pHLIP-Mediated Carbohydrate Tethering at Cell Surfaces to Induce Immune Response
20200262881 ยท 2020-08-20
Inventors
- Yana K. Reshetnyak (Saunderstown, RI, US)
- Oleg A. Andreev (Saunderstown, RI, US)
- Anna Moshnikova (Warwick, RI, US)
- Donald M. Engelman (New Haven, CT)
Cpc classification
A61K9/0019
HUMAN NECESSITIES
A61K47/65
HUMAN NECESSITIES
C07K14/705
CHEMISTRY; METALLURGY
A61K47/646
HUMAN NECESSITIES
C07K16/44
CHEMISTRY; METALLURGY
International classification
A61K47/65
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The invention features a compositions and methods for inducing an immune response to targeted cells. The compositions induce targeting of a cell by positioning carbohydrate epitopes on the surface of the cell by conjugation of the epitope to a pH-triggered membrane peptide (pHLIP).
Claims
1. A composition comprising a purified carbohydrate epitope and a pHLIP peptide.
2. The composition of claim 1, wherein said composition comprising the formula of Carb-Linker-Pept wherein Carb is a carbohydrate epitope; wherein Linker is a non-cleavable linker compound or a membrane non-inserting end of the pHLIP peptide further comprises an amino acid extension; wherein Pept is a pHLIP peptide comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO:_) or AXDQDNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO:_), where X is a functional group, selected from a lysine, a cysteine, a serine, a threonine, or an Azido-containing amino acid; wherein each - is a covalent bond.
3. The composition of claim 1, wherein said carbohydrate epitope and said peptide are connected by a non-cleavable linker or by an extension of the pHLIP peptide membrane non-inserting terminus.
4. The composition of claim 2, wherein said pHLIP peptide extension is a poly-Glycine peptide.
5. The composition of claim 2, wherein said linker comprises a polyethylene glycol (PEG) polymer, wherein the PEG polymer ranges from 4 to 24 PEG units.
6. The composition of claim 5, wherein said linker comprises a polyethylene glycol polymer.
7. The composition of claim 5, wherein said polymer ranges in size from 200 Daltons to 20 kiloDaltons.
8. The composition of claim 1, wherein said carbohydrate epitope comprises a glycan comprising an N-linked glycan, an O-linked glycan, or any combination thereof.
9. The composition of claim 8, wherein the glycan comprises Galactose--1,3-Galactose or derivatives thereof.
10. The composition of claim 8, wherein the glycan comprises tri-Gal or derivatives thereof.
11. The composition of claim 7, wherein the N-linked glycan and the O-linked glycan comprises the core structure GlcNAc2Man3, Mannose-N-acetylgalactosamine [(Man).sub.3(GlcNAc).sub.2], -rhamnose, Globo H, or sialic acid or derivatives thereof.
12. The composition of claim 1, wherein said carbohydrate epitope comprises a blood antigen.
13. The composition of claim 1, wherein said composition comprises 2 or more pHLIP peptides.
14. The composition of claim 1, wherein said composition comprises 2 or more carbohydrate epitopes.
15. The composition of claim 13, wherein 2 carbohydrate epitopes are linked to a single pHLIP peptide.
16. The composition of claim 13, wherein said composition comprising the formula of Carb-Linker-Pept-Linker-Carb wherein Carb is a carbohydrate epitope; wherein Linker is a polyethylene glycol linker; wherein Pept is a pHLIP peptide comprising the sequence Ac-AKQNDDQNKPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 470) or Ac-AKQNDNDNKPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 479) or ACQNDDQNCPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 471) or ACQNDNDNCPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 480) wherein each - is a covalent bond.
17. A method of inducing an immune response in a diseased tissue in a subject, comprising administering to a subject a composition comprising a carbohydrate epitope and a pHLIP peptide.
18. The method of claim 17, wherein said subject comprises a solid tumor.
19. The method of claim 17, wherein said composition is injected directly into a tumor mass.
20. The method of claim 17, wherein said composition is systemically administered.
21. The method of claim 17, wherein a biological effect of said composition is at least 20% greater than that delivered in the absence of said composition.
22. The method of claim 17, wherein said composition targets preferentially to a diseased tissue compared to a healthy tissue, thereby minimizing damage to said healthy tissue.
23. A method for promoting an immune response in a subject, comprising administering to a subject the composition of claim 1, wherein said method comprises placement of said carbohydrate epitope on tumor cell of said subject.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0088] Using the immune system to combat disease is a therapeutic strategy that is finding increasingly wide applications, including in the treatment of cancers. Endogenous antibodies circulate in serum of healthy humans naturally, without previous immunization. These antibodies can be directed against the individual's own antigens as well as against foreign antigens. Endogenous antibodies are polyreactive and mostly react with low affinity but high avidity. For example, when a cancer cell is decorated with multiple carb epitopes, then the affinity for the antibody binding to the cell will. Moreover, pHLIP (as well as other proteins) has free lateral movement in the membrane bilayer, and the affinity of a single epitope-pHLIP to antibody in blood is low. They target cells by recognizing specific signaling molecules (antigens) found on a cell surface. Carbohydrate (saccharide) antigens recruit endogenous (natural) antibodies to initiate CDC (humoral immunity) and ADCC (cellular immunity). In human serum, endogenous (natural) antibodies in total constitute approximately 10% of total serum and about 1% of circulating B lymphocytes in adults are capable of producing these antibodies.
[0089] Decoration of target cells with carbohydrate epitopes that can recruit endogenous (natural) antibodies and proteins lead to activation of antibody-dependent cellular cytotoxicity and/or activation of the classical complement cascade to assemble a membrane attack complex that promoted the formation of pores in the target cell membrane and resulted in cell death.
[0090] To enhance the abundance of natural antibodies, an immune boost may be performed and the titer can be established by ELISA prior to treatment to ensure the presence of a sufficient amount of specific antibodies in the blood. Also, if needed, immunization followed by a boost is performed to produce sufficient amounts of antibodies against specific carbohydrate epitopes.
[0091] Decoration of target cells comprises the addition of purified epitopes, e.g., purified carbohydrates, to the target cell, e.g., to the surface of the target cell. The target cells, e.g., tumor cells or other diseased cells, are modified by the pHLIP-mediated delivery of such purified carbohydrate epitopes to the cells such that the cells are characterized by the presence of a purified carbohydrate moiety on the cell surface, e.g., decorated. The carbohydrate moiety may be different from those carbohydrate moieties that are present on the target cell prior to modification of the cells as described herein. By virtue of the presence of the delivery mediated by pHLIP, a heterologous carbohydrate is put/displayed at the surface of the target cell.
[0092] A heterologous carbohydrate is one that the target cell did not display on its cell surface prior to treatment using the methods and compositions described herein. For example, the target cell does not make, express, or present on its surface in a naturally-occurring state. Alternatively, the target cell makes, expresses, or displays/presents the carbohydrate prior to intervention/modification and/or treatment; however, the expression or presence on the cell surface is low or undetectable. Treatment according to the invention renders the cell with at least 10%, 20%, 50%, 75%, 2-fold, 5-fold, 10-fold or more of the carbohydrate moiety (purified epitope or antigen) on the surface of the treated (tumor or otherwise diseased) cell. Exemplary carbohydrates are those to which the subject/patient comprises antibodies, e.g., IgG or IgM isotype antibodies, which could are identified by an antibody titer test before administration of the construct.
Decoration of Diseased Cells with Carbohydrate Antigens
[0093] Specific decoration of target cells (diseased cells characterized by acidic cell surface microenvironment) with carbohydrate epitopes to recruit endogenous (natural) antibodies and proteins, to induce an immune response is a useful approach in the treatment of tumors and other diseased tissues. However, the importance for successful treatment (and an advantage of the system described herein) is in the ability to activate an immune response predominantly in diseased tissue (tumors) and guiding the immune reaction away from normal tissues, thereby avoiding adverse/undesirable side effects.
[0094] A selected carbohydrate epitope is delivered to and positioned on cell surfaces (decoration) predominantly in targeted diseased tissues, such as tumors. The delivery to and addition of the carbohydrate epitopes to tumor cells and other diseased (acidic) cells and tissues is a great advantage, because the system selectively induces immune responses predominantly within the diseased tissues. The carbohydrate antigens are preferentially inserted into the cell membranes of diseased, e.g., tumor cells compared to surrounding or bordering normal, e.g., non-tumor, cells. pHLIP peptides conjugated to carbohydrate molecules reliably and effectively accomplish this task.
[0095] Acidic diseased cells (tumor cells) are decorated, e.g., modified, using carbohydrate (saccharide) epitopes conjugated to the pHLIP, so that the pHLIP will target tumors by responding to cell surface acidity, insert into tumor cell membranes, and locate enough amount of specified carbohydrate epitopes on the cell surface for efficient recruitment of natural antibodies and proteins, and induction of immune response predominantly in the diseased tissue.
[0096] pHLIP conjugated to polar carbohydrate molecules are typically cleared by the kidney, where there is minimal or no risk of developing an immune reaction since antibodies (large molecules like IgM and IgG antibodies) are excluded from the kidney by their size.
Carbohydrate-pHLIP Constructs
[0097] General representations of pHLIP compositions/constructs comprising pHLIP peptide and a carbohydrate antigen/epitope for cell surface delivery of the carbohydrate antigen/epitope is shown in
[0098] Compositions include those with the following general structure: [0099] Carb-Linker-Peptide
[0100] Carb comprises a carbohydrate epitope to induce immune response by attracting of endogenous antibodies, which then mediate an immune response that leads to killing of the tumor or otherwise diseased cell. Non-limiting examples of carbohydrate epitopes are described below. Peptide is a pHLIP peptide (a non-limiting example is a pHLIP comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 3), or AXDQDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 475), or AX(Z).sub.nXPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 5), where X is a functional group (e.g., for conjugation purposes), selected from lysine (Lys), cysteine (Cys), serine (Ser), threonine (Thr) an Azido-containing amino acid, and Z indicates any amino acid residue and n is any integer between 1 and 10 and including 1 and 10 (e.g., n
[0101] For example, (Z).sub.n could be QDNDQN (SEQ ID NO: 6) or any combination of polar residues, e.g., D, E, N or Q. In some cases, Peptide is a pHLIP conjugate, where a pHLIP peptide is linked with a therapeutic or drug molecule for intracellular delivery. In some cases, Peptide is a linear or cyclic pH-sensitive peptide.
[0102] Linker comprises a covalent bond or a chemical linker. A non-limiting example of linker is a PEG polymer or a flexible extension of the pHLIP peptide membrane non-inserting end ranging in size from 200 Da to 20 kDa. In some examples, the pHLIP peptide membrane non-inserting end can comprise of, for example from 3 to about 20-30 glycine residues (a poly-Gly). The purpose of a polymer or a polypeptide extension is to position epitope at surface of cells and enhance access of antibodies or proteins for binding with the epitope. The size and hydrophobicity of the linker ensures renal clearance of the construct and does not promote hepatic clearance. Linkers include non-cleavable linkers that are stable in the blood. The carbohydrate epitope(s) is preferably linked to pHLIP peptide(s) via non-cleavable link(s), e.g., covalent bond. In examples, the linker is preferably polar or moderately hydrophobic.
[0103] A compound is characterized as polar if it has a measured log P of less than about 1. For example, polarity and hydrophobicity are characterized as follows. Polar: Log P<0.4; Moderately hydrophobic: 2.5<Log P<0.4; and Hydrophobic: Log P>2.5. The polarity and/or hydrophobicity of a drug or compound to be delivered is measured using methods known in the art, e.g., by determining Log P, in which P is octanol-water partition coefficient. A substance is dissolved into octanol-water mixture, mixed and allowed to come to equilibration. The amount of substance in each (or one) phases is then measured. The measurements themselves can be made in a number of ways known in the art, e.g., by measuring absorbance, or determining the amount using NMR, HPLC, isotopic labeling or other known methods.
[0104] An exemplary construct with a carbohydrate epitope linked to a pHLIP is shown in
[0105] Aspects of the present subject matter relate to the surprising discovery that pH-triggered peptides specifically interact with the lipid bilayer of liposomal and cellular membranes and, as such, when conjugated to a carbohydrate epitope, can decorate the liposome or cell with these carbohydrate epitopes. Moreover, pH-triggered peptides can target acidic tissue, and as such, when conjugated to a carbohydrate epitope, can target it to the surface of cells in acidic diseased tissue. The carbohydrate epitopes can recruit endogenous (natural) antibodies and proteins, and induce an immune response.
[0106] The compositions and methods described herein are a very attractive approach in the treatment of tumors and other diseased tissues. The importance for successful treatment rests in the ability to activate an immune response predominantly in diseased tissue (tumors) and guiding the immune reaction away from normal tissues and avoiding side effects.
[0107] The compositions and methods described herein provide a solution and strategy in which selected carbohydrate (saccharide) epitopes are positioned on cell surfaces predominantly in targeted diseased tissues, such as tumors. The invention provides decoration of targeted acidic diseased cells (tumor cells) using carbohydrate (saccharide) epitopes conjugated to the pHLIP, so that the pHLIP targets tumors by responding to cell surface acidity, inserting into tumor cell membranes, and locating enough of the specified carbohydrate epitopes on the cell surface for efficient recruitment of endogenous or natural antibodies and proteins, and induction of immune response predominantly in the diseased tissue. This can provide a great advantage to selectively induce immune responses predominantly within the diseased tissues.
