleukin. Aldesleukin differs from native interleukin-2 in that is it not glycosylated (since it is produced from E. coli ), it has no N-terminal alanine, and has a serine substituted for cysteine at amino acid position 125.

[0073] Conventional approaches, such as those involving radiolabeling a compound, administering it in vivo , and determining its clearance, can be used to determine whether a compound proposed to be a long-acting IL-2R.p biased agonist is "long-acting". For the purposes herein, the long acting nature of an IL-2RP biased agonist is typically determined using flow cytometry to measure STAT5 phosphorylation in lymphocytes at various time points after administration of the agonist to be evaluated in mice. As a reference, the signal is lost by around 24 hours with IL-2 but is sustained for a period greater than that for a long-acting IL-2R.p-biased agonist. As an illustration, the signal is sustained over several days for the RSLAIL-2 compositions. [0074] The disclosure is not limited to any particular long acting, IL-2RP-biased agonist so long as the agonist exhibits an in vitro binding affinity for IL-2RP that is at least 5 times greater (more preferably at least 10 times greater) than the binding affinity for IL-2Ra.p in the same in vitro model, and has at least an effective 10-fold in vivo half-life greater than IL-2 (half- life based on the in-vivo disappearance of IL-2). By way of example, it is possible to measure binding affinities against IL-2 as a standard. RSLAIL-2 is considered to be an IL-2RP-biased (i.e., preferential) agonist. In this regard, the RSLAIL-2 employed in the illustrative supporting examples, (2,7-(bis-methoxyPEGio_kD-carboxyamide)(9h-fluorene-9-yl)methyl N- carbamate)_6avginterleukin-2, exhibits about a 60-fold decrease in affinity to IL-2Ra.p relative to IL-2, but only about a 5-fold decrease in affinity IL-2RP relative to IL-2 as previously described. See, for example, Example 20 in PCT Publication No. WO 2018/132496 describing the binding affinity of RSLAIL-2 to IL-2Ra and IL-2Rp.

Methods, Compositions, Combinations and Kits

[0075] In accordance with the methods, compositions, and kits described herein, the long-acting, IL-2RP-biased agonist is provided in a humoral immunoenhancing amount, that is, in an amount sufficient to enhance a humoral immune response (including both primary and secondary immune responses), such as, for example, an IgM- antibody response, when combined with vaccination, e.g., with a vaccine that confers antibody-mediated immunity. Methods of assessing humoral immunity are well known to those of skill in the art and include, without limitation, antibody titer measurements, for example, by ELISA, hemagglutination, neutralization assays, measuring B cells using, for example, a CTL assay, antibody profiling (Burbelo, P.D., etal, Expert Rev. Vaccines. 2010; 9(6): 567-578), and/or the use of biomarkers such as activation-induced cytidine deaminase (AID) (Frasca, D., etal, Int. Immunol. 2012. Mar 24(3); 175-182).

[0076] In one or more instances, however, the amount is an amount encompassed by one or more of the following ranges expressed in amount of protein: from about 0.01 to 100 mg/kg; from about 0.01 mg/kg to about 75 mg/kg; from about 0.02 mg/kg to about 60 mg/kg; from about 0.03 mg/kg to about 50 mg/kg; from about 0.05 mg/kg to about 40 mg/kg; from about 0.05 mg/kg to about 30 mg/kg; from about 0.05 mg/kg to about 25 mg/kg; from about 0.05 mg/kg to about 15 mg/kg; from about 0.05 mg/kg to about 10 mg/kg; from about 0.05 mg/kg to about 5 mg/kg; from about 0.05 mg/kg to about 1 mg/kg.

[0077] In certain embodiments, the long acting IL-2R.p-biased agonist, e.g., multi(2,7-

(bis-methoxyPEG-carboxyamide)(9H-fluorene-9-yl)methyl N-carbamate)interleukin-2 used in the compositions and methods provided herein, is administered at a dosage amount of from about 0.0005 to 0.3 mg/kg; from about 0.001 mg/kg to about 0.3 mg/kg; from about 0.001 mg/kg to about 0.25 mg/kg; from about 0.001 mg/kg to about 0.15 mg/kg; from about 0.001 mg/kg to about 0.05 mg/kg; from about 0.001 mg/kg to about 0.01 mg/kg; from about 0.001 mg/kg to about 0.008 mg/kg; from about 0.001 mg/kg to about 0.005 mg/kg; from about 0.002 mg/kg to about 0.005 mg/kg; from about 0.002 mg/kg to about 0.004 mg/kg, or from about 0.003 to about 0.009 mg/kg.

[0078] In some embodiments, the long acting åL-2R]3-biased agonist, e.g., multi(2,7-(bis- methoxyPEG-carboxyamide)(9H-fluorene-9-yl)methyl N-carbamate)interleukin-2, is administered at a dose that is less than or equal to 0.003 mg/kg. In certain other embodiments, the long acting IL-2RP-biased agonist is administered at a dose that is less than or equal to about 0.006 mg/kg. In yet some further embodiments, the long acting IL-2R.p-biased agonist is administered at a dose that is less than or equal to about 0.009 mg/kg. Additional exemplary dosing ranges include for example, from about 0.001 mg/kg to about 0.01 mg/kg, or from about 0.002 mg/kg to about 0.008 mg/kg or from about 0.002 mg/kg to less than about 0.006 mg/kg.

[0079] For confirmation, with respect to the long-acting, IL-2RP-biased agonist, the amount, extent, and manner of humoral immune activation/enhancement can vary widely and still be effective when coupled with administration of a vaccine, e.g., a traditional vaccine.

