阅读说明：本技术 恢复对抗癌症的nkg2d通路功能的疫苗组合物和方法 (Vaccine compositions and methods for restoring function of the NKG2D pathway against cancer ) 是由 G.德拉诺夫 K.W.乌彻尔普芬宁 C.哈维 F.S.霍迪 于 2015-03-16 设计创作，主要内容包括：本发明涉及恢复对抗癌症的NKG2D通路功能的疫苗组合物和方法。本发明提供用于治疗癌症的疫苗组合物和方法,其可通过引发针对MIC多肽的免疫响应治疗受试者的癌症。(The present invention relates to vaccine compositions and methods for restoring function of the NKG2D pathway against cancer. The present invention provides vaccine compositions and methods for treating cancer, which can treat cancer in a subject by eliciting an immune response against a MIC polypeptide.)

1. A vaccine composition for the treatment of cancer, said composition comprising

(i) An effective amount of a peptide consisting of:

(a) 1, or a peptide having at least 80% sequence identity thereto;

(b) 2, 11, 12, 13, 21, 22 or 23, or a peptide having at least 80% sequence identity to any one thereof;

(c) 11, 12, 13, 21, 22 or 23 further comprising 2, 4, 6, 8 or 10 flanking amino acids at its N-terminus or C-terminus, or both;

(d) 11, 12, 13, 21, 22 or 23, further comprising one or more flanking amino acids such that the entire peptide consists of about 25 to 30 amino acids; or

(e) 14, or a peptide having at least 80% sequence identity thereto;

wherein the flanking amino acids are amino acids adjacent to the peptide epitope sequence in the full length reference sequence SEQ ID NO 1 or SEQ ID NO 14;

the effective amount is an amount effective to elicit an immune response against the cancer; and

(ii) one or more pharmaceutically acceptable adjuvants.

2. The vaccine composition of claim 1, wherein the composition comprises

(i) An effective amount of a peptide consisting of:

(a) 1, or a peptide having at least 80% sequence identity thereto.

3. The vaccine composition of claim 1, wherein the composition comprises

(i) An effective amount of a peptide consisting of:

(a) 1, or a peptide having 10 or fewer conservative amino acid substitutions;

(b) 2, 11, 12, 13, 21, 22 or 23, or a peptide having 2 or fewer conservative amino acid substitutions;

(c) 5,6, 7, 8, 9, 10, 15, 16, 17, 18, 19, or 20, or a peptide having 5 or fewer conservative amino acid substitutions; or

(d) Amino acids 170 to 268 of SEQ ID NO. 14, or a peptide having 10 or fewer conservative amino acid substitutions.

4. The vaccine composition of claim 1, wherein the composition comprises

(i) An effective amount of a peptide consisting of:

(a) amino acids 179 to 274 of SEQ ID NO 1;

(b) 2, 11, 12, 13, 21, 22 or 23;

(c) 5,6, 7, 8, 9, 10, 15, 16, 17, 18, 19 or 20; or

(d) Amino acids 170 to 268 of SEQ ID NO. 14.

5. The vaccine composition of any one of the preceding claims, wherein the composition is effective to elicit an immune response against cancer cells that express a MIC polypeptide.

6. The vaccine composition of claim 5, wherein the MICA or MICB polypeptide is not attached to a cell.

7. The vaccine composition of any one of the preceding claims, wherein the cancer is melanoma.

8. The vaccine composition of any one of the preceding claims, wherein the vaccine composition comprises a plurality of peptides selected from two or more of SEQ ID NOs 5-10, or SEQ ID NOs 15-20, or peptides having 95% amino acid sequence identity to any one thereof; or selected from two or more of SEQ ID NOs 2-13, or SEQ ID NOs 21-23, or a peptide having 90% amino acid sequence identity to any one thereof.

9. The vaccine composition of any one of the preceding claims, wherein the adjuvant is selected from the group consisting of oil-based adjuvants, CpG DNA adjuvants, mineral salt adjuvants, particulate adjuvants, mucosal adjuvants, cytokines, microbial derivatives, emulsions and Toll-like receptor agonists.

10. The vaccine composition of any one of the preceding claims, wherein the peptide is conjugated to a carrier protein.

11. The vaccine composition of claim 10, wherein the carrier protein is selected from the group consisting of tetanus toxin and diphtheria toxin.

12. The vaccine composition of any one of the preceding claims, wherein the composition comprises a viral capsid protein engineered so as to display at least one of the peptide or peptides on its surface.

13. The vaccine composition of claim 12, wherein the viral capsid protein is a hepatitis b capsid protein.

14. The vaccine composition of any one of the preceding claims, wherein the composition is in the form of a polymer scaffold comprising at least one of the peptide or peptides.

15. The vaccine composition of claim 14, wherein the polymer scaffold is a porous, poly-lactide-co-glycolide (PLG) polymer scaffold.

Technical Field

The present invention relates to methods and compositions for inducing an anti-cancer immune response in a human subject.

Background

MICA is a ligand for NKG2D, a C-type lectin-like, type II transmembrane receptor expressed on most human NK cells, γ δ T cells, and CD8+ T cells. In the ligation reaction, NKG2D signals via the adaptor protein DAP10 to evoke perforin-dependent cytolysis and provide co-stimulation. In humans, NKG2D ligands include MHC class I chain-associated protein A (MICA), closely related MICB, UL-16 binding protein (ULBP)1-4, and RAE-1G.

Although NKG2D ligands are not commonly found on healthy tissues, various forms of cellular stress (including DNA damage) may be expressed up-regulated, leading to their frequent detection in a variety of solid and hematologic malignancies, including melanoma. Activation of NKG2D by ligand-positive transformed cells contributes to exogenous tumor immunity, as NKG 2D-deficient mice show increased tumor susceptibility. However, NKG 2D-mediated tumor immunity is ineffective in many cancer patients. In part, immune escape can be achieved by shedding of NKG2D ligand from tumor cells, which triggers internalization of surface NKG2D and impaired function of cytotoxic lymphocytes. See, e.g., Wu et al, "Presence Expression of the immunological MHC Class I Chain-related molecules is managed by the expressed protein in State Cancer", J Clin Invest 114:560-8 (2004); groh et al, "moving-derived solvent MIC Ligands Expression of NKG2D and T-cell Activation," Nature 419:734-8 (2002); doubrovina et al, "evolution from NK Cell Immunity by MHC Class ICHain-related Molecules Expressing Colon Adenocerloma", J Immunol 171:6891-9 (2003). Shedding of MIC from a tumor results in a decrease in the density of MIC expressed on the surface of tumor cells, which is also one of the mechanisms of tumor escape. See Marten et al, "simple MIC is elongated in the Serum of Patients with functional Gamma Delta T Cell cytotoxin", Int J Cancer 119:2359-65 (2006). Soluble NKG2D ligand may also stimulate expansion of regulatory NKG2D + CD4+ Foxp3-T cells (which may antagonize anti-tumor cytotoxicity by Fas ligand, IL-10 and TGF- β).

MICA is an NKG2D ligand shed from tumor cells, i.e. released from the cell surface into the surrounding medium, and serum from a subpopulation of cancer patients contains elevated levels of soluble form (sMICA). MIC (the term "MIC" refers to MICA and MICB) shedding is achieved by interaction with the protein disulfide isomerase ERp5, which ERp5 cleaves disulfide bonds in the MIC α 3 domain, rendering it susceptible to proteolysis (proteolysis) by ADAM-10/17 and MMP 14. Methods of treating cancer by administering anti-MIC antibodies or antigen-binding peptide fragments have been described. For example, US 8,182,809 describes such methods which utilise a purified antibody or polypeptide comprising an antigen-binding fragment thereof which specifically binds to the amino acid sequence NGTYQT located in the α 3 ectodomain of the MIC polypeptide, in order to inhibit the interaction of the MIC polypeptide with ERp5 and inhibit shedding of the MIC. And US 7,959,916 describes methods of inhibiting shedding of MIC polypeptides from cancer cells using anti-MIC α 3 domain antibodies. Tumor-derived soluble MIC polypeptides, MICA or MICB, or both, have also been suggested as biomarkers for the diagnosis and prognosis of cancer, and anti-MICA or anti-MICB antibodies as therapeutic agents for the treatment of cancer and autoimmune diseases. For example, US 7,771,718 describes a method of mitigating MIC-induced NKG2D inhibition in lymphocytes using anti-MIC antibodies to bind soluble MIC polypeptides.

In practice, methods of treating cancer or other diseases using therapeutic antibodies are relatively expensive because of the need to manufacture large quantities of such antibodies of sufficient purity to be infused into a patient. Given the complexity of large-scale antibody production and the specialized requirements for antibody infusion protocols, alternative approaches are needed to target MIC polypeptides in a more efficient and cost-effective manner. The present invention provides a solution to this problem by providing a vaccine for inducing anti-MIC antibodies in a subject.

Tumor vaccines typically consist of a tumor antigen and an immunostimulatory molecule (e.g., a cytokine or TLR ligand) that act together to induce antigen-specific cytotoxic T Cells (CTLs) that recognize and lyse tumor cells. At this point, almost all vaccines contain shared tumor antigens or whole tumor cell preparations (Gilboa, 1999). Shared tumor antigens are immunogenic proteins that have selective expression in tumors across multiple individuals and are typically delivered to patients as synthetic peptides or recombinant proteins (Boon et al, 2006). In contrast, whole tumor cell preparations are delivered to patients in the form of autologous irradiated cells, cell lysates, cell fusions, heat shock protein preparations, or total mRNA (Parmiani et al, 2007). Because whole tumor cells are isolated by the patient, the cells express both the patient-specific tumor antigen as well as the shared tumor antigen. Finally, there is a third class of tumor antigens that are rarely used in vaccines due to the technical difficulties in recognizing them (Sensi et al, 2006). This class consists of proteins with tumor-specific mutations that lead to altered amino acid sequences. Such muteins have the following potential: (a) uniquely marking tumors (as opposed to non-tumor cells) for recognition and destruction by the immune system (Lennerz et al, 2005); (b) central and sometimes peripheral T cell tolerance is avoided and thus recognized by more potent high affinity T cell receptors (Gotter et al, 2004).