[0108] The pHLIP peptide conjugated to carbohydrate epitopes (e.g., polar carbohydrate molecules) are typically cleared by kidney, where there is little to no risk of developing an immune reaction since antibodies (e.g., large molecules like IgM and IgG) are excluded from the kidney by their size.
Cellular Immune Responses
[0109] Humoral immunity is an aspect of immunity that is mediated by macromolecules found in extracellular fluids, such as secreted antibodies and complement proteins. The immune system is divided into a more primitive innate immune system and an acquired or adaptive immune system of vertebrates, each of which contains humoral and cellular components. Humoral immunity refers to antibody production and the accessory processes that accompany it, including: Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. It also refers to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and promotion of phagocytosis and pathogen elimination.
[0110] The complement system is a biochemical cascade of the innate immune system that helps clear pathogens from an organism. It is derived from many small blood plasma proteins that work together to disrupt the target cell's plasma membrane leading to cytolysis of the cell. The complement system is involved in the activities of both innate immunity and acquired immunity.
[0111] Activation of this system leads to cytolysis, chemotaxis, immune clearance, and inflammation, as well as the marking of pathogens for phagocytosis. The proteins account for 10% of the serum globulin fraction.
[0112] Three biochemical pathways activate the complement system: the classical complement pathway, the alternate complement pathway, and the mannose-binding lectin pathway. The classical complement pathway typically requires antibodies for activation and is a specific immune response, while the alternate pathway can be activated without the presence of antibodies and is considered a non-specific immune response.
[0113] Immunoglobulins are glycoproteins in the immunoglobulin superfamily that function as antibodies. The terms antibody and immunoglobulin are often used interchangeably. They are found in the blood and tissue fluids, as well as many secretions. In structure, they are large Y shaped globular proteins. In mammals there are five types of antibody: IgA, IgD, IgE, IgG, and IgM. Each immunoglobulin class differs in its biological properties and has evolved to deal with different antigens. Antibodies are synthesized and secreted by plasma cells that are derived from the B cells of the immune system.
[0114] An antibody is used by the acquired immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. By binding their specific antigens, antibodies can cause agglutination and precipitation of antibody-antigen products, prime for phagocytosis by macrophages and other cells, block viral receptors, and stimulate other immune responses, such as the complement pathway.
[0115] An incompatible blood transfusion causes a transfusion reaction, which is mediated by the humoral immune response. This type of reaction, called an acute hemolytic reaction, results in the rapid destruction (hemolysis) of the donor red blood cells by host antibodies. The cause is usually a clerical error, such as the wrong unit of blood being given to the wrong patient. The symptoms are fever and chills, sometimes with back pain and pink or red urine (hemoglobinuria). The major complication is that hemoglobin released by the destruction of red blood cells can cause acute renal failure.
[0116] Another example is organ transplant rejection. The major problem of xenotransplantation (transplantation of organs between different species, such as, for example, pigs to humans) is strong reaction of natural antibodies, predominantly IgG and IgM, to a terminal carbohydrate (Gal) on glycolipids or glycoproteins, which appears to be ubiquitously expressed in most tissues in all species except man, the great apes and old world monkeys. Hyperacute rejection of an organ is mediated by antibody and complement, and results in rapid destruction of the organ.
[0117] The compositions and methods described herein (e.g., the pHLIP peptide conjugated to a carbohydrate epitope on the surface of target cells) can recruit endogenous (natural) antibodies to initiate complement-dependent cytotoxicity. This can lead to the activation of the classical complement cascade to assemble a membrane attack complex that promotes the formation of pores in the target cell membrane and results in cell death. The activation of the complement cascade can further amplify immune response through the release of cytokines and inflammatory mediates. These signaling molecules attract immune cells involved in antibody-dependent cell-mediated toxicity, described below.
Cellular Immunity
[0118] The antibody-dependent cellular cytotoxicity (ADCC), also referred to as antibody-dependent cell-mediated cytotoxicity, is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies. It is one of the mechanisms through which antibodies, as part of the humoral immune response, can act to limit and contain infection.
[0119] ADCC is independent of the immune complement system that also lyses targets but does not require any other cell. ADCC requires an effector cell which classically is known to be natural killer (NK) cells that typically interact with IgG antibodies. However, macrophages, neutrophils and eosinophils can also mediate ADCC, such as eosinophils killing certain parasitic worms known as helminths via IgE antibodies. ADCC is part of the adaptive immune response due to its dependence on a prior antibody response.
[0120] The compositions and methods described herein (e.g., the pHLIP peptide conjugated to a carbohydrate epitope on the surface of target cells) can recruit natural antibodies to initiate ADCC. This recruitment of antibodies leads to the activation of the classical complement cascade to assemble a membrane attack complex that promotes the formation of pores in the target cell membrane and results in cell death. The activation of the complement cascade can further amplify immune responses through the release of cytokines and inflammatory mediators. These signaling molecules attract immune cells involved in ADCC such as neutrophils, macrophages, and NK cells. Immune effector cells, recognizing surface-bound antibodies, initiate ADCC through activating Fc receptors. In human serum, natural antibodies in total constitute approximately 10% of total serum and about 1% of circulating B lymphocytes in adults is capable of producing these antibodies.
Blood Group Antigens
[0121] Blood is classified into different groups according to the presence or absence of molecules called antigens. As described by Dean L., an antigen is any substance to which the immune system can respond (Dean L Blood Groups and Red Cell Antigens: Chapter 2Blood group antigens are surface markers on the red blood cell membrane; National Center for Biotechnology Information; 2005). If the immune system encounters an antigen that is not found on the body's own cells, it will launch an attack against that antigen. Conversely, antigens that are found on the body's own cells are known as self-antigens, and the immune system does not normally attack these. When patients receive blood transfusions, their immune systems will attack any donor red blood cells that contain antigens that differ from their self-antigens. Therefore, ensuring that the antigens of transfused red blood cells match those of the patient's red blood cells is essential for a safe blood transfusion.
[0122] Blood group antigens are either sugars or proteins, and they are attached to various components in the red blood cell membrane. In examples, the antigens of the ABO blood group are sugars. The ABO blood type is controlled by a single gene (the ABO gene) with three types of alleles. The gene encodes a glycosyltransferasethat is, an enzyme that modifies the carbohydrate content of the red blood cell antigens. The antigens of the ABO blood group are produced by a series of reactions in which enzymes catalyze the transfer of sugar units. A person's DNA determines the type of enzymes they have, and, therefore, the type of sugar antigens that end up on their red blood cells.
[0123] Blood group antigens include (A, B, and O (H)). The blood group antigens are specific for all the blood group subtypes. Patients with blood group A have B antibodies in their blood, patients with blood group B have A antibodies in their blood, patients with blood group AB have no antibodies against A and B antigens in their blood, and patients with blood group O have both A and B antibodies in their blood.
[0124] The human ABO blood group system is defined by the presence or absence of specific antigens at blood cell surface. These unique carbohydrate or carbohydrate combinations found on the membrane of red blood cells (RBCs) define a person's blood type. The RBCs of a blood type O individual have on their surface the O-antigen, the sugar fucose, arranged in a long repeating chain. The RBCs of a blood type A individual have on their surface the base sugar fucose plus the carbohydrate N-acetyl galactosamine, the A antigen. The RBCs of a blood type B individual have on their surface the base sugar fucose plus the carbohydrate galactose (also called D-galactosamine), the B antigen. The RBCs of a blood type AB individual have on their surface the base sugar fucose plus both the A and B antigens, i.e. both N-acetyl galactosamine and galactose. These RBC antigens are called isoantigens, a term for proteins or other substances that are present in only some members of a species and therefore able to stimulate antibody production in other members of the same species who lack the antigen. Humans who are exposed to foreign isoantigens, and antigens very similar thereto, produce antibodies that respond to the A and/or B antigens absent from their own RBCs. These are termed isoantibodies, and more specifically, isoagglutinins, the term for antibodies normally present in the sera of individuals that cause agglutination of the RBCs of another individual of the same species.
[0125] The immune system of a person of blood type A recognizes as foreign and will react to exposure to the B antigen, galactose, and produce anti-galactose antibody, called anti-B antibody or anti-B isoagglutinin. Likewise, the immune system if a person of blood type B will react to exposure to the A antigen, n-acetyl galactosamine, and will produce anti-N-acetyl galactosamine antibody, called anti-A antibody or anti-A isoagglutinin. The immune system of a person of blood type AB will not react to exposure to either the A or B antigens, galactose or n-acetyl galactosamine, and produces no antibodies to them. In contrast, a person of blood type O recognizes both the A or B antigens, galactose or n-acetyl galactosamine, as foreign, and will produce both anti-A and anti-B antibodies/isoagglutinins.
[0126] The A and B antigens found in the molecules of human RBCs also exist in other biological entities, notably, bacterial cell walls, plants, and other foodstuffs. Bacteria are widespread in the environment, are present in intestinal flora, dust, food and other widely distributed agents, ensuring a constant exposure of individuals to A and B antigens and antigens that are extremely similar to each of these antigens. Many antigens or proteins in foods, such as lectins, have A-like or B-like characteristics and may likewise trigger an immune response and isoagglutinin production. This may explain why individuals who have not been otherwise exposed to antigen, for instance to incompatible blood via transfusion, will have a detectable isoagglutinin level in the blood stream. Isoagglutinin production may be a reaction to environmental provocations of antigens. Small amounts of A and B antigens may enter the body in food, bacteria, or by other means, and these substances initiate the development of isoagglutinins, e.g. the anti-A antibodies and/or anti-B antibodies. See, e.g., Guyton, A. C., Textbook of Medical Physiology 8th ed., W. B. Saunders Co., 1990.
[0127] Isoagglutinin production is generally seen after the first few months of life and continues throughout an individual's life, remaining fairly constant until late in adult life. See, e.g., Liu, Y J et. al., The development of ABO isohemagglutinins in Tawanese. Hum. Hered. July/August, 1996, 46(4):181-4. In the elderly, isoagglutinin production has been found to diminish and it is believed that this is due to the gradual reduction in efficiency of the immune defenses as the cells age. Recent studies measuring isoagglutinin levels suggest that the baseline isoagglutinin levels in children have risen over time. See, e.g., Godzisz, J., Synthesis of natural allohemagglutinins of the ABO blood system in healthy children aged 3 months to 3 years, Rev. Fr. Tranfus. Immunohematol, September, 1979, 22(4): 399-412.
[0128] In addition to sugars (carbohydrate antigens), the antigens of the Rh blood group are proteins. The Rh blood group system consists of 49 defined blood group antigens, among which the five antigens D, C, c, E, and e are the most important. Rh(D) status of an individual is normally described with a positive or negative suffix after the ABO type (e.g., someone who is A Positive has the A antigen and the Rh(D) antigen, whereas someone who is A Negative lacks the Rh(D) antigen). The terms Rh factor, Rh positive, and Rh negative refer to the Rh(D) antigen only. Antibodies to Rh antigens can be involved in hemolytic transfusion reactions and antibodies to the Rh(D) and Rh(c) antigens confer significant risk of hemolytic disease of the fetus and newborn. Rh antibodies are IgG antibodies which are acquired through exposure to Rh-positive blood (generally either through pregnancy or transfusion of blood products). The D antigen is the most immunogenic of all the non-ABO antigens. Approximately 80% of individuals who are D-negative and exposed to a single D-positive unit will produce an anti-D antibody. The percentage of alloimmunization is significantly reduced in patients who are actively exsanguinating. All Rh antibodies except D display dosage (antibody reacts more strongly with red cells homozygous for an antigen than cells heterozygous for the antigen (EE stronger reaction vs Ee). If anti-E is detected, the presence of anti-c should be strongly suspected (due to combined genetic inheritance). It is therefore common to select c-negative and E-negative blood for transfusion patients who have an anti-E. Anti-c is a common cause of delayed hemolytic transfusion reactions.
Galactose--1,3-galactose (-Gal)
[0129] Galactose--1,3-galactose (-Gal) is an oligosaccharide, which, if present on the transplanted organ, can induce hyperacute (immediate), acute vascular (delayed) and cellular (chronic) xenograft transplant rejection. -Gal moiety (di-Galto Gal moieties connected or Tri-Galthree Gal moieties connected and has a higher affinity to an antibody) is added to cell-surface sugars in animals (e.g., swine) by -1,3-galactosyltransferase (GalT). Due to a frame shift mutation, this enzyme is not functional in humans or Old World monkeys, and these species make anti-Gal antibodies likely as a response to Gal-positive bacteria that inhabit the gastrointestinal tract. IgM, IgG.sub.2, and IgA are antibodies specific to -Gal presented as both glycoprotein and glycolipid. Glycoconjugates range from a single terminal -Gal epitope up to eight branches with terminal -Gal epitopes.
[0130] Hyperacute xenograft rejection is a response mediated by human natural IgM antibodies that cross-react with -Gal expressed on the animal endothelial cells and activates the recipient's complement system and destroying the graft.
[0131] Acute vascular rejection results in the loss of the xenograft in a few days or weeks after transplantation. The antibody induces constant activation of the vascular endothelium, which leads to elevated expression of procoagulant proteins, cell adhesion molecules, and cytokines. Clinically, microvascular thrombosis results in focal ischemia (local loss of blood supply) and xenograft rejection.
[0132] Cellular xenograft rejection is due to the vigorous attack of human cytotoxic T-cells and natural killer (NK) cells such that the graft is lost several weeks after transplantation. In addition, human NK cells have activatory receptors that recognize -Gal epitope.