[0080] The actual doses of the vaccine and the long-acting, IL-2RP-biased agonist, as well as the dosing regimen associated with the methods, compositions, combinations, and kits described herein will vary depending upon the age, weight, and general condition of the subject, prior antigen exposure, the judgment of the healthcare professional, the particular vaccine, and long-acting, IL-2RP-biased agonist to be administered. It is believed that the methods, uses, combinations and compositions provided herein may be particularly advantageous for subjects that are elderly and/or are immunocompromised.

[0081] With regard to the frequency and schedule of administering the vaccine (i.e., the immunization schedule) and the long acting, IL-2RP-biased agonist, one of ordinary skill in the art will be able to determine an appropriate frequency and dosing regimen. For example, in a vaccination cycle, a clinician may administer the vaccine, either as a single dose or in a series of doses, e.g., over the course of several days or weeks or months) based upon recommended vaccination schedules. Recommended immunization schedules for routine immunization of children, adolescents, and adults are published by the Centers for Disease Control and Prevention, as well as by the World Health Organization. The long acting, IL-2R.p-biased agonist is administered concurrently with the vaccine, prior to vaccination, and/or following administration of the vaccine. For example, in some treatment modalities, the long acting, IL- 2RP-biased agonist is administered within 7 days of vaccine administration (e.g., on any one of days 1, 2, 3, 4, 5, 6, or 7). In some instances, the long acting, IL-2RP-biased agonist is administered within 4 days of vaccination, e.g., on any one of days 1, 2, 3, or 4. In some additional embodiments, the long acting, IL-2R.p-biased agonist is administered prior to vaccination, for example on any one of days 1, 2, 3, 4, 5, 6, or 7 prior to vaccination. Based upon the long acting nature of the IL-2R.p-biased agonist, the IL-2R.p-biased agonist compounds and compositions described herein may be administered relatively infrequently (e.g., once every four months, once every three months, once every two months, once per month, once every three weeks, once every two weeks, once every 8-10 days, once every week, etc.). In some embodiments, the IL-2R.p-biased agonist is administered on the same day that vaccination occurs. It will be appreciated that the method may include a single cycle of administering the IL- 2RP-biased agonist or multiple cycles of administration. Further, in some embodiments, the method comprises administering one or more doses of the IL-2R.p-biased agonist per cycle of vaccine administration. In some additional embodiments, the method comprises administering one or fewer doses of the IL-2R.p-biased agonist per cycle of vaccine administration (for example, the IL-2R.p-biased agonist may be administered only once over the course of multiple vaccinations comprised in the vaccination schedule). [0082] Exemplary lengths of time associated with the course of vaccination therapy include about one week; about two weeks; about three weeks; about four weeks; about five weeks; about six weeks; about seven weeks; about eight weeks; about nine weeks; about ten weeks; about eleven weeks; about twelve weeks; about thirteen weeks; about fourteen weeks; about fifteen weeks; about sixteen weeks; about seventeen weeks; about eighteen weeks; about nineteen weeks; about twenty weeks; about twenty-one weeks; about twenty -two weeks; about twenty -three weeks; about twenty four weeks; about seven months; about eight months; about nine months; about ten months; about eleven months; about twelve months; about thirteen months; about fourteen months; about fifteen months; about sixteen months; about seventeen months; about eighteen months; about nineteen months; about twenty months; about twenty one months; about twenty -two months; about twenty -three months; about twenty -four months; about thirty months; about three years; about four years and about five years, depending upon factors such as, for example, the type of vaccine, vaccination target, the particular vaccine, the recommended vaccination schedule, patient antibody titers and the like.

[0083] The methods provided herein are useful for (among other things) enhancing humoral immunity in a patient that is administered a vaccine. For example, patients may be responsive to the vaccine alone (e.g., as evidenced by protective antibody titers or other suitable diagnostic assay indicative of protective immunity), as well as to a combination with a long acting, IL-2R.p-biased agonist, but are more responsive to the combination. By way of further example, patients may be non-responsive or only minimally responsive to either the vaccine or the long acting, IL-2R.p-biased agonist, but are responsive to the combination. By way of still further example, patients may be non-responsive to both the vaccine and the long acting, IL-2R.p- biased agonist alone, but are responsive to the combination.

[0084] Administration, e.g., of the vaccine and/or the long acting, IL-2R.p-biased agonist, is typically via injection. Other modes of administration are also contemplated, such as pulmonary, nasal, buccal, rectal, sublingual and transdermal. As used herein, the term "parenteral" includes subcutaneous, intravenous, intra-arterial, intratumoral, intralymphatic, intraperitoneal, intracardiac, intrathecal, and intramuscular injection, as well as infusion injections. As described previously, the vaccine and the long acting, IL-2RP-biased agonist can be administered separately. Alternatively, if administration of the vaccine and the long acting, IL-2RP-biased agonist, is desired to be simultaneous, either as an initial dose or throughout the course of treatment or at various stages of the dosing regimen — and the vaccine and the long acting, IL-2RP-biased agonist are compatible together and in a given formulation — then the simultaneous administration can be achieved via administration of single dosage form/formulation (e.g., intravenous administration of an intravenous formulation that contains both immunological components). One of ordinary skill in the art can determine through routing testing whether two such components are compatible together and in a given formulation. For example, administration to a patient can be achieved through injection of a composition comprising an IL-2RP-biased agonist and a diluent. In addition, administration to a patient can be achieved through injection of a vaccine and a diluent. Further, administration can be achieved through injection of a composition comprising both an IL-2R.pb-biased agonist, a vaccine, and a diluent. With respect to possible diluents, the diluent can be selected from the group consisting of bacteriostatic water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's solution, lactated Ringer's solution, saline, sterile water, deionized water, and combinations thereof. One of ordinary skill in the art can determine through routing testing whether two given pharmacological components are compatible together in a given formulation.

