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1. WO2007149343 - PROTEASES FOR TREATMENT OF VENOMOUS BITES

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PROTEASES FOR TREATMENT OF VENOMOUS BITES
Inventors: Martin Metz, Stephen J. Galli

CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. Provisional Patent Application No.
60/814,432, filed 06/16/2006, which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT
This invention was made with U.S. Government support under NIH Grant R37 A1239990, ROl CA 727074 and P50 HL67674. The U.S. Government has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING
Applicants assert that the paper copy of the Sequence Listing is identical to the Sequence Listing in computer readable form found on the accompanying computer disk. Applicants incorporate the contents of the sequence listing by reference in its entirety.

BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the field of protein-based therapeutics and to treatment of venomous stings and bites.
Related Art
1. Introduction
Venomous reptiles and their prey have co-existed for ~ 200 million years (B. G. Fry et ai, Nature 439, 584 (2006), and snake envenomation still accounts for significant human morbidity and mortality world-wide. It is estimated that more than 2.5 million people are envenomated by snakes each year and that the global snakebite mortality ranges from 50,000 to 125,000 persons annually (J. P. Chippaux, Bull World Health Organ 76, 515 (1998); R. D. Theakston, D. A. Warrell, E. Griffiths, Toxicon 41, 541 (2003)). The mechanisms by which snake envenomation can produce tissue injury and death have been studied extensively (Theakston, et al., supra; A. Rucavado, T. Escalante, J. M. Gutierrez, Toxicon 43, (2004); J. White, Toxicon 45, 951 (2005)), and it is known that many components of snake venoms can
I of 58 induce mammalian mast cells (MCs) to release potent biologically-active mediators (N. K. Dutta, K. G. Narayanan, Nature 169, 1064 (1952); A. Weisel-Eichler, F. Libersat, / Comp Physiol A Neuroethol Sens Neural Behav Physiol 190, 683 (2004)). These MC products in turn can promote enhanced vascular permeability, local inflammation, abnormalities of the clotting and fibrinolysis systems, and shock (D. D. Metcalfe, D. Baram, Y. A. Mekori, Physiol Rev 77, 1033 (1997); M. F. Gurish, K. F. Austen, J Exp Med 194, Fl (2001)).
Accordingly, it has been proposed that the activation of tissue MCs can contribute
significantly to the local tissue injury, systemic distribution of venom components, and death associated with snake envenomation (N. K. Dutta, K. G. Narayanan, supra; A. Weisel-Eichler, F. Libersat, supra).

Findings that MC activation during snake envenomation contributes to pathology are consistent with the well-understood role of MCs in the pathology of allergic disorders such as anaphylaxis and asthma (D. D. Metcalfe, D. Baram, Y. A. Mekori, supra; M. F. Gurish, K. F. Austen, supra; H. Turner, J. P. Kinet, Nature 402, B24 (1999); F. D. Finkelman, M. E.
Rothenberg, E. B. Brandt, S. C. Morris, R. T. Strait, J Allergy Clin Immunol 115, 449 (2005)). However, in certain models of innate immunity to bacterial infection, MCs can enhance survival (B. Echtenacher, D. N. Mannel, L. Hϋltner, Nature 381, 75 (1996); R. Malaviya, T. Ikeda, E. Ross, S. N. Abraham, Nature 381, 77 (1996); A. P. Prodeus, X. Zhou, M. Maurer, S. J. Galli, M. C. Carroll, Nature 390, 172 (1997); M. Maurer et al., J Exp Med 188, 2343 (1998)). In one such model, MCs can reduce morbidity and mortality in part by promoting the degradation of the potent endogenous vasoconstrictor peptide, endothelin-1 (ET-I) (M. Maurer et al., Nature 432, 512 (2004)). We observed that the most toxic components of the venom of Atractaspis engaddensis (the burrowing asp, Israeli mole viper, or, in Hebrew, Saraf Ein Gedi) are the sarafotoxins, which exhibit a very high homology (-70%, at the amino acid level) to ET-I (Y. Kloog et al., Science 242, 268 (1998)). We therefore hypothesized that MCs, although they are thought to contribute to pathology, also might perhaps confer protection against the toxicity of Atractaspis engaddensis venom (Ae. v.), and perhaps might confer protection against the toxins of other venomous animals (such as snakes, lizards, stinging insects, spiders, etc.) as well.
2. Patents and Publications
US 4,012,502 to Philpot, Jr., issued March 15, 1977 entitled "Snake venom inhibitor material and method of purification," discloses a material extracted from snakes such as snake serum, which is purified to obtain an inhibitor for snake venom toxicity.

US 5,714,344 to Ollert, et al., issued February 3, 1998, entitled "Protease-derivatized CVF" discloses cobra venom factor derivatives which exhibit substantially the same complement- activating activity of natural CVF and are prepared using proteases, including serine proteases such as chymotrypsin and trypsin.

5 Maurer et al., "Mast cells promote homeostasis by limiting endothelin-1 induced toxicity," Nature 432:512-516 (2004) discloses that ET-I toxicity during bacterial infections can be diminished by mast cell proteases, particularly chymase. Human endothelin-1 (ET-I) is a 21-amino acid peptide derived from vascular endothelial cells, which is elicited during bacterial sepsis.

0 Chen ZQ, et al., "Cloning and expression of human colon mast cell
carboxypeptidase," World J Gastroenterol, 2004 Feb l;10(3):342-7 discloses the cloning and expression of the human colon mast cell carboxypeptidase (MC-CP) gene. Three amino acids are indicated as different from skin MC-CP (mast cell carboxypeptidase), and the sequence was reported to be 100% identical to lung MC-CP.

5 Grimbaldeston et al., "Mast Cell-Deficient W-sash c-kit Mutant fCitw'sll/w'sh Mice as a

Model for Investigating Mast Cell Biology in Vivo," American Journal of Pathology, 2005; 167:835-848) describes strains of mice used in the present experiments. They describe mice carrying certain mutations in the white spotting (W) locus (i.e., c-kit) which exhibit reduced c-kit tyrosine kinase-dependent signaling that results in mast cell deficiency and 0 other phenotypic abnormalities. The c-kit mutations in Kitw/W'v mice impair melanogenesis and result in anemia, sterility, and markedly reduced levels of tissue mast cells. In contrast, KitW-sh/W-sh mice, bearing the W-sash (Wsh) inversion mutation, have mast cell deficiency but lack anemia and sterility. The authors reported that adult κitw'sh/w'sh mice had a profound deficiency in mast cells in all tissues examined but normal levels of major classes of other 5 differentiated hematopoietic and lymphoid cells.

BRIEF SUMMARY OF THE INVENTION
The following brief summary is not intended to include all features and aspects of the present invention, nor does it imply that the invention must include all features and aspects discussed in this summary.

i0

Express Mail #: EM0861 fi7"WT TS 3 of 58
E The present invention, in certain aspects, comprises a method, material and kit for treating venom toxicity, comprising a protease polypeptide in a stable formulation in a sterile container, wherein said protease is one which cleaves the venom. The kit is intended for use in the field, or may be in a presentation for use in a medical setting. Preferably the kit is self-contained and maintains the protease in a stable condition for a reasonable shelf life. It may maintain the protease in liquid form if appropriate stabilizers are used, or may maintain the protease in powdered (lyophilized) form and further contain a diluent. It may further comprise a syringe for injecting the protease. The kit may comprise a protease selected from the group consisting of carboxypeptidase A, chymotrypsin and papain, carboxypeptidase B and chymase. The protease may be in the form of modified polypeptides, polypeptide fragments, or pro-enzymes. In one aspect, the protease is a human enzyme, e.g., human carboxypeptidase A. Engineered or related enzymes may be used as well, such as a carboxypeptidase which has at least 80% sequence identity to carboxypeptidase A, as set forth in the sequence given under "Specifically Exemplified Proteases," CPBA3_HUMAN. The kit may comprise more than one different protease, that is, a mixture of different proteases that have activity against a single venom protein component, against venom components commonly found in a single species, or against multiple venom components from different species, such as species commonly found in a single geographical area. Papain may be selected for use in the present methods and compositions because of its availability, but is a plant enzyme, whereas human enzymes, or other enzymes whose composition is closer than that of papain to human enzymes, may be less likely to cause immune responses in treated patients, in that the protease is delivered internally, rather than topically. In a preferred embodiment, the protease is selected from the group including mast cell proteases, such as carboxypeptidase A, tryptase and chymase, and proteases of similar enzymatic activity, such as chymotrypsin, papain and carboxypeptidase B.

In other aspects, the present invention comprises a method for treating a venomous bite (it being understood that the term "bite" is not strictly limited to bites). The method comprises administering to the bite victim a material having protease activity against a venom protein. The treatment is suitable for serious bites involving systemic reactions, as further described below.