[0133] The compositions (e.g., Carb-pHLIP peptide constructs) and methods described herein utilize one or a plurality of -Gal epitope(s) selectively tethered via a pHLIP peptide to the surface of cells within diseased tissues (tumors) and induce immune activation and tumor rejection.
Glycosylation
[0134] Glycosylation is the reaction in which a carbohydrate is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor). Glycosylation refers in particular to the enzymatic process that attaches glycans to proteins, or other organic molecules. This enzymatic process produces one of the fundamental biopolymers found in cells (along with DNA, RNA, and proteins). Glycosylation is a form of co-translational and post-translational modification. Glycans serve a variety of structural and functional roles in membrane and secreted proteins. The majority of proteins synthesized in the rough endoplasmic reticulum undergo glycosylation. It is an enzyme-directed site-specific process, as opposed to the non-enzymatic chemical reaction of glycation. Glycosylation is also present in the cytoplasm and nucleus as the O-GlcNAc modification. Five classes of glycans are produced:
[0135] N-linked glycans attached to a nitrogen of asparagine or arginine side-chains. N-linked glycosylation requires participation of a special lipid called dolichol phosphate.
[0136] O-linked glycans attached to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains, or to oxygens on lipids such as ceramide phosphoglycans linked through the phosphate of a phosphoserine;
[0137] C-linked glycans, a rare form of glycosylation where a sugar is added to a carbon on a tryptophan side-chain glypiation, which is the addition of a GPI anchor that links proteins to lipids through glycan linkages.
N-Linked Carbohydrate Antigens or Epitopes Thereof
[0138] N-linked glycosylation, is the attachment of the sugar molecule oligosaccharide known as glycan to a nitrogen atom (the amide nitrogen of an asparagine (Asn) residue of a protein), in a process called N-glycosylation. This type of linkage is important for both the structure and function of some eukaryotic proteins. The N-linked glycosylation process occurs in eukaryotes and widely in archaea, but very rarely in bacteria. The nature of N-linked glycans attached to a glycoprotein is determined by the protein and the cell in which it is expressed.
[0139] All N-linked glycans are based on the common core pentasaccharide, Man.sub.3GlcNAc.sub.2. Further processing in the Golgi results in three main classes of N-linked glycan classes: 1) High-mannose, 2), and Hybrid 3) Complex. High-mannose glycans contain unsubstituted terminal mannose sugars. These glycans typically contain between five and nine mannose residues attached to the chitobiose (GlcNAc2) core. Hybrid glycans are characterized as containing both unsubstituted terminal mannose residues (as are present in high-mannose glycans) and substituted mannose residues with an N-acetylglucosamine linkage (as are present in complex glycans). These GlcNAc sequences added to the N-linked glycan core in hybrid and complex N-glycans are called antennae. A biantennary glycan comprises two GlcNAc branches linked to the core, whereas a triantennary glycan comprises with three GlcNAc branches. Complex N-linked glycans differ from the high-mannose and hybrid glycans by having added GlcNAc residues at both the -3 and -6 mannose sites. Unlike the high-mannose glycans, complex glycans do not contain mannose residues apart from the core structure. Additional monosaccharides may occur in repeating lactosamine (GlcNAc-(1.fwdarw.4)Gal) units. Complex glycans exist in bi-, tri- and tetraantennary forms and make up the majority of cell surface and secreted N-glycans. Complex glycans commonly terminate with sialic acid residues. Additional modifications such as the addition of a bisecting GlcNAc at the mannosyl core and/or a fucosyl residue on the innermost GlcNAc are also possible.
O-Linked Glycosylation
[0140] O-linked glycosylation is the attachment of a sugar molecule to an oxygen atom in an amino acid residue in a protein. O-linked glycosylation is a form of glycosylation that occurs in the Golgi apparatus in eukaryotes. It also occurs in archaea and a number pathogenic bacteria including Burkholderia cenocepacia, Neisseria gonorrhoeae and Acinetobacter baumannii.
ON-acetylgalactosamine (O-GalNAc)
[0141] O-linked glycosylation occurs at a later stage during protein processing, probably in the Golgi apparatus. This is the addition of N-acetyl-galactosamine to serine or threonine residues by the enzyme UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (EC number 2.4.1.41), followed by other carbohydrates (such as galactose and sialic acid). This process is important for certain types of proteins such as proteoglycans, which involves the addition of glycosaminoglycan chains to an initially unglycosylated proteoglycan core protein. These additions are usually serine O-linked glycoproteins, which seem to have one of two main functions. One function involves secretion to form components of the extracellular matrix, adhering one cell to another by interactions between the large sugar complexes of proteoglycans. GlcNAc--Ser/Thr, which are found in nuclear and cytoskeletal proteins, were the first reported example of glycosylated proteins found in a location other than secretory channels.
O-fucose
[0142] O-fucose is added between the second and third conserved cysteines of EGF-like repeats in the Notch protein, and other substrates by GDP-fucose protein O-fucosyltransferase 1, and to Thrombospondin repeats by GDP-fucose protein O-fucosyltransferase 2 (commonly referred to as POFUT2). In the case of EGF-like repeats, the O-fucose may be further elongated to a tetrasaccharide by sequential addition of N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and for Thrombospondin repeats, may be elongated to a disaccharide by the addition of glucose. Both of these fucosyltransferases have been localized to the endoplasmic reticulum, which is unusual for glycosyltransferases, most of which function in the Golgi apparatus.
O-glucose
[0143] O-glucose is added between the first and second conserved cysteines of EGF-like repeats in the Notch protein, and possibly other substrates by protein:O-glucosyltransferase. This enzyme is localized to the ER like the O-fucosyltransferases. The O-glucose modification appears to be necessary for proper folding of the EGF-like repeats of the Notch protein, and increases secretion of this receptor.
O-mannose
[0144] During O-mannosylation, a mannose residue is transferred from mannose-p-dolichol to a serine/threonine residue in secretory pathway proteins. O-mannosylation is common to both prokaryotes and eukaryotes.
Sialic Acid
[0145] Sialic acid is a generic term for the N- or O-substituted derivatives of neuraminic acid, a monosaccharide with a nine-carbon backbone. It is also the name for the most common member of this group, N-acetylneuraminic acid (Neu5Ac or NANA). Sialic acids are found widely distributed in animal tissues and to a lesser extent in other organisms, ranging from fungi to yeasts and bacteria, mostly in glycoproteins and gangliosides (they occur at the end of sugar chains connected to the surfaces of cells and soluble proteins).
[0146] The sialic acid family includes 43 derivatives of the nine-carbon sugar neuraminic acid, but these acids rarely appear free in nature. Normally they can be found as components of oligosaccharide chains of mucins, glycoproteins and glycolipids occupying terminal, non-reducing positions of complex carbohydrates on both external and internal membrane areas where they are very exposed and develop important functions.
[0147] Exemplary sialic acid derivatives include:
##STR00010##
[0148] Sialic acids are found at all cell surfaces of vertebrates and some invertebrates, and also at certain bacteria that interact with vertebrates. Many viruses such as some adenoviruses (Adenoviridae), rotaviruses (Reoviridae) and influenza viruses (Orthomyxoviridae) can use host-sialylated structures for binding to their target host cell. Sialic acids provide a good target for these viruses since they are highly conserved and are abundant in large numbers in virtually all cells. Unsurprisingly, sialic acids also play an important role in several human viral infections. The influenza viruses have hemagglutinin (HA) glycoproteins on their surfaces that bind to sialic acids found on the surface of human erythrocytes and on the cell membranes of the upper respiratory tract.
[0149] In hemagglutination, viruses are mixed with blood cells, and the virus enters into cells of the upper respiratory tract. Widely used anti-influenza drugs (oseltamivir and zanamivir) are sialic acid analogs that interfere with release of newly generated viruses from infected cells by inhibiting the viral enzyme neuraminidase.
[0150] Some bacteria also use host-sialylated structures for binding and recognition. For example, free sialic acid can behave as a signal to some specific bacteria, like Pneumococcus, and can help the bacterium recognize that it has reached a vertebrate environment suitable for its colonization. Modifications of sialic acids, such as the N-glycolyl group at the 5 position or O-acetyl groups on the side chain, may reduce the action of bacterial sialidases.
pHLIP peptides
[0151] In the schematic, Carb-Linker-Peptide, Peptide is a pHLIP peptide (a non-limiting example is pHLIP comprising the Var3 sequence AXDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 3), or AXDQDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 4) or AX(Z).sub.nXPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 5), where X is a functional group for conjugation purposes, selected from lysine (Lys), cysteine (Cys), Azido-containing amino acid or other modified amino acids, and Z indicates any amino acid residue and n is any integer between (and including) 1-10 (e.g., 1n10).
[0152] For example, (Z).sub.n could be QDNDQN (SEQ ID NO: 6) or any combination of polar residues, e.g., D, E, N or Q. The membrane non-inserting N-terminal flanking sequence of pHLIP peptide can optionally be extended. For example, the N terminus of any of these sequences can be extended by the addition of amino acids to space the epitope away from the cell surface, e.g. by including a (glycine) extension. Non-limiting examples of such an extension include a peptide sequence with a poly-Gly motif
[0153] An example of a wild type (WT) pHLIP peptide is AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT (SEQ ID NO: 7) in which AEQNPIY (SEQ ID NO: 8) represents a flanking sequence, WARYADWLFTTPLLLLDLALLV (SEQ ID NO: 9) represents a membrane-inserting sequence, and DADEGT (SEQ ID NO: 10) represents a flanking sequence.
[0154] Other exemplary pHLIP peptides are shown in the Tables below.