[0085] The humoral immunoenhancing combination described herein, i.e., the long acting IL-2RP-biased agonist and vaccine, may be provided in the form of a kit. As described above, the components may be comprised in a single composition, optionally accompanied by one or more pharmaceutically acceptable excipients, or may be provided in separate containers, where the kit typically includes instructions for use. Suitable pharmaceutically acceptable excipients include those described, for example, in the Handbook of Pharmaceutical Excipients, 7^th ed., Rowe, R.C., Ed., Pharmaceutical Press, 2012. The kit components, e.g., compositions comprising the vaccine and the long acting IL-2RP-biased agonist, may be in either liquid or in solid form. In certain preferred embodiments, both the vaccine and the long acting IL-2RP- biased agonist are in solid form. Preferred solid forms are those that are solid dry forms, e.g., containing less than 5 percent by weight water, or preferably less than 2 percent by weight water. The solid forms are generally suitable for reconstitution in an aqueous diluent.

[0086] The present methods, kits and compositions are useful for enhancing the therapeutic effectiveness of a vaccine, for example, by improving the subject’s humoral response to the vaccine. An enhanced response may be evaluated at any suitable time point during treatment (over the course of the vaccination schedule), after a single round of treatment, after 2- 3 cycles of treatment, etc., and by any of a number of suitable methods as described herein.

[0087] In yet some further embodiments, the methods, kits, compositions and the like provided herein are effective to enhance the IgM response of a subject to vaccination. For instance, as can be seen from the supporting examples, administration of the exemplary long acting IL-2Ra.p-biased agonist, multi(2,7-(bis-methoxyPEG-carboxyamide)(9H-fluorene-9- yl)methyl N-carbamate)interleukin-2, was effective to enhance the IgM response in mice when combined with traditional vaccination when compared to the IgM response observed for traditional vaccination alone. (Similar results are described in Example 2, based upon a non human primate model). The enhancement in the IgM immune response at even the lowest dose administered (0.4 IU) suggests that IL-2Ra.p-biased agonist, multi(2,7-(bis-methoxyPEG- carboxyamide)(9H-fluorene-9-yl)methyl N-carbamate)interleukin-2, may be particularly effective when used in combination with vaccines which require greater antigen exposures to elicit a desired immune response. In addition, multi(2,7-(bis-methoxyPEG-carboxyamide)(9H- fluorene-9-yl)methyl N-carbamate)interleukin-2 may be particularly advantageous when used during post-exposure prophylaxis for treating conditions in which attainment of high IgM titers is important for therapeutic success.

[0088] Further, as evidenced by the results from Example 2, within one week of

RSLAIL-2 administration, an enhanced anti-KLH IgM response was observed in all dosed animals, and was maintained until the end of the study in several monkeys following the second KLH immunization. At both doses of RSLAIL-2 administered in the study described in Example 2, immunoenhancement of the TDAR to the first KLH immunization occurred in most monkeys, compared to controls, peaking at 1 week post-dose (regardless of day of dose administration) in most monkeys for IgM, notably with IgM titers increased from approximately 64 times to 128 times in comparison to immunized control animals in which RSLAIL-2 administration was absent. These striking results provide evidence of the universality of the ability of an IL-2Ra.p- biased agonist such as RSLAIL-2 to significantly enhance a humoral IgM response, e.g., when combined with a vaccine in a general immunization process, and demonstrate that the noted effect is conserved not only across different species but is also conserved across various vaccine types.

[0089] Thus, in some embodiments, the methods and combinations provided herein are effective to increase IgM antibody titers in a subject, i.e., are effective to provide immunoenhancement of an IgM response in a vaccinated subject. In specific but non-limiting embodiments, the method is effective to increase the subject’s IgM antibody titers by at least about 2-fold to about 100-fold or more as compared to the subject’s IgM antibody titers in the absence of administering the IL-2Ra.p-biased agonist. For example, in some specific embodiments, the treatment method is effective to increase the subject’s IgM antibody titers at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold or at least about 110-fold as compared to the subject’s IgM antibody titers in the absence of administering the IL-2Ra.p-biased agonist. The increase can be measured at any suitable timepoint during the course of the immunization schedule that can be suitably used as a basis for comparison. In one or more embodiments, the increase is measured at the peak of the IgM antibody titer response.