Disclosure of Invention

The present invention provides compositions and methods for treating cancer in a subject by eliciting an immune response against a MIC polypeptide. The term "MIC" as used herein refers to MICA and/or MICB. In one embodiment, the invention provides a vaccine composition for the treatment of cancer, the composition comprising as an immunogenic component an effective amount of a peptide comprising or consisting of one or more of SEQ ID NOs 1 to 23, said effective amount being an amount effective to elicit an immune response against a MIC polypeptide or cancer. In another embodiment, the vaccine composition comprises as an immunogenic component an effective amount of a peptide comprising or consisting of one or more of SEQ ID NOs 1-4 or 2-4, one or more of SEQ ID NOs 5-7, one or more of SEQ ID NOs 8-10, or one or more of SEQ ID NOs 5-13. In another embodiment, the vaccine composition comprises an effective amount of a peptide comprising or consisting of one or more of SEQ ID NOs 14-23, one or more of SEQ ID NOs 15-23, one or more of SEQ ID NOs 18-23, or one or more of SEQ ID NOs 21-23 as an immunogenic component.

In one embodiment, the vaccine composition is effective to elicit an in vitro immune response against a MIC polypeptide. In another embodiment, the vaccine composition is effective to elicit an in vivo immune response against a MIC polypeptide.

In one embodiment, the immune response is directed against a MIC polypeptide that is not attached to the cell, also referred to as a soluble MIC polypeptide. The soluble MIC may be in monomeric or multimeric form. In another embodiment, the immune response is directed against a cancer cell expressing a MIC polypeptide. The cancer cell may be in vitro or in vivo. In one embodiment, the vaccine composition is effective to elicit an immune response against cancer cells that express a MIC polypeptide. The cancer cell may be in vitro or in vivo.

In one embodiment, the MIC polypeptide is a MICA or MICB polypeptide, or a fusion protein comprising the α 3 domains of MICA and MICB.

Any cancer cell expressing a MIC can be treated using the compositions and methods of the invention. In one embodiment, the cancer is selected from the group consisting of prostate cancer, multiple myeloma, glioblastoma multiforme, and melanoma. In one embodiment, the cancer is melanoma.

In one embodiment, the peptide comprises or consists of: one or more of SEQ ID NOs 8-13, or a peptide having 90% or 95% amino acid sequence identity thereto. In one embodiment, the peptide comprises or consists of: one or more of SEQ ID NOs 15-23, or a peptide having 90% or 95% amino acid sequence identity to any one thereof.

In one embodiment, the vaccine composition comprises a plurality of peptides selected from two or more of SEQ ID NOs 5-10, or a peptide having 95% amino acid sequence identity to any one thereof; or selected from two or more of SEQ ID NO 8-13, or a peptide having 90% amino acid sequence identity to any one thereof. In one embodiment, the vaccine composition comprises a plurality of peptides selected from two or more of SEQ ID NOs 15-20, or a peptide having 95% amino acid sequence identity to any one thereof; or selected from two or more of SEQ ID NO 21-23, or a peptide having 90% amino acid sequence identity to any one thereof.

In one embodiment, the peptide is conjugated to a carrier protein. In one embodiment, the carrier protein is selected from tetanus toxin and diphtheria toxin.

In one embodiment, the vaccine composition comprises a viral capsid protein engineered to display the at least one peptide or plurality of peptides on its surface. In one embodiment, the viral capsid protein is a hepatitis b capsid protein.

In one embodiment, the vaccine composition is in the form of a polymeric scaffold comprising the at least one peptide or plurality of peptides. In one embodiment, the polymer scaffold is a porous poly-lactide-co-glycolide (PLG) polymer scaffold. In one embodiment, the polymeric scaffold further comprises one or both of a GM-CSF protein and a Toll-like receptor agonist. In one embodiment, the polymer scaffold further comprises autologous tumor cell lysate of a subject to be treated for cancer with the composition.

The present invention also provides methods of treating cancer in a subject by administering to the subject a vaccine composition described herein. In one embodiment, the vaccine composition of the invention is administered as part of a treatment regimen. In one embodiment, the treatment regimen further comprises one or more of radiation therapy, immunotherapy, chemotherapy, or targeted therapy. In one embodiment, the method comprises administering at least two, preferably three, independent vaccine compositions of the invention, each having an immunogen that is different from the other, as part of a prime-boost (prime-boost) strategy.

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

Drawings

FIG. 1: mapping of epitopes on MICA x 100 reference structures. Epitope mapping was performed using overlapping peptide arrays. Each peptide is a 20 amino acid linear sequence with a 10 amino acid offset (offset) per peptide.

Fig. 2A and 2B: epitope protection in common MICA and MICB alleles.

FIG. 3 includes FIGS. 3A-3C; wherein: fig. 3A and 3B: chimeric protein design with appropriately placed epitopes for MIC antibodies. Epitopes of MICA Abs 28 and 29 (highlighted in blue and red) were placed into an unrelated protein with a similar Ig domain structure, human CMV protein UL 18. Comparing the structures of MICA a3(a) and chimeric protein (B) shows protection against epitopes of MICA antibodies 28 and 29.

FIG. 3C: the sequence of the chimeric protein was aligned to the MICA and UL18 sequences (C). Residues of UL18 that bind to human LIR were mutated (shown in white). Residues 206 and 210 of MICA are polymorphic (G/S and W/R, respectively).

FIG. 4 includes FIGS. 4A-4C; wherein: fig. 4A and 4B: design of the-MICA to show minimal disulfide stability of MICA epitopes. The min-MICA protein was designed to focus the B cell response on a key part of the protein. Disulfide bonds (green) were introduced to stabilize the conformation of the MICA Ab29 epitope. The beta chain linking the Ab28 and Ab29 epitopes was deleted (deleted) to reduce protein flexibility and improve solubility. It is noted that the N and C termini of the mini-MICA protein are closely adjacent, which enables display on the surface of the hepatitis b nucleocapsid. blue-Ab 28 epitope, red-Ab 29 epitope.

FIG. 4C: the min-MICA sequence was aligned to MICA.

FIG. 5 includes FIGS. 5A-5D; wherein: figures 5A-5D are a series of graphs depicting the therapeutic activity of human anti-MICA antibodies. Fig. 5A is a graph depicting the improved survival of AML Ab2 implanted into mice with SCIDs of human U937 tumor cells (3 × 100 micrograms Ab per week). The days elapsed are shown on the x-axis and the percent survival is shown on the y-axis. Fig. 5B is a graph depicting that antibody treatment significantly reduced sMICA concentration in the serum of treated mice as measured by ELISA. The duration of treatment is shown on the x-axis and sMICA concentration in serum is shown on the y-axis. Figures 5C and 5D show that after one week of treatment, MICA antibodies reduced sMICA in tumor homogenates (normalized to tumor mass; see figure 5C) and increased MICA expression on the tumor cell surface as analyzed by flow cytometry (see figure 5D). The x-axis in fig. 5C shows experimental conditions and the y-axis shows sMICA concentration in tumor homogenates. The x-axis in fig. 5D shows experimental conditions and the y-axis shows Mean Fluorescence Intensity (MFI).

FIG. 6 includes FIGS. 6A-6F; wherein: FIGS. 6A-6F are a series of graphs depicting human antibodies enhancing NK cell accumulation and function in tumors. For these data, SCID mice bearing U937 tumors were treated with MICA mAbs (3 × 100 μ g) for one week and evaluated for NK cell function. FIGS. 6A, 6B and 6C show that this antibody treatment increased CD45 penetration by tumor⁺NK1.1⁺NK cell surface levels of NKG2D (see fig. 6A) and NKp46 (see fig. 6B) and induced NK cell accumulation in tumors (see fig. 6C, normalized to 1 × 10⁵An individual CD45⁺A cell). FIGS. 6D and 6E show that treatment increased CD45 penetration by tumors⁺NK.1⁺NK cells expressed IFN γ (see fig. 6D) and perforin (see fig. 6E). FIG. 6F depicts that all three human MICA antibodies increased progression through splenocytes⁵¹Indirect in vivo (ex vivo) killing of Cr-labeled YAC-1 cells.

Detailed Description

The present invention provides compositions and methods for treating cancer in a subject by eliciting an immune response against a MIC polypeptide. The terms "elicit," "stimulate," and "induce" are used interchangeably to mean to generate a nascent immune response in a subject or to increase the intensity or persistence of an existing immune response. The compositions of the invention contain at least one MIC peptide as an immunogenic component (also referred to herein as an "immunogen") that comprises or consists of the full-length α 3 domain [ SEQ ID NO:1] of MICA [ SEQ ID NO:1] or MICB [ SEQ ID NO:14 ]. In certain embodiments, the MIC peptide is an epitope selected from SEQ ID NOS: 2-13 or SEQ ID NOS: 15-23.

In the context of the present invention, an epitope is a part of an antigenic molecule that is capable of eliciting an immune response (preferably a cytotoxic T cell response or an antibody secreting B cell mediated response) to the molecule or that can be bound by an antibody. The inventors identified the minimal epitope represented by SEQ ID NO 11-13 and 21-23 as the antibody binding epitope for CM33322 Ab4, CM33322 Ab28 and CM33322 Ab29, described in U.S. provisional application Nos. 61/792,034 and 61/913,198 and U.S. application No. 14/025,573. These antibodies are isolated from cancer patients who respond to immunotherapy. These antibodies enhance the activity of NK cells and CD 8T cells against cancer cells by inhibiting the cleavage of MIC proteins from cancer cells. The antibodies bind to the α 3 domain of the MIC protein and have strong anti-tumor activity in relevant animal models. These clinical immunological studies demonstrated that induction of antibodies against the α 3 domain of the MIC protein restored anti-tumor immune function against cancer. According to the invention, epitopes recognized by these antibodies can be used as immunogenic components of cancer vaccines to stimulate antibody production against the MIC α 3 domain. An important element of the present invention is that antibodies are raised against the alpha 3 domain of the MIC, but not against the alpha 1-alpha 2 domain of the MIC, as long as the NKG2D receptor on NK cells and CD 8T cells binds to said alpha 1-alpha 2 domain. Accordingly, the present invention provides epitopes of MICA and B proteins that are important for an effective anti-MIC immune response in humans, as well as methods and compositions relating to their use as immunogenic components of cancer vaccines.