TABLE-US-00001 TABLE1 ExemplarypHLIPpeptides Name Sequence SEQIDNo. Var3-1a ACDQDNPWRAYLDLLFPTDTLLLDLLWA SEQ.IDNO.11 Var3-1b AKDQDNPWRAYLDLLFPTDTLLLDLLWA SEQ.IDNO.12 Var3-2a ACQDNDQNCPWRAYLDLLFPTDTLLLDLLWA SEQ.IDNO.13 Var3-2b AKQDNDQNKPWRAYLDLLFPTDTLLLDLLWA SEQ.IDNO.14 WT-1 GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.15 WT-2 AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.16 Var3-WT-Cys ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG SEQ.IDNO.17 Cys-Var3-WT ACDDQNPWRAYLDLLFPTDTLLLDLLWDADEG SEQ.IDNO.18 Lys-Var3-WT AKDDQNPWRAYLDLLFPTDTLLLDLLWDADEG SEQ.IDNO.19 WT-Cys1 AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG SEQ.IDNO.20 WT-Cys2 Ac-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGCT SEQIDNO.21 WT-Cys3 GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG SEQ.IDNO.22 Cys-WT1 Ac-ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG SEQ.IDNO.23 Var0-NT ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.24 Lys-WT1 AKEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.25 Lys-WT2 Ac-AKEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG SEQIDNO.26 WT-KC AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG SEQ.IDNO.27 K-WT-C AKEQNPIYWARYADWLFTTPLLLLDLALLVDADECT SEQ.IDNO.28 N-pHLIP ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGTG SEQ.IDNO.29 N-pHLIP-b ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGT SEQIDNO.30 K-pHLIP ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG SEQ.IDNO.31 NNQ GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT SEQ.IDNO.32 D25A AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGT SEQ.IDNO.33 D25A-KC Ac-AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG SEQIDNO.34 D14A AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.35 P20A AAEQNPIYWARYADWLFTTALLLLDLALLVDADEGT SEQ.IDNO.36 D25E AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGT SEQ.IDNO.37 D14E AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.38 3D AAEQNPIIYWARYADWLFTDLPLLLLDLLALLVDADEGT SEQ.IDNO.39 R11Q GEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG SEQ.IDNO.40 D25Up GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG SEQ.IDNO.41 D25Down GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG SEQ.IDNO.42 D14Up GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG SEQ.IDNO.43 D14Down GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEGTCG SEQ.IDNO.44 P20G AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT SEQ.IDNO.45 H1-Cys DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT SEQ.IDNO.46 H1 DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADET SEQIDNO:47 H2-Cys DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADEGCT SEQ.IDNO.48 Cys-H2 CDDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADET SEQIDNO:49 H2 DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADEGT SEQIDNO:50 H2N-Cys DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT SEQ.IDNO.51 H2N DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADEGT SEQIDNO:52 H2N2-Cys DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT SEQ.IDNO.53 H2N2 DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANEGT SEQIDNO:54 1a-Trp AEQNPIYWARYADFLFTTPLLLLDLALLVDADET SEQ.IDNO.55 1b-Trp AEQNPIYFARYADWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.56 1c-Trp AEQNPIYFARYADFLFTTPLLLLDLALLWDADET SEQ.IDNO.57 Fast-1orVar1 AKEDQNPYWARYADWLFTTPLLLLDLALLVDG SEQ.IDNO.58 Var1-2D1D ACEDQNPYWARYADWLFTTPLLLLDLALLVDG SEQ.IDNO.59 Fast1-Cysor AEDQNPYWARYADWLFTTPLLLLDLALLVDCG SEQ.IDNO.60 Var1-2D1D-Cys Fast1-E-Cysor AEDQNPYWARYADWLFTTPLLLLELALLVECG SEQ.IDNO.61 Var1E Fast1-E-Lys AKEDQNDPYWARYADWLFTTPLLLLDLALLVG SEQIDNO:62 Fast2orVar2 AKEDQNPYWRAYADLFTPLTLLDLLALWDG SEQ.IDNO.63 Fast2-E-Cysor AEDQNPYWARYADWLFTTPLLLLELALLVCG SEQIDNO:64 Var2E Var2-2D1D ACEDQNPYWRAYADLFTPLTLLDLLALWDG SEQ.IDNO.65 Var3-3D ACDDQNPWRAYLDLLFPTDTLLLDLLW SEQ.IDNO.66 Var3-3D-cys AKDDQNPWRAYLDLLFPTDTLLLDLLWC SEQIDNO:67 Var4-3E ACEEQNPWRAYLELLFPTETLLLELLW SEQIDNO:68 Var5-3Da ACDDQNPWARYLDWLFPTDTLLLDL SEQIDNO:69 Var6-3Db CDNNNPWRAYLDLLFPTDTLLLDW SEQIDNO:70 Var8-3Eb CEEQQPWAQYLELLFPTETLLLEW SEQIDNO:71 Var9-3Ec CEEQQPWRAYLELLFPTETLLLEW SEQIDNO:72 Var15-2N CDDDDDNPNYWARYANWLFTTPLLLLNGALLVEAEET SEQIDNO:73 Var16-2P CDDDDDNPNYWARYAPWLFTTPLLLLPGALLVEAEE SEQIDNO:74
TABLE-US-00002 TABLE2 ExemplarypHLIPpeptides Name Sequence SEQIDNo. Var14-Rev Ac-TEDADVLLALDLLLLPTTFL SEQ.IDNO.75 WDAYRAWYPNQECA-Am Sh AEQNPIYWARYADWLFTTPL SEQ.IDNO.76 Sh-Cys AEQNPIYWARYADWLFTTPCL SEQ.IDNO.77 Cys-Sh ACEQNPIYWARYADWLFTTPL SEQ.IDNO.78 Sh-1Trp AEQNPIYFARYADWLFTTPL SEQ.IDNO.79 Sh-W2 AEQNPIYFARYADLLFPTTLAW SEQIDNO.80 Sh-W1 AEQNPIYWARYADLLFPTTLAF SEQIDNO.81 Sh-2W AEQNPIYWARYADLLFPTTLAW SEQIDNO.82 Sh-1D KEDQNPWARYADLLFPTTLAW SEQ.IDNO.83 Sh-1Db KEDQNPWARYADLLFPTTLW SEQIDNO.84 Var12-1D ACEDQNPWARYADLLFPTTLAW SEQ.IDNO.85 Var10-2D ACEDQNPWARYADWLFPTTLLLL SEQ.IDNO.86 D Var13-1E ACEEQNPWARYAELLFPTTLAW SEQ.IDNO.87 Var11-2E ACEEQNPWARYAEWLFPTTLLLL SEQ.IDNO.88 E Var7-3E ACEEQNPWARYLEWLFPTETLLL SEQ.IDNO.89 EL Var7-3Eb ACEEQNPQAEYAEWLFPTTLLLL SEQIDNO.90 E Acmeans Acetylated N-terminus Ammeans Amidated C-terminus
TABLE-US-00003 TABLE 3 Coded and exemplary non-coded amino acids including L-isomers, D- isomers, alpha-isomers, beta-isomers, glycol-, and methyl- modifications. No. Abbrev Name 1 Ala Alanine 2 Arg Arginine 3 Asn Asparagine 4 Asp Aspartic acid 5 Cys Cysteine 6 Gln Glutamine 7 Glu Glutamic acid 8 Gly Glycine 9 His Histidine 10 Ile Isoleucine 11 Leu Leucine 12 Lys Lysine 13 Met Methionine 14 Phe Phenylalanine 15 Pro Proline 16 Ser Serine 17 Thr Threonine 18 Trp Tryptophan 19 Tyr Tyrosine 20 Val Valine 21 Sec Selenocysteine 22 Sem Selenomethionine 23 Pyl Pyrrolysine 24 Aad Alpha-aminoadipic acid 25 Acpa Amino-caprylic acid 26 Aecys Aminoethyl cysteine 27 Afa Aminophenyl acetate 28 Gaba Gamma-aminobutyric acid 29 Aiba Aminoisobutyric acid 30 Aile Alloisoleucine 31 AIg Allylglycine 32 Aba Amino-butyric acid 33 Aphe Amino-phenylalanine 34 Brphe Bromo-phenylalanine 35 Cha Cyclo-hexylalanine 36 Cit Citrulline 37 Clala Chloroalanine 38 Cie Cycloleucine 39 Clphe Fenclonine (or chlorophenylalanine) 40 Cya Cysteic acid 41 Dab Diaminobutyric acid 42 Dap Diaminopropionic acid 43 Dap Diaminopimelic acid 44 Dhp Dehydro-proline 45 Dhphe DOPA (or 3,4-dihydroxyphenylalanine) 46 Fphe Fluorophenylalanine 47 Gaa Glucosaminic acid 48 Gla Gamma-carboxyglutamic acid 49 Hag Homoarginine 50 Hlys Hydroxylysine 51 Hnvl Hydroxynorvaline 52 Hog Homoglutamine 53 Hoph Homophenylalanine 54 Has Homoserine 55 Hse Homocysteine 56 Hpr Hydroxyproline 57 Iphe Iodo-phenylalanine 58 Ise Isoserine 59 Mle Methyl-leucine 60 Msmet Methionine-methylsulfonium chloride 61 Nala Naphthyl-alanine 62 Nle Norleucine (or 2-aminohexanoic acid) 63 Nmala N-methyl-alanine 64 Nva Norvaline (or 2-aminopentanoic acid) 65 Obser O-benzyl-serine 66 Obtyr O-benzyl-tyrosine 67 Oetyr O-ethyl-tyrosine 68 Omser O-methyl-serine 69 Omthr O-methy-threonine 70 Omtyr O-methyl-tyrosine 71 Orn Ornithine 72 Pen Penicillamine 73 Pga Pyroglutamic acid 74 Pip Pipecolic acid 75 Sar Sarcosine 76 Tfa Trifluoro-alanine 77 Thphe Hydroxy-Dopa 78 Vig Vinylglycine 79 Aaspa Amino-aminoethylsulfanylpropanoic acid 80 Ahdna Amino-hydroxy-dioxanonanolic acid 81 Ahoha Amino-hydroxy-oxahexanoic acid 82 Ahsopa Amino-hydroxyethylsulfanylpropanoic acid 83 Tyr(Me) Methoxyphenyl-methylpropanyl oxycarbonylamino propanoic acid 84 MTrp Methyl-tryptophan 85 pTyr Phosphorylated Tyr 86 pSer Phosphorylated Ser 87 pThr Phosphorylated Thr 88 BLys BiotinLys 89 Hyp Hydroproline 90 Phg Phenylglycine 91 Cha Cyclohexyl-alanine 92 Chg Cyclohexylglycine 93 Nal Naphthylalanine 94 Pal Pyridyl-alanine 95 Pra Propargylglycine 96 Gly(allyl) Pentenoic acid 97 Pen Penicillamine 98 MetO Methionine sulfoxide 99 Pca Pyroglutamic acid 100 Ac-Lys Acetylation of Lys
TABLE-US-00004 TABLE 4 Non-limiting examples of protonatable residues and their substitutions including L-isomers, D- isomers, alpha-isomers, and beta-isomers. Original Residue Exemplary amino acids substitution Asp (D) Glu (E); Gla (Gla); Aad (Aad) Glu (E) Asp (D); Gla (Gla); Aad (Aad)
TABLE-US-00005 TABLE 5 Examples of coded amino acid substitutions Original Residue Substitution Ala (A) Gly; Ile; Leu; Met; Phe; Pro; Trp; Tyr; Val Arg (R) Lys Asn (N) Gln; His Asp (D) Glu Cys (C) Ser; Met Gln (Q) Asn; His Glu (E) Asp Gly (G) Ala; Ile; Leu; Met; Phe; Pro; Trp; Tyr; Val His (H) Asn; Gln Ile (I) Ala; Gly; Leu; Met; Phe; Pro; Trp; Tyr; Val Leu (L) Ala; Gly; Ile; Met; Phe; Pro; Trp; Tyr; Val Lys (K) Arg Met (M) Ala; Gly; Leu; Ile; Phe; Pro; Trp; Tyr; Val Phe (F) Ala; Gly; Leu; Ile; Met; Pro; Trp; Tyr; Val Pro (P) Ala; Gly; Leu; Ile; Met; Phe; Trp; Tyr; Val Ser (S) Thr Thr (T) Ser Trp (W) Ala; Gly; Leu; Ile; Met; Pro; Phe; Tyr; Val Tyr (Y) Ala; Gly; Leu; Ile; Met; Pro; Phe; Trp; Val Val (V) Ala; Gly; Leu; Ile; Met; Pro; Phe; Trp; Tyr
