WO2019070991A1 - Enhancing the efficacy of cancer vaccines - Google Patents

Abstract

Human CD27 agonist antibody (?hCD27) enhances the antitumor CD8+ T cell response preferentially in the setting of vaccines containing linked class I and II epitopes. CD27 agonist antibodies have the potential to broaden the clinical benefit of peptide vaccines targeting class I-restricted tumor antigens for cancer immunotherapy.

Description

ENHANCING THE EFFICACY OF CANCER VACCINES

[01] This invention was made with Government support under Federal Grant Nos. F31- CA210535-01, R01-CA 177476-04 and P50-CA190991-02 awarded by the NIH/NCI and R01-NS099463-01, R01-NS085412-04, R01-NS086943-03, and U01-NS090284- 02 awarded by the NIH/NINDS. The Federal Government has certain rights to this invention.

[02] This application claims the priority benefit of U.S. Application Serial No. 62/568,414 filed October 5, 2017. The disclosure of the application is expressly incorporated herein.

TECHNICAL FIELD OF THE INVENTION

[03] This invention is related to the area of anti-tumor therapy. In particular, it relates to anti-tumor immunotherapy.

BACKGROUND OF THE INVENTION

[04] Tumor immunotherapy has emerged as a promising treatment modality for advanced malignancies. Specifically, peptide vaccines derived from MHC class I-restricted tumor antigens offer the promise of inducing robust tumor-specific CD8⁺ T cell responses (1,2) to promote effective antitumor immunity (3-6). Unfortunately, the efficacy of class I-restricted peptide vaccines has proven to be limited (7,8), with overall clinical response rates as low as 3% (9). Class II CD4⁺ T cell helper epitopes are also capable of inducing potent antitumor immune responses (10-13), but peptide vaccines consisting of co-administered class Ι/II epitopes have also not yet experienced widespread clinical success (7). Novel adjuvant strategies are clearly needed to enhance the clinical utility of peptide vaccines in cancer immunotherapy.

[05] Immunomodulatory antibodies targeting T cell checkpoint molecules have emerged as promising therapies that may be capable of promoting robust tumor-specific immunity in the setting of tumor vaccines that are otherwise ineffective (14-16). CD27, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a costimulatory molecule expressed on naive and activated CD4⁺ and CD8⁺ T cells (17) and is known to be important in T cell activation (18), maturation (19), cytokine secretion (20), and survival (21), making it a promising target for T cell-based immunomodulation. Recently, a novel, fully human anti-human CD27 monoclonal antibody (αhCD27) was developed which binds with high affinity to induce potent human T cell responses in the context of T cell receptor stimulation (22). Because CD27 stimulation on both CD4⁺ T cells and CD8⁺ T cells can lead to their enhanced effector function and concomitant vaccine-induced CD4⁺ T cell help strengthens CD8⁺ T cell vaccine responses, we hypothesized that αhCD27 could be leveraged as an adjuvant for peptide vaccines and that it would provide a therapeutic benefit preferentially in the setting of peptide vaccines comprised of class I and II epitopes.

[06] In this study, we evaluated the therapeutic effect of αhCD27 as a vaccine adjuvant in the setting of a highly aggressive intracranial tumor model. We demonstrate that αhCD27 enhances the immune response to class I-restricted tumor antigens, and that its adjuvant effect is potentiated in the setting of linked class I- and class II-restricted peptides. Additionally, we show that a universal CD4⁺ epitope is sufficient to enhance the tumor-specific CD8⁺ T cell response and increase the efficacy of tumor-derived class I-restricted peptide vaccines in the setting of adjuvant αhCD27, eliminating the need for class Il-restricted tumor antigens. Taken together, our data suggest that αhCD27 coordinates CD8⁺ and CD4⁺ T cell responses to enhance the antitumor immune response. These findings highlight the potential for CD27 agonist antibodies to improve the clinical benefit of peptide vaccines for cancer immunotherapy.

[07] There is a continuing need in the art to improve treatment results for patients with cancers, and particularly for patients with poorly immunogenic or inaccessible cancers.

SUMMARY OF THE INVENTION

According to one aspect of the invention a method is provided for treating a tumor in a human. An agonist anti-CD27 antibody is administered to the human. A peptide vaccine comprising an MHC-I epitope linked to an MHC-II epitope is administered to the human. The MHC-I epitope is expressed as part of a first protein by the tumor.

[09] According to another aspect of the invention, a kit for treating a tumor in a human is provided. The kit comprises an agonist anti-CD27 antibody; and a peptide vaccine comprising an MHC-I epitope linked to an MHC-II epitope. The MHC-I epitope is expressed as part of a first protein by the tumor.

[10] Another aspect of the invention is a composition for treating a tumor in a human. The composition comprises an agonist anti-CD27 antibody and a peptide vaccine comprising an MHC-I epitope linked to an MHC-II epitope. The MHC-I epitope is expressed as part of a first protein by the tumor.

[11] According to still another aspect of the invention, a kit is provided for treating a tumor in a human. The kit comprises an agonist anti-CD27 antibody, a first peptide comprising an MHC-II epitope, and a linking moiety for linking the peptide to a second peptide comprising an MHC-I epitope.

[12] Another kit for treating a tumor in a human comprises an agonist anti-CD27 antibody, a vector comprising a first region encoding a first peptide comprising an MHC-II epitope, and a cloning site adjacent to the first region for insertion of a second region encoding a second peptide comprising an MHC-I epitope, wherein the first region is under the transcri ptional control of a promoter and upon insertion of the second region in the cloning site a fusion peptide is formed and expressed.

[13] These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with methods and products for treating tumors in a human or other mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

[01] Figs. 1A-1D. αhCD27 enhances the ovalbumin protein response and promotes antitumor efficacy. Groups of hCD27 mice (n = 5 per group) received intraperitoneal ovalbumin protein alongside αhCD27 or hlgGl isotype control (3 days prior to and on the day of whole Ova protein vaccination), and Ova(I)-specific CD8+ T cell responses were evaluated by H2-Kb(SIINFEKL)-PE tetramer staining of peripheral blood cells one week after vaccination (Fig. 1A). Vaccine-induced CD8⁺ T cell responses were also evaluated by ex vivo re-stimulation of splenocytes with Ova(I) peptide in an IFNy ELISPOT assay (Fig. IB). hCD27 mice bearing intracranial B16.0VA tumors received αhCD27 (or hlgGl) isotype control at days 3 and 6 after tumor implantation, with our without whole ovalbumin protein on day 6 after tumor implantation; tumors were harvested on day 14 after implantation and analyzed for the frequency of tumor- infiltrating Ova(I)-specific CD8⁺ T cells by H2-Kb(SIINFEKL)-PE tetramer staining (Fig. 1C). Additionally, the efficacy of whole ovalbumin protein +/- αhCD27 (n = 7 per group) was evaluated in hCD27 mice bearing 3-day established intracranial B 16. OVA (Fig. ID). Statistical analyses were performed using Student's unpaired t- test (Fig. 1 and Fig. IB), one-way ANOVA with Tukey post-hoc comparisons (Fig. IC), or the Gehan-Breslow-Wilcoxon test (Fig. ID). Statistical significance was determined at a *P value < 0.05.