Table 1: the antibody binding epitope is at the position in the amino acid sequence of the MICA 001 reference sequence (SEQ ID NO: 1). Epitopes are shown in bold and underlined.

Table 2: MICA epitopes recognized by human antibodies from patients responding to cancer immunotherapy (epitopes underlined)

Table 3: MICA epitopes with short flanking sequences

Table 4: minimum MICA epitopes

Table 5: MICB epitopes recognized by human antibodies from patients responding to cancer immunotherapy in the MICB reference sequence (SEQ ID NO:14) (epitope underlined)

Table 6: MICB epitopes with short flanking sequences

Table 7: minimum MICB epitope

The present invention provides a vaccine composition suitable for administration to a human comprising at least one MIC peptide as an immunogenic component. In one embodiment, the at least one MIC peptide comprises or consists of the full-length α 3 domain of MICA or MICB, which corresponds to amino acids 181 to 274 of the reference sequence, [ SEQ ID NO:1 ]. In another embodiment, the at least one peptide comprises or consists of a peptide epitope of a MIC peptide selected from any one of SEQ ID NOs 2-13 or SEQ ID NOs 15-23. In one embodiment, the at least one peptide consists of a peptide epitope selected from SEQ ID NOS: 11-13 or SEQ ID NOS: 21-23 and one or more flanking amino acids. In this regard, the term "flanking amino acids" refers to the amino acids adjacent to the peptide epitope sequence in the full-length reference sequence [ SEQ ID NO:1 for MICA, or SEQ ID NO:14 for MICB ]. In certain embodiments, at least one peptide epitope comprises 2, 4, 6, 8, or 10 flanking amino acids at its N-terminus or C-terminus, or both. In one embodiment, the at least one peptide consists of a peptide epitope selected from SEQ ID NOS: 11-13 or SEQ ID NOS: 21-23 and one or more flanking amino acids such that the peptide consists of about 25 to 30 amino acids, or a length suitable to effectively induce an antibody that responds to a MIC protein.

In one embodiment, the vaccine composition comprises as its immunogenic components at least two peptide epitopes of a MIC peptide selected from SEQ ID NO:2-13 or SEQ ID NO: 15-23. In one embodiment, the vaccine composition comprises as its immunogenic components at least two peptide epitopes of a MIC peptide selected from SEQ ID NO:2-4 or SEQ ID NO: 15-23. In one embodiment, the vaccine composition comprises as its immunogenic components at least two peptide epitopes of a MIC peptide selected from SEQ ID NO 5-10. In one embodiment, the vaccine composition comprises as its immunogenic components at least two peptide epitopes of a MIC peptide selected from SEQ ID NO:11-13 or SEQ ID NO: 21-23.

In one embodiment, the vaccine composition comprises as an immunogenic component thereof one or more peptide epitopes of a MIC peptide selected from SEQ ID NOs 2-13 or SEQ ID NOs 15-23, wherein said peptide epitopes are in the form of linear sequences. In one embodiment, the peptide epitope is in the form of a structural constraint ring. In one embodiment, the peptide retains its native secondary structure, e.g., in the form of one or more loops. In one embodiment, the loop is created using a disulfide bond or a chemical linker. Preferably, the loop is adapted to mimic a three-dimensional conformation of a MIC epitope on a human protein.

In another embodiment, the vaccine composition comprises a nucleic acid encoding one or more of the peptides of SEQ ID NOS: 2-13 or SEQ ID NOS: 15-23. The nucleic acid may be in the form of an expression vector, such as a plasmid or viral vector, or the nucleic acid may be packaged into a nanoparticle. In one embodiment, the nucleic acid is delivered to the subject by injection. In one embodiment, the nucleic acid is injected as purified DNA or in the form of nanoparticles. In one embodiment, a modified immune cell that has been modified to express the nucleic acid is injected. In one embodiment, the immune cell is modified via in vitro transfection or infection with a vector comprising the nucleic acid.

In one embodiment, the vaccine composition comprises as its immunogenic component a plurality of peptides comprising or consisting of two or more peptides selected from SEQ ID NO:2-13 or SEQ ID NO: 15-23. In one embodiment, the plurality of peptides comprises or consists of at least two peptides selected from SEQ ID NOS: 2-4 or SEQ ID NOS: 15-23. In one embodiment, the plurality of peptides comprises or consists of at least two selected from SEQ ID NOS 5-10. In one embodiment, the plurality of peptides comprises or consists of at least two selected from SEQ ID NOS: 11-13 or SEQ ID NOS: 21-23.

In one embodiment, the at least one peptide or the plurality of peptides is conjugated to a second peptide comprising an MHC-II epitope. Preferably, the amino acid sequence of the second peptide consists of 25 amino acids or less, or 15 amino acids or less. In particular embodiments, the second peptide consists of 9-12 amino acids, 10-18 amino acids, or 8-18 amino acids. Preferably, the second peptide contains a T cell epitope or a B cell epitope. In one embodiment, the T cell epitope is a T helper cell epitope, or a cytotoxic T cell epitope, effective to enhance differentiation of B cells into antibody-producing plasma cells. In one embodiment, the epitope is a covered epitope of different MHC alleles, or an epitope presented by a number of MHC allotypes. In another embodiment, the epitope is a peptide presented by different MHC alleles.

The peptides comprising or incorporated into the vaccine compositions of the invention are preferably purified to remove contaminating chemical precursors (if chemically synthesized), or are substantially free of cellular material from the cell or tissue source from which they are derived. In particular embodiments, the peptide is 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% free of contaminating chemical precursors, proteins, lipids, or nucleic acids. In a preferred embodiment, the peptide is substantially free of contaminating viruses. Preferably, each composition for administration to a subject is at least 95%, at least 97%, or at least 99% free of contaminating viruses.

In one embodiment, the at least one peptide or peptides of the vaccine composition of the invention comprises or consists of one or more peptides that are at least 90%, at least 95%, at least 98%, or at least 99% identical to a peptide selected from any one of SEQ ID NO2-13, SEQ ID NO 5-10, SEQ ID NO 11-13, SEQ ID NO 15-20, and SEQ ID NO 21-23.

In one embodiment, the at least one peptide or plurality of peptides comprises or consists of one or more peptides that are at least 90%, at least 95%, at least 98%, or at least 99% similar to a peptide selected from any one of SEQ ID NOs 2-13 or SEQ ID NOs 15-23. In this regard, the term "similar" refers to amino acid sequence similarity defined in terms of the number of conservative and non-conservative amino acid changes in a query sequence relative to a reference sequence. Conservative and non-conservative amino acid changes are known in the art. See, for example, W.R.Taylor, The Classification of Amino Acid Conservation, J.Theor.biol.1986119: 205-. In general, a conservative amino acid change refers to the substitution of one amino acid for another having substantially similar chemical properties, particularly with reference to the amino acid side chain. Non-conservative changes refer to the replacement of one amino acid for another with a significantly different chemical property. In general, conservative substitutions are those in the art that are considered less likely to affect the overall structure or biological function of a polypeptide, while non-conservative changes are considered more likely to affect structure and function.

Non-limiting examples of conservative amino acid changes include substitutions of amino acids in the following groups: aliphatic, aromatic, polar, non-polar, acidic, basic, phosphorylatable hydrophobic, hydrophilic, small non-polar, small polar, large non-polar and large polar. Non-limiting examples of non-conservative amino acid changes include amino acid substitutions between the aforementioned groups.

In one embodiment, a conservative amino acid change is a substitution wherein the substitution matrix of the pair of residues has a positive value. Examples of amino acid substitution matrices are known in the art, such as the BLOSUM50 matrix or the PAM250 matrix (see W.A. Pearson, Rapid and Sensitive Sequence company with FASTP and FASTA, meth.enzymology, 1990183: 63-98, edited by R.Doolittle, Academic Press, San Diego). Other examples of Scoring matrices (Scoring matrices) and Comparisons between them are found in M.S. Johnson and J.P.Overington,1993, A Structural Basis for Sequence Comparisons: An evaluation of Scoring methods, J.mol.biol.233: 716-738.

In a preferred embodiment, a conservative amino acid change is the substitution of one amino acid for another in the same chemical group, wherein said group is selected from the group consisting of neutral and polar amino acids (Ser, Thr, Pro, Ala, Gly, Asn, Gln), negatively charged and polar amino acids (Asp, Glu), positively charged and polar amino acids (His, Arg, Lys), non-polar amino acids lacking a loop structure (Met, Ile, Leu, Val), non-polar amino acids with a loop structure (Phe, Tyr, Trp) and cysteine.

In one embodiment, the vaccine composition of the invention comprises as its immunogenic component a chimeric protein consisting of two or more MIC peptide epitopes independently selected from SEQ ID NO2-13 or SEQ ID NO:15-23, wherein said epitopes are linked. In one embodiment, the two or more MIC peptide epitopes are the same epitope. In another embodiment, the two or more MIC peptide epitopes comprise at least two different MIC peptide epitopes. In one embodiment, the vaccine composition comprises as an immunogenic component thereof a chimeric protein displayed on the surface of a viral capsid, such as a hepatitis b nucleocapsid.

In one embodiment, the vaccine composition of the invention comprises as its immunogenic component a chimeric protein consisting of two or more MIC peptide epitopes selected from SEQ ID NO2-13 or SEQ ID NO:15-23 placed into an immunoglobulin (Ig) domain having a similar overall immunoglobulin fold as compared to MICA. In one embodiment, the Ig domain is an Ig domain selected from one of: UL18 (human CMV), the C-terminal Ig domain of IFN-. alpha./β binding protein C12R (Poxviry decoy receptor, PDB ID:3OQ3), the N-terminal Ig domain of the outer capsid protein from T4-like phage (Hoc, PDB ID:3SHS), and the human CMV protein US2(PDB ID:1IM 3).