TABLE-US-00006 TABLE6 Non-limitingexamplesofmembrane- insertingsequencesbelongingtodifferent groupsofpHLIPpeptides.Eachprotonatable residue(showninbold)couldbereplacedby itssubstitutionfromTable4.Eachnon-polar residuecouldbereplacedbyitscodedamino acidsubstitutionfromTable5,and/ornon- codedaminoacidsubstitutionsfromTable3. SEQ Groups Sequences IDNO: WTBRC WARYADWLFTTPLLLLDLALL 91 YARYADWLFTTPLLLLDLALL 92 WARYSDWLFTTPLLLYDLGLL 93 WARYTDWFTTPLLLYDLALLA 94 WARYTDWLFTTPLLLYDLGLL 95 WARYADWLFTTPLLLLDLSLL 96 WT-BRC LLALDLLLLPTTFLWDAYRAW 97 Reverse LLALDLLLLPTTFLWDAYRAY 98 LLGLDYLLLPTTFLWDSYRAW 99 ALLALDYLLLPTTFWDTYRAW 100 LLGLDYLLLPTTFLWDTYRAW 101 LLSLDLLLLPTTFLWDAYRAW 102 ATRAM GLAGLLGLEGLLGLPLGLLEGLWLGL 103 ATRAM LGLWLGELLGLPLGLLGELGLLGALG 104 Reverse Var3 WRAYLDLLFPTDTLLLDLLW 105 Var3 WLLDLLLTDTPFLLDLYARW 106 Reverse Var7 WARYLEWLFPTETLLLEL 107 WAQYLELLFPTETLLLEW 108 Var7 LELLLTETPFLWELYRAW 109 Reverse WELLLTETPFLLELYQAW 110 Single WLFTTPLLLLNGALLVE 111 D/E WLFTTPLLLLPGALLVE 112 WARYADLLFPTTLAW 113 Single EVLLAGNLLLLPTTFLW 114 D/E EVLLAGPLLLLPTTFLW 115 Reverse WALTTPFLLDAYRAW 116 pHLIP- NLEGFFATLGGEIALWSLVVLAIE 117 Rho EGFFATLGGEIALWSDVVLAIE 118 EGFFATLGGEIPLWSDVVLAIE 119 pHLIP- EIALVVLSWLAIEGGLTAFFGELN 120 Rho EIALVVDSWLAIEGGLTAFFGE 121 Reverse EIALVVDSWLPIEGGLTAFFGE 122 pHLIP-CA9 ILDLVFGLLFAVTSVDFLVQW 123 pHLIP-CA9 WQVLFDVSTVAFLLGFVLDLI 124 Reverse
TABLE-US-00007 TABLE7 Non-limitingexamplesofpHLIPsequences.Acysteine, alysine,anazido-modifiedaminoacid,oranalkynyl modifiedaminoacidcanbeincorporatedattheN- terminal(first6residues)orC-terminal(last6 residues)partsofthepeptidesforconjugation withacargo,andalinker. SEQIDNO Name Sequence SEQIDNO:125 WT-2D AEQNPIYWARYADWLFTTPLLLLDLALLVDADET SEQIDNO:126 WT-6E AEQNPIYWARYAEWLFTTPLLLLELALLVEAEET SEQIDNO:127 WT-3D ADDQNPWRAYLDLLFPDTTDLLLLDLLWDADET SEQIDNO:128 WT-9E AEEQNPWRAYLELLFPETTELLLLELLWEAEET SEQIDNO:129 WT-GlaD AEQNPIYWARYAGlaWLFTTPLLLLDLALLVDADET SEQIDNO:130 WT-DGla AEQNPIYWARYADWLFTTPLLLLGlaLALLVDADET SEQIDNO:131 WT-2Gla AEQNPIYWARYAGlaWLFTTPLLLLGlaLALLVDADET SEQIDNO:132 WT-AadD AEQNPIYWARYAAadWLFTTPLLLLDLALLVDADET SEQIDNO:133 WT-DAad AEQNPIYWARYADWLFTTPLLLLAadLALLVDADET SEQIDNO:134 WT-2Aad AEQNPIYWARYAAadWLFTTPLLLLAadLALLVDADET SEQIDNO:135 WT-GlaAad AEQNPIYWARYAGlaWLFTTPLLLLAadLALLVDADET SEQIDNO:136 WT-AadGla AEQNPIYWARYAAadWLFTTPLLLLGlaLALLVDADET SEQIDNO:137 WT-1 GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQIDNO:138 WT-2 GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQIDNO:139 WT-3 AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQIDNO:140 WT-4 AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQIDNO:141 WT-2N AEQNPIYWARYANWLFTTPLLLLNLALLVDADEGT SEQIDNO:142 WT-2K AEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGT SEQIDNO:143 WT-2DNANQ GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT SEQIDNO:144 WT-D25A AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGT SEQIDNO:145 WT-D14A AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGT SEQIDNO:146 WT-P20A AAEQNPIYWARYADWLFTTALLLLDLALLVDADEGT SEQIDNO:147 WT-D25E AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGT SEQIDNO:148 WT-D14E AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGT SEQIDNO:149 WT-3D-2 AAEQNPIIYWARYADWLFTDLPLLLLDLLALLVDADEGT SEQIDNO:150 WT-R11Q GEQNPIYWAQYADWLFTTPLLLLDLALLVDADEG SEQIDNO:151 WT-D25Up GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEG SEQIDNO:152 WT-D25Down GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEG SEQIDNO:153 WT-D14Up GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGT SEQIDNO:154 WT-D14Down GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEG SEQIDNO:155 WT-P20G AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT SEQIDNO:156 WT-DH DDDEDNPIYWARYADWLFTTPLLLLHGALLVDAD SEQIDNO:476 WT-2H DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADE SEQIDNO:157 WT-L16H CEQNPIYWARYADWHFTTPLLLLDLALLVDADE SEQIDNO:158 WT-1Wa AEQNPIYWARYADFLFTTPLLLLDLALLVDADET SEQIDNO:159 WT-1Wb AEQNPIYFARYADWLFTTPLLLLDLALLVDADE SEQIDNO:160 WT-1Wc AEQNPIYFARYADFLFTTPLLLLDLALLWDADET SEQIDNO:161 WT-W6 ADNNPWIYARYADLTTFPLLLLDLALLVDFDD SEQIDNO:162 WT-W17 ADNNPFIYARYADLTTWPLLLLDLALLVDFDD SEQIDNO:163 WT-W30 ADNNPFIYARYADLTTFPLLLLDLALLVDWDD SEQIDNO:164 WT-W17-P7 ADNNPFPYARYADLTTWILLLLDLALLVDFDD SEQIDNO:165 WT-W39-R11 ADNNPFIYAYRADLTTFPLLLLDLALLVDWDD SEQIDNO:166 WT-W30-R15 ADNNPFIYATYADLRTFPLLLLDLALLVDWDD SEQIDNO:167 WT-Rev Ac-TEDADVLLALDLLLLPTTFLWDAYRAWYPNQEA-Am SEQIDNO:168 Var1-3D AEDQNPYWARYADWLFTTPLLLLDLALLVD SEQIDNO:169 Var1-1D2E AEDQNPYWARYADWLFTTPLLLLELALLVE SEQIDNO:170 Var2-3D AEDQNPYWRAYADLFTPLTLLDLLALWD SEQIDNO:171 Var3-3D ADDQNPWRAYLDLLFPTDTLLLDLLW SEQIDNO:172 Var3-WT ADDQNPWRAYLDLLFPTDTLLLDLLWDADE SEQIDNO:173 Var3-Gla2D ADDQNPWRAYLGlaLLFPTDTLLLDLLW SEQIDNO:174 Var3-DGlaD ADDQNPWRAYLDLLFPTGlaTLLLDLLW SEQIDNO:175 Var3-2DGla ADDQNPWRAYLDLLFPTDTLLLGlaLLW SEQIDNO:176 Var3-2GlaD ADDQNPWRAYLGlaLLFPTGlaTLLLDLLW SEQIDNO:177 Var3-GlaDGla ADDQNPWRAYLGlaLLFPTDTLLLGlaLLW SEQIDNO:178 Var3-D2Gla ADDQNPWRAYLDLLFPTGlaTLLLGlaLLW SEQIDNO:179 Var3-3Gla ADDQNPWRAYLGlaLLFPTGlaTLLLGlaLLW SEQIDNO:180 Var3-Aad2D ADDQNPWRAYLAadLLFPTDTLLLDLLW SEQIDNO:181 Var3-DAadD ADDQNPWRAYLDLLFPTAadTLLLDLLW SEQIDNO:182 Var3-2DAad ADDQNPWRAYLDLLFPTDTLLLAadLLW SEQIDNO:183 Var3-2AadD ADDQNPWRAYLAadLLFPTAadTLLLDLLW SEQIDNO:184 Var3-AadDAad ADDQNPWRAYLAadLLFPTDTLLLAadLLW SEQIDNO:185 Var3-D2Aad ADDQNPWRAYLDLLFPTAadTLLLAadLLW SEQIDNO:186 Var3-3Aad ADDQNPWRAYLAadLLFPTAadTLLLAadLLW SEQIDNO:187 Var3-GlaAadD ADDQNPWRAYLGlaLLFPTAadTLLLDLLW SEQIDNO:188 Var3-GlaDAad ADDQNPWRAYLGlaLLFPTDTLLLAadLLW SEQIDNO:189 Var3-2GlaAad ADDQNPWRAYL LLFPT
TLLL
LLW SEQIDNO:190 Var3-AadGlaD ADDQNPWRAYL
LLFPT
TLLLDLLW SEQIDNO:191 Var3-AadDGla ADDQNPWRAYL
LLFPTDTLLL
LLW SEQIDNO:192 Var3-GlaAadGla ADDQNPWRAYL
LLFPT
TLLL
LLW SEQIDNO:193 Var3-GLL GEEQNPWLGAYLDLLFPLELLGLLELGLW SEQIDNO:194 Var3-M ADDDDDDPWQAYLDLLFPTDTLLLDLLW SEQIDNO:195 Var4-3E AEEQNPWRAYLELLFPTETLLLELLW SEQIDNO:196 Var5-3Da ADDQNPWARYLDWLFPTDTLLLDL SEQIDNO:197 Var6-3Db DNNNPWRAYLDLLFPTDTLLLDW SEQIDNO:198 Var7-3E AEEQNPWARYLEWLFPTETLLLEL SEQIDNO:199 Var7-M DDDDDDPWQAYLDLFPTDTLALDLW SEQIDNO:200 Var8-3E EEQQPWAQYLELLFPTETLLLEW SEQIDNO:201 Var9-3E EEQQPWRAYLELLFPTETLLLEW SEQIDNO:202 Var10-2D AEDQNPWARYADWLFPTTLLLLD SEQIDNO:203 Var11-2E AEEQNPWARYAEWLFPTTLLLLE SEQIDNO:204 Var12-1D AEDQNPWARYADLLFPTTLAW SEQIDNO:205 Var13-1E AEEQNPWARYAELLFPTTLAW SEQIDNO:206 Var15-2N DDDDDNPNYWARYANWLFTTPLLLLNGALLVEAEET SEQIDNO:207 Var16-2P DDDDDNPNYWARYAPWLFTTPLLLLPGALLVEAEET SEQIDNO:208 Var17 AEQNPIYFARYADFLFTTPLLLLDLALLWDADET SEQIDNO:209 Var18 AEQNPIYWARYADFLFTTPLLLLDLALLVDADET SEQIDNO:210 Var19a AEQNPIYWARYADWLFTTPL SEQIDNO:211 Var20 AEQNPIYFARYADLLFPTTLAW SEQIDNO:212 Var21 AEQNPIYWARYADLLFPTTLAF SEQIDNO:213 Var22 AEQNPIYWARYADLLFPTTLAW SEQIDNO:214 Var23 AEQNPIYFARYADWLFTTPL SEQIDNO:215 Var24 EDQNPWARYADLLFPTTLAW SEQIDNO:216 ATRAM GLAGLAGLLGLEGLLGLPLGLLEGLWLGLELEGN SEQIDNO:217 pHLIP-CA9 EQNPIYILDLVFGLLFAVTSVDFLVQWDDAGD SEQIDNO:218 pHLIP-Rho NLEGFFATLGGEIALWSLVVLAIE SEQIDNO:219 pHLIP-RhoM1 NNEGFFATLGGEIALWSDVVLAIE SEQIDNO:220 pHLIP-RhoM2 DNNEGFFATLGGEIPLWSDVVLAIE
[0155] Carbohydrate epitopes may also be delivered to the cell surface of target cells (tumor cells and other diseased tissues/cells) using cyclic pHLIP peptides. A cyclic peptide is one that comprises a circle geometry or structure. For example, the entire structure of the peptide is circular or a portion of the structure is circular. For example, in the latter case the peptide comprises a cyclic portion and a linear (or tail) portion. In various embodiments, a pH triggered peptide comprises at least 4 amino acids, where (a) at least 2 of the at least 4 amino acids of the peptide are non-polar amino acids, (b) at least 1 of the at least 4 amino acids of the peptide is a protonatable amino acid, and (c) the peptide has a higher affinity to a membrane lipid bilayer at pH 5.0 compared to at pH 8.0. Such pHLIP peptides are described in International Patent Application No. PCT/US2017/023458 (PCT publication no. WO2017/165452A1, hereby incorporated by reference.
[0156] Exemplary cyclic pHLIP peptides are described and shown below. A lowercase c at the beginning of a sequence herein denotes a cyclic peptide (e.g., as in c[(WE).sub.3WC]) (Peptide 1), and a lowercase 1 denotes a linear peptide (e.g., as in 1(CW(EW).sub.4)) (Peptide 188). In the case of cyclic structures that comprise a tail, the cyclic portion of the compound is within brackets, and the tail portion follows (is to the right of) the brackets. For example, in the compound c[E.sub.5K]W.sub.5C, c[E.sub.5K] is the cyclic peptide portion, and W.sub.5C (SEQ ID NO: 477) is the peptide tail portion. As another example, in c[E.sub.5K]W.sub.4C, the cyclic peptide portion is c[E.sub.5K] and the peptide tail portion is W.sub.4C (SEQ ID NO: 478).
[0157] With respect to cyclic peptides, the amino acids within brackets may be present in the order listed in brackets from left to right, or in any order. For example, a cyclic peptide c[X.sub.2Y.sub.2] may have the corresponding linear sequence: XXYY, XYXY, YXXY, XYYX, or YXYX. In some cases, multiple examples of corresponding linear sequences for an exemplary cyclic peptide are listed in Table 3.