Figs. 2A-2E. The adjuvant effect of αhCD27 on the class I peptide response is enhanced by a linked class II epitope. Groups of hCD27 mice (n = 5 per group) received Ova(I) peptide, co-administration of Ova(I) and Ova(II^A) peptides, the linked Ova(I-II^A) peptide, or whole ovalbumin protein, alongside αhCD27 or hlgGl isotype control administered 3 days prior to and on the day of vaccination; CD8⁺ T cell responses were evaluated by re-stimulation with the Ova(I) peptide in a IFNy ELISPOT (Fig. 2A), representative ELISPOT results are shown in (Fig. 2B). For survival experiments, groups of hCD27 mice (n = 7 per group) bearing established intracranial B 16. OVA tumors were treated with Ova(I) peptide (Fig. 2C), coadministered Ova(I) + Ova(II^A) peptide (Fig. 2D), or the linked Ova(I-II^A) peptide (E) 6 days after tumor implantation, alongside αhCD27 or hlgGl isotype control administered 3 days prior to and on the day of vaccination. Statistical analyses were performed using two-way ANOVA with Tukey post-hoc comparisons (Fig. 2 A) or the Gehan-Breslow-Wilcoxon test (Figs. 2C, 2D, 2E). Statistical significance was determined at a *P value < 0.05. Figs. 3A-3E. CD8⁺ and CD4⁺ T cells are required for the adjuvant effect and survival benefit of αhCD27. hCD27 mice (n = 5 per group) received whole ovalbumin protein +/- αhCD27, as previously described; CD8⁺ cells were depleted by aCD8 antibody (2.43) administered intraperitoneally for 3 consecutive days immediately prior to the first αhCD27 administration, and CD4⁺ cells were depleted by aCD4 antibody (GK1.5) administered intraperitoneally for 3 consecutive days beginning 5 days after vaccination (effector), or 3 consecutive days immediately prior to the first αhCD27 administration (priming), and CD8⁺ T cell responses were determined 8 days after vaccination by ex vivo re-stimulation of splenocytes with Ova(I) peptide in an IFNy ELISPOT (Fig. 3A). The effect of T cell depletion on antitumor efficacy was evaluated in a tumor challenge experiment in which hCD27 mice (n = 7 per group) received whole ovalbumin protein combined with αhCD27 prior to intracranial B16.0VA tumor implantation (on day -7 and days -10,-7, respectively); CD8⁺ T cells were depleted by aCD8 administered for 3 consecutive days immediately after tumor implantation (red), and CD4⁺ T cells were depleted by aCD4 administered for 3 consecutive days immediately prior to αhCD27 treatment (priming phase, blue) or immediately after tumor implantation (effector phase, green) (Fig. 3B). The immune responses to two class II ovalbumin epitopes were evaluated in hCD27 mice (n = 5 per group) vaccinated with intraperitoneal whole ovalbumin protein alongside αhCD27 or hlgGl isotype control (as described previously) one week after vaccination by ex vivo re-stimulation of splenocytes with Ova(II^A) or Ova(II^B) peptide in an IFNy ELISPOT assay (Fig. 3C). To evaluate the impact of CD27 stimulation on vaccine-induced ovalbumin-specific T cells, an adoptive transfer was employed, in which CD8⁺ or CD4⁺ T cells were isolated from OTI or OTIxhCD27 mice and ΟΤII or OTIIxhCD27, respectively, and intravenously infused into wildtype C57BL/6 hosts (n = 5 per group) one day prior to αhCD27 and whole ovalbumin protein administration (as described previously) (D); CD8⁺ T cell responses were measured by IFNy ELISPOT upon ex vivo re-stimulation with Ova(I) peptide (Fig. 3E). Statistical analyses were performed using a one-way ANOVA with Tukey post-hoc comparisons (Fig. 3A), the Gehan-Breslow-Wilcoxon test (Fig. 3B), Student's unpaired t-test (Fig. 3C), and two-way ANOVA with Tukey post-hoc comparisons (Fig. 3E). Statistical significance was determined at a *P value < 0.05. [04] Fig. 4A-4C. A universal CD4⁺ T cell helper epitope is sufficient to enhance CD8⁺ tumor-specific vaccine responses in the setting of adjuvant αhCD27. CD8⁺ T cell responses were evaluated in mice vaccinated with intradermal Ova(I) or Ova(I)-P30 peptides, with combined αhCD27 or hlgGl, one week after vaccination by ex vivo re- stimulation of splenocytes with Ova(I) peptide in an IFNy ELISPOT assay; CD4⁺ cells were depleted by aCD4 antibody (GK1.5) administered intraperitoneally for 3 consecutive days immediately prior to the first αhCD27 administration (Fig. 4A). Representative ELISPOT images from mice vaccinated with Ova(I) or Ova(I)-P30 with αhCD27 or hlgGl are shown in (Fig. 4B). For survival analysis, hCD27 mice bearing 3-day established intracranial B16.0VA tumors were treated with Ova(I)-P30 and combined αhCD27 or hlgGl isotype control (n = 7 per group), as described previously (Fig. 4C). Statistical analyses were performed using two-way ANOVA with Tukey post-hoc comparisons (Fig. 4 A) or the Gehan-Breslow-Wilcoxon test (Fig. 4C). Statistical significance was determined at a *P value < 0.05.

[05] Figs. 5A-5D. CD27 stimulation coordinates CD4⁺ T cell help and vaccine-induced CD8⁺ T cell responses. hCD27 mice (n = 5 per group) were immunized with intradermal Ova(I-II^A) or Ova(I)-P30 alongside αhCD27 or hlgGl isotype control, as described previously. Immune responses to class I and II peptide epitopes were evaluated one week after vaccination by ex vivo re-stimulation of splenocytes with the peptides shown in an IFNy ELISPOT. Correlation analyses were performed comparing ex vivo responses to re-stimulation with Ova(I) and Ova(II^A) in mice that received Ova(I-II^A) vaccination in the setting of hlgGl (Fig. 5 A) or αhCD27 (Fig. 5B) or Ova(I) and P30 in mice that received Ova(I)-P30 vaccination, in the setting of hlgGl (Fig. 5C) or αhCD27 (Fig. 5D). Statistical analyses were performed using Pearson correlation analysis, and statistical significance was determined at a *P value < 0.05.

[06] Figs. 6A-6E. αhCD27 enhances the T cell response to a linked class Ι/II peptide vaccine derived from Trp2. Mice were vaccinated with intradermal Trp2(I) or Trp2(I)-P30 peptides alongside αhCD27 or hlgGl isotype control (as described previously), and CD8⁺ T responses were evaluated one week after vaccination by ex vivo re-stimulation of splenocytes with Trp2(I) peptide in an IFNy ELISPOT (Fig. 6A); representative ELISPOT images are shown in (Fig. 6B). Correlation analyses were performed comparing ex vivo responses to re-stimulation with Trp2(I) and P30 peptides in the setting of hlgGl (Fig. 6C) or αhCD27 (Fig. 6D). For survival analysis, groups of hCD27 mice (n = 7 per group) bearing 3-day established intracranial B16.F10 tumors were treated with Trp2(I) or Trp2(I)-P30 with αhCD27 or hlgGl isotype control (results of two separate experiments for each vaccine are shown) (Fig. 6E). Statistical analyses were performed using two-way A NOVA with Tukey post-hoc comparisons (Fig. 6A), Pearson correlation analysis (Fig. 6C, Fig. 6D), or the Gehan- Breslow-Wilcoxon test (Fig. 6E). Statistical significance was determined at a *P value < 0.05.

DETAILED DESCRIPTION OF THE INVENTION

[07] The inventors have developed a method for stimulating an increased immune response that may be sufficient to affect survival rate positively. The minimum elements for observing this increased immune response are a vaccine that comprises both an MHC- I epitope and an MHC-II epitope and an agonist anti-CD27 antibody. The MHC-I epitope is represented in the tumor, whether in a tumor associated protein or in a tumor neo-antigen.