In one embodiment, the vaccine composition of the invention comprises two separate components suitable for separate administration, the first component comprising an immunogen consisting of a first MIC peptide comprising or consisting of the full-length α 3 domain of MICA [ SEQ ID NO:1] or MICB; the second component comprises an immunogen consisting of one or more MIC peptide epitopes selected from SEQ ID NO2-13 or SEQ ID NO 15-23. In one embodiment, the vaccine composition comprises a first component comprising an immunogen consisting of a first MIC peptide comprising or consisting of the full-length α 3 domain of MICA [ SEQ ID NO:1 ]; and one or more additional components each comprising an immunogen consisting of one or more MIC peptide epitopes selected from SEQ ID NOs 2-13 or SEQ ID NOs 15-23. Preferably, the first component is applied in a prime-boost fashion according to methods known in the art before the second or additional component.

In one embodiment consistent with any of the preceding embodiments, the vaccine composition of the present invention may comprise one or more polynucleotide sequences encoding MIC epitopes of SEQ ID NOs 1-23. In a further embodiment, the DNA encoding one or more MIC epitopes is in the form of a nanoparticle comprising the DNA.

Peptide variants

In some cases, the amino acid sequence of a peptide disclosed herein can be modified and altered to produce, for example, a peptide variant (e.g., a peptide having defined sequence homology to a peptide disclosed herein) so long as the antigen binding properties of the peptide variant are maintained or improved relative to the unmodified peptide (the antigen binding properties of any modified peptide can be assessed using in vitro and/or in vivo assays described herein and/or techniques known in the art).

While peptide variants are generally observed and discussed at the amino acid level, actual modifications are often introduced or performed at the nucleic acid level. For example, variants having 80%, 85%, 90%, 95%, 96%, 97%, 98, or 99% amino acid sequence identity to a peptide of the invention can be generated by modifying a nucleic acid encoding the peptide, or a portion/fragment thereof, using techniques known in the art (e.g., cloning techniques).

Amino acid sequence modifications typically fall into one or more of three categories: a substitution modification, an insertion modification or a deletion modification. Insertions include amino and/or terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions are generally smaller insertions than those of amino or carboxy termini fusions, for example, about one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about 2 to 6 residues are deleted at any one position in the protein molecule. Amino acid substitutions are typically single residues, but can occur at multiple different positions at once; insertions will typically be of approximately about 1 to 10 amino acid residues; and deletions will range from about 1 to 30 residues. Deletions or insertions may be made in adjacent pairs, i.e. 2 residues are deleted or 2 residues are inserted. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at the final construct (construct). The mutation must not place the sequence out of reading frame and preferably will not generate a complementary region capable of producing secondary mRNA structure. Substitutional modifications are those in which at least one residue has been removed and a different residue inserted in its place. In some cases, the substitution may be a conservative amino acid substitution. In some cases, a peptide herein may include one or more conservative amino acid substitutions relative to a peptide of the invention. For example, a variant may include 1, 2,3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-30, 30-40, or 40-50 conservative amino acid substitutions relative to the peptides shown in table 1. Alternatively, a variant may include 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer conservative amino acid substitutions relative to the peptides shown in table 1. Such substitutions are generally made according to table 2 below and are referred to as conservative substitutions. Methods for predicting resistance to protein modification are known in the art (see, e.g., Guo et al, proc.natl.acad.sci., USA,101(25): 9205-.

Table 2: conservative amino acid substitutions

In some cases, the substitutions are not conservative. For example, amino acids in the peptides shown in table 1 may be substituted with amino acids that may alter certain properties or aspects of the peptides. In some cases, non-conservative amino acid substitutions may be made, for example, to alter the structure of the peptide, to alter the binding characteristics of the peptide (e.g., to increase or decrease the binding affinity of the peptide to the antigen and/or to alter the binding specificity of the peptide to the antigen).

In some cases, the peptide and/or peptide variant may include or may be a fragment of a peptide shown in table 1. Such fragments may comprise fewer, e.g., 1, 2,3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 3637, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50-100, 101-150 amino acids than the CDRs, FRs and/or AAs shown in table 1, e.g., as long as the fragment retains at least a portion of the binding properties of the full-length peptide (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the binding properties of the full-length peptide). Truncation may be at the amino terminus, the carboxyl terminus, and/or within the peptides herein.

In some cases, the interaction surface of a peptide variant may be the same (e.g., substantially the same) as the unmodified peptide, e.g., to alter (e.g., increase or decrease), retain, or maintain the binding properties of the peptide variant relative to the unmodified peptide. Methods for identifying the interaction surface of peptides are known in the art (Gong et al, BMC: Bioinformatics,6: 1471-.

One skilled in the art would readily understand how to determine the identity of two polypeptides (e.g., unmodified peptides and peptide variants). The identity may be calculated, for example, after aligning the two sequences so that the identity is at its highest level. Another way of calculating identity can be done by published algorithms. Optimal alignment of sequences for comparison can be performed by: the local identity algorithm of Smith and Waterman, adv.Appl.Math,2:482(1981), the identity alignment algorithm of Needleman and Wunsch, J.mol.biol.48:443(1970), the search similarity method of Pearson and Lipman, Proc.Natl.Acad.Sci.USA 85:2444(1988), the computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin genetics software package, genetics computer group, 575Science Dr., Madison, Wis) or by detection (instraction).

By e.g., Zuker, Science 244:48-52 (1989); jaeger et al, Proc.Natl.Acad.Sci.USA 86:7706-10 (1989); the algorithms disclosed in Jaeger et al, Methods Enzymol.183:281-306(1989), which are incorporated herein by reference, can achieve the same type of identity to nucleic acids for at least the materials involved in nucleic acid alignment. It is understood that generally any method may be used, and in some cases the results of these various methods may differ, but those skilled in the art understand that if identity is found using at least one of these methods, the sequence will be said to have that identity and is intended to be disclosed herein.

In some cases, as described in more detail in the method section below, the therapeutic compositions disclosed herein can be manufactured using genetic material (e.g., DNA and/or mRNA) isolated and/or purified from immune cells (e.g., B cells, including memory B cells) obtained using the methods disclosed herein. Once such genetic material is obtained, methods of using it to make the therapeutic compositions disclosed herein are known in the art and/or summarized below.

In some cases, the peptide may include a detectable label. As used herein, "label" refers to a moiety having at least one element, isotope, or functional group incorporated into the moiety that enables the peptide to which the label is attached to be detected. The labels may be directly linked (i.e., via a bond) or may be linked by a linker (e.g., such as, for example, a cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkylene group, a cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenylene group, a cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynylene group, a cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkylene group, a cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenylene group, a cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynylene group, a substituted or unsubstituted arylene group, a substituted or unsubstituted heteroarylene group, or a substituted or unsubstituted acylene group, or any combination thereof, which may constitute a linker). The label may be attached to the peptide at any position that does not interfere with the biological activity or properties of the polypeptide of the invention being detected.

The marker may include: a label comprising an isotopic moiety,the isotope may be a radioactive isotope or a heavy isotope, including but not limited to²H、³H、¹³C、¹⁴C、¹⁵N、³¹P、³²P、³⁵S、⁶⁷Ga、^99mTc(Tc-99m)、¹¹¹In、¹²³I、¹²⁵I、¹⁶⁹Yb and¹⁸⁶re; a label comprising an immunological or immunoreactive moiety, which may be an antibody or antigen, which may be bound to an enzyme (e.g. horseradish peroxidase); a label that is colored, luminescent, phosphorescent, or includes a fluorescent moiety (e.g., the fluorescent label FITC); a label having one or more photoaffinity moieties; a label having a ligand moiety with one or more known binding partners (e.g., biotin-streptavidin, FK506-FKBP, etc.).

In some cases, the label may comprise one or more photoaffinity moieties in order to directly resolve intermolecular interactions in a biological system. A variety of known luminophores (photophores) may be employed, most of which rely on the photoconversion of diazo compounds, azides, or diazirines to nitrenes or carbenes (see, e.g., bayer, h., photophered Reagents in Biochemistry and Molecular Biology (1983), Elsevier, Amsterdam, the entire contents of which are incorporated herein by reference). In certain embodiments of the invention, the photoaffinity labels used are ortho-, meta-and para-azidobenzoyl substituted with one or more halogen moieties, including but not limited to 4-azido-2, 3,5, 6-tetrafluorobenzoic acid.

The label may also be or may act as an imaging agent. Exemplary imaging agents include, but are not limited to, those used for Positron Emission Tomography (PET), Computer Assisted Tomography (CAT), single photon emission computed tomography, x-ray, fluoroscopy, and Magnetic Resonance Imaging (MRI); an antiemetic agent; and a contrast agent. Exemplary diagnostic agents include, but are not limited to, fluorescent moieties, luminescent moieties, magnetic moieties; gadolinium chelates (e.g. gadolinium chelates containing DTPA, DTPA-BMA, DOTA and HP-DO 3A), iron chelates, magnesium chelates, manganese chelates, copper chelates, iron chelates, and iron chelates,Chromium chelates, iodine-based materials useful for CAT and x-ray imaging, and radionuclides. Suitable radionuclides include, but are not limited to¹²³I、¹²⁵I、¹³⁰I、¹³¹I、¹³³I、¹³⁵I、⁴⁷Sc、⁷²As、⁷²Se、⁹⁰Y、⁸⁸Y、⁹⁷Ru、¹⁰⁰Pd、¹⁰¹mRh、¹¹⁹Sb、¹²⁸Ba、¹⁹⁷Hg、²¹¹At、²¹²Bi、²¹²Pb、¹⁰⁹Pd、¹¹¹In、⁶⁷Ga、⁶⁸Ga、⁶⁷Cu、⁷⁵Br、⁷⁷Br、⁹⁹mTc、¹⁴C、¹³N、¹⁵O、³²P、³³P and¹⁸F。

fluorescent and luminescent moieties include, but are not limited to, a variety of different organic or inorganic small molecules commonly referred to as "dyes", "labels" or "indicators". Examples include, but are not limited to, fluorescein, rhodamine, acridine dyes, Alexa dyes, cyanine dyes, and the like. The fluorescent and luminescent moieties may include a variety of naturally occurring proteins and derivatives thereof, such as genetically engineered variants. For example, fluorescent proteins include Green Fluorescent Protein (GFP), enhanced GFP, red, blue, yellow, cyan, and sapphire fluorescent proteins, reef coral fluorescent proteins, and the like. Luminescent proteins include luciferase, aequorin, and derivatives thereof. Many Fluorescent and luminescent dyes and proteins are known in the art (see, e.g., U.S. patent publication 2004/0067503; Valeur, B., "Molecular Fluorescence: Principles and Applications", John Wiley and Sons, 2002; and Handbook of Fluorescence Probes and Research Products, Molecular Probes, 9 th edition, 2002).