TABLE-US-00008 TABLE8 providesasummaryofpeptidesequences Peptide Sequence LinearSequence SEQIDNO 1 c[(WE).sub.3WC] WEWEWEWC 221 2 c[(WE).sub.4WC] WEWEWEWEWC 222 3 c[(WE).sub.5WC] WEWEWEWEWEWC 223 4 c[(LE).sub.4WC] LELELELEWC 224 5 c[E.sub.4W.sub.5C] EEEEWWWWWC 225 6 l(CW(EW).sub.4) CWEWEWEWEW 226 7 c[R.sub.4W.sub.5C] RRRRWWWWWC 227
TABLE-US-00009 TABLE9 providesadditionalnon-limitingexamplesofpeptidesequences. Cyclic Circular Linear SEQ Peptide Sequence Sequence IDNO 1 c[E.sub.3W.sub.5C] EEEWWWWWC 228 2 c[E.sub.3W.sub.5C] EWEWWWWEC 229 3 c[E.sub.3W.sub.5C] EWWEWWWEC 230 4 c[E.sub.3W.sub.5C] EWWWEWWEC 231 5 c[E.sub.3W.sub.5C] EWWWWEWEC 232 6 c[E.sub.3W.sub.5C] EWWWWWEEC 233 7 c[E.sub.3W.sub.5C] EWEEWWWWC 234 8 c[E.sub.3W.sub.5C] EWWEEWWWC 235 9 c[E.sub.3W.sub.5C] EWWWEEWWC 236 10 c[E.sub.3W.sub.5C] EWWWWEEWC 237 11 c[E.sub.3W.sub.5C] WEEEWWWWC 238 12 c[E.sub.3W.sub.5C] WWEEEWWWC 239 13 c[E.sub.3W.sub.5C] WWWEEEWWC 240 14 c[E.sub.3W.sub.5C] WWWWEEEWC 241 15 c[E.sub.3W.sub.5C] WEWEEWWWC 242 16 c[E.sub.3W.sub.5C] WEWWEEWWC 243 17 c[E.sub.3W.sub.5C] WEWWWEEWC 244 18 c[E.sub.3W.sub.5C] WEWWWWEEC 245 19 c[E.sub.3W.sub.5] EEEWWWWW 246 20 c[E.sub.3W.sub.5] EWEWWWWE 247 21 c[E.sub.3W.sub.5] EWWEWWWE 248 22 c[E.sub.3W.sub.5] EWWWEWWE 249 23 c[E.sub.3W.sub.5] EWWWWEWE 250 24 c[E.sub.3W.sub.5] EWWWWWEE 251 25 c[E.sub.3W.sub.5] EWEEWWWW 252 26 c[E.sub.3W.sub.5] EWWEEWWW 253 27 c[E.sub.3W.sub.5] EWWWEEWW 254 28 c[E.sub.3W.sub.5] EWWWWEEW 255 29 c[E.sub.3W.sub.5] WEEEWWWW 256 30 c[E.sub.3W.sub.5] WWEEEWWW 257 31 c[E.sub.3W.sub.5] WWWEEEWW 258 32 c[E.sub.3W.sub.5] WWWWEEEW 259 33 c[E.sub.3W.sub.5] WEWEEWWW 260 34 c[E.sub.3W.sub.5] WEWWEEWW 261 35 c[E.sub.3W.sub.5] WEWWWEEW 262 36 c[E.sub.3W.sub.5] WEWWWWEE 263 37 c[D.sub.3W.sub.5C] DDDWWWWWC 264 38 c[D.sub.3W.sub.5C] DWDWWWWDC 265 39 c[D.sub.3W.sub.5C] DWWDWWWDC 266 40 c[D.sub.3W.sub.5C] DWWWDWWDC 267 41 c[D.sub.3W.sub.5C] DWWWWDWDC 268 42 c[D.sub.3W.sub.5C] DWWWWWDDC 269 43 c[D.sub.3W.sub.5C] DWDDWWWWC 270 44 c[D.sub.3W.sub.5C] DWWDDWWWC 271 45 c[D.sub.3W.sub.5C] DWWWDDWWC 272 46 c[D.sub.3W.sub.5C] DWWWWDDWC 273 47 c[D.sub.3W.sub.5C] WDDDWWWWC 274 48 c[D.sub.3W.sub.5C] WWDDDWWWC 275 49 c[D.sub.3W.sub.5C] WWWDDDWWC 276 50 c[D.sub.3W.sub.5C] WWWWDDDWC 277 51 c[D.sub.3W.sub.5C] WDWDDWWWC 278 52 c[D.sub.3W.sub.5C] WDWWDDWWC 279 53 c[D.sub.3W.sub.5C] WDWWWDDWC 280 54 c[D.sub.3W.sub.5C] WDWWWWDDC 281 55 c[D.sub.3W.sub.5] DDDWWWWW 282 56 c[D.sub.3W.sub.5] DWDWWWWD 283 57 c[D.sub.3W.sub.5] DWWDWWWD 284 58 c[D.sub.3W.sub.5] DWWWDWWD 285 59 c[D.sub.3W.sub.5] DWWWWDWD 286 60 c[D.sub.3W.sub.5] DWWWWWDD 287 61 c[D.sub.3W.sub.5] DWDDWWWW 288 62 c[D.sub.3W.sub.5] DWWDDWWW 289 63 c[D.sub.3W.sub.5] DWWWDDWW 290 64 c[D.sub.3W.sub.5] DWWWWDDW 291 65 c[D.sub.3W.sub.5] WDDDWWWW 292 66 c[D.sub.3W.sub.5] WWDDDWWW 293 67 c[D.sub.3W.sub.5] WWWDDDWW 294 68 c[D.sub.3W.sub.5] WWWWDDDW 295 69 c[D.sub.3W.sub.5] WDWDDWWW 296 70 c[D.sub.3W.sub.5] WDWWDDWW 297 71 c[D.sub.3W.sub.5] WDWWWDDW 298 72 c[D.sub.3W.sub.5] WDWWWWDD 299 73 c[Gla.sub.3W.sub.5] GlaGlaGlaWWWWW 300 74 c[Gla.sub.3W.sub.5] GlaWGlaWWWWGla 301 75 c[Gla.sub.3W.sub.5] GlaWWGlaWWWGla 302 76 c[Gla.sub.3W.sub.5] GlaWWWGlaWWGla 303 77 c[Gla.sub.3W.sub.5] GlaWWWWGlaWGla 304 78 c[Gla.sub.3W.sub.5] GlaWWWWWGlaGla 305 79 c[Gla.sub.3W.sub.5] GlaWGlaGlaWWWW 306 80 c[Gla.sub.3W.sub.5] GlaWWGlaGlaWWW 307 81 c[Gla.sub.3W.sub.5] GlaWWWGlaGlaWW 308 82 c[Gla.sub.3W.sub.5] GlaWWWWGlaGlaW 309 83 c[Gla.sub.3W.sub.5] WGlaGlaGlaWWWW 310 84 c[Gla.sub.3W.sub.5] WWGlaGlaGlaWWW 311 85 c[Gla.sub.3W.sub.5] WWWGlaGlaGlaWW 312 86 c[Gla.sub.3W.sub.5] WWWWGlaGlaGlaW 313 87 c[Gla.sub.3W.sub.5] WGlaWGlaGlaWWW 314 88 c[Gla.sub.3W.sub.5] WGlaWWGlaGlaWW 315 89 c[Gla.sub.3W.sub.5] WGlaWWWGlaGlaW 316 90 c[Gla.sub.3W.sub.5] WGlaWWWWGlaGla 317 91 c[E.sub.3W.sub.4C] EEEWWWWC 318 92 c[E.sub.3W.sub.4C] EWEWWWEC 319 93 c[E.sub.3W.sub.4C] EWWEWWEC 320 94 c[E.sub.3W.sub.4C] EWWWEWEC 321 95 c[E.sub.3W.sub.4C] EWWWWEEC 322 96 c[E.sub.3W.sub.4C] EWEEWWWC 323 97 c[E.sub.3W.sub.4C] EWWEEWWC 324 98 c[E.sub.3W.sub.4C] EWWWEEWC 325 99 c[E.sub.3W.sub.4C] EWWWWEEC 326 100 c[E.sub.3W.sub.4C] WEEEWWWC 327 101 c[E.sub.3W.sub.4C] WWEEEWWC 328 102 c[E.sub.3W.sub.4C] WWWEEEWC 329 103 c[E.sub.3W.sub.4C] WWWWEEEC 330 104 c[E.sub.3W.sub.4C] WEWEEWWC 331 105 c[E.sub.3W.sub.4C] WEWWEEWC 332 106 c[E.sub.3W.sub.4C] WEWWWEEC 333 107 c[E.sub.3W.sub.4] EEEWWWW 334 108 c[E.sub.3W.sub.4] EWEWWWE 335 119 c[E.sub.3W.sub.4] EWWEWWE 336 110 c[E.sub.3W.sub.4] EWWWEWE 337 111 c[E.sub.3W.sub.4] EWWWWEE 338 112 c[E.sub.3W.sub.4] EWEEWWW 339 113 c[E.sub.3W.sub.4] EWWEEWW 340 114 c[E.sub.3W.sub.4] EWWWEEW 341 115 c[E.sub.3W.sub.4] EWWWWEE 342 116 c[E.sub.3W.sub.4] WEEEWWW 343 117 c[E.sub.3W.sub.4] WWEEEWW 344 118 c[E.sub.3W.sub.4] WWWEEEW 345 119 c[E.sub.3W.sub.4] WWWWEEE 346 120 c[E.sub.3W.sub.4] WEWEEWW 347 121 c[E.sub.3W.sub.4] WEWWEEW 348 122 c[E.sub.3W.sub.4] WEWWWEE 349 123 c[D.sub.3W.sub.4C] DDDWWWWC 350 124 c[D.sub.3W.sub.4C] DWDWWWDC 351 125 c[D.sub.3W.sub.4C] DWWDWWDC 352 126 c[D.sub.3W.sub.4C] DWWWDWDC 353 127 c[D.sub.3W.sub.4C] DWWWWDDC 354 128 c[D.sub.3W.sub.4C] DWDDWWWC 355 129 c[D.sub.3W.sub.4C] DWWDDWWC 356 130 c[D.sub.3W.sub.4C] DWWWDDWC 357 131 c[D.sub.3W.sub.4C] DWWWWDDC 358 132 c[D.sub.3W.sub.4C] WDDDWWWC 359 133 c[D.sub.3W.sub.4C] WWDDDWWC 360 134 c[D.sub.3W.sub.4C] WWWDDDWC 361 135 c[D.sub.3W.sub.4C] WWWWDDDC 362 136 c[D.sub.3W.sub.4C] WDWDDWWC 363 137 c[D.sub.3W.sub.4C] WDWWDDWC 364 138 c[D.sub.3W.sub.4C] WDWWWDDC 365 139 c[D.sub.3W.sub.4] DDDWWWW 366 140 c[D.sub.3W.sub.4] DWDWWWD 367 141 c[D.sub.3W.sub.4] DWWDWWD 368 142 c[D.sub.3W.sub.4] DWWWDWD 369 143 c[D.sub.3W.sub.4] DWWWWDD 370 144 c[D.sub.3W.sub.4] DWDDWWW 371 145 c[D.sub.3W.sub.4] DWWDDWW 372 146 c[D.sub.3W.sub.4] DWWWDDW 373 147 c[D.sub.3W.sub.4] DWWWWDD 374 148 c[D.sub.3W.sub.4] WDDDWWW 375 149 c[D.sub.3W.sub.4] WWDDDWW 376 150 c[D.sub.3W.sub.4] WWWDDDW 377 151 c[D.sub.3W.sub.4] WWWWDDD 378 152 c[D.sub.3W.sub.4] WDWDDWW 379 153 c[D.sub.3W.sub.4] WDWWDDW 380 154 c[D.sub.3W.sub.4] WDWWWDD 381 155 c[Gla.sub.3W.sub.4] GlaGlaGlaWWWW 382 156 c[Gla.sub.3W.sub.4] GlaWGlaWWWGla 383 157 c[Gla.sub.3W.sub.4] GlaWWGlaWWGla 384 158 c[Gla.sub.3W.sub.4] GlaWWWGlaWGla 385 159 c[Gla.sub.3W.sub.4] GlaWWWWGlaGla 386 160 c[Gla.sub.3W.sub.4] GlaWGlaGlaWWW 387 161 c[Gla.sub.3W.sub.4] GlaWWGlaGlaWW 388 162 c[Gla.sub.3W.sub.4] GlaWWWGlaGlaW 389 163 c[Gla.sub.3W.sub.4] GlaWWWWGlaGla 390 164 c[Gla.sub.3W.sub.4] WGlaGlaGlaWWW 391 165 c[Gla.sub.3W.sub.4] WWGlaGlaGlaWW 392 166 c[Gla.sub.3W.sub.4] WWWGlaGlaGlaW 393 167 c[Gla.sub.3W.sub.4] WWWWGlaGlaGla 394 168 c[Gla.sub.3W.sub.4] WGlaWGlaGlaWW 395 169 c[Gla.sub.3W.sub.4] WGlaWWGlaGlaW 396 170 c[Gla.sub.3W.sub.4] WGlaWWWGlaGla 397 171 c[(WE).sub.3WC] WEWEWEWC 398 172 c[(EW).sub.3WC] EWEWEWWC 399 173 c[(WD).sub.3WC] WDWDWDWC 400 174 c[(DW).sub.3WC] DWDWDWWC 401 175 c[(WGla).sub.3WC] WGlaWGlaWDWC 402 176 c[(GlaW).sub.3WC] DWDWDWDC 403 177 c[(WE).sub.4] WEWEWEWE 404 178 c[(EW).sub.4] EWEWEWEW 405 179 c[(WD).sub.4] WDWDWDWD 406 180 c[(DW).sub.4] DWDWDWDW 407 181 c[(WGla).sub.4] WGlaWGlaWGlaWGla 408 182 c[(GlaW).sub.4] GlaWGlaWGlaWGlaW 409 183 c[CW(EW).sub.4] CWEWEWEWEW 410 184 c[(WGla).sub.2WDWC] WGlaWGlaWDWC 411 185 c[(EW).sub.3EC] EWEWEWEC 412 186 c[(DW).sub.3DC] DWDWDWDC 413 187 c[E.sub.5K]W.sub.5C Cyclic:EEEEEK 414(cyclicportion), Tail:WWWWWC 415(Tail) 188 c[E.sub.4K]W.sub.5C Cyclic:EEEEK 416(cyclicportion), Tail:WWWWWC 417(Tail) 189 c[E.sub.5K]W.sub.4C Cyclic:EEEEEK 418(cyclicportion), Tail:WWWWC 419(Tail) 190 c[E.sub.4K]W.sub.4C Cyclic:EEEEK 420(cyclicportion), Tail:WWWWC 421(Tail) 191 c[E.sub.5K]W.sub.5 Cyclic:EEEEEK 422(cyclicportion), Tail:WWWWW 423(Tail) 192 c[E.sub.4K]W.sub.5 Cyclic:EEEEK 424(cyclicportion), Tail:WWWWW 425(Tail) 193 c[E.sub.5K]W.sub.4 Cyclic:EEEEEK 426(cyclicportion), Tail:WWWW 427(Tail) 194 c[E.sub.4K]W.sub.4 Cyclic:EEEEK 428(cyclicportion), Tail:WWWW 429(Tail) 195 c[D.sub.5K]W.sub.5C Cyclic:DDDDDK 430(cyclicportion), Tail:WWWWWC 431(Tail) 196 c[D.sub.4K]W.sub.5C Cyclic:DDDDK 432(cyclicportion), Tail:WWWWWC 433(Tail) 197 c[D.sub.5K]W.sub.4C Cyclic:DDDDDK 434(cyclicportion), Tail:WWWWC 435(Tail) 198 c[D.sub.4K]W.sub.4C Cyclic:DDDDK 436(cyclicportion), Tail:WWWWC 437(Tail) 199 c[D.sub.5K]W.sub.5 Cyclic:DDDDDK 438(cyclicportion), Tail:WWWWW 439(Tail) 200 cW.sub.5 Cyclic:DDDDK 440(cyclicportion), Tail:WWWWW 441(Tail) 201 cW.sub.4 Cyclic:DDDDDK 442(cyclicportion), Tail:WWWW 443(Tail) 202 c[D.sub.4K]W.sub.4 Cyclic:DDDDK 444(cyclicportion), Tail:WWWW 445(Tail) 203 c[Gla.sub.5K]W.sub.5C Cyclic:GlaGlaGlaGlaGlaK 446(cyclicportion), Tail:WWWWWC 447(Tail) 204 c[Gla.sub.4K]W.sub.5C Cyclic:GlaGlaGlaGlaK 448(cyclicportion), Tail:WWWWWC 449(Tail) 205 c[Gla.sub.5K]W.sub.4C Cyclic:GlaGlaGlaGlaGlaK 450(cyclicportion), Tail:WWWWC 451(Tail) 206 c[Gla.sub.4K]W.sub.4C Cyclic:GlaGlaGlaGlaK 452(cyclicportion), Tail:WWWWC 453(Tail) 207 c[Gla.sub.5K]W.sub.5 Cyclic:GlaGlaGlaGlaGlaK 454(cyclicportion), Tail:WWWWW 455(Tail) 208 c[Gla.sub.4K]W.sub.5 Cyclic:GlaGlaGlaGlaK 456(cyclicportion), Tail:WWWWW 457(Tail) 209 c[Gla.sub.5K]W.sub.4 Cyclic:GlaGlaGlaGlaGlaK 458(cyclicportion), Tail:WWWW 459(Tail) 210 c[Gla.sub.4K]W.sub.4 Cyclic:GlaGlaGlaGlaK 460(cyclicportion), Tail:WWWW 461(Tail) 211 c[E.sub.5W.sub.5C] EEEEEWWWWWC 462 212 c[E.sub.4W.sub.4C] EEEEWWWWC 463 213 c[(WE).sub.4CW] WEWEWEWECW 464 214 c[(WR).sub.4WC] WRWRWRWRWC 465
Examples
[0158] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
Example 1
[0159] pHLIP Peptide
[0160] pHLIP peptides are described here and in U.S. Pat. Nos. 9,814,781 and 9,289,508 (hereby incorporated by reference in their entireties) as well as U.S. Patent Publication 20180117183, 20180064648, 20180221500, 20180117183, 20180064648, 20160256560, 20150191508, 20150051153, and 20120142042, 20120039990, and 20080233107, each of which is hereby incorporated by reference in their entireties.