Definitions

[08] Articles "a" and "an" are used to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means at least one element and can include more than one element.

[09] "About" is used to provide flexibility to a numerical range endpoint by providing that a given value may be "slightly above" or "slightly below" the endpoint without affecting the desired result.

[10] The terms "including," "comprising," or "having," and variations thereof, are meant to encompass the elements listed thereafter and equivalents as well as additional elements. Embodiments recited as "including," "comprising" or "having" certain elements are also contemplated as "consisting essentially" of and "consisting of those certain elements.

[11] Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise- Indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[12] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[13] "Treatment," "therapy" and/or "therapy regimen" refer to the clinical intervention made in response to a disease, disorder, or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder, or condition.

[14] The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.

[15] The term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. The term "nonhuman animals" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. Preferably, the subject is a human patient is suffering from, or at risk of developing, cancer. [16] The term "biological sample" as used herein includes, but is not limited to, a sample containing tissues, cells, and/or biological fluids isolated from a subject. Examples of biological samples include, but are not limited to, tissues, cells, biopsies, blood, lymph, serum, plasma, urine, saliva, mucus and tears. In one embodiment, the biological sample is a biopsy (such as a tumor biopsy). A biological sample may be obtained directly from a subject (e.g., by blood or tissue sampling) or from a third party (e.g., received from an intermediary, such as a healthcare provider or lab technician).

[17] The term "disease" as used herein includes, but is not limited to, any abnormal condition and/or disorder of a structure or a function that affects a part of an organism. It may be caused by an external factor, such as an infectious disease, or by internal dysfunctions, such as cancer, cancer metastasis, and the like.

[18] An epitope that is described as not adjacent to a second epitope means that the two epitopes (or their sequences) are not immediately adjacent in the primary sequence of the protein from which they are derived. Thus the sequence of the first epitope does not abut the sequence of the second epitope, either in a sequential fashion or in an overlapping fashion or in an inclusion fashion. Thus the sequence of the two epitopes when joined together in a peptide vaccine form a new primary sequence. In some embodiments the two epitopes may be adjacent in the originating protein. An adjacent region of a vector requires proximity such that upon insertion of a second region in a cloning site adjacent to a first coding sequence, a fusion protein is formed upon expression.

[19] A vaccine for administration to a tumor-bearing human along with an agonist anti- CD27 antibody will comprises at least one MHC-I (MHC class I) epitope and at least one MHC-II (MHC class II) epitope, linked together. Typically, these will be covalently linked, such as when they are synthesized together as part of a single polypeptide chain. Alternatively, they may non-covalently linked, for example, using a tight binding pair such as avidin-streptavidin. They may be chemically cross-linked if desired. Multiple of either type of such epitopes may be used in a single peptide vaccine. The MHC-I epitope may be a personal epitope unique to the patient's tumor (or very rare in the population). Alternatively, the MHC-I epitope may be an epitope that is commonly found on tumors of the type found in the patient. Rare tumor mutations or epitopes are typically found in 0.5 % or less, 1% or less, 2% or less, 3% or less, or 4% or less of the relevant population. Common tumor mutations are typically found in 5% or greater, 10% or greater, 15% or greater, 20% or greater of the relevant population.

[20] A peptide vaccine will typically be of a size suitable for complete chemical synthesis.

However, such vaccines may also be formed by expression by a cloning vector in a host cell. Semi-synthetic synthesis may include a combination of synthesis routes, such as expression, cleavage and chemical conjugation, for example. A typical size suitable for complete chemical synthesis is a peptide of less than 50 amino acid residues, less than 45 amino acid residues, less than 50 amino acid residues, less than 50 amino acid residues, less than 40 amino acid residues, less than 35 amino acid residues, less than 30 amino acid residues, less than 25 amino acid residues, or less than 20 amino acid residues. A minimum size must be sufficient to comprise two epitopes. This may be at least 10, 12, 14, 16, 18, or 20 amino acids residues.

[21] Various types of tumors may be treated, including, for example, glioblastoma, medulloblastomas, carcinoma, adenocarcinoma, etc. Other examples of tumors include, adrenocortical carcinoma, anal cancer, appendix cancer, grade I (anaplastic) astrocytoma, grade II astrocytoma, grade ΙII astrocytoma, grade IV astrocytoma, atypical teratoid/rhabdoid tumor of the central nervous system, basal cell carcinoma, bladder cancer, breast sarcoma, bronchial cancer, bronchoalveolar carcinoma, cervical cancer, craniopharyngioma, endometrial cancer, endometrial uterine cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, fibrous histiocytoma, gall bladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic tumor, gestational trophoblastic tumor, glioma, head and neck cancer, hepatocellular cancer, Hilar cholangiocarcinoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, Langerhans cell histiocytosis, large-cell undifferentiated lung carcinoma, laryngeal cancer, lip cancer, lung adenocarcinoma, malignant fibrous histiocytoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, endocrine neoplasia, nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian clear cell carcinoma, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, papillomatosis, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pineal parenchymal tumor, pineoblastoma, pituitary tumor, pleuropulmonary blastoma, renal cell cancer, respiratory tract cancer with chromosome 15 changes, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous non- small cell lung cancer, squamous neck cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymic carcinoma, thymoma, thyroid cancer, cancer of the renal pelvis, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor.

[22] MHC-II epitopes which can be used may or may not be related to a tumor antigen or the tumor antigen from which the MHC-I epitope is derived. Although not bound by any theory or mechanism of action, the MHC-II epitope is understood to act to prime the CD4⁺ T cell response. An MHC-II epitope may be derived from common vaccines, including but not limited to poliovirus, diphtheria toxin, influenza virus, pertussis, smallpox, shingles, hepatitis A, B, or E, human papillomavirus, measles, mumps, rabies, rubella, rotavirus, anthrax, tetanus, tuberculosis, meningitis, pneumococcal pneumonia, and cholera. It may be desirable to pretreat the patient with the vaccine from which the MHC-II epitope has been derived. Pretreatment may also be accomplished with the MHC-II epitope in a simplified format than the original vaccine, such as a single protein or peptide.

[23] The two agents for treating a subject should be administered within a limited time frame. In some embodiments they are administered together in admixture or separately. In other embodiments a length time intervenes between the two administrations. This time period may be at least 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, or 24 hours. They should be close enough together that the stimulating function of one administration should be available to affect and synergize with the other administration's effect. This will be typically be less than 1 month, less than 3 weeks, less than 2 weeks, less than 1 week, less than 5 days, or less than 3 days.

[24] Kits may be formulated to contain more than one element in a single package, whether divided internally or undivided. Additional reagents, devices, buffers, instructions, warnings, etc. may also be provided. Typically, components will be separately packaged within a kit. Kits of the invention may comprise any of the following components without limitation: an agonist anti-CD27 antibody, a peptide vaccine comprising an MHC-I epitope and an MHC-II epitope, as described, a peptide with either an MHC-I or MHC-II epitope, a linking reagent, a linking moiety on a peptide with either an MHC-I or MHC-II epitope, and a vector for expressing a peptide with an MHC-I epitope and an MHC-II epitope, including a site for insertion of a coding sequence for the MHC-I epitope. Optionally, linkers between two epitopes may comprise a cleavage site to facilitate processing in cells for antigen presentation.

[25] A composition that comprises two or more components will be an admixture of the two or more components. These will typically be in a single container that is not divided, although a divided container may be used in which the divider is breached to form the admixture. In some embodiments, the composition is only formed when the components which have been separately administered form in the recipient body.