The peptides used in the vaccine compositions of the present invention may be manufactured synthetically. In certain embodiments, one or more peptide bonds are replaced, for example to improve the physiological stability of the peptide, by: a retro-inverso bond (C (O) -NH); reduction of amide bond (NH-CH)₂) (ii) a Thiomethylene linkage (S-CH)₂Or CH₂-S); oxygen methylene bond (O-CH)₂Or CH₂-O); ethylene linkage(CH₂-CH₂) (ii) a Thioamide bond (C (S) -NH); trans-olefinic linkage (CH ═ CH); a fluoro-substituted trans-olefinic linkage (CF ═ CH); ketomethylene linkage (C (O) -CHR or CHR-C (O), wherein R is H or CH₃) (ii) a And fluoro-ketomethylene linkage (C (O) -CFR or CFR-C (O), wherein R is H or F or CH₃)。

In certain embodiments, the peptide is modified by one or more of acetylation, amidation, biotinylation, cinnamoylation, farnesylation, luciferin (fluorination), formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr, or Thr), stearoylation, succinylation, and sulfonylation.

In one embodiment, the at least one peptide or plurality of peptides is conjugated to a carrier protein. In one embodiment, the carrier protein is selected from tetanus toxin and diphtheria toxin. In another embodiment, the peptide is modified to extend in vivo half-life by protection against peptidase activity, for example as described in US 2009/0175821. In one embodiment, the peptide or modified peptide is further conjugated to polyethylene glycol (PEG), alkyl groups (e.g., C1-C20 linear or branched alkyl groups), fatty acid groups, and combinations thereof.

In one embodiment, the plurality of peptides retain native secondary structure, e.g., are short disulfide-linked loops. In another embodiment, the secondary structure is generated in the form of a loop using a disulfide bond or by exposing the peptide to a chemical linker or cross-linking agent.

In one embodiment, the vaccine composition comprises a viral capsid protein engineered to display the at least one peptide or plurality of peptides on its surface. In one embodiment, the viral capsid protein is a hepatitis B capsid protein, such as described in Proc Natl Acad Sci U S A.1999Mar 2; 96(5): 1915-20.

In one embodiment, the at least one peptide or plurality of peptides is contained in a micelle or nanoparticle structure. The use of micelles may be advantageous, for example, in maintaining the secondary structure of the peptide, as described in J.Am.chem.Soc.,1998,120(39), p.9979-9987.

Stent embodiments

In one embodiment, the vaccine composition comprises or is in the form of a protein scaffold and the at least one peptide or plurality of peptides is contained within the scaffold. A particularly preferred scaffold is a porous poly-lactide-co-glycolide (PLG) polymer scaffold. In one embodiment, the scaffold further comprises one or both of a GM-CSF protein and a Toll-like receptor agonist. In one embodiment, the Toll-like receptor agonist comprises or consists of an unmethylated CpG oligonucleotide (TLR9 agonist). The scaffold may also contain autologous tumor cell lysates, wherein the subject is treated with autologous reference (i.e., the subject's own tumor cell lysates). In one embodiment, the stent is a WDVAX stent as described in US 2013/0202707, WO 2011/063336, and US 2012/0100182. The scaffold also describes Nature Materials, DOI:10.1038/NMAT2357 and Science transformation Medicine, Sci Transl Med 1,8ra19(2009), published online on 1/11/2009; 10.1126/scitranslim.3000359.

Additives and adjuvants

The vaccine composition of the present invention may further comprise one or more pharmaceutically acceptable additives or adjuvants. In one embodiment, the vaccine composition does not comprise an adjuvant. In one embodiment, the one or more adjuvants are selected from the group consisting of oil-based adjuvants, CpG DNA adjuvants, mineral salt gel adjuvants, particulate adjuvants, mucosal adjuvants, and cytokines.

Adjuvants may comprise any number of delivery systems, such as mineral salts, surfactants, synthetic microparticles, oil-in-water emulsions, immunostimulatory complexes, liposomes, virosomes, and virus-like particles. The adjuvant further comprises one or more immune response potentiators such as microbial derivatives (e.g., bacterial products, toxins such as cholera toxin and thermolabile toxins from e.coli, lipids, lipoproteins, nucleic acids, peptidoglycans, carbohydrates, peptides), cells, cytokines (e.g., dendritic cells, IL-12, and GM-CSF), hormones, and small molecules. Contemplated adjuvants include, but are not limited to, oil-based adjuvants (e.g., Freund's adjuvant), CpG oligonucleotides (see Klinman 2003 Expert Rev. vaccines 2:305-15), aluminum salt adjuvants, calcium salt adjuvants, emulsions, and surfactant-based formulations (e.g., MF59, ASO2, Montanide, ISA-51, ISA-720, and QA 21). A review of the improvements in vaccine adjuvants is found in Pashine et al 2005, Nature Med.11(4): S63-S68.

In one embodiment, the adjuvant comprises or consists of one or more Toll-like receptor (TLR) agonists. In one embodiment, the TLR agonist is a pathogen-associated agonist selected from the group consisting of triacylglycerides (gram-positive bacteria), peptidoglycans (gram-positive bacteria), bacterial lipoproteins, lipoteichoic acids, LPS (porphyromonas gingivalis, leptospira interrogans), GPI-anchored proteins (trypanosoma cruzi), neisserial porins (neisserial porins), hemagglutinin (MV), phosphomannan (phosphomannans) (candida), LAM (mycobacteria), ssRNA viruses (WNV), dsRNA viruses (RSV, MCMV), LPS (gram-negative bacteria), F-proteins (RSV), mannans (candida), phosphoinositide (trypanosoma), envelope proteins (RSV and MMTV), flagellins (flagellar), phenol-soluble regulatory proteins (staphylococcus epidermidis), diacyllipopeptides (mycoplasma), LTA (streptococcus), zymosan (saccharomyces), and (zymosan), Viral ssRNA (influenza, VSV, HIV, HCV), ssRNA from RNA viruses, dsDNA viruses (HSV, MCMV), hemozoin (plasmodium), and unmethylated CpG DNA (bacteria and viruses).

In one embodiment, the TLR agonist is selected from the group consisting of Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I: C; u, AGP, MPL A, RC-529, MDF2 β, CFA, flagellin, MALP-2, Pam2Cys, FSL-1, guanosine analogs, imidazoquinolines (e.g., imiquimod, guanylquinoline, and so on),R848, resiquimod) Loxoribine, imidazoquinoline, loxoribine, ssPolyU, 3M-012 and CpG-oligonucleotides.

Preparation

The vaccine compositions of the present invention may be formulated using one or more physiologically acceptable carriers or excipients. For example, when the composition is formulated as a liquid, it may comprise sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid. In one embodiment, the formulation is for intradermal or subcutaneous administration. In one embodiment, the formulation is for inhalation or insufflation (either through the mouth or nose). In one embodiment, the formulation is for oral, buccal, parenteral, vaginal or rectal administration. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

Preferably, the vaccine composition is formulated to provide improved chemical stability of the peptide component during storage and transport. For example, in one embodiment, the formulation prevents or reduces oligomerization of the peptide. In another embodiment, the agent prevents or reduces oxidation of an amino acid residue of the peptide. The formulation may be lyophilized or may be a liquid formulation.

In one embodiment, the composition is formulated for injection. In a preferred embodiment, the composition is a sterile lyophilized formulation, substantially free of contaminating cellular material, chemicals, viruses, or toxins. In particular embodiments, the formulation for injection is provided in a sterile single dose container. The formulation may or may not contain added preservatives. Liquid preparations may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents (formulating agents) such as suspending, stabilizing and/or dispersing agents.

In one embodiment, the formulation comprises liposomes.

In one embodiment, the vaccine composition of the invention is formulated with one or more other therapeutic agents for the treatment of cancer.

The vaccine compositions of the present invention are pharmaceutical compositions and may include one or more pharmaceutically acceptable carriers, additives or vehicles. In one embodiment, the one or more pharmaceutically acceptable carriers, additives or vehicles are selected from the group consisting of ion exchangers, alumina, aluminum stearate, lecithin, Self Emulsifying Drug Delivery Systems (SEDDS) such as D-I-tocopheryl polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tween (Tween) or other similar polymer delivery matrices, serum proteins such as human serum albumin, buffer substances such as phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene-polyoxyethylene-glycol-based polymers, polyethylene glycol-polyoxyethylene glycol-1000, sodium stearate, sodium stearate, sodium stearate, sodium stearate, sodium stearate, polyethylene glycol and lanolin. Cyclodextrins, such as I-, theta-, and K-cyclodextrins, may also be advantageously used to enhance delivery of the compounds of the formulations described herein.

The vaccine compositions of the invention may also contain pharmaceutically acceptable acids, bases or buffers to improve the stability of the formulated compound or its delivery form.

In one embodiment, the vaccine composition of the invention is in the form of a solution or powder for inhalation and/or nasal administration. Such compositions may be formulated according to techniques known in the art using suitable dispersing or wetting agents such as, for example, tween 80 and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that may be used are, inter alia, mannitol, water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially the polyoxyethylated versions thereof. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and/or suspensions. Other commonly used surfactants such as tweens and spans (Span) and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes.

In one embodiment, the vaccine composition of the present invention is in the form of an orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral administration, carriers which are commonly used include lactose and corn starch. Lubricating agents such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

Methods of treatment and administration

The vaccine composition of the present invention can be used for preventing and treating cancer. Accordingly, the present invention provides methods of preventing cancer in a subject at risk of developing cancer and methods of treating cancer in a subject in need of such treatment. In one embodiment, the cancer is selected from the group consisting of prostate cancer, multiple myeloma, glioblastoma multiforme, and melanoma. In one embodiment, the cancer is melanoma.