[0090] In some additional embodiments, the methods and combinations provided herein are useful for enhancing the antibody response to a vaccine that mediates protection against disease through the induction of serum antibodies by prolonging the duration (persistence) of the antibody response, e.g., IgM response, at or above a threshold level effective to prevent or fight against infection. For example, in some illustrative embodiments, the methods and uses are effective to prolong or improve the persistence of a threshold level of IgM antibody protection in a vaccinated subject by at least about 10% to at least about 400% when compared to the duration of the subject’s threshold level of protection in the absence of administration of the IL-2Ra.p- biased agonist. In further, but non-limiting embodiments, the treatment method is effective to increase a vaccinated subject’s threshold level of IgM antibody protection by at least about 10- 400%, at least about 10-300%, at least about 10-200%, at least about 10-100%, at least about 10- 90%, at least about 10-80%, at least about 10-70%, at least about 10-60%, at least about 10-50%, at least about 10-40%, at least about 10-30%, at least about 10-20%, at least about 20-400%, 20- 300%, at least about 20-200%, at least about 20-100%, at least about 20-90%, at least about 20- 80%, at least about 20-70%, at least about 20-60%, at least about 20-50%, at least about 20-40%, at least about 20-30%, at least about 30-400%, 30-300%, at least about 30-200%, at least about 30-100%, at least about 30-90%, at least about 30-80%, at least about 30-70%, at least about 30- 60%, at least about 30-50%, at least about 30-40%, at least about 40-400%, 40-300%, at least about 40-200%, at least about 40-100%, at least about 40-90%, at least about 40-80%, at least about 40-70%, at least about 40-60%, at least about 40-50%, at least about 50-400%, 50-300%, at least about 50-200%, at least about 50-100%, at least about 50-90%, at least about 50-80%, at least about 50-70%, at least about 50-60%, at least about 60-400%, 60-300%, at least about 60- 200%, at least about 60-100%, at least about 60-90%, at least about 60-80%, at least about 60- 70%, at least about 70-400%, 70-300%, at least about 70-200%, at least about 70-100%, at least about 70-90%, at least about 70-80%, at least about 80-400%, 80-300%, at least about 80-200%, at least about 80-100%, at least about 80-90%, at least about 90-400%, 90-300%, at least about 90-200%, at least about 90-100%, at least about 100-400%, 100-300%, at least about 100-200%, at least about 200-400%, 200-300%, or at least about 300-400% in comparison to duration of the subject’s threshold level of protection in the absence of administration of the IL-2Ra.p-biased agonist.

[0091] All articles, books, patents, patent publications and other publications referenced herein are incorporated by reference in their entireties. In the event of an inconsistency between the teachings of this specification and the art incorporated by reference, the meaning of the teachings and definitions in this specification shall prevail (particularly with respect to terms used in the claims appended herein). For example, where the present application and a publication incorporated by reference defines the same term differently, the definition of the term shall be preserved within the teachings of the document from which the definition is located.

EXAMPLES

[0092] It is to be understood that the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention(s) provided herein.

Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. Materials and Methods

[0093] Multi(2,7-(bis-methoxyPEG-carboxyamide)(9H-fluorene-9-yl)methyl N- carbamate)interleukin-2 used in the following examples: Recombinant human IL-2 having an amino acid sequence identical to that of aldesleukin (des-alanyl-1, serine- 125 human interleukin- 2) was cloned and expressed and used to prepare the exemplary long acting IL-2Ra.p-biased agonist referred to herein generally as RSLAIL-2. RSLAIL-2 refers to a composition obtainable upon following the procedures of Example 1 in PCT Int. Pat. Appl. Pub. No. WO 2015/125159, and generically refers to a composition comprising multi -PEGylated forms of IL-2, wherein attachment of the PEG reagent used to form the conjugates is releasable following administration to a subject.

[0094] More particularly, the composition comprising multi -PEGylated forms of IL-2 used in the following example is (2,7-(bis-methoxyPEGio_kD-carboxyamide)(9H-fluorene-9- yljmethyl N-carbamate)_4-6interleukin-2, which may also be referred to as (2,7-(bis- methoxyPEGio_kD-carboxyamide)(9H-fluorene-9-yl)methyl N-carbamate)_6avginterleukin-2 (CAS No. 1939126-74-5), also referred to as bempegaldesleukin. Briefly, (2,7-(bis-methoxyPEGio_ko- carboxyamide)(9H-fluorene-9-yl)methyl N-carbamate)_6avginterleukin-2 was prepared by reacting a reactive releasable polyethylene glycol reagent, mPEG2-C2-fmoc-20K-NHS,

, with purified recombinant human IL-2 as described PCT Int. Pat. Appl. Pub. No. WO 2015/125159 (Example 1) to provide a composition comprising compounds encompassed by the following formula:

wherein IL-2 is des-alanyl-1, serine-125 human interleukin-2 (aldesleukin), “n” in each of the polyethylene glycol chains is about 227 (i.e., where each polyethylene glycol chain extending from the central fluorenyl core has a weight average molecular weight of about 10,000 daltons, such that the weight average molecular weight of the overall branched PEG moiety is about 20,000 daltons), and conjugates comprised in the composition possesses on average about six branched FMOC-polyethylene glycol moieties attached to IL-2.

[0095] Tetanus toxoid adsorbed onto aluminum phosphate was obtained from Serum

Institute of India, Ltd.

[0096] Sterile normal saline was used for dilutions of tetanus vaccine.

[0097] KLH (keyhole limpet hemocyanin) was supplied by Thermo Fisher Scientific and stored at 4° C prior to use. KLH was diluted in 0.9% Sodium Chloride for Injection (USP) to a concentration of 1 mg/mL and allowed to warm to ambient temperature prior to administration.

EXAMPLE 1

Investigation of Multi(2,7-(bis-methoxyPEG-carboxyamide)(9H-fluorene-9-yl)methyl N- carbamate)interleukin-2 for Enhancing Antibody Titers Following Exemplary Vaccine

Administration in Rats

[0098] Male Wistar rats aged 7-8 weeks were randomized based on body weights into six groups with N=3 for each group as shown in the Table 1 below. Tetanus toxoid adsorbed onto aluminum phosphate was administered subcutaneously to rats (at doses of 40 IU, 4.0 IU or 0.4 IU diluted in saline as required) on Day 0, and used as a model vaccine. (2,7-(bis- methoxyPEGio_kD-carboxyamide)(9H-fluorene-9-yl)methyl N-carbamate)_4-6interleukin-2 (“RSLAIL-2”) was administered on Days 0, 9 & 18 by intravenous injection for study groups D, E & F, while control groups (A, B & C) received only saline. Blood was collected from the retro orbital plexus of each of the rats on Day 0 prior to tetanus toxoid vaccine or (2,7-(bis- methoxyPEGio_kD-carboxyamide)(9H-fluorene-9-yl)methyl N-carbamate)_4-6interleukin-2 administration for pre-immunization serum preparation. A summary of the study groups is provided in Table 1 below.