The material having protease activity may be selected from the group consisting of carboxypeptidase A, chymotrypsin, papain, carboxypeptidase B and chymase. It may be not an enzyme per se, but may only have carboxypetidase activity against the venom. The venom protein may be selected from Table 1 and Table 2. The venom may be from a snake, a bee, a spider, a platypus, a scorpion, and ant, a Portuguese man-of-war, or a microorganism. In one aspect, it is particularly preferred that the venom is from a venomous snake dangerous to humans.

The material used in the present method or kit may be a material having protease activity or carboxypeptidase activity, or a protease having at least 40% identity to human carboxypeptidase of CPB A3JHUMAN. The material having protease activity may be selected from the group consisting of: carboxypeptidase, chymotrypsin, chymase, or papain, or sequences at least 80% identical thereto. In addition, the carboxypeptidase used may be carboxypeptidase A, or a sequence at least 80% identical thereto.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a series of graphs A-F plotting mouse temperature change versus time after injection of Sarafotoxin 6b (S6b) or Atractaspis engaddensis venom (A.e.v.), indicating that S6b and A.e.v. toxicity is limited by Carboxypeptidase A (CPA) and possibly other proteases released from mast cells (MCs) after activation of the MCs through the Endothelin A receptor (ETA);

Figure 2 A-C is a series of graphs; D is a series of photographs; and E -F are graphs of temperature change; 2A shows change in absorbance of a specific substrate for
Carboxypeptidase A (CPA) for normal wildtype (WT) mast cells (MCs) and MCs infected with either empty vector or CPA shRNA, indicating that shRNA treatment successfully knocked down CPA activity; 2B shows numbers of peritoneal MCs (PMCs) for wildtype mice (C57BL/6) and MC-deficient Kitw'sh/W'sh mice engrafted intraperitoneal^ (i.p.) with empty vector- or CPA shRNA-infected MCs, indicating that engraftment with shRNA MCs results in normalization of PMC numbers to WT levels;2C is two graphs showing PMC degranulation after S6b injection, and after A.e.v. injection, indicating that engraftment with shRNA MCs results in similar PMC responsiveness to S6b and A.e.v. as in WT mice; 2D is a series of photographs of mesentery showing that engraftment with shRNA MCs results in the appearance of MCs in the mesentery; and 2E-F are graphs and tables for temperature change, sarafotoxin levels and survival for WT mice, MC-deficient κitw~MW'Λ mice, and κitw'sll/w'sh mice engrafted with empty vector or CPA shRNA MCs;

Express Mail #: EMO861673931 IS 5 of 58 Figure 3 is a series of graphs A-D showing temperature changes in mice injected with Agkistrodon contort Hx contortrix (Southern Copperhead) venom (A.c.c. venom) or Crotalus atrox (Western Diamondback Rattlesnake) venom (CM.V.), indicating that MCs protect mice against toxic effects of these venoms by releasing proteases, including CPA;

Figure 4 is a bar graph showing sizes of hemorrhagic lesions induced by Crotalus atrox venom (Ca. v) in WT mice (C57BL/6), MC deficient mice (Kitw-sh/w-sh) and Kitw-sh/w'sh mice engrafted with MCs in the skin, indicating that toxic effects of C.a.\ in the skin are also limited by MCs;

Figure 5 is a graph (5A) and table (5B) showing the toxicity of honey bee (A.
mellifera) venom and brown recluse spider venom;

Figure 6 is a series of bar graphs A-F showing PMC degranulation after i.p. injection of S6b (left side) or Ae.v. (right side) in WT mice (Kit+/+), MC-deficient Kitw/Kitw'v mice .engrafted i.p. with ETA+/- (ETA+/- ESCMCs-* Kitw/Kitw-V) or ETA-/- (ETA
ESCMCs^ Kitw/Kitw'v) mouse embryonic stem cell-derived MCs (ESCMCs), or C57BL/6 mice that received i.p. injections of vehicle, chymostatin or potato carboxypeptidase before S6b or Ae.v. injection, indicating: 1) that S6b and Ae. v. induce the degranulation of PMCs via ETA expressed by MCs, and 2) that the protease inhibitors used (chymostatin and carboxypeptidase inhibitor [PCI]),do not alter the responsiveness of PMCs to S6b or Ae.v;

Figure 7 is a bar graph showing the degree of degradation of S6b by peritoneal lavage cells (PLCs, containing 2x104 PMCs) which were co- incubated with vehicle alone, chymostatin or PCI, indicating that Carboxypeptidase A (CPA) is mainly responsible for the degradation of S6;

Figure 8 (S5) is a series of graphs A-F showing changes in rectal temperature (A,D) sarafotoxin levels (B,E) and PMC degranulation (C,F) in wild type (WT) mice vs. mouse MC protease 4-deficient (mMCP4-deficient) mice;

Figure 9 is a series of graphs A-H that show results for wild type (WT) vs. mMCP4 -/-(mMCP4-defϊcient or "mMCP4 knockout") mice. The graphs show (A,D) body temperature, (B,E) intra-peritoneal ET-I levels (measured at 60 min), and (C,F) levels of PMC
degranulation between WT and mMCP4-/- mice. (G) compares the effects of vehicle (Veh), chymostatin (Chy, 120 μg) or potato carboxypeptidase inhibitor (PCI, 150 μg) added to lysates from 3 x 106 C57BL/6 peritoneal cells, and (H) shows degradation of ET-I in vitro);

Express Mail #: EM086167393T IS 6 of 58 Figure 10 is a bar graph showing remaining activity of S6b after incubation with various proteases;

Figure 11 is a bar graph showing percent mast cell degranulation after treatment with different venom and venom components; and

Figure 12 is a graph showing changes of temperature in mice injected with Apis mellifera venom incubated with human tryptase and incubated with human carboxypeptidase A.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Overview
Snake envenomation, and envenomation by stinging insects such as honeybees and wasps, or by spiders, causes significant morbidity and mortality. Many snake insect and spider venom components can induce mast cells to release potent biologically-active mediators. Accordingly, it has been thought that the activation of mast cells by snake or insect venom can contribute significantly to the local tissue injury, and death, associated with snake or insect envenomation, or the pathology associated with spider envenomation. We show that mast cells, in contrast to the widely held belief, can significantly reduce snake venom-induced pathology by releasing carboxypeptidase A and perhaps other proteases, which can degrade venom components. Mast cells also can reduce the pathology and mortality associated with envenomation by honeybee venom or the venom of the brown recluse spider. These findings identify a new biological function for mast cells: conferring resistance to the morbidity and mortality induced by animal venoms.

The mast cell proteases can be recombinant human proteins, which may be prepared as lyophilized powders that can be solubilized in the field with a suitable vehicle. If the agent is based on human proteins, the likelihood of patients treated with the agent developing a potentially fatal allergic reactivity to the product (i.e., anaphylaxis upon subsequent exposure to the product) would be greatly reduced or eliminated. This is in contrast to current "anti-venom" shots for snake envenomation, which contain foreign immunoglobulins to which anaphylaxis reactivity often develops. An additional advantage of this new method, is that one therapeutic agent (comprising mast cell proteases) can reduce the toxicity of venoms from different types of snakes from different taxonomic groups, whereas "anti-venom" therapy consisting of antibodies to the venom of a single species of snake often is useful only for the treatment of bites from that snake or closely related species. The new method also may be useful to reduce the pathology associated with stings of stinging insects or venomous spiders.

Definitions
As described below in the heading "Polypeptide preparations," the present methods and compositions will comprise one or more proteases, which may be selected from a wide variety of proteases. The term "protease" is used in its scientifically accepted sense of an enzyme that degrades proteins; the present proteases, also called peptidases, may be endopeptideases or exopeptidasese. The present proteases may be fragments of protease proteins, and are thus referred to as "polypeptides," meaning either a full-length protein or a fragment thereof having catalytic activity in hydrolyzing venom proteins, e.g., S6b. It is preferred that the present protease be of human origin, or of mammalian origin, in order to minimize immune or other adverse reactions when administered to a human subject. Non-human proteases, which are tolerable to humans, e.g., papain, are also preferred. The term "protease" includes proteases that are in active form, as well is in an inactive form, such as a proenzyme, which can become activated in vivo.

The term "venom toxicity" is used in its scientifically accepted sense, and refers to venom or venom components, as described below, from various organisms, which comprise a peptide or mixture of peptides or proteins causing cellular injury or pathological response in the recipient of the venom. It is intended that the recipient is a "subject in need" of the present protease preparation, i.e. a subject suffering severe or life threatening symptoms of envenomation, such as exemplified below by increase in body temperature, but further exemplified by one or more of severe pain in the chest and abdomen, anxiety, raised blood pressure, breathing difficulties and heart palpitations, nausea and vomiting, sweating, excessive salivation and watery eyes. The body temperature could either fall or rise above normal and the blood pressure may rise with an increased pulse rate.