[0161] Linker
[0162] A linker could be relatively small, e.g., only a few atoms, to a rather large polar (or moderately hydrophobic) polymer or an N-terminal lengthening of the pHLIP peptide by the addition of amino acids, e.g., glycine residues (poly-Gly). In some examples, a linker can be part of membrane non-inserting pHLIP peptide sequence, such as those with a poly-Gly motif. In some examples, a linker could be PEG polymer. The purpose of a polymer or pHLIP extension is to position epitopes at the surfaces of cells to enhance the access of antibodies or proteins for binding to the epitope. The size and hydrophobicity of the linker should ensure renal clearance of the construct and should not promote hepatic clearance. For example, a linker comprises a covalent bond or a chemical linker. Non-limiting example of linker is a PEG polymer in size ranging from 200 Da up to 20 kDa.
[0163] In some examples the following linkers and their derivatives could be used N--maleimidoacet-oxysuccinimide ester (AMAS); N--maleimidobutyryl-oxysuccinimide ester (GMBS); N--maleimidopropyl-oxysuccinimide ester (BMPS); N--malemidocaproyl-oxysuccinimide ester (EMCS); m-maleimidobenzoyl-n-hydroxysuccinimide ester (MBS); succinimidyl 3-(bromoacetamido)propionate (SBAP); succinimidyl (4-iodoacetyl)aminobenzoate (SIAB); N--maleimidocaproic acid (EMCA); succinimidyl 4-(n-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC); succinimidyl iodoacetate (SIA); succinimidyl (4-iodoacetyl)aminobenzoate (SIAB); succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB); succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH); 3-propargyloxypropanoic acid, succinimidyl ester (alkyne, succinimidyl ester); 1,4-bismaleimidobutane (BMB); bismaleimidohexane (BMH); bismaleimidoethane (BMOE); tris(2-maleimidoethyl)amine (TMEA); N--maleimidopropionic acid hydrazide; (BMPH); N--maleimidocaproic acid hydrazide (EMCH); N--maleimidoundecanoic acid hydrazide (KMUH); 4-(4-n-maleimidophenyl)butyric acid hydrazide (MBPH); or p-maleimidophenyl isocyanate (PMPI).
[0164] Carbohydrate Epitope
[0165] As shown in
[0166] Alpha Gal Epitope (Gal)
[0167] In all mammals except man, apes and old world monkeys, specific carbohydrate linkages are present, such as Galactose--1,3-Galactose (Gal):
##STR00011##
[0168] In humans, the Gal-(alpha)-1,3-Gal link is recognized as foreign and a significant immune response against it is developed. Gal and its derivatives could be linked to pHLIP to induce immune response predominantly within diseased tissues.
##STR00012##
[0169] Other carbohydrates include Gal--1,4-Gal; Gal--1,6-Gal; Gal--1,3-Glc; Fuc--1,2-Gal; Gal--1,2-Gal and their derivatives.
-Rhamnose and its Derivatives Thereof
[0170] Shown below is an example of an unusual sugar occurring in L-form, -rhamnose and its derivatives, which induces a significant immune response: Naturally occurring L-rhamnose does not appear in the body, and an antibody against it is available, as in case of Gal.
##STR00013##
Blood Group Antigens and Derivatives Thereof
[0171] Blood group antigens and their derivatives are used for the compositions and methods described herein. An O (or H) antigen includes Fucose-Galactose-N-acetylglucosamine-Galactose-Glucose or its epitope is Fuc(1-2)Gal; an A antigen includes N-acetylgalactosamine (GalNAc) glycosidically bonded to the O antigen or its epitope is GalNAc(1-3)[Fuc(1-2)]Gal; and a B antigen includes Galactose glycosidically bonded to the O antigen or its epitope is Gal(1-3)[Fuc(1-2)]Gal. The structures of epitopes are depicted below:
##STR00014##
[0172] These antigens can be further divided into six subtypes based on linkage arrangement
[0173] The examples of A and B type 2 saccharides for conjugation with pHLIP are shown below:
##STR00015##
[0174] The epitope of A antigen conjugated with membrane non-inserting part of pHLIP is used in patients with blood groups B and O; the epitope of B antigen conjugated with membrane non-inserting part of pHLIP could be used in patients with blood groups A and O, and patients with blood group AB needs to get infusion of antibodies (isohemagglutinins). Structures below are examples of derivatives of synthetic epitopes of type 2 A antigen, B antigen and O antigen ready for conjugation with membrane non-inserting part of pHLIP:
##STR00016##
N-Linked Carbohydrates:
[0175] Mannose-N-acetylgalactosamine [(Man).sub.3(GlcNAc).sub.2] attached to pHLIP
##STR00017##
[0176] Specific examples might include three types: high mannose, complex, and hybrid (
Globo H Carbohydrate Epitope
[0177] Globo H is a hexasaccharide, which is expressed on the surface of some cancers cells, specifically lung cancer, breast cancer and prostate cancer, and in some tumors (depicted below):
##STR00018##
Sialic Acid Antigens/Epitopes
[0178] Sialic acid antigen (and its derivatives) for binding with hemagglutinin is also used as the carbohydrate epitope for the compositions and methods described herein:
##STR00019##
[0179] The sialic acid family includes 43 derivatives of the nine-carbon sugar neuraminic acid, but these acids rarely appear free in nature. Normally they can be found as components of oligosaccharide chains of mucins, glycoproteins and glycolipids occupying terminal, non-reducing positions of complex carbohydrates on both external and internal membrane areas where they are very exposed and develop important functions. Exemplarily sialic acid derivatives include:
##STR00020##
Example 2: Tethering Rhamnose to Cancer Cells by pHLIP
[0180] Two different pHLIP constructs were synthesized with L-rhamnose: [0181] i) Ser residue coupled with rhamnose (Rha) was added to the pHLIP sequence during the peptide synthesis to obtain:
TABLE-US-00010 Rha-pHLIP: (SEQIDNO:468) AS(Rha)DDQNPWRAYLDLLFPTDTLLLDLLWA (constructwassynthesizedandpurifiedby IrisBiotech,GmbH) [0182] ii) Rhamnose-PEG12-malemide was synthesized and purified by Iris Biotech, GmbH (
[0183] Progression of the reaction was monitored by (RP-HPLC) (the gradient: water and acetonitrile with 0.05% TFA). Purification of Rha-PEG12-pHLIP was conducted using RP-HPLC followed by lyophilization. The construct purity and identity was established by analytical RP-HPLC and surface-enhanced laser desorption/ionization time of flight (SELDI-TOF) mass spectroscopy, respectively. Constructs concentration were calculated by absorbance at 280 nm using pHLIP extinction coefficient.
[0184] Induction of an immunological response requires a proper positioning of carbohydrate epitope at the surface of tumor cells. This was verified on 3-D tumor cancer cell culture (tumor spheroids). Briefly, a 2% agarose solution was made by dissolving in pH 7.4 PBS. 150 L of the solution is pipetted into each well of a 48-well flat bottom tissue culture plate. After the agarose gel sufficiently settled (1 h), 150 L of DMEM supplemented with 10% FBS and ciprofloxacin.HCl was added to each well. The covered plate was left in a humidified atmosphere at 37 C. and 5% CO.sub.2 in cell culture incubator for 24 h. On the next day, the excess medium was removed from the agarose layer. HeLa cells (10,000 cells) in 200 L of DMEM containing 2% matrigel were added into each well and incubated for 3-4 days to allow the formation of spheroids. Matrigel was dissolved on ice overnight and added in ice cold DMEM at a concentration of 2.5% (to obtain a final concentration of 2% once added to the wells). Then the mixture was heated to 37 C. before being combined with the cells. Tumor spheroids were incubated in 50 L of PBS buffer, pH 6.3 containing 0-2 Rha-pHLIP or Rha-PEG12-pHLIP in a humidified atmosphere of 5% CO.sub.2 at 37 C. for 30 min. After treatment, the spheroids were washed several times in 1 mL of PBS. Next, spheroids were treated at pH 7.4 with Lectin conjugated with Cy3, which recognizes rhamnose followed by washing. The spheroids were imaged using a fluorescent inverted confocal microscope. The representative images are shown in
[0185] Some non-specific binding of lectin-Cy3 to cancer cells was observed; however when tumor spheroids were pre-treated with Rha-pHLIP or Rha-PEG12-pHLIP, a much stronger binding of lectin-Cy3 was observed, and thus, fluorescence, was observed. This data indicated that pHLIP positioned carbohydrate epitope at the surface of cancer cells in 3-D cell culture, and epitope was recognized by the corresponding antibody.
Example 3: Tethering -Gal to Cancer Cells by pHLIP and Activating Immune Response in Animals
[0186] Several different pHLIP constructs were synthesized with the -Gal epitope: [0187] i) di-Gal-SH was synthesized by Synthose, Inc. (
[0189] To obtain di-Gal-pHLIP, di-Gal-PEG4-pHLIP and di-Gal-PEG12-pHLIP, first, N--maleimidoacet-oxysuccinimide ester (AMAS), NHS-PEG4-malemide, NHS-PEG12-malemide or cross-linkers were conjugated with single Lys residue at the N-terminal end of pHLIP peptide to obtain AMAS-pHLIP and malemide-PEGs-pHLIP. The progression of the reactions and purification were carried out using the reverse phase HPLC (the gradient: water and acetonitrile with 0.05% TFA).
[0190] At the second step, di-Gal-malemide was coupled with AMAS-pHLIP malemide-PEG4-pHLIP or malemide-PEG12-pHLIP. Progressions of the reactions and purification was conducted using RP-HPLC the gradient: water and acetonitrile with 0.05% TFA) followed by lyophilization. The constructs purity and identity were established by analytical RP-HPLC and surface-enhanced laser desorption/ionization time of flight (SELDI-TOF) mass spectroscopy, respectively. Constructs concentration were calculated by absorbance at 280 nm using pHLIP extinction coefficient.
[0191] Effect on Length of Linker
[0192] di-Gal epitopes conjugated to pHLIP using different lengths of linkers (di-Gal-pHLIP, di-Gal-PEG4-pHLIP and di-Gal-PEG12-pHLIP) were investigated on tumor spheroids. Briefly, a 2% agarose solution was made by dissolving in pH 7.4 PBS. 150 L of the solution was pipetted into each well of a 48-well flat bottom tissue culture plate. After the agarose gel sufficiently settled (1 h), 150 L of DMEM supplemented with 10% FBS and ciprofloxacin.HCl was added to each well. The covered plate was left in a humidified atmosphere at 37 C. and 5% CO.sub.2 in cell culture incubator for 24 h. On the next day, the excess medium was removed from the agarose layer. HeLa cells (10,000 cells) in 200 L of DMEM containing 2% matrigel were added into each well and incubated for 3-4 days to allow the formation of spheroids. Matrigel was dissolved on ice overnight and added in ice cold DMEM at a concentration of 2.5% (to obtain a final concentration of 2% once added to the wells). Then the mixture was heated to 37 C. before being combined with the cells. Tumor spheroids were incubated in 50 L of PBS buffer, pH 6.3 containing 0-2 M di-Gal-pHLIP, di-Gal-PEG4-pHLIP or di-Gal-PEG12-pHLIP in a humidified atmosphere of 5% CO2 at 37 C. for 30 min. After treatment, the spheroids were washed several times in 1 mL of PBS. Next, spheroids were treated with anti-alpha-Gal human IgG antibody (clone m86) conjugated with 647 nm fluorescent dye, at pH 7.4 followed by washing. The spheroids were imaged using a fluorescent inverted confocal microscope.