Table 1. Study Group Protocols

[0099] To assess the anti-tetanus toxoid antibody titers of IgM and IgG, blood was collected from the rats on day 7 (Weekl), day 14 (Week 2) & day 28 (Week 4). Anti-tetanus toxoid IgM titers were analyzed using Rat Anti-Tetanus Toxoid IgM ELISA kit (ThermoFisher Scientific) while Anti Tetanus Toxoid IgG titers were analyzed using Rat Anti-Tetanus Toxoid IgG ELISA kit (ThermoFisher Scientific) according to the manufacturer recommendations.

[00100] Absolute quantities of antibody titers were derived from the standard curves generated according to the manufacturer recommendations. The vehicle and RSLAIL-2 treated groups were compared by plotting antibody titers across the time points collected using Graph pad Prizm ver. 6.0, while statistical significance was determined using two-way ANOVA Turkeys’ multiple comparison tests.

[00101] Results: Administration of the tetanus toxoid vaccine at 40 IU showed significant up-regulation of IgM titers both with and without RSLAIL-2 administration on Day 7; the titers reached basal levels by week 2 (Day 14) and week 4 (Day 28) in the absence of administration of RSLAIL-2. However, administration of the tetanus toxoid vaccine at a dose of either 4 IU or 0.4 IU demonstrated no significant upregulation of IgM titers in comparison to basal levels at all evaluated time points in the absence of RSLAIL-2.

[00102] In contrast, rats administered RSLAIL-2 showed a significant increase in IgM levels over basal levels at all three time points at tetanus toxoid vaccine doses of both 4 IU and even 0.4 IU as illustrated in FIG. 1.

[00103] Sera from rats administered the tetanus toxoid were analyzed for anti-tetanus toxoid IgG titers on weeks 2 & 4, and the values then compared between the treatment groups and vehicle as shown in the FIG 2. Based on these preliminary results, it appears that in the vaccine animal model used in this study, administration of RSLAIL-2 does not result in a significant increase or enhancement of IgG titers over basal levels.

[00104] As illustrated in the model system above, the illustrative long acting IL-2Ra.p- biased agonist, multi(2,7-(bis-methoxyPEG-carboxyamide)(9H-fluorene-9-yl)methyl N- carbamate)interleukin-2, was effective to enhance the IgM response in mice when combined with traditional vaccination when compared to the IgM response observed for traditional vaccination alone. The enhancement in the IgM immune response at even the lowest dose administered (0.4 IU) suggests that IL-2Ra.p-biased agonist, multi(2,7-(bis-methoxyPEG- carboxyamide)(9H-fluorene-9-yl)methyl N-carbamate)interleukin-2 may be particularly effective when used in combination with vaccines with require greater antigen exposures to elicit a desired immune response. In addition, multi(2,7-(bis-methoxyPEG-carboxyamide)(9H-fluorene-9- yl)methyl N-carbamate)interleukin-2 may be particularly advantageous when used during post exposure prophylaxis for treating conditions in which attainment of high IgM titers is important for therapeutic success.

[00105] The results from the above model suggest that multi(2,7-(bis-methoxyPEG- carboxyamide)(9H-fluorene-9-yl)methyl N-carbamate)interleukin-2 may be used in combination with any of a number of vaccines for improving a humoral immune response thereto.

EXAMPLE 2

Investigation of Multi(2,7-(bis-methoxyPEG-carboxyamide)(9H-fluorene-9-yl)methyl N- carbamate)interleukin-2 for Ability to Enhance T-cell Dependent Antibody Response (TDAR) Following Administration to Cynomolgus Monkeys

[00106] The objective of this study was to further explore the immunopharmacology characteristics of IL-2RP-biased agonists such as RSLAIL-2 by evaluating immune cell populations after treatment to further explore the ability of such molecules/compositions to enhance T-cell dependent antibody response (TDAR) in non-human primates.

[00107] Five male monkeys (plus 1 alternate) and five female monkeys (plus 1 alternate), each 2 to 4 years of age, were used in the study. The KLH formulation was allowed to warm to ambient temperature prior to administration. On Days 1 and 22, animals received 1 mg of KLH by subcutaneous injection into the right caudal thigh region. RSLAIL-2 was administered once by intravenous injection to cynomolgus monkeys (Charles River Laboratories) per the protocol summarized below in Table 2. RSLAIL-2 was formulated in Formulation Buffer (concentration of 0.9 mg/mL) and was administered once on Day 1 or Day 4 by intravenous injection as described in Table 2. Groups 1 to 3 were dosed on Day 1; Groups 4 and 4 were dosed on Day 4; administration was via intravenous (bolus) injection to the tail vein. Dose levels in Table 2 are based on amount protein.

[00108] The study plan and any amendment(s) to or procedures involving the care and use of animals were reviewed and approved by CR-SEN Institutional Animal Care and Use Committee (IACUC). During the study, the care and use of animals was conducted with guidance from the USA National Research Council and the Canadian Council on Animal Care (CCAC).

Table 2: Study Design

^a Based on the most recent body weight measurement. ^b Animals were released from the study on Day 36. ^c On Day 1 KLH immunization (1 mg/dose) was performed pre-dose. ^d RSLAIL-2 Formulation Buffer (10 mM citrate, 7% trehalose buffer, pH 4.0). [00109] The following parameters and endpoints were evaluated: mortality, clinical observations, body weights, appetence, clinical pathology parameters (hematology), immunophenotyping, and T-cell dependent antibody response (TDAR) analyses.