The preferred proteases include mast cell proteases, which are released upon mast cell activation, preferably human mast cell proteases. The term "mast cell protease" means essentially one or more of "carboxypeptidase A", "chymase," or "tryptases" alpha and beta. Other preferred proteases include those with enzymatic activity that is similar to that of mast cell proteases, such as carboxypeptidase B or chymotrypsin.

The term "carboxypeptidase" is used generally to refer to the M14 family of enzymes in the MEROPS peptidase database (http://merops.sanger.ac.uk/). The family is very widely distributed, being known from almost all kinds of organisms except viruses. Most of the peptidases in the family are carboxypeptidases, hydrolyzing single, C-terminal amino acids from polypeptide chains. They have a recognition site for the free C-terminal carboxyl group, which is a key determinant of specificity. Two main types of specificity are illustrated by carboxypeptidase A (Ml 4.001), which favors residues with aromatic or branched side chains, and carboxypeptidase B (M 14.003), which prefers basic amino acids. An exceptional type of activity in the family (in subfamily C) is the dipeptidyl-peptidase activity of gamma-glutamyl-(L)-meso-diaminopimelate peptidase I (M14.008) from Bacillus sphaericus, involved in bacterial cell wall metabolism. Another exemplary carboxypeptidase is
Carboxypeptidase U, which is s activated by thrombin and once activated, potently attenuates fibrinolysis.

The term "carboxypeptidase A" is used in its scientifically accepted sense and is defined herein as EC 3.4.17.1. It causes release of a C-terminal amino acid, but little or no action with -Asp, -GIu, -Arg, -Lys or —Pro. This is a particularly preferred and exemplified enzyme. It includes the pro-enzyme, a representative sequence of which is given as Human Procarboxypeptidase A2, GenBank/EMBL IAYE.

The term "carboxypeptidase activity" is defined herein as a peptidase activity that catalyzes the removal of amino acids from the C-terminus of peptides, oligopeptides or proteins. Defined in a general manner, the carboxypeptidase activity is capable of cleaving the amino acid X from the C-terminus of a peptide, polypeptide, or protein, wherein X represents any amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Cys, GIn, GIu, GIy, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and VaI. It will be understood that the isolated polypeptides having carboxypeptidase activity of the present invention are unspecific as to the amino acid sequence of the peptide, polypeptide, or protein to be cleaved. Further clarification is found in US 6,951,749 to Blinkovsky, et al., issued October 4, 2005.

The term "chymotrypsin" means EC 3.4.21.1, which has a preferential cleavage at Tyr, Trp, Phe, and Leu. It is also known as chymotrypsins A and B; a-chymar ophth;
avazyme; chymar; chymotest; enzeon; quimar; quimotrase; a-chymar; a-chymotrypsin A; or

ExDress Mail #: EM086 J 67393T IS 9 of 58 a-chymotrypsin. Sample sequences include GenBank/EMBL P17538, Chymotrypsinogen, and CAA74031, chymotrypsin [Homo sapiens].

The term "chymase" means EC 3.4.21.39, which has the preferential cleavage:
Phe/Xaa > Tyr/Xaa > Trp/Xaa > Leu/Xaa. It is also known as mast cell protease I; skeletal muscle protease; skin chymotryptic proteinase; mast cell serine proteinase, chymase; or skeletal muscle (SK) protease. It is found in mast cell granules. A representative sequence is given at GenBank/EMBL P23946, "Chymase precursor (Mast cell protease I)."

The term "tryptase" is used in its conventional sense, and is EC 3.4.21.59. As defined at OMIM, 191080, tryptases are serine proteases implicated in asthma and are highly expressed in human mast cells. They are derived from at least 4 nonallelic genes clustered on chromosome 16pl3.3: TPSABl (191080), which represents the alpha and beta-I tryptase alleles; TPSB2 (191081), which represents the beta-II and beta-Ill tryptase alleles; TPSGl (609341); and TPSDl (609272).

The term "papain" means EC 3.4.22.2, which carries out hydrolysis of proteins with broad specificity for peptide bonds, but preference for an amino acid bearing a large hydrophobic side chain at the P2 position. It does not accept VaI in Pl1. It is also known as papayotin; summetrin; velardon; papaine; Papaya peptidase I. A representative sequence is given at GenBank/EMBL P00784, "Papain precursor (Papaya proteinase I)."

The term "kit" as used herein refers to a unit-packaged combination of the elements of a formulated protease, e.g., carboxypeptidase or other protease(s). Such kits promote the proper use and formulation of the materials when used in combination and avoid improper dosing. The formulated elements of the kit may be in ready to use or precursor form (e.g., lyophilized form) requiring reconstitution in a solution. The kit preferably contains the appropriate carriers and solvents to reconstitute a precursor or active form of the
carboxypeptidase. The kit may thus include a container having a sterile, lyophilized powder of carboxypeptidase in a sugar stabilizer, along with water for injection, and an applicator such as a syringe or a spring-loaded needle that shoots through a membrane in the tip and into the recipient's body to deliver the medication, such as a device used in the EpiPen. The container may also have other injectable active ingredients, such as antibodies to venom, antihistamines, neuroprotective agents, anesthetics, or a venom-binding agent. See, Nobuhisa I, "Structural elements of Trimeresurus flavoviridis serum inhibitors for recognition of its venom phospholipase A2 isozymes," FEBS Lett 1998 Jun 16;429(3):385-9 for a description of an five inhibitors (PLI-I-V) against Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) venom phospholipase A2 (PLA2) isozymes which were isolated from its serum. PLI-I, which is composed of two repeated three-finger motifs, and PLI-IV and PLI-V, which contain a sequence similar to the carbohydrate recognition domain (CRD) of C-type lectins, showed ability to bind to three Tf venom PLA2 isozymes. The present kits may be in "unit dosage form," i.e., having a container, such as a vial containing enough active ingredient (protease) for a single treatment of a venomous bite.

The term "carrier" refers to compounds commonly used on the formulation of pharmaceutical compounds used to enhance stability, sterility and deliverability of the therapeutic compound. When the viral, non-viral or protein delivery system is formulated as a solution or suspension, the delivery system is in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The term "bite" is used herein generically to refer to any form of envenomation, including stings, accidental injection, poisoning, etc.
The following terms are used to describe the sequence relationships between two or more polypeptides: "reference sequence," "comparison window," "sequence identity," "percent identity," and "substantial identity." A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, such as a polypeptide sequence, or may comprise a complete translated cDNA or gene sequence. Generally, a reference sequence is at least peptides in length, frequently at least 25 residues in length, and often at least 50 residues in length. Since two polypeptides may each (1) comprise a sequence (i.e., a portion of the complete polypeptide sequence) that is similar between the two polypeptides, and (2) may further comprise a sequence that is divergent between the two polypeptides, sequence comparisons between two (or more) polypeptides are typically performed by comparing sequences of the two
polypeptides over a "comparison window" to identify and compare local regions of sequence similarity.

A "comparison window," as used herein, refers to a conceptual segment of at least 20 contiguous peptide positions wherein a polypeptide sequence may be compared to a reference sequence of at least 20 contiguous amino acid residues and wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) /. MoI. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected, using default gap and insertion penalties. In addition, sequence identity may be based on homology, using PAMF or Blossom comparison tables, whereby conservative amino acid substitutions are scored higher than non-conservative substitutions.

The term "sequence identity" means that two polypeptide sequences are identical (i.e., on an amino acid-by-amino acid basis) over the window of comparison. The term
"percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid 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 (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a polypeptide sequence, wherein the polypeptide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 residue positions, frequently over a window of at least 25-50 amino acids, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 26 percent or less of the reference sequence over the window of comparison. The term "injectable formulation" means a formulation suitable for injection into humans and/or animals, wherein the injection is intradermal, subcutaneous, intramuscular or intravenous. The formulation may be used in a field setting, or in a hospital setting, which is preferred for intravenous administration. In a filed setting, the formulation may be comprised in a kit for self- administration, as described, for example in US 5,114,406 to Gabriel, et al., issued May 19, 1992, entitled, "Injection device for injection, especially self-administered injection, of medicament, including mechanisms for nulling and for selecting dosage, especially for use with multi-dose ampules." US 4,839,341 to Massey, et al., issued June 13, 1989, entitled "Stabilized insulin formulations," further described formulations adaptable according to the present methods for formulating the present proteases for use in a kit. According to this patent, one may prepare a formulation stabilized against aggregation containing a hydroxybenzene and a polyethylene glycol-polypropylene glycol polymer. The present injectable formulations comprise the proteases described in a purified state, i.e., free of contaminants, pyrogens, or unrelated activity. In a preferred form, the proteases are prepared by recombinant DNA methods and purified from cell culture by methods known in the art.