[26] We show below that αhCD27 preferentially enhances the T cell response to peptide vaccines containing linked class Ι/II epitopes. Similar to clinical experiences with single-epitope class I-restricted peptide vaccines (9), we found that αhCD27 was not efficacious as a vaccine adjuvant in the setting of single class I-restricted peptides. In contrast, when administered alongside linked class Ι/II peptide vaccines designed to target both CD8⁺ and CD4⁺ T cells, we show that adjuvant αhCD27 leads to robust vaccine-induced CD8⁺ T cell responses in a CD4-dependent manner. Indeed, we found that CD27 stimulation on both antigen-specific CD4⁺ and CD8⁺ T cells, rather than either T cell compartment alone, is required for the adjuvant activity of αhCD27. As such, we concluded that αhCD27 preferentially enhances vaccine responses in the setting of linked class I/II-epitopes, which distinctively allows for increased tumor- specific CD8⁺ T cell responses and prolonged survival in mice bearing aggressive and poorly immunogenic intracranial tumors. Most importantly for clinical translation, we show that the linked class II epitope can be a universal helper epitope and does not need to be derived from a tumor-specific antigen to give rise to antitumor efficacy. We demonstrate the clinical potential of this approach using a synthetic peptide vaccine targeting both CD4⁺ and CD8⁺ T cells against a clinically-relevant tumor antigen, Trp2. Our findings represent a significant improvement to class I-restricted tumor-derived peptide vaccines and highlight an increased potential for their clinical translation.

[27] Here, we report a novel use of αhCD27 as a combination therapy with a vaccine, in which its adjuvant effect is most pronounced in the setting of vaccines that target both CD4⁺ and CD8⁺ T cells. We believe that αhCD27 enhances vaccine immunogenicity by coordinating CD4⁺ and CD8⁺ T cell responses, thereby increasing the helper function of vaccine-specific CD4⁺ T cells and the resultant antitumor effector function of tumor-specific CD8⁺ T cells. Indeed, neither adjuvant αhCD27 nor a linked class II epitope alone is sufficient to promote antitumor efficacy, while their combination has a substantial therapeutic effect, thus indicating a synergistic relationship between CD27 stimulation and CD4⁺ T cell help. CD27 signaling has been previously shown to drive T_H1 polarization in mouse (33) and human (34) CD4⁺ T cells by upregulating IL-12RP2 and T-bet expression and was also found to enhance their helper activity towards CD8⁺ T cells (35). Additionally, we surmise that CD4⁺ T cell help may render CD8⁺ T cells more sensitive to CD27 signaling, thus amplifying the effect of αhCD27 on the CD8⁺ T cell response compared to the effect that αhCD27 would have in the absence of CD4⁺ T cell help. We believe that these characteristics of CD27 biology make αhCD27 uniquely poised as a promising vaccine adjuvant for enhancing the response to peptide vaccines containing linked class I/II epitopes.

[28] There are a variety of clinically-available peptide vaccines comprised of a tumor- derived class I epitope linked to a universal helper epitope or tumor-derived class II epitope (13,36,37), which would make for promising vaccine candidates for combination with adjuvant αhCD27. In addition to peptide vaccines targeting high- frequency tumor mutations, we believe that adjuvant αhCD27 may enhance the clinical benefit of peptide vaccines derived from class I-restricted tumor neo-antigens, especially when linked to immunogenic class II epitopes. The therapeutic promise of such neo-epitope peptide vaccines has recently been demonstrated in the setting of advanced melanoma, where the administration of long peptides derived from melanoma neo-antigens has resulted in effective antitumor T cell responses (38,39). To broaden the scope of this approach, we envision that putatively any class I tumor neo-antigen could be linked to a helper epitope (e.g., P30) and administered alongside adjuvant αhCD27 to enhance its immunogenicity and efficacy, giving rise to a customizable vaccine/adjuvant treatment modality.

[29] αhCD27 was previously shown to promote effective tumor immune responses as a single agent in mice bearing immunogenic tumors (23). We demonstrate that this antibody can also be employed as a vaccine adjuvant with a preferential effect on linked class Ι/II peptide vaccines, a treatment strategy more relevant in the setting of weak endogenous tumor immune responses and particularly useful for potentiating the immunogenicity of class I-restricted neo-antigens. Importantly, the addition of a class II epitope alone was not sufficient to promote efficacy of a class I-restricted peptide vaccine in the absence of adjuvant αhCD27, indicating the requirement for both CD27 stimulation and CD4⁺ T cell help in promoting the efficacy of class I-restricted peptides. We believe that our study serves as a proof-of-principle demonstration of the adjuvant potential of exogenous CD27 stimulation. As such, our findings warrant further clinical investigation into αhCD27 as an adjuvant for peptide vaccines consisting of a class I-restricted tumor antigen linked with a class II helper epitope for instilling effective tumor-specific T cell immunity.

[30] The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLE 1 Material and Methods Study Design

[31] The goal of this study was to characterize the adjuvant activity of a clinically available immunomodulatory agonist anti-CD27 antibody and its therapeutic potential in a mouse model of advanced stage intracranial malignancy. The experimental design involves studies of vaccine-induced immunogenicity and survival of mice bearing intracranial B16 melanoma tumors. All mice were of the C57BL/6 background, aged 6-12 weeks, and female; naive or tumor-bearing animals were randomized into treatment groups before the start of each experiment. Immunogenicity experiments were performed in groups of 5 mice each, while survival studies were performed with group sizes in excess of 7 mice each. Sample sizes were calculated using F-power analysis (a = 0.05) to yield at least 80% power to detect interactions, based on pilot data. For survival studies, pre-defined humane endpoints were used, according to the Duke University Institutional Animal Care and Use Committee (IACUC) guidelines. All experimental protocols and procedures were approved by the Duke University IACUC. All experiments were performed at least three times, and all outliers were included in the data analysis.

Mice and tumor cell lines

[32] All mice were bred and maintained under pathogen-free conditions at Duke University Medical Center (DUMC). C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, NC, USA), and transgenic OT-I and ΟΤ-II mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Human CD27 transgenic (hCD27) mice, which express both murine and human CD27 molecules under the native murine CD27 promoter (23), were obtained from Celldex Therapeutics (Hampton, NJ, USA) and bred at DUMC. Homozygous hCD27 males were bred with C57BL/6, OT-I, or ΟΤ-II females to generate heterozygous hCD27, hCD27xOT-L or hCD27xOT-II mice, respectively, for use in experiments. All animal experiments were performed according to protocols approved by the Duke University IACUC (Protocol Number: A283-15-1 1). The tumor cell lines B16.F10 and B16.0VA were a kind gift from Dr. Richard G. Vile at Mayo Clinic (24,25). All cell lines used were submitted for cell line authentication (Cell Check) and pathogen testing including mycoplasma testing (IMPACT) at IDEXX BioResearch prior to use. We confirmed species of origin, cell line specific markers and assessed for possible cross- contamination with other cell lines or pathogens

Evaluation of vaccine-induced Tcell responses

[33] Whole ovalbumin (Ova) protein was purchased from Sigma Aldrich (St. Louis, MO, USA), and custom peptides were purchased from JPT (Berlin, Germany) (Table 1). hCD27 mice were vaccinated on day 0 with 2.5 mg intraperitoneal whole Ova protein resuspended at 10 mg/mL in water or intradermally with the indicated peptide emulsified in incomplete Freund's adjuvant (TFA) (ThermoFisher, Waltham, MA, USA). Mice were dosed intraperitoneally on days -3 and 0 with 100 μg αhCD27 (Celldex Therapeutics) or recombinant human IgGl Fc isotype control (Bio X Cell, West Lebanon, NET, USA). On day 7, whole blood (50-100 uL) was collected by retro-orbital puncture for flow cytometric analysis of Ova(I)-specific cells, and spleens were harvested for ELISPOT assays.