[00110] There were no RSLAIL-2-related unscheduled deaths or any effects on body weights, body weight gains, or appetence.

[00111] Blood samples were processed to serum. The resultant serum was aliquoted (3 aliquots of target 50 pL) and stored on dry ice and transferred in a freezer set to maintain -80°C until analysis.

[00112] Anti-KLH IgM and anti-KLH IgG antibodies were determined for all samples using validated titer-based ELISA methods. Results are reported as titers (the highest dilution factor that produced an A450nm value > plate specific cut-point [PSCP]). If samples were < PSCP at the minimum required dilution (MRD), then < MRD is reported for individual animals. Samples testing < MRD was assigned a value equal to MRD/2 for the purpose of group median calculation. If the calculated median was < MRD, the group median value is reported as < MRD.

[00113] White blood cell count (absolute and differential) were determined from whole blood using an AD VIA hematology analyzer. Cellular antigens and cell populations were quantified using specific antibodies against marker antigens and reported as relative percentages and/or absolute cell counts (cells/pL blood). The lymphocyte counts obtained from the hematology assessment were used for the purpose of absolute count calculations and reported as total lymphocyte counts.

[00114] Following KLH (keyhole limpet hemocyanin) immunization on Day 1 and administration of RSLAIL-2 on Day 1 at > 0.03 mg/kg and on Day 4 at 0.06 mg/kg, soft/liquid feces were noted in all animals, generally appearing around Day 6 or 7 after administration of RSLAIL-2. In addition, between Day 4 and Day 8, decreased activity, partly closed eyes, hunched posture, tremors, dehydration and/or pale gums were noted on 1 animal administered RSLAIL-2 at 0.06 mg/kg on Day 1 (Animal No. 3001). As no improvement of clinical signs were noted, additional support was given to this animal starting on Day 11 (lactated Ringer’s solution, fruits and vegetables, and FiberBites) and anti -diarrhea medication (Pepto Bismol, bismuth subsalicylate) until Day 31. Decreased activity was also noted on Day 6 and 7 for 1 animal (Animal No. 5501), and hunched posture on Day 7 and 8 for 2 animals (Nos. 5001 and 5501) administered 0.06 mg/kg on Day 4. Animal No. 5001 was given FiberBites from Day 7 to Day 18 to alleviate abnormal feces. All these observations generally resolved within a week following the administration, with the exception of the soft/liquid feces noted until the end of the study.

[00115] As described below, an initial decrease in lymphocyte count was observed 1 to 2 days post-dose (down to 0.8 times when compared to pre-study values), followed by an increase on Days 8 and 12 (up to 1.2 times when compared to pre-study values) through Day 15 but at a lesser magnitude (up to 0.4 times when compared to pre-study values). These changes were associated with transient increases in monocytes and basophils (up to 0.9 times and 3.2 times, respectively, when compared to pre-study values), following 1 or 2 weeks after administration.

In addition, a persistent increase in eosinophils (up to 13 times when compared to pre-study values) was noted.

[00116] At both doses of RSLAIL-2, transient RSLAIL-2 -related decreases in total lymphocyte counts (TLC) and absolute counts of total T, helper T, cytotoxic T, follicular helper T and PD1+ follicular helper T lymphocytes occurred following dosing compared to pre-dose values. These decreases were noted on Day 3 (down to 0.10 times) for the animals dosed on Day 1 (Groups 2 and 3) or on Day 5 (down to 0.23 times) for the animals dosed on Day 4 (Groups 4 and 5). These decreases were followed by RSLAIL-2-related increases in the TLC and absolute counts of all lymphocyte subsets on Day 8 (up to 4.25 times, for Day 1 dosing) or Day 12 (up to 6.77 times, for Day 4 dosing), which generally reversed by Day 15 or 21, respectively. The absolute counts of cytotoxic T cells tended to be slightly above the pre-dose values for the rest of the study in all groups administered RSLAIL-2. There were generally no clear RSLAIL-2- related changes of the lymphocyte populations assessed, except for decreases of helper T cells (down to 0.52x), and increases in cytotoxic T cells (up to 1.63 times) on Days 12 to 36 in animals dosed on Day 4 with 0.06 mg/kg RSLAIL-2.

[00117] In the control monkeys, there were minimal anti-KLH antibodies detected in one (IgM) or both (IgG) control monkeys following the first KLH immunization on Day 1, likely due to the use of suboptimal KLH dose level to detect any RSLAIL-2-mediated immuno- enhancement. However, anti-KLH IgG antibodies were detected in both control monkeys by Day 21. At both doses of RSL AIL-2, immunoenhancement of the TDAR to the first KLH immunization occurred in most monkeys, compared to controls, peaking at 1 week post-dose (regardless of day of dose administration) in most monkeys for IgM (up 64 times to 128 times) and 1-3 weeks post-dose in some monkeys for IgG (8 to 16 times). However, there was no clear immuno-enhancement of the IgG response to the KLH immunization. Following the second KLH immunization on Day 22, the anti-KLH IgM levels decreased gradually (monkeys dosed on Day 1 (both doses) or persisted (monkeys dosed on Day 4 (both doses) until Day 36). Both control animals had an increased IgG response after the second KLH injection, whereas there were no clear increases in the anti-KLH IgG responses in any RSLAIL-2 -treated monkeys compared to controls.