METHODS AND COMPOSITIONS
Animals
C-kit mutant genetically mast cell-deficient (WBfReS-W/+ x C57BL/6J- Wv/+)~ Fr Kitw/Kitw-V (WBB6F1-Kitw/Kitw'v) (Kitw/Kitw-V) mice (Y. Kitamura, S. Go, K. Hatanaka, Blood 52, 447 (1978)) and the congenic normal WBB6F1-+/+ (Kit+/+) mice, and C57BL/6J mice, were purchased from Jackson Laboratories, Bar Harbor, Maine. Mast cell-deficient C57BL/6- Kitw-Sh/Kitw'sh (Kitw-sh/Kitw~sh) mice and the congenic normal C57BL/6 mice, which were originally provided by Peter Besmer (Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, NY) (R. Duttlinger et al., Development 118, 705 (1993)) and mouse mast cell protease 4-deficient (mMCP4-/-) mice on the C57BL/6 background (E. Tchougounova, G. Pejler, M. Abrink, J Exp Med 198, 423(2003)), were bred and maintained at the Stanford University

Research Animal Facility. Both adult Kitw/Kitw'v mice and adult Kitw~sh/Kitw~sh mice have a profound deficiency of mast cells, including virtually no mast cells in the peritoneal cavity or within the mesentery, and <1% wild type levels of mast cells in the skin (S. J. Galli, K. M. Zsebo, E. N. Geissler, Adv Immunol 55, 1 (1994); P. J. Wolters et al., Clin Exp Allergy 35, 82 (2005); M. A. Grimbaldeston et al., Am J Pathol 167, 835 (2005)). All animal care and experimentation was conducted in accord with current National Institutes of Health and Stanford University Institutional Animal Care and Use Committee guidelines.

Mast-cell engraftment of Kit^/Kit" "" and Kttw-s/!/Kitw'sA mice
Some Kitw/KitW v and some Kitw'sh/Kitw'sh mice (female, 4-6-week-old) were repaired of their mast cell deficiency selectively and locally by the injection of growth factor-dependent BMCMCs, of WBB6F1-Kit+/+ (Kit+/+) or C57BL/6 mouse origin, respectively, into the peritoneal cavity (T. Nakano et al., J Exp Med 162, 1025 (1985); M. Maurer et al., / Exp Med 188, 2343 (1998)). Briefly, femoral bone marrow cells from Kit+/+ or C57BL/6 mice were maintained in vitro for ~4 weeks in IL-3 -containing medium until mast cells represented >95% of the total cells according to staining with May-Griinwald-Giemsa. 1.0 x 106 mast cells in 200 μl of PBS, were injected i.p. (via a 26 gauge needle) into each mouse and the mice were used for experiments, together with gender- and age-matched mast cell-deficient Kitw/Kitw'v mice and Kit+/+ WT mice, or mast cell-deficient KitW-sh/KitW-sh mice and C57BL/6 WT mice, 4-6 weeks after adoptive transfer of BMCMCs. Other Kitw/Kitw'v mice received, 4-6 weeks before injection of sarafotoxin 6b or Atractaspis engaddensis venom, i.p. injections of 1.0 x 106 ESCMCs that did (ETA+/-) or did not (ETA-/-) express ETA; ESCMCS were generated in vitro from ETA+/- or ET A-/- Strain 129 ES cells (D. E. Clouthier et al., Development 125, 813 (1998)) as previously described (M. Tsai et al., Proc Natl Acad Sci USA 97, 9186 (2000); M Maurer et al., Nature 432, 512 (2004)). Other Kitw~ sh/KitW sh mice received, 4 weeks before injection of sarafotoxin 6b, i.p. injections of 1.0 x 106 BMCMCs expressing an MC-CPA-targeting shRNA or empty vector (see below). Other Kitw" sh/Kitw'sh mice received, 6 weeks before s.c. injection of venom either one i.d. injection (via a 30 gauge needle) of 2.0 x 106 BMCMCs in 50 μl of PBS (Crotalus atrox venom-injected mice) or 5 i.d. injections distributed over the length of the back skin (3 injections) and the belly skin (2 injections), with each injection containing 1.5 x 106 BMCMCs in 50 μl of PBS (Apis mellifera venom-injected mice). Other Kitw/Kitw~v mice received, 6 weeks before i.d. injection of Loxosceles reclusa venom 2.0 x 106 BMCMCs in 4 x 50 μl of PBS, injected (via a 30 gauge needle) into the back skin over an area of approximately 1.5 cm x 1.5 cm.

Lentiviral vector production.
pLentiLox 3.7 (pLL3.7) (kindly provided by Dr. L. Van Parijs (D. A. Rubinson et al., Nat Genet 33, 401 (2003)), a vector engineered to co-express enhanced green fluorescent protein (GFP) as a reporter gene, permitting infected cells to be tracked by flow cytometry, was digested with Xhol and Hpal and the annealed oligos 5'-t- GAAACAGTTTGATGTGAAA-ttcaagaga-TTTCACATCAAACTGTTTC-ttttttc-3' SEQ ID NO: 1 and 5'-tcgagaaaaaa-GAAACAGTTTGATGTGAAA-tctcttgaa- TTTCACATCAAACTGTTTC-a-31 SEQ ID NO: 2 were ligated into pLL3.7 to yield a CPA-directed shRNA-producing vector. The 19 nt CPA target sequences are indicated in capitals in the oligonucleotide sequence. Active viral stocks were created and concentrated as previously described (L. Allies et al., MoI Ther ό, 615 (2002)). Briefly, 293T cells were transfected with the transfer vector plasmid pLL3.7-MC-CPA or pLL3.7 (empty vector), the WSV-G envelope-encoding plasmid pMD.G, and the packaging plasmid CMVΔR8.74 (L. Ailles et al., supra) using the calcium phosphate method. The supernatants were harvested 48 and 72 h post-traπsfectioπ, pooled, passed through a 0.45 μm filter, ultracentrifuged for 2 h 20 min at 19,200 rpm in an SW28 rotor, re-suspended in 100 μl of 0.1% BSA in PBS and stored at -800C.

Lentiviral infection and characterization of CPA shRNA-containing mast cells
2-5 week old BMCMCs were infected with virus carrying the CPA-targeting shRNA or the empty vector. Since pLL3.7 carries a CMV-GFP cassette, BMCMCs were sorted for GFP expression at 72-96 h after infection using FACS Aria (Becton Dickinson) and then were cultured in IMDM + 10 ng/ml IL-3 (Peprotech) + 10 ng/mL SCF (Amgen). 1.0 x 106 infected BMCMCs were injected i.p. into Kitw'sh/Kitw'sh mice and experiments were performed 4 weeks later. Since, to our knowledge, this is the first time shRNA-targeted mast cells have been used in vivo, we assessed the CPA enzymatic activity in the peritoneal mast cells (PMCs) which arose from the adoptively-transferred CPA shRNA- or empty vector-containing mast cells by measuring reduction of absorbance of a chromogenic substrate (N-[4-Methoxyphenylazoformyl]-Phe-OH) specific for CPA. We also counted the numbers of mast cells in the peritoneum (as assessed by counting toluidine blue-positive PMCs in an improved Neubauer chamber), and evaluated the appearance of mast cells in the mesentery. The latter point is important, since the migration of adoptively-transferred mast cells into the mesentery appears to be required for such mast cells to be able to provide protective function in a model of innate immunity to bacterial infection (T. Jippo, E. Morii, A. Ito, Y. Kitamura, J Exp Med 197, 1417 (2003)). Mesenteric windows were prepared and stained with Csaba stain for mast cell detection (M. A. Grimbaldeston et al., supra). We also evaluated levels of mMCP-5 in Western blots of CPA shRNA PMCs, which revealed that levels of mMCP-5 protein are greatly reduced in such cells, a finding which has also been reported in mast cells derived from CPA-deficient mice (T. B. Feyerabend et al., MoI Cell Biol 25, 6199 (2005)).

Sarafotoxin and ET-I measurements
Sarafotoxin concentrations were measured using an endothelin EIA with 100% cross -reactivity for sarafotoxins (Cayman Chemical, Ann Arbor, MI), and ET-I levels were measured by ELISA (Biomedica, Vienna). All measurements of amounts of sarafotoxins or ET-I in peritoneal fluid were made at 60 min after injection or at time of death, if that occurred sooner.