[0193] The representative images are shown in
[0194] In Vivo Experimental Data
[0195] Tri-Gal-PEG4-pHLIP was used in animal studies described herein. The -Gal epitope is absent only in humans, apes and Old World monkeys, however it is profusely generated in non-primate mammals, prosimians and New World monkeys. Glycosylation enzyme 1,3 galactosyltransferases (1,3GT) allows transfer of galactose from uridine diphosphate (UDP)-gal to N-acetyllactosamine, producing the -Gal epitope. Since humans and Old World primates lack the -Gal epitope, they are not immunotolerant to it, and produce large quantities of anti-Gal antibodies. The presence of -Gal epitope on the surface of animal cells (mouse cells) requires use of knockout animals, where the 1,3GT gene locus is disrupted and 1,3 galactosyltransferases is not produced and therefore synthesis of -Gal epitope is not occurring.
[0196] Mice deficient in 1-3 galactosyltransferase 2 (A3galt2) on 129/SvEv-057BL/6J background heterozygous breeding pairs was obtained from Taconic Biosciences. The knockout mouse model is described in the article entitled Normal development and function of invariant natural killer T cells in mice with isoglobotrihexosylceramide (iGb3) deficiency by Porubsky et al. PNAS 2007 Apr. 3; 104(14): 5977-5982, incorporated herein by reference in its entirety.
[0197] Mice were bred such that a male was housed with two females in harem. Breeding males were separated after the sperm plug was noted or before parturition day. To obtain DNA for mouse genotyping tail biopsies were done on days 10-21 of animal age. The genotyping assay was performed on samples by Taconic. The colony of homozygous mice was established.
[0198] All animal studies with the Gal-pHLIP construct were conducted according to the animal protocol AN1920-003 approved by the Institutional Animal Care and Use Committee at the University of Rhode Island, in compliance with the principles and procedures outlined by the National Institutes of Health for the care and use of animals. Homo- and heterozygous A3galt2-knockout female and male mice on 129/SvEv-057BL/6J background were used in the study.
[0199] First, immunization of mice was performed to develop antibodies against tri-Gal epitope. Briefly, at day 1 mice were immunized with Gal1-3Gal1-4Glc-HSA (HSA: human serum albumin) from Dextra Laboratories, 20 g/mouse emulsified in complete Freund's adjuvant. Booster injections of Gal1-3Gal1-4Glc-HSA emulsified in incomplete Freund's adjuvant were administered at days 13, 20 and 27. The blood samples were collected at day 1 (prior immunization) and day 31 (after completion of immunization), serum was isolated and kept at 80 C before use in ELISA.
[0200] ELISA assay was performed to confirm presence of antibodies against tri-Gal epitope. Briefly, 25 l of 4 g/m1Gal1-3Gal1-4Glc-BSA (BSA: bovine serum albumin) in 100 mM bicarbonate/carbonate buffer was plated to 96-well half-area plates and incubated overnight at 37 C. Solution was removed, and wells were treated with 1% BSA/PBS buffer for 1 h at 37 C. 25 l of mouse serum in 2% BSA/PBS at different dilution ratios was added to wells and incubated for 24 h at +4 C. Wells were washed 5 times with wash solution, and incubated with peroxidase-conjugated donkey anti-mouse IgG in 1% BSA/PBS buffer for 2 hours at RT. Wells were washed 5 times with wash solution. TMB (3,3,5,5-Tetramethylbenzidine) substrate solution was added to wells, and plates were incubated for 10-15 min. After sufficient color development, reaction was stopped by adding the equal volume of 10% sulfuric acid to the wells. Absorbance was measured at 450 nm using plate reader. The average absorbance reading in the serum samples of all animals with dilution of 1:500 as OD=1.2 (subtracting values of OD obtained on samples prior to immunization), and the average absorbance reading in the serum samples with dilutions of 1:5000 is OD=0.5, which indicated that antibodies against tri-Gal was developed in animals (a titer of 1:5000).
[0201] B16-F10 melanoma murine cancer cells were used in the study, since it is known that these murine cells are lacking expression of Gal epitope on their surface. At the same time, LLC (Lewis Lung Carcinoma) cells were used as a positive control, which have higher natural expression of Gal epitope. The control group of animals (negative control) developed B16-F10 tumor in the flank (1 million cells/mouse) and did not receive any treatment. The positive control was a group of mice with implanted LLC cancer cells (1 million cells/mouse). The treated group was a group of animals with B16-F10 tumors, which obtained tri-Gal-PEG4-pHLIP construct for 10 consecutive days in a form of intraperitoneal (IP) injections (450 l of 80 M).
[0202] The overall total dose of tri-Gal received in the course of multiple IP injections was 60 mg/kg. When the tumor reached about 1 cm.sup.3 (about 1 g) in the control (non-treated) group, the animals were sacrificed; tumors were collected (
[0203] About 65% of tumor weight reduction was observed after IP administration of tri-Gal-PEG4-pHLIP. In a positive control group, where animals were developing LLC tumor, the tumor development was suppressed and on day 12th after cancer cells implantation tumor was 60% smaller compared to the LLC tumors developed in the wild-type animals.
Example 4: Tethering Two Carbohydrate Epitopes by pHLIP to Cancer Cells to Bind Two Heads of an Ig Antibody
[0204] To enhance performance of antibodies and enhance immune response, it is important to promote binding of both heads of IgG with 2 epitopes coupled to the same pHLIP peptide. To achieve this goal a carbohydrate epitope (described above) is conjugated with PEG12 or PEG24 links, which then, is coupled with one of the following pHLIP peptides:
TABLE-US-00011 (SEQIDNO:470) Ac-AKQNDDQNKPWRAYLDLLFPTDTLLLDLLWA (SEQIDNO:471) ACQNDDQNCPWRAYLDLLFPTDTLLLDLLWA
[0205] PEG12 and PEG24 can be used to introduce a spacer for 5 nm and 10 nm, respectively. The six residues (QNDDQN (SEQ ID NO: 472) between points of PEG conjugation to pHLIP provides additional space of a few nanometers. Alternatively, QDNDQN (SEQ ID NO. 6) may be used. Thus, two epitopes at the single pHLIP construct binds two heads of Ig antibody, since the distance between heads is 5-25 nm, and thus achieves enhanced avidity, enhanced affinity and immune response. Alternatively, the distance may be about 10 nm, or 10-15 nm, which corresponds to a typical distance between the two antigen binding sites binding sites of an antibody.
Example 5
[0206] In aspects, provided herein is a composition comprising a purified carbohydrate epitope and a pHLIP peptide. For example, the composition has the formula of Carb-Linker-Pept wherein Carb is a carbohydrate epitope; wherein Linker is a non-cleavable linker compound or a membrane non-inserting end of the pHLIP peptide further comprises an amino acid extension; wherein Pept is a pHLIP peptide comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 3) or AXDQDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 4), where X is a functional group, selected from a lysine, a cysteine, a serine, a threonine, or an Azido-containing amino acid; wherein each - is a covalent bond.
[0207] In embodiments, the carbohydrate epitope and peptide are connected by a non-cleavable linker or by an extension of the pHLIP peptide membrane non-inserting terminus.
[0208] In embodiments, the pHLIP peptide extension is a poly-Glycine peptide.
[0209] In other embodiments, the linker has a polyethylene glycol (PEG) polymer, wherein the PEG polymer ranges from 4 to 24 PEG units. In embodiments, the linker has a polyethylene glycol polymer. For example, the polymer ranges in size from 200 Daltons to 20 kiloDaltons.
[0210] In embodiments, the carbohydrate epitope has a glycan comprising an N-linked glycan, an O-linked glycan, or any combination thereof. For example, the glycan includes Galactose--1,3-Galactose or derivatives thereof. In other examples, the glycan includes tri-Gal or derivatives thereof.
[0211] In embodiments, the N-linked glycan and the O-linked glycan have the core structure GlcNAc2Man3, Mannose-N-acetylgalactosamine [(Man).sub.3(GlcNAc).sub.2], -rhamnose, Globo H, or sialic acid or derivatives thereof.
[0212] In other examples, the carbohydrate epitope has a blood antigen.
[0213] In embodiments, the composition described herein has 2 or more pHLIP peptides. For example, the composition has 2 or more carbohydrate epitopes. In examples, the 2 carbohydrate epitopes are linked to a single pHLIP peptide.
[0214] In embodiments, the composition has the formula of Carb-Linker-Pept-Linker-Carb wherein Carb is a carbohydrate epitope; wherein Linker is a polyethylene glycol linker; wherein Pept is a pHLIP peptide comprising the sequence Ac-AKQNDDQNKPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 470) or Ac-AKQNDNDNKPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 479) or ACQNDDQNCPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 471) or ACQNDNDNCPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 480) wherein each - is a covalent bond.
[0215] In aspects, provided herein is a method of inducing an immune response in a diseased tissue in a subject, including administering to a subject a composition comprising a carbohydrate epitope and a pHLIP peptide. In embodiments, the subject has a solid tumor.
[0216] In embodiments, the composition is injected directly into a tumor mass. In other embodiments, the composition is systemically administered. In embodiments, a biological effect of the composition is at least 20% greater than that delivered in the absence of said composition.
[0217] In other embodiments, the composition targets preferentially to a diseased tissue compared to a healthy tissue, thereby minimizing damage to said healthy tissue.
[0218] In embodiments, provided herein is a method for promoting an immune response in a subject, including administering to a subject the composition described herein, wherein said method comprises placement of said carbohydrate epitope on tumor cell of said subject.
General Definitions
[0219] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry).
[0220] As used herein, the term about in the context of a numerical value or range means 10% of the numerical value or range recited or claimed, unless the context requires a more limited range.
[0221] In the descriptions above and in the claims, phrases such as at least one of or one or more of may occur followed by a conjunctive list of elements or features. The term and/or may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases at least one of A and B; one or more of A and B; and A and/or B are each intended to mean A alone, B alone, or A and B together. A similar interpretation is also intended for lists including three or more items. For example, the phrases at least one of A, B, and C; one or more of A, B, and C; and A, B, and/or C are each intended to mean A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together. In addition, use of the term based on, above and in the claims is intended to mean, based at least in part on, such that an unrecited feature or element is also permissible.
[0222] It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, 0.2-5 mg is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.
[0223] A small molecule is a compound that is less than 2000 daltons in mass. The molecular mass of the small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons.
[0224] As used herein, an isolated or purified carbohydrate molecule, nucleic acid molecule, polynucleotide, polypeptide, or protein, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) or polypeptide is free of the amino acid sequences, or nucleic acid sequences that flank it in its naturally-occurring state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally-occurring state. A purified or isolated polypeptide is free of the amino acids or sequences that flank it in its naturally-occurring state.
[0225] Similarly, by substantially pure is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.
[0226] The transitional term comprising, which is synonymous with including, containing, or characterized by, is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase consisting of excludes any element, step, or ingredient not specified in the claim. The transitional phrase consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
[0227] The terms subject, patient, individual, and the like as used herein are not intended to be limiting and can be generally interchanged. That is, an individual described as a patient does not necessarily have a given disease, but may be merely seeking medical advice.
[0228] As used herein, the singular forms a, an, and the include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to a disease, a disease state, or a nucleic acid is a reference to one or more such embodiments, and includes equivalents thereof known to those skilled in the art and so forth.
[0229] As used herein, treating encompasses, e.g., inhibition, regression, or stasis of the progression of a disorder. Treating also encompasses the prevention or amelioration of any symptom or symptoms of the disorder. As used herein, inhibition of disease progression or a disease complication in a subject means preventing or reducing the disease progression and/or disease complication in the subject.
[0230] As used herein, a symptom associated with a disorder includes any clinical or laboratory manifestation associated with the disorder, and is not limited to what the subject can feel or observe.
[0231] As used herein, effective when referring to an amount of a therapeutic compound refers to the quantity of the compound that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this disclosure.
[0232] As used herein, pharmaceutically acceptable carrier or excipient refers to a carrier or excipient that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. It can be, e.g., a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the subject.
[0233] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
[0234] Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
[0235] The term identical or percent identity, in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity over a specified region, e.g., of an entire polypeptide sequence or an individual domain thereof), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection. Such sequences that are at least about 80% identical are said to be substantially identical. In some embodiments, two sequences are 100% identical. In certain embodiments, two sequences are 100% identical over the entire length of one of the sequences (e.g., the shorter of the two sequences where the sequences have different lengths). In various embodiments, identity may refer to the complement of a test sequence. In some embodiments, the identity exists over a region that is at least about 10 to about 100, about 20 to about 75, about 30 to about 50 amino acids or nucleotides in length. In certain embodiments, the identity exists over a region that is at least about 50 amino acids in length, or more preferably over a region that is 100 to 500, 100 to 200, 150 to 200, 175 to 200, 175 to 225, 175 to 250, 200 to 225, 200 to 250 or more amino acids in length.
[0236] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. In various embodiments, when using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
[0237] A comparison window refers to a segment of any one of the number of contiguous positions (e.g., least about 10 to about 100, about 20 to about 75, about 30 to about 50, 100 to 500, 100 to 200, 150 to 200, 175 to 200, 175 to 225, 175 to 250, 200 to 225, 200 to 250) in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. In various embodiments, a comparison window is the entire length of one or both of two aligned sequences. In some embodiments, two sequences being compared comprise different lengths, and the comparison window is the entire length of the longer or the shorter of the two sequences. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0238] In various embodiments, an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 may be used, with the parameters described herein, to determine percent sequence identity for nucleic acids and proteins. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, as known in the art. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
OTHER EMBODIMENTS
[0239] 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.
[0240] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
[0241] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.