Table 3A. Individual T-cell Dependent Antibody Response (Anti-KLH IgM) Values (Males)

Group 1 - Reference Item Group 2 - RSLAIL-2 0.03 mg/kg/dose Group 3 - RSLAIL-2 0.06 mg/kg/dose Group 4 - RSLAIL-2 0.03 mg/kg/dose Group 5 - RSLAIL-2 0.06 mg/kg/dose

TITER

Group Animal Day -2 Day 5 Day 8 Day 12 Day 15 Day 21 Day 26 Day 29 Day 33 Day 36 No.

1 1101 125 125 125 125 125 125 125 125 125 125

(<250) (<250) (<250) (<250) (<250) (<250) (<250) (<250) (<250) (<250)

2 2101 125 250 8000 4000 4000 2000 1000 1000 500 500

(<250)

3 3001 125 250 4000 2000 1000 1000 500 500 500 250

(<250)

4 4001 125 125 125 500 500 1000 1000 1000 500 500

(<250) (<250) (<250)

5 5001 125 125 1000 16000 8000 4000 4000 4000 4000 2000

(<250) (<250) Table 3B. Individual T-cell Dependent Antibody Response (Anti-KLH IgM) Values (Females)

Group 1 - Reference Item Group 2 - RSL AIL-2 0.03 mg/kg/dose Group 3 - RSL AIL-2 0.06 mg/kg/dose Group 4 - RSL AIL-2 0.03 mg/kg/dose Group 5 - RSL AIL-2 0.06 mg/kg/dose

TITER

1 1601 125 125 125 250 125 250 250 1000 2000 1000

(<250) (<250) (<250) (<250)

2 2601 125 250 4000 2000 2000 1000 500 1000 500 500

(<250)

3 3501 125 250 4000 2000 2000 500 250 500 250 250

(<250)

4 4501 125 125 500 4000 2000 2000 2000 4000 2000 2000

(<250) (<250)

5 5501 125 125 500 4000 2000 2000 2000 8000 4000 4000

(<250) (<250)

Table 4A. Individual T-cell Dependent Antibody Response (Anti-KLH IgG) Values (Males)

TITER

1 1101 250 250 250 500 500 500 1000 4000 4000 8000

(<500) (<500) (<500)

2 2101 250 250 4000 8000 8000 8000 4000 4000 4000 4000

(<500) (<500) 3 3001 250 250 500 500 500 500 500 4000 4000 4000

(<500) (<500)

4 4001 250 250 250 500 500 500 500 1000 500 1000

(<500) (<500) (<500)

5 5001 250 250 250 4000 4000 4000 1000 4000 4000 4000

(<500) (<500) (<500)

Table 4B. Individual T-cell Dependent Antibody Response (Anti-KLH IgG) Values (Females)

TITER

1 1601 250 250 250 250 250 1000 2000 8000 16000 16000

(<500) (<500) (<500) (<500) (<500)

2 2601 250 250 500 500 500 250 250 500 500 2000

(<500) (<500) (<500) (<500)

3 3501 250 250 500 500 250 250 250 2000 4000 4000

(<500) (<500) (<500) (<500) (<500)

4 4501 250 250 250 4000 2000 4000 2000 8000 8000 16000

(<500) (<500) (<500)

5 5501 250 250 250 4000 2000 4000 2000 32000 16000 32000

(<500) (<500) (<500)

[00118] Single intravenous administration of RSLAIL-2 to monkeys was tolerated at doses up to 0.06 mg/kg, with transient clinical signs at 0.06 mg/kg. At all dose levels, RSLAIL- 2 induced transient decreases in absolute counts of total lymphocytes and T cell subsets at 1- or 2-days post dose, followed by increases, which peaked at approximately one-week post-dose in all animals. Within one week of RSLAIL-2 administration, an enhanced anti-KLH IgM response was observed in all dosed animals, and was maintained until the end of the study in several monkeys following the second KLH immunization. There were no clear increases in the primary or secondary anti-KLH IgG responses in any of the RSLAIL-2-treated monkeys.

[00119] The results from this study further support the results in Example 1 and demonstrate the universality of the ability of RSLAIL-2 to notably enhance a humoral IgM response, e.g., when combined with vaccines in a general protein immunization process. The results are consistent across both the tetanus toxoid rat model described in Example 1 and the non-human primate KLH protein antigen-based model described here in Example 2. The data provided herein demonstrates that the noted effect is conserved not only across different species but is also conserved across various vaccine types. Compositions such as RSLAIL-2 have been discovered to be effective in notably enhancing IgM responses to vaccination, e.g., to elicit an initial heightened IgM response, e.g., to bolster a patient’s acute response to vaccination.

Claims

S CLAIMED:

1. A method comprising administering to a subject a vaccine and a long acting IL-2RP- biased agonist to generate or enhance humoral immunity in the subject.

2. The method of claim 1, wherein the administering of the long acting IL-2RP-biased agonist is effective to enhance the humoral immune response of the subject when compared to the humoral immune response of the subject upon administration of the vaccine in the absence of the long acting IL-2RP-biased agonist.

3. A method for modulating (i.e., enhancing) a humoral immune response to vaccination by administering to a subject that has been vaccinated or is to be vaccinated, a humoral immunoenhancing amount a long acting IL-2RP-biased agonist.

4. A method for providing protection against infection by administering to a subject a vaccine directed to a pathogen for which protection is antibody-mediated, the improvement comprising further administering to the subject a humoral immunoenhancing amount of a long acting IL-2RP-biased agonist to generate or enhance humoral immunity in the subject.

5. A combination for administration to a subject comprising a vaccine and a long acting IL- 2R.p-biased agonist for generating or enhancing humoral immunity in the subject.

6. A kit comprising a humoral immunoenhancing amount of a long acting IL-2RP-biased agonist and a vaccine, accompanied by instructions for use.