Assessment of peritoneal mast cell degranulation
Cytospins were prepared from cells collected by peritoneal lavage, stained with May-Grimwald-Giemsa stain and examined under a light microscope at x 1,000 magnification to quantify the extent of peritoneal MC (PMC) degranulation. The slides were coded so that the observers assessing the extent of PMC degranulation were not aware of the identity of the individual slides. MC degranulation is expressed as the % of the PMCs examined (at least 60 PMCs per cytospin) in which >50% ("Extensive" degranulation of that cell), 10-50%
("Moderate" degranulation of that cell) or <10% ("None", indicative of no significant evidence of degranulation of that cell) of the cytoplasmic granules of that PMC exhibited morphological evidence of degranulation, namely, alterations in the staining characteristics, size or distribution of the granules (M Maurer et al., supra; B. K. Wershil, T. Murakami, S. J. Galli, J Immunol 140, 2356 (1988); A. P Prodeus, X. Zhou, M. Maurer, S. J. Galli, M. C. Carroll, Nature 390, 172 (1997)).

Statistical analysis
The differences in percentages of mice exhibiting death by 24 h after injection with S6b or various venoms was compared by Fisher's exact test, whereas analysis of variance (ANOVA) for repeated measures was used to assess differences in body temperature responses. All other data were tested for statistical significance using the unpaired two-tailed Student's t-test or, to compare values for the extent of mast cell degranulation, the Chi-square test. P<0.05 is considered statistically significant. Unless otherwise specified, all data are presented as mean ± s.e.m. or mean + s.e.m.

Venoms
Venom from Atractaspis engaddensis, obtained as described (C. Takasaki, N.
Tamiya, A. Bdolah, Z. Wollberg, E. Kochva, Toxicon 26, 543 (1988)) and stored at -200C, was kindly provided by Elazar Kochva and Avner Bdolah (Department of Zoology, Tel Aviv University, Israel). Crotalus atrox and Agkistrodon contortrix contortrix venoms were purchased from Natural Toxins Research Center (Kingsville, TX), Apis mellifera venom was purchased from Vespa Laboratories (Spring Mills, PA), and Loxosceles reclusa venom was purchased from Spiderpharm (Yarnell, AZ).

The venom used in the examples is sarafotoxin 6b. This is snake venom peptide, and is a vasoconstrictor from Atractaspis engaddensis (Israeli burrowing asp) having the
Sequence:

Cys-Ser-Cys-Lys-Asp-Met-Thr-Asp-Lys-Glu-Cys-Leu-Tyr-Phe-Cys-His-Gln-Asp-Val-Ile-Trp (disulfide bonds between residues 1 and 15, 3 and 11). SEQ ID NO: 3

Sarafotoxins are short peptide toxins found in the venoms of snakes from Atractaspis spp., which display potent vasoconstriction properties. These peptides, which share a high degree of sequence identity with endothelins, recognize and bind to endothelin receptors. Snakes have also evolved toxins that block L-type Ca2+ currents (e.g., calciseptine, FS2 toxins, C10S2C2 and S4C8). Snake venom proteins have also been shown to increase vascular permeability. One such protein, increasing capillary permeability protein (ICPP) has recently been isolated from the venom of Vipera lebetina. ICPP is an extremely potent permeability factor with a structure similar to vascular endothelial growth factor (VEGF). Thus there is a vast array of snake toxins with potent cardiovascular activity.

Swissprot contains the following sarafotoxin sequences: SRTD ATREN (P13211),

Sarafotoxin-D (S6D) (SRTX-D). - Atractaspis engaddensis (Israeli burrowing asp);
SRTX ATREN (Pl 3208), Sarafotoxins precursor [Contains: Sarafotoxin A, Ser-isoform (Sarafotoxin A) (S6A) (SRTX-A); Sarafotoxin C (S6C) (SRTX-C); Sarafotoxin B (S6B) (SRTX-B); Sarafotoxin E (S6E) (SRTX-E); Sarafotoxin A, Thr-isoform]. - Atractaspis engaddensis (Israeli burrowing asp); and O6RY98 9SAUR Long-sarafotoxins (Fragment) { GENE:Name=l-SRTXs } - Atractaspis microlepidota.
- ™ -'-«'• '"•- 17 of 58 As discussed in US 6,555,116 to Buchanan, et al., April 29, 2003, entitled
"Alleviation of the allergenic potential of airborne and contact allergens by thioredoxin," Venoms from snakes are characterized by active protein components (generally several) that contain disulfide (S--S) bridges located in intramolecular (intrachain) cystines and in some cases in intermolecular (interchain) cystines. The position of the cystine within a given toxin group is highly conserved. The importance of intramolecular S-S groups to toxicity is evident from reports showing that reduction of these groups leads to a loss of toxicity in mice (Yang, C. C. (1967) Biochim. Biophys. Acta. 133:346-355; Howard, B. D. et al., (1977) Biochemistry 16:122-125). The neurotoxins of snake venom are proteins that alter the release of neurotransmitter from motor nerve terminals and can be presynaptic or postsynaptic. Common symptoms observed in individuals suffering from snake venom neurotoxicity include swelling, edema and pain, fainting or dizziness, tingling or numbing of affected part, convulsions, muscle contractions, renal failure, in addition to long-term necrosis and general weakening of the individual, etc.

The presynaptic neurotoxins are classified into two groups. The first group, the beta-neurotoxins, includes three different classes of proteins, each having a phospholipase A2 component that shows a high degree of conservation. The proteins responsible for the phospholipase A2 activity have from 6 to 7 disulfide bridges. Members of the β-neurotoxin group are either single chain (e.g., caudotoxin, notexin and agkistrodotoxin) or multichain (e.g., crotoxin, ceruleotoxin and Vipera toxin) β-bungarotoxin, which is made up of two subunits, constitutes a third group. One of these subunits is homologous to the Kunitz-type proteinase inhibitor from mammalian pancreas. The multichain β-neurotoxins have their protein components linked ionically whereas the two subunits of β-bungarotoxin are linked covalently by an intermolecular disulfide. The B chain subunit of β-bungarotoxin, which is also homologous to the Kunitz-type proteinase inhibitor from mammalian pancreas, has 3 disulfide bonds.

The second presynaptic toxin group, the facilitatory neurotoxins, is devoid of enzymatic activity and has two subgroups. The first subgroup, the dendrotoxins, has a single polypeptide sequence of 57 to 60 amino acids that is homologous with Kunitz-type trypsin inhibitors from mammalian pancreas and blocks voltage sensitive potassium channels. The second subgroup, such as the fasciculins (e.g., fasciculin 1 and fasciculin 2), is cholinesterase inhibitors and have not been otherwise extensively studied.

Sequences for the following venom proteins are given in SWISSPROT:



Aside from neurotoxins, other types of venom toxins will be susceptible to the present protease treatments, such as hemotoxins (found in pit vipers) or necrotoxins (found in brown recluse spiders). Venoms from snakes of various families, including Crotalidae (a family of venomous snakes, the pit vipers, characterized by front, movable, hollow fangs and a depression or pit between the nostril and the eye, sometimes considered a subfamily of Viperidae) Elapidae (fixed-fang snakes such as cobras, kraits, and coral snakes),
Hydrophiidae (Sea snakes of several different species belong to a group related to the cobras), and Viperidae (e.g., copperhead, canebrake or timber rattlesnake, Eastern
diamondback rattlesnake, pigmy rattlesnake, cottonmouth) may be treated. Also included are lizard venom, e.g., Heloderma suspectum (containing neurotoxin), stinging insects (discussed below) and microbial toxins (as discussed below). Venomous snakes dangerous to humans include the following: Adder, Asp, Black snake, Black mamba, Boomslang, Brown snake (Australian), Bushmaster, Cobra, Common lancehead, Coral snake, Cottonmouth, American Copperhead, Australian Copperhead, Death adder, Diamondback, Fer-de-lance, Fierce Snake, Gaboon Viper, King Cobra, Krait Lancehead, Mamba, Philippine Cobra, Philippine Spitting Cobra, Pit Viper, Rattlesnake, Russell's Viper, Saw-scaled Viper, Sea snake, Taipan, Tiger snake, and Urutu.

Thus there are a number of venom proteins, which may be treated by the present methods and materials.

The following exemplary venoms, toxins and toxoids are available from Calbiochem/EMD and given with the catalog number:









Treatment of bee and brown recluse spider envenomation is also specifically contemplated and exemplified here. Although there are thousands of species of bees, only the honeybee, Apis mellifera, is a significant cause of allergic reactions. The response ranges from local discomfort to systemic reactions such as shock, hypotension, dyspnea, loss of consciousness, wheezing and/or chest tightness that can result in death. The only treatment that is used in these cases is the injection of epinephrine.