In one embodiment, the vaccine composition of the invention is administered to a subject suffering from a cancer associated with MICA overexpression. In one embodiment, the cancer is selected from melanoma, lung, breast, kidney, ovary, prostate, pancreas, stomach, and colon cancers, lymphomas, or leukemias. In one embodiment, the cancer is melanoma. In one embodiment, the cancer is a plasma cell malignancy, e.g., Multiple Myeloma (MM) or a precancerous condition of plasma cells. In some embodiments, the subject has been diagnosed with or is predisposed to cancer.

The vaccine compositions of the present invention may be administered alone or as part of a therapeutic regimen or combination therapy as described below. The vaccine compositions of the invention may also be administered alone, or in multiple administrations, for example in a prime-boost strategy. In this respect, the term "prime-boost" refers to the sequential use of two different immunogens. The two different immunogens are typically administered sequentially after a period of time, such as 10 to 30 days or 10 to 60 days. In one embodiment, the period of time is 2 to 4 weeks. Thus, for example, in one embodiment, a vaccine composition of the invention is administered at time 0 and a second vaccine composition of the invention (comprising a different immunogen) is administered after a period of time, e.g., 10 to 30 days, 10 to 60 days, or 2 to 4 weeks.

In one embodiment, one or more different vaccine compositions of the invention are administered to a subject at multiple sites as described in US 8,110,196. Preferably, each site drains to a lymph node or group of lymph nodes. In one embodiment, the vaccine composition of the invention is administered to multiple sites draining to two or more lymph nodes selected from the group consisting of head and neck lymph nodes, axillary lymph nodes, tracheobronchial lymph nodes, parietal lymph nodes, gastric lymph nodes, ileocecal lymph nodes, and inguinal and subgingoinal (subinguinal) lymph nodes. In another embodiment, the site is selected from the group consisting of right arm, left arm, right thigh, left thigh, right shoulder, left shoulder, right breast, left breast, abdomen, right hip, and left hip. In one embodiment, the site is or drains to a non-enveloped lymph tissue cluster selected from the group consisting of tonsils, adenoids, appendices, and peyer's patches. In one embodiment, the vaccine composition of the invention is administered to a site draining into the spleen.

In one embodiment, each vaccine composition is administered by a route independently selected from the group consisting of intradermal, subcutaneous, transdermal, intramuscular, oral, rectal, vaginal, by inhalation, and combinations thereof. In one embodiment, at least one composition is injected directly into lymph nodes, lymph node clusters, or non-enveloped lymph tissue clusters at different anatomical sites.

The methods of the invention include any suitable route of administration, such as intradermal, subcutaneous, intravenous, intramuscular, or mucosal. Mucosal routes of administration include, but are not limited to, oral, rectal, vaginal and intranasal. In a preferred embodiment, at least one composition is administered transdermally, intradermally, subcutaneously, orally, rectally, vaginally, or by inhalation. Any route approved by the U.S. Food and Drug Administration (FDA) may be used in the vaccine compositions of the present invention. An exemplary method of administration is described in the FDA CDER Data Standards Manual, version number 004 (which is available at FDA. grave/CDER/dsm/DRG/drg00301. htm).

Preferably, the route of administration is selected so as to target the composition to a specific site, for example by direct injection to a lymph node or lymph node cluster, by oral administration to target the gastric lymph node, by anal administration to target the rectal lymph node, by inhalation or aerosol to target the pulmonary lymph node, or by any other suitable route of administration.

Where the method of the invention comprises administration of the vaccine composition to multiple sites, the compositions are preferably administered substantially simultaneously, for example within one to eight hours or during the same visit. In one embodiment, each composition is administered within one to two hours, within one to three hours, within one to four hours, or within one to five hours.

When the vaccine composition is in the form of a scaffold, the method of vaccinating a subject comprises implanting the scaffold composition into the subject, preferably subcutaneously. In certain embodiments, the method of vaccinating a subject may comprise implanting or injecting the scaffold vaccine composition in two or more regions of the subject's anatomy.

In one embodiment, the methods of the invention further comprise administering to the subject an antigen presenting cell that has been primed with at least one MIC peptide selected from SEQ ID NOS: 2-13. In a preferred embodiment, the antigen presenting cell is a dendritic cell.

In one embodiment, the method further comprises administering one or more adjuvants to the subject. In one embodiment, the one or more adjuvants are selected from the group consisting of oil-based adjuvants, CpG DNA adjuvants, mineral salt gel adjuvants, particulate adjuvants, mucosal adjuvants, and cytokines. Such adjuvants may be formulated with the compositions of the invention, or administered separately from the composition, e.g., prior to, concurrently with, or subsequent to administration of the composition to a subject.

The methods disclosed herein can be applied to a wide range of species, such as humans, non-human primates (e.g., monkeys), horses, cows, pigs, sheep, deer, elk, goats, dogs, cats, ferrets, rabbits, guinea pigs, hamsters, rats, and mice.

The term "treating" or "treating" as used herein refers to partially or completely alleviating, inhibiting, ameliorating and/or soothing a disease or disorder in a subject. In some cases, the treatment can result in the sustained absence of the disease or disorder from which the subject suffers.

Generally, the method comprises selecting a subject susceptible to or at risk of a disorder or disease. In some cases, the subject may be treated for a disorder or disease with a pharmaceutical composition disclosed herein. For example, in some cases, the method comprises selecting a subject having cancer, e.g., wherein the subject's cancer can be treated by targeting one or both of MICA and/or angiopoietin-2 (angioetin-2).

In some cases, the method of treatment can include a single administration, multiple administrations, and repeat administrations as needed to prevent or treat the disease or disorder from which the subject is suffering. In some cases, a method of treatment can include assessing a subject's disease level prior to, during, and/or after treatment. In some cases, treatment may continue until a decrease in the subject's disease level is detected.

The term "administration" as used herein refers to implanting, absorbing, ingesting, injecting or inhaling the peptide of the present invention, in whatever form. In some cases, one or more of the peptides disclosed herein can be administered to a subject topically (e.g., nasally) and/or orally. For example, the methods herein comprise administering an effective amount of a compound or compound composition to achieve a desired or specified effect. The specific dose and treatment regimen for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, the condition or symptom, the patient's disposition to the disease, condition or symptom, and the judgment of the treating physician.

After administration, the subjects can be evaluated to detect, assess or determine their disease level. In some cases, treatment can be continued until a change (e.g., a decrease) in the level of disease in the subject is detected.

Upon improvement of a patient's condition (e.g., alteration (e.g., reduction) of a subject's disease level), a maintenance dose of a compound, composition, or combination of the invention can be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced to a level that maintains an improved condition, depending on the symptoms. However, patients may require intermittent treatment for extended periods of time when any recurrence of disease symptoms occurs.

In some cases, the present disclosure provides methods of detecting immune cells, e.g., B cells and/or memory B cells, from a human subject. Such methods may be used, for example, to monitor the level of immune cells, e.g., B cells and/or memory B cells, in a human subject, e.g., after an event. Exemplary events may include, but are not limited to, detection of disease, infection; administering a therapeutic composition, administering a therapeutic agent or regimen, administering a vaccine, inducing an immune response, as disclosed herein. Such methods may be used clinically and/or for research.

Effective amount and dosage

In one embodiment, an effective amount of a vaccine composition of the invention is an amount sufficient to reduce the severity of cancer in a subject having cancer, or an amount sufficient to reduce or ameliorate the severity of one or more symptoms thereof, an amount sufficient to prevent cancer progression, an amount sufficient to prevent further metastasis of cancer, an amount sufficient to cause clinical regression of cancer, or an amount sufficient to enhance or improve the therapeutic effect of another therapy or therapeutic agent administered concurrently with, prior to, or subsequent to a vaccine composition of the invention.

Symptoms of cancer are well known to those skilled in the art and include, but are not limited to, unusual nevus characteristics, changes in the appearance of the nevus (including asymmetry, borders, color, and/or diameter), newly pigmented skin areas, unusual nevus, subungual darkened areas, breast bumps, nipple changes, breast cysts, breast pain, death, wasting, fatigue, excessive fatigue, difficulty eating, loss of appetite, chronic cough, severe dyspnea, hemoptysis, hematuria, hematochezia, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal distension, bloating, abdominal fluid, vaginal bleeding, constipation, abdominal distension, colonic perforation, acute peritonitis (infection, fever, pain), pain, hematemesis, profuse sweating, fever, hypertension, anemia, diarrhea, jaundice, dizziness, chills, muscle spasms, colonic metastases, abdominal distension, abdominal pain, abdominal distension, abdominal pain, abdominal distension, abdominal pain, abdominal distension, abdominal pain, abdominal distension, abdominal pain, abdominal distension, abdominal pain, abdominal distension, abdominal pain, abdominal distension, abdominal pain, abdominal distension, abdominal pain, abdominal distension, abdominal pain, abdominal, Lung, bladder, liver, bone, kidney and pancreas metastases, dysphagia, etc.

In one embodiment, an effective amount of a vaccine composition of the invention is an amount sufficient to generate an antibody secreting B cell or cytotoxic T cell mediated immune response against one or more peptides of a vaccine composition of the invention. In one embodiment, an effective amount of a vaccine composition of the invention is an amount sufficient to generate an antibody-secreting B cell or cytotoxic T cell mediated immune response against a cancer cell. The ability of the vaccine composition of the invention to elicit an immune response can be determined using any conventional method available to those skilled in the art. In one embodiment, an effective amount of each composition is an amount sufficient to produce a cytotoxic T cell response in the subject, e.g., as measured by a mixed lymphocyte T cell assay.

In one embodiment, an effective amount of a vaccine composition administered to a subject or at a particular site of a subject is an amount that delivers 1 to 1000 micrograms of one or more peptides of the composition. In one embodiment, the amount of peptide is 1 to 100 micrograms, 1 to 200 micrograms, 1 to 300 micrograms, 1 to 400 micrograms, 1 to 500 micrograms, 1 to 600 micrograms, 1 to 700 micrograms, 1 to 800 micrograms, or 1 to 900 micrograms. In another embodiment, the amount of peptide is 1 to 10 micrograms, 1 to 20 micrograms, 1 to 30 micrograms, 1 to 40 micrograms, 1 to 50 micrograms, 1 to 60 micrograms, 1 to 70 micrograms, 1 to 80 micrograms, or 1 to 90 micrograms. Preferably, the total amount of peptide administered to the subject does not exceed 5 mg, and most preferably the total amount does not exceed 2 mg.