Table 1. Peptide sequences

Flow cytometric analysis of Ova(I)-specific CD8⁺ T cells

[34] For the detection of circulating Ova(I)-specific CD8⁺ T cells, 50 μL, whole blood was incubated with rat anti-mouse CD8-FITC (BD Biosciences, San Jose, CA, USA) and H2-Kb(SUNFEKL)-PE murine tetramer (MBL International, Woburn, MA, USA) in 100 μL, PBS in the dark for 30 min at room temperature. Red blood cells (RBCs) were lysed, and cells were fixed with 1 mL IX FACS Lysing Solution (BD Biosciences) in the dark for 15 min at room temperature and washed in PBS. All samples were analyzed on a FACSCalibur flow cytometer (BD Bioscences); absolute numbers per μL blood were calculated using Flowcount® beads (Beckman Coulter, Indianapolis, IN, USA), according to the manufacturer's instructions.

IFNy ELISPOT

[35] Vaccine-specific T cell responses were evaluated ex vivo by IFNy ELISPOT.

MultiScreen® 96-well filter plates (EMD Millipore, Billerica, MA, USA) were coated with 10 μg/mL anti-mouse IFNy antibody (Mabtech, Cincinnati, OH, USA) overnight at 4°C. A total of 2.5 x 10⁵ splenocytes/well were incubated in duplicate in RPMI media supplemented with 10% FBS (Gemini Bio-Products, West Sacramento, CA, USA), IX non-essential amino acids (Life Technologies, Carlsbad, CA, USA), 1 mM L-glutamine (Life Technologies), and 100 IU/mL penicillin + 100 μg/mL streptomycin (Life Technologies), in the presence or absence of 1 μg/mL of the indicated peptide overnight at 37°C in a 5% C0₂ incubator. Spots were developed using 1 μg/mL biotinylated anti-mouse IFNy mAb (Mabtech), a VECTASTAIN®

¹ X₁ is selected from the group consisting of W, F, Y. H, D, E, N, Q, I and K; X₂. is selected from the group consisting of F, N, Y and W; X₃ is selected from the group consisting of H and K, and X₄ is selected from the group consisting of A, D and E. Elite ABC horseradish peroxidase kit (Vector Laboratories, Burlingame, CA, USA), and AEC substrate chromagen (Sigma); spots were quantified by ZellNet Consulting (Fort Lee, NJ, USA).

Tumor Implantation

[36] B16.F10 and B16.0VA cells were grown in DMEM (Life Technologies), 10% FBS and 2 mM L-glutamine at 37°C in 5% CO₂. For intracranial tumor implantation, cells were harvested, resuspended at 3 x 10⁶ cells/mL (B 16. OVA) or 2 x 10³ cells/mL (B16.F10), mixed 1 : 1 with 10% methylcellulose in PBS, and loaded into a 250 mL syringe (Hamilton, Reno, NV) with an attached 25-gauge needle. The needle was positioned 2 mm to the right of bregma and 4 mm below the surface of the skull at the coronal suture using a stereotactic frame ( opf Instruments, Tujunga, CA). A dose of 7,500 cells (B16.0VA) or 500 cells (B16.F10) in a total volume of 5 was injected into hCD27 mice. Tumor-bearing mice were monitored daily for morbidity endpoints and survival according to the Duke University IACUC guidelines.

Analysis of tumor-infiltrating lymphocytes

[37] Tumors were harvested at day 14 after implantation and homogenized in a Stomacher® 80 Biomaster (Seward, Islandia, NY) in 6 mL digestion buffer [RPMI 1640 supplemented with 100 IU/mL penicillin + 100 μg/mL streptomycin, 1 mM L- glutamine, IX non-essential amino acids, 1 mM sodium pyruvate (Life Technologies), 25 μΜ β-mercaptoethanol (Therm oFisher), 10% FBS, 133 μg/mL DNase I (Roche, Indianapolis, IN, USA), and 133 units/mL Type IV collagenase (Life Technologies)] for 20 min at 37°C. The resultant cell suspension was filtered through a 40 μm strainer and washed twice with PBS. The cells were stained with LIVE/DEAD® (ThermoFisher), H2-Kb(SHNFEKL) tetramer, and antibodies for CD3, CD4, and CD8 cell surface markers (BD Biosciences), according to the manufacturers' instructions. The cells were resuspended in 150 μL PBS and analyzed on a FACSCalibur flow cytometer.

T cell Depletion Studies [38] For immunogenicity studies, mice were depleted of CD4⁺ or CD8⁺ cells in the priming phase by once daily intraperitoneal doses of 200 g aCD4 (GK1.5, Bio X Cell) or aCD8 (2.43, Bio X Cell), respectively, for three consecutive days prior to vaccine/αhCD27 administration (as previously described), and immune responses were assessed at day 7 after vaccination. For survival studies, CD8⁺ cells were depleted by once daily intraperitoneal administration of 200 μg aCD8 for three consecutive days immediately after intracranial tumor implantation and before Ova/αhCD27 treatment. For CD4 depletion studies in tumor-bearing mice, a tumor challenge model was employed in which mice were implanted with intracranial B 16. OVA tumors one week after vaccination with whole Ova protein and αhCD27; CD4⁺ cells were depleted by once daily intraperitoneal administration of 200 μg aCD4 for three consecutive days prior to Ova/αhCD27 vaccination (priming phase) or for three consecutive days immediately after intracranial tumor implantation (effector phase).

Adoptive lymphocyte transfers

[39] CD4⁺ and CD8⁺ T cells were purified from the spleens of ΟΤII and OTI mice, respectively, by magnetic labeling in an autoMACs® Pro Separator (Miltenyi Biotec, San Diego, CA). Briefly, spleens were disaggregated and filtered into single cell suspensions, and RBCs were removed by incubating for 5 min in IX lysis buffer (BD Biosiences). The splenocytes were then washed once in RPMI media and once in MACs buffer (Miltenyi Biotec) and resuspended at 2.5 x 10⁸ cells per mL. The cells were then subject to magnetic labeling and T cell isolation using CD4⁺ and CD8⁺ T cell isolation kits (Miltenyi Biotec), according to the manufacturer's instructions. The purified CD4⁺ and CD8⁺ T cells were then mixed at a 2: 1 ratio in PBS at a total cell concentration of 3 x 10⁷ per mL, and 100 μL, of the appropriate cell mixture was injected intravenously in the tail veins of wildtype C57BL/6 mice.

Statistical Analysis

[40] Overall survival was computed from the date of tumor implantation to the date of humane endpoint or death. Survival distributions are described using Kaplan-Meier methods, and the Gehan-Breslow-Wilcoxon test was used to compare survival distributions between treatment groups. Student's unpaired t-test was used to compare IFNy SFU and tetramer values upon adjuvant αhCD27 administration versus isotype control. One-way ANOVA was used to analyze TIL levels and the effect of T-cell depletion on the ovalbumin vaccine response. Two-way ANOVA was used to assess the magnitude of the effect of αhCD27 on the CD8⁺ T cell response in animals receiving different vaccine types. Associations of class II responses with class I responses were assessed using the Pearson correlation coefficient.