The treatment of bee stings is important not only for individuals with allergic reactions. The "killer" or Africanized bee, a variety of honeybee is much more aggressive than European honeybees and represents a danger in both South and North America. While the lethality of the venom from the Africanized and European bees appears to be the same (Schumacher, M. I. et al., (1989) Nature 337:413), the behaviour pattern of the hive is completely different. It was reported that Africanized bees respond to colony disturbance more quickly, in greater numbers and with more stinging (Collins, A. M. et al., (1982) Science 218:72-74). A mass attack by Africanized bees may produce thousands of stings on one individual and cause death. The "killer" bees appeared as a result of the interbreeding between the African bee (Apis mellifera scutellatd) and the European bee (Apis mellifera melliferά). African bees were introduced in 1956 into Brazil with the aim of improving honey production being a more tropically adapted bee. Africanized bees have moved from South America to North America, and they have been reported in Texas and Florida.

Bee venom is a complex mixture with at least 40 individual components, that include major components as melittin and phospholipase A2, representing respectively 50% and 12% of the total weight of the venom, and minor components such as small proteins and peptides, enzymes, amines, and amino acids.

Melittin is a polypeptide consisting of 26 amino acids with a molecular weight of 2840. It does not contain a disulfide bridge. Owing to its high affinity for the lipid-water interphase, the protein permeates the phospholipid bilayer of the cell membranes, disturbing its organized structure. Melittin is not by itself a toxin but it alters the structure of membranes and thereby increases the hydrolitic activity of phospholipase A2, the other major component and the major allergen present in the venom.

Bee venom phospholipase A2 is a single polypeptide chain of 128 amino acids, is cross-linked by four disulfide bridges, and contains carbohydrate. The main toxic effect of the bee venom is due to the strong hydrolytic activity of phospholipase A2 achieved in association with melittin.

Bites of the brown recluse spider (Loxosceles reclusa) are thought to be the major reason for spider venom-induced cutaneous injury in the United States (Swanson and Vetter (2005) New Engl J Med 352:700-707) and numerous cases of brown recluse spider envenomations have been reported worldwide (Furbee et al., (2006) Clin Lab Med 26:211-226). The treatment of brown recluse spider bites remains controversial and mainly consists of routine first aid and subsequent supportive care including hyperbaric oxygen, dapsone, antihistamines, antibiotics, dextran, glucocorticoids, vasodilators, heparin, nitroglycerin, electric shock, curettage, surgical excision, and antivenom (Swanson and Vetter (2005) New Engl J Med 352:700-707).

The effects of a brown recluse spider bite can range from rather mild self-limiting and self-healing skin irritations to necrotizing lesions and systemic effects, such as fever, chills, rash, joint pains, and hemolysis. These clinical effects of brown recluse spider envenomations are often referred to as Loxoscelism, necrotic arachnidism, or gangrenocutaneous
arachnidism (Wasserman et al., (1983) J Toxicol Clin Toxicol 21:451-72).

Loxosceles venom contains several enzymes, among them alkaline phosphatase, 59 ribonucleotide phosphohydrolase, esterase, protease, hyalυronidase, and sphingomyelinase D (Furbee et al., (2006) CHn Lab Med 26:211-226).

Figure 11 shows various degrees of mast cell degranulation caused by venom and venom components from honeybee, wasps, one spider and four different snakes. Venom and venom components from honeybee, wasps, one spider (brown recluse spider) and four different snakes (western diamondback rattlesnake, southern copperhead, mole viper and Indian cobra) can induce the degranulation of human mast cells. To assess whether human mast cells have the ability to be activated by venom or venom components, human mast cell line LAD-2 and human cord blood-derived mast cells were stimulated with the indicated venoms and, where commercially available, with venom components. Calcium Ionophore A23187 was used as a positive control, NaCl as negative control. Data is derived from 3-6 independent experiments but only from one batch of cells (i.e., hCBMCs from one patient). *** = p<0.005, * = p<0.05 vs. negative control.

It can be seen that A mellifera venom causes more mast cell degranulation than other components; that in wasp venom, the Vespula spp. Venom causes more mast cell
degranulation than other components.

Polypeptide Preparations

The present invention contemplates the use of protease polypeptides, for example, in particular, polypeptides related to (1) carboxypeptidase A, (2) carboxypeptidase B, (3) chymotrypsin, (4) chymase, and (5) papain.
Specifically exemplified proteases
(1) carboxypeptidase A
Carboxypeptidases A and B are the principal mammalian representatives of the metalloprotease family. Both are exopeptidases of similar structure and active sites.
Carboxypeptidase A, like chymotrypsin, prefers C-terminal aromatic and aliphatic side chains of hydrophobic nature, whereas carboxypeptidase B is directed toward basic arginine and lysine residues. The amino acid sequence and nucleic acid sequence of carboxypeptidase A was described in Natsuaki, M., et al., "Human skin mast cell carboxypeptidase: functional characterization, cDNA cloning, and genealogy," J. Invest. Dermatol. 99 (2), 138-145 (1992).

The amino acid sequence of this protein is as follows, taken from GenBank Accession No. AAB22578 (Swiss prot CBPA3_HUMAN):
SEQ ID NO: 4

1 ipgrhsyaky nnwekivawt ekmmdkypem vsrikirstv ednplyvlki geknerrkai

61 fmdcgihare wvspafcqwf vyqatktygr πkimtklldr mnfyilpvfn vdgyiws wtk

121 nrmwrknrsk nqnskcigtd Inrnfnaswn sipntndpca dnyrgsapes eketkavtnf

181 irshlneikv yttfhsysqm llfpygytsk lppnhedlak vakigtdvls tryetryiyg

241 piestnypis gssldwaydl gikhtfafel rdkgkfgfll pesrikptcr etmlavkfia

301 kyilkhts

Further examples of carboxypeptidase polypeptides and nucleic acids encoding same are found in US 6,951,749 to Blinkovsky, et al., issued October 4, 2005, entitled
"Carboxypeptidases and nucleic acids encoding same."
Further guidance in the design or selection of an appropriate enzyme may be found in Guasch, A., et al., "Three-dimensional structure of porcine pancreatic procarboxypeptidase A. A comparison of the A and B zymogens and their determinants for inhibition and activation," /. MoI. Biol. 224 (1), 141-157 (1992).

Another example of a suitable carboxypeptidase is Human Procarboxypeptidase A2, GenBank Accession No. IAYE. The following SwissProt entries also contain exemplary sequences for use in the present polypeptide preparations: BRS1_ARATH (Q9M099)

Another example is serine carboxypeptidase 2 precursor; other examples are
CAC3_BOVIN (P05805) Proproteinase E precursor (Procarboxypeptidase A complex component III) (Procarboxypeptidase A-S6 subunit III) (PROCP A-S6 HI). - Bos taurus (Bovine); CBAL_BACST (P13722), Alanine carboxypeptidase (EC 3.4.17.6) (Fragment). -Bacillus stearothermophilus (Geobacillus stearothermophilus); CBPl-HORVU (P07519) Serine carboxypeptidase 1 precursor (EC 3.4.16.5) (Serine carboxypeptidase I)
(Carboxypeptidase C) (CP-MI) [Contains: Serine carboxypeptidase 1 chain A (Serine carboxypeptidase I chain A); Serine carboxypeptidase 1 chain B (Serine carboxypeptidase I chain B)]. {GENE: Name=CBPl; Synonyms=CXP; 1 } - Hordeum vulgare (Barley);
CBP1_ORYSA (P37890); Serine carboxypeptidase 1 precursor (EC 3.4.16.5) (Serine carboxypeptidase I) (Carboxypeptidase C). {GENE: Name=CBPl ; Ordered
LocusNames=Osl2gl5470} - Oryza sativa (Rice); CBPA1_HUMAN (P15085)
• Carboxypeptidase Al precursor (EC 3.4.17.1). {GENE: Name=CPAl; Synonyms=CPA} - Homo sapiens (Human); CBPA2_HUMAN (P48052); Carboxypeptidase A2 precursor (EC 3.4.17.15). {GENE: Name=CPA2} - Homo sapiens (Human); CBPA3_HUMAN (P15088) -- - Mast cell carboxypeptidase A precursor (EC 3.4.17.1) (MC-CPA) (Carboxypeptidase A3). {GENE: Name=CPA3} - Homo sapiens (Human); CBPA3_MOUSE (P15089) Mast cell carboxypeptidase A precursor (EC 3.4.17.1) (MC-CPA) (Carboxypeptidase A3). {GENE: Name=Cpa3} - Mus musculus (Mouse); CBPA3_RAT (P21961) Mast cell carboxypeptidase A precursor (EC 3.4.17.1) (RMC-CP) (Carboxypeptidase A3) (R-CPA) (Fragment). {GENE: Name=Cpa3} - Rattus norvegicus (Rat) Carboxypeptidase A4 precursor (EC 3.4.17.-) (Carboxypeptidase A3). {GENE: Name=CPA4; Synonyms=CPA3;
ORFNames=UNQ694/PRO1339} - Homo sapiens (Human); CBPA4_HUMAN (Q9UI42) Carboxypeptidase A4 precursor (EC 3.4.17.-) (Carboxypeptidase A3). {GENE:
Name=CPA4; Synonyms=CPA3; ORFNames=UNQ694/PRO1339 } - Homo sapiens
(Human); CBPA6_HUMAN (Q8N4T0); and Carboxypeptidase A6 precursor (EC 3.4.17.1) (Carboxypeptidase B). {GENE: Name=CPA6; Synonyms=CPAH} - Homo sapiens (Human).