Combination therapy

The invention also provides a method for treating or preventing cancer, the method comprising administering to a subject in need thereof a vaccine composition of the invention, and one or more additional therapeutic agents or treatment regimens. In one embodiment, the vaccine composition of the invention is administered as part of a treatment regimen comprising surgery, a chemotherapeutic agent, or radiation therapy, immunotherapy, or any combination of the foregoing.

In one embodiment, the treatment regimen comprises or further comprises one or more immunostimulatory agents. In one embodiment, the one or more immunostimulatory agents are selected from anti-CTLA-4 antibodies or peptides, anti-PD-1 antibodies or peptides, anti-PDL-1 antibodies or peptides, anti-OX 40 (also known as CD134, TNFRSF4, ACT35 and/or TXGP1L) antibodies or peptides, anti-GITR (also known as TNFRSF18, AITR and/or CD357) antibodies or peptides, anti-LAG-3 antibodies or peptides and/or anti-TIM-3 antibodies or peptides.

In one embodiment, the one or more immunostimulatory agents are selected from the group consisting of anti-MICA antibodies described in WO 2013/049517 or WO 2008/036981. In one embodiment, the one or more immunostimulatory agents are selected from CM33322 Ab4, CM33322 Ab28, and CM33322 Ab29, which are described in U.S. provisional application nos. 61/792,034 and 61/913,198 and U.S. application No. 14/025,573.

In one embodiment, the therapeutic regimen comprises or further comprises one or more cytokines. In one embodiment, the vaccine composition of the invention comprises one or more cytokines. In one embodiment, the at least one cytokine is an interleukin or an interferon. In one embodiment, the at least one cytokine is an interleukin selected from the group consisting of IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, and IL-18. In another embodiment, the at least one cytokine is an interferon selected from IFN α, IFN β and IFN γ.

In one embodiment, the vaccine composition of the present invention is administered as part of a treatment regimen comprising administering to a subject at least one chemotherapeutic agent selected from the group consisting of a histone deacetylase inhibitor ("HDAC") inhibitor, a proteasome inhibitor, an alkylating agent, and a topoisomerase inhibitor.

In one embodiment, the chemotherapeutic agent is AN inhibitor selected from the group consisting of hydroxamic acid, vorinostat (zoinuza), suberoylanilide hydroxamic acid (SAHA) (Merck), trichostatin a (tsa), LAQ824(Novartis), panobinostat (LBH589) (Novartis), belinostat (PXD101) (CuraGen), ITF 7 italfaco SpA (Cinisello), cyclotetrapeptide, depsipeptide (demipide) (romidepsin, FK228) (glucose Pharmaceuticals), benzamide, entinostat (sn275-275/MS-275) (Syndax Pharmaceuticals), MGCD0103(Celgene), short chain aliphatic acids, valproic acid, phenyl butyrate, AN-9, pivanex (pigment pharmaceutical), CHR-96 (therapeutic 3996 (therapy), and chroramics 2845 (HDACs).

In one embodiment, the chemotherapeutic agent is a proteasome inhibitor selected from the group consisting of bortezomib (Millennium Pharmaceuticals), NPI-0052(Nereus Pharmaceuticals), carfilzomib (PR-171) (Onyx Pharmaceuticals), CEP 18770, and MLN 9708.

In one embodiment, the chemotherapeutic agent is an alkylating agent, such as melphalan.

In one embodiment, the chemotherapeutic agent is a topoisomerase inhibitor such as doxorubicin (doxorubicin).

In one embodiment, the therapeutic regimen comprises or further comprises one or more of chemotherapy, radiation therapy, cytokines, chemokines and other biological signaling molecules, tumor specific vaccines, cellular cancer vaccines (e.g., GM-CSF transduced cancer cells), tumor specific monoclonal antibodies, autologous and allogeneic stem cell salvage (e.g., to increase graft versus tumor effect), other therapeutic antibodies, molecular targeted therapies, anti-angiogenic therapies, infectious agents with therapeutic purposes (such as tumor-localized bacteria), and gene therapy.

Reagent kit

The invention provides a pharmaceutical pack or kit for carrying out the methods or treatment regimens of the invention. In one embodiment, the kit comprises a vaccine composition of the invention in lyophilized form. In one embodiment, the kit comprises the vaccine composition of the invention in the form of a protein scaffold.

In another embodiment, the kit further comprises a cytokine or adjuvant in one or more additional containers.

The composition in each container may be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution or buffered solution, or other pharmaceutically acceptable sterile fluid. Alternatively, the composition may be lyophilized or dried; in such cases, the kit optionally further comprises, in a separate container, a preferably sterile pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.) for reconstituting the composition to form a solution for injection purposes.

In another embodiment, the kit further comprises one or more reusable or disposable devices for administering the drug (e.g., syringes, needles, dispensing pens), preferably packaged in sterile form and/or packaged alcohol tablets. Instructions for use are optionally included for administration of the composition by a clinician or by a patient. The kit may also contain other materials, for example metal or plastic foil, such as a blister pack.

In some embodiments, the present disclosure provides methods of using any one or more of the vaccine compositions disclosed herein (hereinafter denoted as "X") in the following methods:

substance X is used as a medicament in the treatment of one or more diseases or disorders disclosed herein (e.g., cancer, referred to as "Y" in the examples below). Use of substance X for the manufacture of a medicament for the treatment of Y; and substance X for the treatment of Y.

In some cases, the therapeutic compositions disclosed herein can be formulated for sale in the united states, import into the united states, and/or export from the united states.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Examples

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1: design of chimeric proteins with appropriately placed epitopes for MICA antibodies

Two designs are shown in figures 3 and 4. In the first design (figure 3), two key epitopes recognized by human MICA antibodies were placed into unrelated proteins (UL 18 from human CMV) that had similar total immunoglobulin folds. This protein should be particularly useful in cases where the primary immunization with the human MICA α 3 domain is followed by a boost.

In the second design (fig. 4), the smallest protein in which two key epitopes are linked was generated. Because of the close proximity of the N-and C-termini, the protein can be displayed on the surface of a viral capsid, such as a hepatitis b nucleocapsid.

Example 2: therapeutic Activity of human MICA antibodies

Method

The study designApproval by the institutional animal care and use committee (IACUC, protocol ID 08-049) was obtained. Six-week-old male SCID (ICR-Prkdc)^scid) Mice were obtained from Taconic (Hudson, NY). U937 cells were obtained from the American Type Culture Collection (ATCC, Manassas, Va.). For survival experiments, 2X 10⁶Individual cells were injected into the peritoneal cavity of mice first used for the experiment. Tumors were allowed to grow for ten days before mice were randomly selected into the double-blind treatment group. Each treatment group contained ten mice, sufficient to discern survival benefits based on prior laboratory experience. Random selection was based on in vivo imaging of mice, and the treatment groups included mice with similar overall mean signal intensity, showing similar tumor burden. Double blindness will be treated by external laboratory members who have not undergone survival experiments. The investigators administered treatment with a syringe labeled "group a" or "group B". At the end of each survival experiment, the studies were not mixed. Antibody treatment was administered intravenously at 100 micrograms/dose. Animals received three doses per week for a total of three weeks. Mice were bled weekly to detect circulating sMICA. All mice were included in the analysis.

For short-term treatment, 2X 10 subcutaneous implants⁶U937 cells and allowed tumors to grow (establish) for ten days. Mice with palpable tumors were then treated with fully human antibodies (isotypes AML Ab2, Mel Ab28, or Mel Ab29) for one week (3 × 100 μ g). On day eight after the initial treatment, mice were sacrificed and tumors and spleens were excised and tumor mass was recorded. Tumors were cut into small pieces in petri dishes (petri dish) containing 5 ml of digestion medium containing RPMI medium with 2% FBS, 50U/ml collagenase type IV (Invitrogen) and 10U/ml dnase (Roche). The tissue was incubated in digestion medium at 37 ℃ for 2 hours. The tumors were then further dissociated with a mild MACS dispenser (Miltenyi Biotech). The supernatant of the tumor cell suspension was stored for measurement of local sMICA concentration. The cell suspension was filtered through a 70 micron filter and washed three times with PBS. Single cell suspensions were then treated with Zombie Yellow (reactive dye, BioLegend), NKG2D-APC (CX5), perforin-PE (eBioOMAK-D), CD45-PacBlue (30-F11), NKp46-PerCP/Cy5.5(29A1.4), IFN γ -BV711(XMG1.2), NK1.1-BV510(PK136), CD16-APC/Cy7(93) and CD49b-FITC (DX5) for NK cell analysis. All antibodies were from BioLegend except perforin (eBiosciences). Additional separate aliquots of cells were stained with anti-MICA-PE (clone 6D4, BioLegend) for MICA expression.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Human MICA/B antibodies effectively inhibit MICA shedding in the tumor microenvironment

A mouse model for evaluating the therapeutic efficacy of human MICA antibodies was established. Mice do not have MICA or MICB orthologs (orthologs), but the mouse NKG2D receptor recognizes human MICA/B. See Liu et al, 2013,JCI123(10):4410-4422. We implanted human U937 cells, an AML cell line, in SCID mice with NK cells but lacking T cells and B cells. This model enabled us to determine the effect of human MICA antibodies on NK cell-mediated immunity against human tumor cells, but CD 8T cell responses could not be evaluated in this model. MICA antibody AML Ab2 was expressed with a murine IgG2a Fc fragment to enable proper interaction with the murine Fc receptor. The patient antibody has a human IgG1 isotype, which is functionally similar to mouse IgG2 a. Mice were implanted with U937 cells and randomized into double-blind treatment groups ten days later. Treatment over a three week period (3 x 100 micrograms/week) provided significant survival benefit, with 55% survival at day 45 in the treatment group (AML Ab2) compared to 0% in the control group (isotype). See fig. 5A. Mechanistic studies indicate that sMICA becomes undetectable in serum after only two weeks of antibody treatment, whereas sMICA levels rise in the control group. See fig. 5B.