EXAMPLE 2 αhCD27 enhances the immune response to a whole protein vaccine and promotes antitumor efficacy

[41] To explore the use of CD27 costimulation as a vaccine adjuvant, we first evaluated the effect of αhCD27 on the CD8⁺ T cell response to vaccination with whole protein using ovalbumin as a model antigen. Groups of hCD27 mice received ovalbumin combined with αhCD27 or hlgGl, and vaccine responses were evaluated by the frequency (as determined by tetramer staining of peripheral blood leukocytes) and effector function (as determined by ex vivo re-stimulation in a IFNy ELISPOT) of T cells specific for the immunodominant ovalbumin class I epitope (Ova(I)) (Fig. 1). We found that the frequency of peripheral blood Ova(I)-specific CD8+ T cells increased from -0.5% to -7% in mice that received whole ovalbumin protein combined with αhCD27 compared to human IgGl (hlgGl) controls (Fig. 1A, P = 0.0035). Additionally, we observed an increase in the level of IFNy-producing splenic lymphocytes upon ex vivo re-stimulation with Ova(I) peptide, from -110 + 37 IFNy+ spot forming units (SFUs) per 10⁶ splenocytes in control mice to 1,656 + 139 SFUs per 10⁶ splenocytes in αhCD27-treated mice (Fig. 1 B, P < 0.0001).

[42] We next asked whether adjuvant αhCD27 combined with whole ovalbumin protein would potentiate the tumor immune response and be efficacious against intracranial B 16. OVA, a highly aggressive melanoma model that is consistently resistant to vaccine immunotherapy (26). hCD27 mice bearing 3-day established intracranial B 16. OVA tumors were treated with whole ovalbumin protein, with or without adjuvant αhCD27. Flow cytometric analysis of their tumor-infiltrating lymphocytes (TILs) revealed a marked increase in the level of Ova(I)-specific CD8+ T cells, from -20% of the CD8⁺ TIL population in mice that received ovalbumin alone to -50% in mice treated with ovalbumin + αhCD27 (Fig. 1C, Ova vs Ova + αhCD27: P - 0.0162). This enhanced level of tumor-infiltrating CD8⁺ T cells corresponded to an 18-day increase in median survival in mice that received ovalbumin + αhCD27 compared to control animals (Fig. ID, Ova + αhCD27 vs. Ova + hlgGl : P = 0.0024).

EXAMPLE 3

The adjuvant effect of αhCD27 on the class I peptide response is enhanced by a linked class II epitope

[43] Given the potent therapeutic effect of αhCD27 combined with whole ovalbumin protein and to inform the use of αhCD27 in combination with peptide vaccines, we next examined which peptide components of whole ovalbumin protein were contributing to the enhanced ovalbumin-specific CD8⁺ T cell response in the setting of adjuvant αhCD27. We compared the adjuvant effect of αhCD27 when combined with four different ovalbumin-derived vaccines (Table 1): 1) the single immunodominant class I-restricted ovalbumin peptide epitope, SIINFE L (Ova(I)); 2) Ova(I) co-administered with an immunodominant class II-restricted peptide epitope, TEWT S SNVMEERKIK V (Ova(II^A), located immediately downstream of Ova(I); 3) a long peptide comprised of the continuous Ova(I) and Ova(II^A) sequences (Ova(I-II^A)); and 4) the whole ovalbumin protein. We administered each of these four vaccines with combined αhCD27 or hlgGl isotype control and evaluated the CD8⁺ T cell response one week after vaccination by ex vivo restimulation with the Ova(I) peptide in a IFNy ELISPOT assay. We observed only slight (but statistically insignificant) increases in the Ova(I)-specific CD8⁺ T cell response in the settings of αhCD27 alongside the single Ova(I) peptide vaccine or Ova(I) co-administered with Ova(II^A) (Fig. 2A and 2B). In contrast, αhCD27 in the setting of Ova(I-II^A) and whole ovalbumin protein resulted in a 5-fold increase in the level of Ova(I)-specific CD8⁺ T cells compared to controls (Fig. 2A, linked Ova(I-II^A) + hlgGl vs linked Ova(I-II^A) + αhCD27: P = 0.00045; whole Ova + hlgGl vs whole Ova + αhCD27: P = 0.0016).

[44] These results were mirrored in our tumor efficacy experiments with the three peptide vaccines. hCD27 mice bearing 3-day established intracranial tumors were treated with Ova(I), Ova(I)+Ova(II^A), or Ova(I-II^A), with or without αhCD27. None of these vaccines were therapeutic in the absence of adjuvant αhCD27, and αhCD27 combined with Ova(I) (Fig. 2 A) or Ova(I)+Ova(II^A) (Fig. 2B) had no effect on survival. In contrast, we found that adjuvant αhCD27 in the setting of linked Ova(I-II^A) resulted in a significant therapeutic benefit (Fig. 2C, Ova(I-II^A) + hlgGl vs Ova(I-II^A) + αhCD27: P = 0.0004), reminiscent of our previous findings with whole ovalbumin protein. Taken together, these results suggest that linked class I and II epitopes drive the adjuvant activity of αhCD27 on the CD8⁺ T cell response such that αhCD27 prolongs survival preferentially in the setting of vaccines comprised of linked class I and II epitopes.

EXAMPLE 4

CD8⁺ T cells and CD4⁺ T cells during the priming phase of the vaccine response are required for the adjuvant effect of ahCD27

[45] The failure of a class I-restricted peptide vaccine to induce effective antitumor immune responses is consistent with prior clinical experience with peptide vaccines (9), but we found that αhCD27 could enhance the tumor-specific CD8⁺ T cell response and resultant antitumor efficacy when combined with vaccines containing linked class I and II epitopes. We thus hypothesized that effector CD8⁺ T cells and CD4⁺ T cells, in either the priming or effector phase, were necessary for the antitumor effect of adjuvant αhCD27. To test this hypothesis, we first examined the effect of CD4⁺ T cell depletion on the CD8⁺ T cell response to whole ovalbumin protein vaccination, during the priming phase (depletion prior to vaccination) and effector phase (depletion 6 days after vaccination) of the vaccine response. We found that a lack of CD4⁺ T cells in the priming phase significantly reduced the adjuvant effect of αhCD27 on the CD8⁺ T cell response to whole ovalbumin protein vaccination, as determined by ex vivo re-stimulation with Ova(I) peptide in a ΙFΝγ ELISPOT (Fig. 3A, Ova + αhCD27 vs Ova + αhCD27 + aCD4 priming: P - 0.00316), while the depletion of CD4⁺ T cells later in the vaccine response had only a slight but statistically non-significant effect (Fig. 3A). Additionally, the depletion of CD8⁺ T cells at the effector phase completely abrogated the ex vivo response to Ova(I) peptide re-stimulation (Fig. 3A, Ova + αhCD27 vs Ova + αhCD27 + aCD8: P - 0.00272).

[46] We next examined the effect of CD8⁺ or CD4⁺ T cell depletion on the antitumor effect of adjuvant αhCD27 in a challenge setting of intracranial B 16. OVA, in which mice were pretreated with αhCD27 + ovalbumin one week prior to tumor implantation. To distinguish between the priming and effector phases of the vaccine response, CD8⁺ T cells were depleted one week after vaccination, at the time of tumor implantation (effector phase), and CD4⁺ T cells were depleted immediately prior to vaccination (priming phase) or at the time of tumor implantation (effector phase). We found that the depletion of CD8⁺ T cells one week after vaccination completely abrogated the efficacy of combined αhCD27 + whole ovalbumin protein (Fig. 3B, red). In contrast, the depletion of CD4⁺ T cells one week after vaccination had no significant effect on the efficacy of αhCD27 + ovalbumin (Fig. 3B, green); however, CD4⁺ T cell depletion prior to vaccination completely abrogated antitumor efficacy (Fig. 3B, blue, Ova + αhCD27 vs Ova + αhCD27 + aCD4 priming: P = 0.0004). These data demonstrate that both CD8⁺ and CD4⁺ T cells are necessary for the therapeutic effect of adjuvant αhCD27 but that CD4⁺ T cells contribute to this effect only during the priming phase of the vaccine response. Consistent with our own data and previous reports showing that class II epitopes are integral for robust class I-restricted vaccine responses (11), these results suggest that CD4⁺ T cell help is critical for the adjuvant effect of αhCD27.