It is also known that the active site zinc ion in carboxypeptidase A is bound to His 69, GIu 72 and His 196 and water molecule, which is hydrogen bound to GIu 270. The other key residues are GIu 270, Arg 127, Asn 144, Arg 145 and Tyr 248. Thus, one would conserve these residues when alternative sequences to native carboxypetidase are made, as discussed further below. The following sequence is from MEROPS, http://merops.sanger.ac.uk/.

SEQUENCE MER01190
>MER01190 - carboxypeptidase Al [M14.001] {peptidase unit: 111-419} (active site residue(s): 237,380; metal ligands: 179,182,306) (Homo sapiens) from P15085.
Bolded residues participate in the active site.
1 MRGLLVLSVLLGAVFGKEDFVGHQVLRISVADEAQVQKVKELEDLEHLQLDFWRGPAHPG 60 61 SPIDVRVPFPSIQAVKIFLESHGISYETMffiD VQSLLDEEQEQMFAFRSRARSTDTFNYA 120 121 TYHTLEEIYDFLDLLV AENPHLVSKIQIGNTYBGRPIYVLKFSTGGSKRPAIWIDTGMΓS 180 181 REWVTQASGVWFAKKITQDYGQDAAFTAILDTLDIFLEIVTNPDGFAFrHSTNRMWΛKTR 240 241 SHTAGSLCIGVDPNRNWDAGFGLSGASSNPCSETYHGKFANSEVEVKSIVDFVKDHGNIK 300 301 AHSIiϊSYSQLLMYPYGYKTEPVPDQDELDQLSKAAVTALASLYGTKFNYGSIIKAIYQA 360 361 SGSTIDWTYSQGIKYSFTFELRDTGRYGFLLPASQIIPTAKET WLALLTIMEHTLNHPY 419 SEQ ID NO: 5
Another exemplary carboxypeptidase is described in US 5,593,674 to Drayna, et al., issued January 14, 1997, entitled "Plasma carboxypeptidase," and hereby incorporated by reference. It describes a polypeptide, designated plasma carboxypeptidase B (PCPB), purified from human plasma. It was cloned from a human liver cDNA library using PCR
amplification. It shares some common structural features and catalytic and substrate binding sites with carboxypeptidase A and with pancreas carboxypeptidase B, and in its human embodiment shares the most sequence identity with known carboxypeptidases A and B.
(2) carboxyppeptidase B
Specific examples of carboxypeptidase B (EC 3.4.17.2) nucleic acid sequences are described in GenBank Accession Number AJ224866, Homo sapiens mRNA for pancreatic procarboxypeptidase B; and GenBank Accession Number BCO15338, Homo sapiens carboxypeptidase B 1 (tissue). Amino acid sequences are given in UniProtKB/S wiss-Prot

P00732, CBPB1_BOVIN; P55261, CBPB1_CANFA; Pl 5086, CBPBl J3UMAN;

P09955, CBPB1_PIG; P19223, CBPB1_RAT; P04069, CBPB_ASTFL; and

P19628, CBPB_PROAT.

(3) Chvmotrvpsin
The present chymotrypsin polypeptides are best exemplified by the following
SwissProt entries:

AACTJSUMAN (POlOl 1), Alpha- 1-antichymotrypsin precursor (ACT) [Contains: Alpha-1-antichymotrypsin His-Pro-less]. {GENE: Name=S ERPIN A3; Synonyms=AACT } - Homo sapiens (Human); CLCRJHUM AN (Q99895) Caldecrin precursor (EC 3.4.21.2)
(Chymotrypsin C). {GENE: Name=CTRC; Synonyms=CLCR} - Homo sapiens (Human)

CTRBl JHUMAN (P17538); Chymotrypsinogen B precursor (EC 3.4.21.1) [Contains:
Chymotrypsin B chain A; Chymotrypsin B chain B; Chymotrypsin B chain CJ. { GENE:

Name=CTRB 1 ; Synonyms=CTRB } - Homo sapiens (Human); CTRLJHUMAN (P40313)

Chymotrypsin-like protease CTRL-I precursor (EC 3.4.21.-). {GENE: Name=CTRL;
Synonyms=CTRLl } - Homo sapiens (Human); LCLPJHUMAN (P34168); Chymotrypsin-like serine proteinase (EC 3.4.21.-) (LCLP) (Fragment). - Homo sapiens (Human).

(4) Chymase
The present chymase polypeptides are exemplified by the following sequences from SwissProt: MCPT1_HUMAN (P23946), Chymase precursor (EC 3.4.21.39) (Mast cell protease I). {GENE: Name=CMAl; Synonyms=CYH, CYM } - Homo sapiens (Human); Q3SY36_HUMAN Chymase 1, mast cell, preproprotein {GENE:Name=CMAl } - Homo sapiens (Human) Q4FEB3_HUMAN; Chymase 1 preproprotein transcript E (Fragment) {GENE:Name=CMAl } - Homo sapiens (Human) ; Q4FEB5_HUMAN Chymase 1 preproprotein transcript I (Fragment) {GENE:Name=CMAl } - Homo sapiens (Human); Q7RTY7_HUMAN Ovochymase precursor {GENE:Name=OVCHl; Synonyms=OVCH} -Homo sapiens (Human); and Q9UDH5_HUMAN Chymase (Fragments) - Homo sapiens (Human).
(5) Papain
Preferred exemplary sequences include the following SwissProt entries:
PAPA1_CARPA (P00784), Papain precursor (EC 3.4.22.2) (Papaya proteinase I) (PPI) (Allergen Car p 1). - Carica papaya (Papaya); PAPA2_CARPA (P14080), Chymopapain precursor (EC 3.4.22.6) (Papaya proteinase II) (PPII). - Carica papaya (Papaya);
POLA_CHPVE (P10941) Polyprotein p69 (ORFA polyprotein) [Contains: Papain-like protease p29 (EC 3.4.22.-); p40 protein]. - Cryphonectria hypovirus 1 (strain EP713) (CHV-1/EP713) (Chestnut blight fungus hypovirulence-associated virus); POLA-CHPVU
(Q9YTU3) Polyprotein p69 (ORFA polyprotein) [Contains: Papain-like protease p29 (EC

3.4.22.-); p40 protein]. - Cryphonectria hypovirus 1 (strain Euro7) (CHV-1/Euro7) (Chestnut blight fungus hypovirulence-associated virus); POLB_CHPVE (Q04350); ORFB
polyprotein [Contains: Papain-like protease p48 (EC 3.4.22.-); Putative RNA-directed RNA polymerase/Helicase (EC 2.7.7.48) (EC 3.6.1.-)]. - Cryphonectria hypovirus 1 (strain EP713) (CHV-1/EP713) (Chestnut blight fungus hypovirulence-associated virus); POLB_CHPVU (Q9YTU2); ORFB polyprotein [Contains: Papain-like protease p48 (EC 3.4.22.-); Putative RNA-directed RNA polymerase/Helicase (EC 2.7.7).]
Additional polypeptide sequences
The polypeptides of the invention include polypeptides having amino acid
substitutions, i.e., variant polypeptides, so long as the polypeptide variant is proteolytic. For example, for carboxypeptidase variants, it is preferred that the variant has at least about 50% of the biological activity of the corresponding non-variant polypeptides, e.g., a polypeptide having a sequence such as listed above. The variant polypeptides include the substitution of

"- * ™ ,,r.n ,- « ~.-.~ j j Ot JO at least one amino acid residue in the polypeptide for another amino acid residue, including substitutions which utilize the D rather than L form, as well as other well known amino acid analogs, e.g., unnatural amino acids such as a, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and the like. These analogs include phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, l,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, a-methyl-alanine, para-benzoyl-phenylalanine, phenylglycine, propargylglycine, sarcosine, e-N,N,N-trimethyllysine, e-N-acetyllysine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids and tert-butylglycine.