7. The method, combination or kit of any one of claims 1-6, wherein the vaccine confers antibody-mediated immunity against infection and/or disease to the subject.

8. The method, combination or kit of claim 7, wherein the vaccine is selected from a live attenuated pathogen, a whole inactivated organism, an inactivated bacterial toxin, a recombinant vaccine, a subunit vaccine, a conjugate vaccine, and a viral vaccine.

9. The method, combination or kit of claim 7, wherein the vaccine is selected from a vaccine against one or more of diphtheria, hepatitis A, hepatitis B, herpes zoster, human papillomavirus, haemophilus influenza type a and/or b, chickenpox, measles, meningococcus, adenovirus, anthrax, cholera, diphtheria, Japanese encephalitis, mumps, pertussis, pneumococcus, polio, rabies, rotavirus, rubella, shingles, whooping cough, smallpox, tetanus, tuberculosis, typhoid fever, varicella, and yellow fever.

10. The method, combination, or kit of any one of claims 1-6, wherein the vaccine is a non cancer vaccine.

11. The method, combination, or kit of any one of claims 1-6, wherein the vaccine is not directed to a coronavirus, e.g., the vaccine is not a SARS-CoV-2 vaccine.

12. The method, combination, or kit of claim 11, wherein the vaccine is not a vaccine for prevention of COVID-19.

13. The method, combination, or kit of any one of claims 1-12, wherein the long acting IL- 2RP-biased agonist comprises aldesleukin (des-alanyl-1, serine-125 human interleukin-2) releasably covalently attached to one or more polyethylene glycol) polymers.

14. The method, combination, or kit of claim 13, wherein the method, combination, or kit comprises aldesleukin (des-alanyl-1, serine-125 human interleukin-2) releasably covalently attached to an average of about six poly(ethylene glycol) polymers.

15. The method, combination, or kit of claim 13 or claim 14, wherein each of the poly(ethylene glycol) polymers has a weight average molecular weight of from about 10,000 daltons to about 55,000 daltons.

16. The method, combination, or kit of claim 13 or claim 14, wherein the long acting IL- 2RP-biased agonist is (2,7-(bis-methoxyPEGio_kD-carboxyamide)(9H-fluorene-9-yl)methyl N- carbamate)4-6interleukin-2.

17. The method, combination, or kit of any one of the foregoing claims, wherein the vaccine and the long acting IL-2R.p-biased agonist are administered concurrently or sequentially and in any order.

18. The method, combination, or kit of claim 17, wherein administration comprises vaccination accompanied by administration of a single dose of the long acting IL-2RP-biased agonist.

19. The method, combination, or kit of claim 17 or claim 18, wherein the vaccine and the long acting IL-2R.p-biased agonist are both administered on day 1 of treatment.

20. The method, combination, or kit of claim 17 or claim 18, wherein the vaccine is administered on day 1 of treatment and the long acting IL-2R.p-biased agonist is administered on any one of days 1 to 7 of treatment (e.g., the long acting IL-2R.p-biased agonist is administered on any one of days 1, 2, 3, 4, 5, 6, or 7 of treatment).

21. The method, combination, or kit of claim 17 or claim 18, wherein the long acting IL- 2RP-biased agonist is administered on day 1 of treatment and the vaccine is administered on any one of days 1 to 7 of treatment (e.g., the vaccine is administered on any one of days 1, 2, 3, 4, 5, 6, or 7 of treatment).

22. The method, combination, or kit of claim 17, wherein administration comprises vaccination accompanied by administration of multiple doses (e.g., two, three, four or more) of the long acting IL-2R.p-biased agonist.

23. The method, combination, or kit of any one of the foregoing claims, wherein the vaccine and the long acting IL-2R.p-biased agonist are administered via the same or different routes of administration.

24. The method, combination, or kit of any one of the foregoing claims, wherein the long acting IL-2R.p-biased agonist is administered at a dose in a range of from about 0.0005 to 0.3 mg/kg, or is administered at a dosage amount of about 0.001 to about 0.01 mg/kg.

25. The method, combination, or kit of claim 24, wherein the long acting IL-2RP-biased agonist is administered at a dose ranging from about 0.003 to about 0.009 mg/kg.

26. The method, combination, or kit of claim 24, wherein the long acting IL-2RP-biased agonist is administered at a dose of 0.003 mg/kg or is administered at a dose of 0.006 mg/kg.

27. The method, combination, or kit of any one of the foregoing claims, wherein the subject is a human subject.

28. The method, combination, or kit of claim 27, wherein the subject is elderly, is immunocompromised, and/or is hyporesponsive to traditional vaccination.

29. The method, combination, or kit of any one of the foregoing claims, wherein the method, combination or kit is effective to provide immunoenhancement of an IgM response in the subject.

30. The method, combination, or kit of any one of the foregoing claims, wherein the method, combination or kit is effective to enhance the antibody response to the vaccine by prolonging the duration (persistence) of the antibody response at or above a threshold level effective to prevent or fight against infection.

31. The method, combination, or kit of claim 30, wherein the antibody response is an IgM antibody response.

32. The method, combination, or kit of claim 29, wherein the method, combination, or kit is effective to increase the subject’s IgM antibody titers by at least about 2-fold to about 100- fold or more when compared to the subject’s IgM antibody titers in the absence of administering the IL-2Ra.p-biased agonist.

33. The method, combination, or kit of claim 32, wherein the method, combination, or kit is effective to increase the subject’s IgM antibody titers at least about 2-fold, at least about 5- fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 50- fold, at least about 75-fold, at least about 100-fold or at least about 110-fold when compared to the subject’s IgM antibody titers in the absence of administering the IL-2Ra.p-biased agonist.