EXAMPLE 5

Direct CD27 stimulation on vaccine-specific CD4⁺ and CD8⁺ T cells is necessary for the adjuvant effect ofahCD27 [47] Our previous data reveals the necessity of CD4⁺ T cells in the priming phase of the vaccine response for αhCD27 to enhance the immunogenicity and antitumor efficacy of a whole ovalbumin protein vaccine, leading us to further investigate the effect of αhCD27 on the ovalbumin-specific CD4⁺ T cell response. We hypothesized that αhCD27 enhances the CD4⁺ T cell response to ovalbumin class II epitopes and that direct stimulation of CD27 expressed on ovalbumin-specific CD8⁺ and CD4⁺ T cells was necessary for the observed adjuvant effect of αhCD27. To test this hypothesis, we first asked if αhCD27 enhances the CD4⁺ T cell response to class II epitopes in the whole ovalbumin protein vaccine. We measured changes in the immune response to Ova(II^A) and Ova(II^B), a class II ovalbumin epitope downstream from Ova(II^A) (Table 1), elicited by whole ovalbumin protein vaccination, with or without adjuvant αhCD27. Indeed, we found that the addition of αhCD27 enhanced the T cell response to both of these class Il-restricted epitopes, as determined by IFNy ELISPOT (Fig. 3C, Ova(II^A): P = 0.0056; Ova(II^B): P = 0.071,).

[48] Next, to determine if direct engagement of CD27 on antigen-specific CD4⁺ and CD8⁺ T cells was necessary for the enhanced CD8⁺ T cell response to whole ovalbumin protein vaccination, we employed an adoptive transfer wherein wildtype C57BL/6 mice received intravenous infusions of CD8⁺ T cells purified from OT-I or OT-I x hCD27 transgenic mice, whose CD8⁺ T cells are specific for Ova(I), and CD4⁺ T cells purified from ΟΤ-II or ΟΤ-II x hCD27 transgenic mice, whose CD4⁺ T cells are specific for Ova(II^B). CD8⁺ and CD4⁺ T cells purified from OT-I or ΟΤ-II mice express only murine CD27 and so would not be responsive to αhCD27 treatment, while those purified from OT-I x hCD27 or ΟΤ-II x hCD27 mice, which express both murine and human CD27, could be responsive to treatment with αhCD27 (Fig. 3D). Of note, the cohort of mice receiving OT-I + OT-II T cells, which do not express hCD27, serve as an experimental control for determining the baseline immune response to whole ovalbumin protein vaccination (i.e., in the absence of exogenous CD27 stimulation) in this adoptive transfer setting. We found that vaccine responses were highest in mice that received OT-I x hCD27 CD8⁺ T cells and ΟΤ-II x hCD27 CD4⁺ T cells, as measured by ex vivo re-stimulation with Ova(I) in a IFNy ELISPOT (Fig. 3E, OTI + OTII vs OTI x hCD27 + OTII x hCD27: P = 0.028), indicating that direct CD27 stimulation on both ovalbumin-specific CD8⁺ and CD4⁺ T cells contributes to the immunogenicity of whole ovalbumin protein in the setting of αhCD27. Taken together, our data suggest that αhCD27 enhances both vaccine- induced CD8⁺ and CD4⁺ T cell responses, resulting in increased CD4⁺ T cell helper function to allow for a more pronounced vaccine-induced CD8⁺ effector response.

EXAMPLE 6

A universal CD4⁺ T cell helper epitope is sufficient to enhance CDS' tumor-specific vaccine responses in the setting of adjuvant ahCD27

[49] In light of our finding that the therapeutic effect of adjuvant αhCD27 requires vaccine- specific CD4⁺ T cells in the priming phase of the immune response, we wondered if non-specific CD4⁺ T cell help would be sufficient to instill antitumor efficacy in the setting of adjuvant αhCD27. To test this hypothesis, we evaluated the adjuvant activity of αhCD27 when combined with a known CD4⁺ T cell universal class II helper epitope, tetanus toxin (TT) P30 (TT(948-968)) (27). We developed a peptide vaccine consisting of Ova(I) covalently linked with the P30 epitope (Ova(I)-P30) (Table 1) and administered it with or without adjuvant αhCD27. A linker sequence consisting of a furin cleavage site (28) was included between the Ova(I) and P30 epitopes to ensure that this synthetic long peptide would be processed into two distinct epitopes. Indeed, we found that αhCD27 enhances the vaccine response to Ova(I)-P30 (Fig. 4A and 4B, Ova(I)-P30 + hlgGl vs Ova(I)-P30 + αhCD27: P = 0.0003) and that the CD8⁺ T cell response to Ova(I) was greater upon vaccination with Ova(I)-P30 compared to Ova(I) in the setting of adjuvant αhCD27 (Fig. 4B, Ova(I) + αhCD27 vs Ova(I)-P30 + αhCD27: P = 0.0327). Consistent with our previous findings, this enhanced Ova(I) response was reduced when CD4⁺ T cells were depleted prior to vaccination (Fig. 4B, Ova(I)-P30 + αhCD27 vs Ova(I)-P30 + αhCD27 + aCD4: P = 0.0001). Moreover, αhCD27 combined with Ova(I)-P30 prolonged survival in hCD27 mice bearing intracranial B16.0VA tumors (Fig. 4C, Ova(I)-P30 + hlgGl vs Ova(I)- P30 + αhCD27: P = 0.0005). These data indicate that a universal class II helper epitope can be the source of vaccine-induced helper CD4⁺ T cells, thereby broadening the utility of this strategy by eliminating the need for a tumor-derived class II epitope in the vaccine. Importantly, however, the addition of the class II P30 epitope alone was not sufficient to enhance the vaccine-induced immune response and promote antitumor efficacy, while combined αhCD27 unveiled the efficacy of this synthetic long peptide vaccine, thus highlighting the synergy between CD4⁺ T cell help and adjuvant αhCD27.