We next investigated the functional effects of treatment with three fully human MICA/B antibodies at an early time point. One week after treatment of SCID mice with subcutaneous tumors, sMICA levels in mice in the AML Ab2, Mel Ab28, and Mel Ab29 treated groups were greatly reduced compared to isotype controls. See fig. 5C. Flow cytometric analysis of tumors also showed significantly increased expression of MICA on the tumor cell surface, reflecting in vitro results. See fig. 5D. These results demonstrate that human MICA/B antibodies effectively inhibit MICA shedding in the tumor microenvironment and thereby increase the density of MICA on tumor cells recognized by cytotoxic lymphocytes.

Human MICA/B antibodies thereby improve both local and systemic NK cell-mediated immunity against tumor cells

We performed further mechanistic studies on tumor infiltrating NK cells at the one week treatment time point. Inhibition of MICA shedding in tumors increased NKG2D surface expression on tumor infiltrating NK cells. See fig. 6A. Antibody treatment also resulted in > 40-fold expansion of tumor infiltrating NK cells, as well as increased expression of NKp46 receptor. See fig. 6B and 6C. Expanded tumor infiltrating NK cells produce greater amounts of IFN γ, a cytokine critical for anti-tumor immunity, and express higher levels of perforin, a key molecule for cytotoxic function. See fig. 6d and 6 e. To determine the cytotoxic potential of NK cells in mice treated with MICA/B antibodies, we evaluated the in vivo killing of YAC-1 cells by splenic NK cells ex vivo. Enhanced killing was observed across all anti-MICA antibody treated mice relative to isotype treated mice. See fig. 6F. The human MICA/B antibody thereby improved NK cell-mediated immunity against tumor cells both locally and systemically.

Sequence listing

<110> Daner-Farber cancer institute Corp

<120> vaccine compositions and methods for restoring function of the NKG2D pathway against cancer

<130> DFCI-082/001WO 322270-2469

<140> PCT/US2015/020694

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Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

85 90 95

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

100 105 110

Pro Ser

<210> 28

<211> 114

<212> PRT

<213> Intelligent people

<400> 28

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

1 5 10 15

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

20 25 30

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

35 40 45

Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

50 55 60

His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

65 70 75 80

Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

85 90 95

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

100 105 110

Pro Ser

<210> 29

<211> 114

<212> PRT

<213> Intelligent people

<400> 29

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

1 5 10 15

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

20 25 30

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

35 40 45

Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

50 55 60

His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

65 70 75 80

Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

85 90 95

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

100 105 110

Pro Ser

<210> 30

<211> 114

<212> PRT

<213> Intelligent people

<400> 30

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Glu Ser Ser Val

1 5 10 15

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

20 25 30

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr

35 40 45

Pro Arg Asn Ile Ile Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser

50 55 60

His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

65 70 75 80

Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Arg Gly Glu Glu Gln Arg

85 90 95

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

100 105 110

Pro Ser

<210> 31

<211> 114

<212> PRT

<213> Intelligent people

<400> 31

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

1 5 10 15

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

20 25 30

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

35 40 45

Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

50 55 60

His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

65 70 75 80

Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

85 90 95

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

100 105 110

Pro Ser

<210> 32

<211> 114

<212> PRT

<213> Intelligent people

<400> 32

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

1 5 10 15

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

20 25 30

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

35 40 45

Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

50 55 60

His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

65 70 75 80

Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

85 90 95

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

100 105 110

Pro Ser

<210> 33

<211> 114

<212> PRT

<213> Intelligent people

<400> 33

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

1 5 10 15

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

20 25 30

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

35 40 45

Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

50 55 60

His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

65 70 75 80

Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

85 90 95

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

100 105 110

Pro Ser

<210> 34

<211> 114

<212> PRT

<213> Intelligent people

<400> 34

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Glu Ser Gly Val

1 5 10 15

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

20 25 30

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr

35 40 45

Pro Arg Asn Ile Ile Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser

50 55 60

His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

65 70 75 80

Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Arg Gly Glu Glu Gln Arg

85 90 95

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

100 105 110

Pro Ser

<210> 35

<211> 100

<212> PRT

<213> Intelligent people

<400> 35

Tyr Leu Lys Ser Gly Val Ala Ile Arg Arg Thr Val Pro Pro Met Val

1 5 10 15

Asn Val Thr Cys Ser Glu Val Ser Glu Gly Asn Ile Thr Val Thr Cys

20 25 30

Arg Ala Ser Ser Phe Tyr Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln

35 40 45

Asp Gly Val Ser Leu Ser His Asn Thr Gln Gln Trp Gly Asp Val Leu

50 55 60

Pro Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg

65 70 75 80

Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn

85 90 95

His Gly Thr His

100

<210> 36

<211> 100

<212> PRT

<213> Intelligent people

<400> 36

Tyr Leu Lys Ser Gly Val Ala Ile Arg Arg Thr Val Pro Pro Met Val

1 5 10 15

Asn Val Ile Cys Ser Glu Val Ser Glu Gly Asn Ile Thr Val Thr Cys

20 25 30

Arg Ala Ser Ser Phe Tyr Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln

35 40 45

Asp Gly Val Ser Leu Ser His Asn Thr Gln Gln Trp Gly Asp Val Leu

50 55 60

Pro Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg

65 70 75 80

Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn

85 90 95

His Gly Thr His

100

<210> 37

<211> 100

<212> PRT

<213> Intelligent people

<400> 37

Tyr Leu Lys Ser Gly Val Ala Ile Arg Arg Thr Val Pro Pro Met Val

1 5 10 15

Asn Val Thr Cys Ser Glu Lys Ser Glu Gly Asn Ile Thr Val Thr Cys

20 25 30

Arg Ala Ser Ser Phe Tyr Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln

35 40 45

Asp Gly Val Ser Leu Ser His Asn Thr Gln Gln Trp Gly Asp Val Leu

50 55 60

Pro Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg

65 70 75 80

Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn

85 90 95

His Gly Thr His

100

<210> 38

<211> 100

<212> PRT

<213> Intelligent people

<400> 38

Tyr Leu Lys Ser Gly Val Ala Ile Arg Arg Thr Val Pro Pro Met Val

1 5 10 15

Asn Val Thr Cys Ser Glu Val Ser Glu Gly Asn Ile Thr Val Thr Cys

20 25 30

Arg Ala Ser Ser Phe Tyr Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln

35 40 45

Asp Gly Val Ser Leu Ser His Asn Thr Gln Gln Trp Gly Asp Val Leu

50 55 60

Pro Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg

65 70 75 80

Gln Gly Glu Glu Gln Lys Phe Thr Cys Tyr Met Glu His Ser Gly Asn

85 90 95

His Gly Thr His

100

<210> 39

<211> 100

<212> PRT

<213> Intelligent people

<400> 39

Tyr Leu Lys Ser Gly Val Ala Ile Arg Arg Thr Val Pro Pro Met Val

1 5 10 15

Asn Val Thr Cys Ser Glu Val Ser Glu Gly Asn Ile Thr Val Thr Cys

20 25 30

Arg Ala Ser Ser Phe Tyr Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln

35 40 45

Asp Gly Val Ser Leu Ser His Asn Thr Gln Gln Trp Gly Asp Val Leu

50 55 60

Pro Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg

65 70 75 80

Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn

85 90 95

His Ser Thr His

100

<210> 40

<211> 100

<212> PRT

<213> Intelligent people

<400> 40

Tyr Leu Lys Ser Gly Val Ala Ile Arg Arg Thr Val Pro Pro Met Val

1 5 10 15

Asn Val Thr Cys Ser Lys Val Ser Glu Gly Asn Ile Thr Val Thr Cys

20 25 30

Arg Ala Ser Ser Phe Tyr Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln

35 40 45

Asp Gly Val Ser Leu Ser His Asn Thr Gln Gln Trp Gly Asp Val Leu

50 55 60

Pro Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg

65 70 75 80

Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn

85 90 95

His Ser Thr His

100

<210> 41

<211> 114

<212> PRT

<213> Intelligent people

<400> 41

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

1 5 10 15

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

20 25 30

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

35 40 45

Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

50 55 60

His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

65 70 75 80

Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

85 90 95

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

100 105 110

Pro Ser

<210> 42

<211> 90

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 42

His Pro Val Val Lys Gly Gly Val Arg Ser Gln Ala Ala Asn Arg Ala

1 5 10 15

Glu Ala Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu

20 25 30

Ser Trp Arg Gln Asp Gly Val Ser Leu Ser His Asp Thr Gln Gln Trp

35 40 45

Gly Asp Val Leu Pro Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala

50 55 60

Thr Arg Ile Cys Ser Asn Gln Asn Tyr Thr Cys Tyr Val Thr His Gly

65 70 75 80

Asn Trp Thr Val Glu Ile Pro Ile Ser Val

85 90

<210> 43

<211> 92

<212> PRT

<213> Intelligent people

<400> 43

Pro Pro Met Val Asn Val Thr Arg Ser Glu Ala Ser Glu Gly Asn Ile

1 5 10 15

Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu

20 25 30

Ser Trp Arg Gln Asp Gly Val Ser Leu Ser His Asp Thr Gln Gln Trp

35 40 45

Gly Asp Val Leu Pro Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala

50 55 60

Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu

65 70 75 80

His Ser Gly Asn His Ser Thr His Pro Val Pro Ser

85 90

<210> 44

<211> 91

<212> PRT

<213> Intelligent people

<400> 44

His Pro Val Val Lys Gly Gly Val Arg Asn Gln Asn Asp Asn Arg Ala

1 5 10 15

Glu Ala Phe Cys Thr Ser Tyr Gly Phe Phe Pro Gly Glu Ile Gln Ile

20 25 30

Thr Phe Ile His Tyr Gly Asp Lys Val Pro Glu Asp Ser Glu Pro Gln

35 40 45

Cys Asn Pro Leu Leu Pro Thr Leu Asp Gly Thr Phe His Gln Gly Cys

50 55 60

Tyr Val Ala Ile Phe Ser Asn Gln Asn Tyr Thr Cys Arg Val Thr His

65 70 75 80

Gly Asn Trp Thr Val Glu Ile Pro Ile Ser Val

85 90

<210> 45

<211> 46

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 45

Ile Thr Val Thr Ser Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr

1 5 10 15

Leu Ser Trp Leu Ser His Asp Thr Gln Cys Trp Gly Asp Val Leu Pro

20 25 30

Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Cys Thr Arg Ile

35 40 45