EXAMPLE 7

CD27 stimulation coordinates CD4⁺ T cell help and vaccine-induced CD8^÷ T cell responses

[50] We show above that vaccination with linked class I and II epitopes increases the capacity for αhCD27 to enhance the vaccine-induced CD8⁺ T cell response. We hypothesized that the adjuvant activity of αhCD27 occurs because the enhanced CD4⁺ T cell response potentiates the CD8⁺ T cell response in the setting of CD27 stimulation. If correct, this hypothesis would predict that the magnitude of the CD4⁺ T cell response dictates the magnitude of the CD8⁺ T cell response. We thus analyzed the relationship between vaccine-induced CD4⁺ and CD8⁺ T cell responses in the settings of αhCD27 and hlgGl isotype control. We plotted the class I and class II T cell responses (as determined by IFNy ELISPOT) of individual mice vaccinated with Ova(I-II^A) or Ova(I)-P30 peptide upon ex vivo re-stimulation with Ova(II^A) versus Ova(I) (Fig. 5A and 5B) or P30 versus Ova(I) (Fig. 5C and 5D), respectively, normalized across cumulative experiments. Indeed, for both of these cases, we found that the class II response positively correlates with the class I response in the presence of adjuvant αhCD27 (Ova(II^A) vs Ova(I) with Ova(I-II^A) vaccination, P = 0.0156 and R = 0.631; P30 vs Ova(I) with Ova(I)-P30 vaccination, P = 0.0299 and R = 0.6817) (Fig. 5), while no statistically significant relationship between the CD4⁺ and CD8⁺ T cell responses was found in the setting of hlgGl isotype control. This analysis suggests that a coordinated CD4/CD8 response is orchestrated upon CD27 stimulation, further highlighting the potential of αhCD27 as a vaccine adjuvant for peptide vaccines comprised of linked class I and class II epitopes. EXAMPLE S

ahCD27 enhances the immune response to a peptide vaccine derived from a tumor- associated antigen

[51] To demonstrate the clinical feasibility of αhCD27 as a peptide vaccine adjuvant, we evaluated the ability of αhCD27 to enhance the vaccine response to a known tumor- associated antigen, tyrosinase-related protein 2 (Trp2) (29,30). Vaccination with the Trp2 immunodominant class I epitope (Trp2(I)) (Table 1) has been shown to promote robust antigen-specific immune responses in C57BL/6 mice (31,32). We found that αhCD27 enhances the vaccine response to both Trp2(I) and a linked Trp2(I)-P30 peptide vaccine (Fig. 6A and 6B, Trp2(I) + hlgGl vs Trp2(I) + αhCD27: P = 0.0220; Trp2(I)-P30 + hlgGl vs Trp2(I)-P30 + αhCD27: P = 0.0009). Additionally, similar to our previous findings, αhCD27 enhances the vaccine-induced CD8⁺ T cell response to a greater degree in the setting of Trp2(I)-P30 compared to Trp2(I) alone (P = 0.00147), leading to an overall higher vaccine response in the setting of Trp2(I)-P30 (Fig. 6A and 6B, Trp2(I) + αhCD27 vs Trp2(I)-P30 + αhCD27: P = 0.0004). Furthermore, the magnitude of the P30-specific CD4⁺ T cell response correlates with the magnitude of the Trp2(I)-specific CD8⁺ T cell response in the setting of αhCD27 (Fig. 6D, P30 vs Trp2(I), P = 0.0012 and R = 0.867), but not in the setting of hlgGl (Fig. 6C), indicating the ability for αhCD27 to coordinate CD4⁺ and CD8⁺ T cell responses in this vaccine setting as well. Our immunogenicity results were mirrored in a tumor efficacy experiment, in which <xhCD27 combined with Trp2(I)-P30 prolonged survival in mice bearing 3-day established intracranial B16.F10 tumors (Fig. 6E, Trp2(I)-P30 + hlgGl vs Trp2(I)-P30 + αhCD27: P = 0.0095), while αhCD27 in combination with Trp2(I) had no effect relative to control groups. Our findings in the setting of αhCD27 combined with a clinically-relevant tumor antigen serve as a proof of principle for the promising translational potential of αhCD27 as a peptide vaccine adjuvant for tumor immunotherapy. References

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Claims

Claims

1. A method of treating a tumor in a human comprising:

administering an agonist anti-CD27 antibody to the human;

administering a peptide vaccine comprising an MHC-I epitope linked to an

MHC-II epitope,

wherein the MHC-I epitope is expressed as part of a first protein by the tumor.

2. The method of claim 1 wherein the MHC-I epitope is a portion of a tumor neo- antigen, said MHC-I epitope comprising a somatic mutation in the tumor.

3. The method of claim 1 wherein the MHC-II epitope is a portion of second protein.

4. The method of claim 1 wherein the MHC-II epitope is a portion of a second protein with which the human has been previously vaccinated.

5. The method of claim 4 wherein the second protein is a bacterial protein.

6. The method of claim 4 wherein the second protein is a viral protein .

7. The method of claim 4 wherein the second protein is tetanus toxoid.

8. The method of claim 4 wherein the second protein is P30 (tetanus toxoid amino acids 948-968; SEQ ID NO: 5).

9. The method of claim 1 wherein the MHC-II epitope is PADRE (SEQ ID NO: 9 or 10).

10. The method of claim 1 wherein the MHC-II epitope is a portion of the first protein.

11. The method of claim 1 wherein the MHC-I epitope is a portion of a tumor antigen expressed by the tumor in excess of expression of the tumor antigen in non-tumor tissues of the human.

12. The method of claim 10 wherein the MHC-II epitope is not an adjacent part of the first protein.

13. A kit for treating a tumor in a human, comprising:

an agonist anti-CD27 antibody; and

a peptide vaccine comprising an MHC-I epitope linked to an MHC-II epitope, wherein the MHC-I epitope is expressed as part of a first protein by the.

14. The kit of claim 13 wherein the first protein is a protein that is expressed by a tumor in excess of expression of the tumor antigen in non-tumor tissues.

15. The kit of claim 13 wherein the first protein is a tumor neo-antigen, said MHC-I

epitope comprising a somatic mutation in the tumor.

16. The kit of claim 14 wherein the first protein is expressed by a tumor in excess of expression of the tumor antigen in non-tumor tissues in at least 10% of tumors of the same type as the tumor.

17. The kit of claim 15 wherein the somatic mutation occurs in at least 10% of tumors of the same type as the tumor.

18. The kit of claim 13 wherein the MHC-II epitope is a portion of second protein.

19. The kit of claim 13 wherein the MHC-II epitope is a portion of a second protein with which the human has been previously vaccinated.

20. The kit of claim 18 wherein the second protein is a bacterial protein .

21. The kit of claim 18 wherein the second protein is a viral protein.

22. The kit of claim 18 wherein the second protein is tetanus toxoid.

23. The kit of claim 18 wherein the second protein is P30 (tetanus toxoid amino acids 948-968; SEQ ID NO: 5).

24. The kit of claim 13 wherein the MHC-II epitope is PADRE (SEQ ID NO: 9 or 10).

25. The kit of claim 11 wherein the MHC-II epitope is a portion of the first protein.

26. The kit of claim 25 wherein the MHC-II epitope is not an adjacent part of the first protein.

27. The kit of claim 1 1 wherein the MHC-I epitope is a private tumor antigen, found in less than 1% of tumors of the same type as the tumor.

28. A composition for treating a tumor in a human, comprising: an agonist anti-CD27 antibody; and

a peptide vaccine comprising an MHC-I epitope linked to an MHC-II epitope, wherein the MHC-I epitope is expressed as part of a first protein by the tumor.

29. A kit for treating a tumor in a human, comprising:

an agonist anti-CD27 antibody;

a first peptide comprising an MHC-II epitope; and

a linking moiety for linking the peptide to a second peptide comprising an

MHC-I epitope.

30. The kit of claim 29 wherein the linking moiety is a chemical agent for cross-linking two peptides.

31. The kit of claim 29 wherein the linking agent is bi-functional agent for linking two peptides.

32. The kit of claim 29 wherein the linking moiety is attached to the first peptide.

33. A kit for treating a tumor in a human, comprising:

an agonist anti-CD27 antibody;

a vector comprising a first region encoding a first peptide comprising an MHC-II epitope, and a cloning site adjacent to the first region for insertion of a second region encoding a second peptide comprising an MHC-I epitope, wherein the first region is under the transcriptional control of a promoter and upon insertion of the second region in the cloning site a fusion peptide is formed and expressed.

34. The kit of claim 33 wherein the cloning site comprises a linker between the first and second peptide when expressed as a fusion peptide.