Conservative amino acid substitutions are preferred—that is, for example, aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids;
leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids;
serine/glycine/alanine/threonine as hydrophilic amino acids. Conservative amino acid substitution also includes groupings based on side chains. Members in each group can be substituted for one another. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine. These may be substituted for one another. A group of amino acids having aliphatic-hydroxyl side chains is serine and threonine. A group of amino acids having amide-containing side chains is asparagine and glutamine. A group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan. A group of amino acids having basic side chains is lysine, arginine, and histidine. A group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid may be accomplished to produce a variant polypeptide of the invention.
Protein engineering
As discussed in US 6,358,726 to Takakura, et al., issued March 19, 2002, entitled "Thermostable protease," a mutation such as deletion, substitution, insertion or addition of one to several amino acid residues in an amino acid sequence may be generated in a naturally occurring protein including the polypeptides exemplified here. Such mutation may be generated due to a polymorphism or a mutation of the gene encoding the protein, or due to a modification of the protein in vivo or during purification after synthesis may occur.
Nevertheless, it is known that such a mutated protein may exhibit physiological and biological activities equivalent with those of a protein without a mutation. This is applicable to a protein in which such a mutation is introduced into its amino sequence artificially, in which case it is possible to generate a wide variety of mutations. For example, it is known that a polypeptide in which a cysteine residue in the amino acid sequence of human interleukin-2 (IL-2) is substituted with a serine residue retains an interleukin-2 activity

(Science, 224:1431 (1984)). Thus, a protease having an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted or added in the amino acid sequences disclosed here and having a protease activity at least equivalent with that of the protease of the present invention is within the scope of the present invention.

It is also possible to use the present teachings to design novel polypeptides that will cleave venom proteins and therefore be useful in the treatment of venomous bites. The candidate peptide is incubated with a toxin protein such as S6b, as described below in Example 11 , or any other toxin or combination of toxins of interest. If less than 50% of the control signal is obtained, preferably less than 10% of the control signal, the peptide may be used n the present method. The signal may be assessed by an ELISA assay, if one exists for that toxin (as it does in the case of S6b) or by any other method which can quantify the amount and/or residual biological activity of that toxin or group of toxins, such as a mouse LD50 assay. The peptide must also be stable in a pharmaceutical preparation, as can be determined by standard stability testing. It must also be non-toxic, as determined by standard testing, beginning in cell culture and progressing to animal testing, including Phase I studies in humans. Since a small dose of the polypeptide is administered and may only be administered one time, it is not necessary that long term safety be addressed.

Means for modifying a known candidate sequence, such as listed here, are know in the art. For example, in Lien et al., "Combinatorial strategies for the discovery of novel protease specificities," Comb Chem High Throughput Screen. 1999 Apr;2(2):73-90. The authors there describe proven and possible ways to generate novel cleavage specificities in serine proteases using combinatorial mutagenesis. The paper further compares the different ways of screening or selecting for desirable mutants, and examines the ways in which combinatorial substrate libraries can be used to gain a more comprehensive insight into protease cleavage preferences. US 6,383,775 to Duff, et al., dated May 7, 2002, entitled "Designer proteases" provides further guidance. In this method, a designer protease is selected using a negative selection procedure, wherein a cell expressing IgAl protease on its surface is depleted. For example, affinity chromatography using an antibody specific for the protease (or artificially introduced epitope tag) can be used. Alternatively, the designer protease is selected using a positive selection procedure, wherein the cleaved protease is detected.

The present enzymes are evolutionarily diverse and tolerate a large degree of sequence variation. For example, a BLAST search was run with human mast cell
carboxypeptidase A, SWISS PROT CBP A3. The results show that even enzymes in this family with identity as low as 36% had similar protease function:

Carboxypeptidase A5 precursor (EC 3.4.17.1) [CPA5] [Homo 436

CBPA5_HUMAN sapiens AA
(Human) ]

Score = 289 bits (739), Expect = le-76
Identities = 149/409 (36%), Positives = 246/409 (60%), Gaps = 13/409 (3%)
Polypeptide modifications and derivatives
Other types of modifications are possible to the specific polypeptides disclosed here. For example, the polypeptides may further by PEGylated, as described in US 5,766,897 to Braxton, issued June 16, 1998, entitled "Cysteine-pegylated proteins," hereby incorporated by reference.

The polypeptides can be synthesized in vitro, e.g., by the solid phase peptide synthetic method or by recombinant cellular approaches (see above), or by in vitro
transcription/translation systems. The synthesis products may be fusion polypeptides, i.e., the polypeptide comprises the polypeptide variant or derivative according to the invention and another peptide or polypeptide, e.g., a His, HA or EE tag. Such methods are described, for example, in U.S. Pat. Nos. 5,595,887; 5,116,750; 5,168,049 and 5,053,133; Olson et al., Peptides, 9, 301, 307 (1988). The solid phase peptide synthetic method is an established and widely used method, which is described in the following references: Stewart et al., Solid

Phase Peptide Synthesis, W. H. Freeman Co., San Francisco (1969); Merrifield, /. Am. Chem. Soc, 85 2149 (1963); Meienhofer in "Hormonal Proteins and Peptides," ed.; C. H. Li, Vol. 2 (Academic Press, 1973), pp. 48-267; Bavaay and Merrifield, "The Peptides," eds. E. Gross and F. Meienhofer, Vol. 2 (Academic Press, 1980) pp. 3-285; and Clark-Lewis et al., Meth. Enzymol., 287, 233 (1997). These polypeptides can be further purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing;

SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography.

Derivatives of a polypeptide of the invention can be readily prepared by methods known in the art. See, for example, Advanced Organic Chemistry, J. March, cited infra. For example, amides of the polypeptide of the invention may also be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide. A preferred method for amide formation at the C-terminal carboxyl group is to enzymatically couple a modified amino acid to the C terminus and convert it to a carboxyamide. For example, use of 2-o-nitrophenylalanine and an exocarboxypeptidase such as carboxypeptidase Y will form the modified alanyl carboxyamide. Photolysis converts the modified alanyl group to an NH.sub.2 group such that an unsubstituted carboxyamide is formed. Another method involves cleaving the polypeptide from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine.

Salts of carboxyl groups of a polypeptide of the invention may be prepared in the usual manner by contacting the polypeptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.

N-acyl derivatives of an amino group of the polypeptide may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected peptide. O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation may be carried out together, if desired.

Formyl-methionine, pyroglutamine and trimethyl-alanine may be substituted at the N-terminal residue of the polypeptide. Other amino-terminal modifications include
aminooxypentane modifications (see Simmons et al., Science, 276, 276 (1997)).

Acid addition salts of the polypeptide or of amino residues of the polypeptide may be prepared by contacting the polypeptide or amine with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid. Esters of carboxyl groups of the peptides may also be prepared by any of the usual methods known in the art.

The polypeptides of the invention may include moieties, e.g., other peptide or polypeptide molecules (fusion polypeptides), such as antibodies or fragments thereof, nucleic acid molecules, sugars, lipids, e.g., cholesterol or other lipid derivatives which may increase membrane solubility, fats, a detectable signal molecule such as a radioisotope, e.g., gamma emitters, small chemicals, metals, salts, synthetic polymers, e.g., polylactide and
polyglycolide, and surfactants which preferably are covalently attached or linked to polypeptide of the invention. The polypeptides of the invention may also be modified in a manner that would increase their stability in vivo, i.e., their resistance to degradation and/or their metabolic half-lives. These modifications typically may be chemical alterations of the N-- and/or C-- termini and/or alterations of side chain groups by such techniques as esterification, amidation, reduction, protection and the like. Methods to prepare such derivatives are well known to the art. See, for example, Advanced Organic Chemistry, 4th ed., Jerry March, 1992, J. Wiley & Sons, New York.
Polypeptide formulations
The present polypeptides may be prepared for intramuscular, subcutaneous, intradermal, intravenous, nasal or inhalation delivery. The polypeptide may be lyophilized, or in liquid form in a stable buffer, such as that described for corticotropin releasing factor in US 5,780,431.

Additional guidance in formulating the present carboxypeptidases is found in
5,759,539 to Whitmire, issued June 2, 1998, entitled "Method for rapid enzymatic alcohol removal" in that there is taught there that the addition of various protease inhibitors will benefit an enzyme formulation. Such inhibitors should be of an amount not to interfere with the activity of the active protease, but merely serve to stabilize the protease during storage. Protease inhibitors will have only minimal use if the present protease polypeptides are stored in inactive form, e.g., in powdered form or as a pro-peptide.

The present polypeptides will have excipients, defined herein as stabilizers of enzyme activity, solubilizing agents which increase the solubility of the enzymes, release modifying agents, viscosity modifiers, matrix modifying agents and pH buffering agents, can be added to the formulation as appropriate to maximize the efficacy of the enzyme formulation.

Enzyme stabilizers include carbohydrates, amino acids, fatty acids, and surfactants and are known to those skilled in the art. Stabilizers are based on a ratio to the protein on a weight basis. Examples include carbohydrates such as sucrose, lactose, mannitol, dextran, r, .., ... «»„»*. ~~Λ,.- 38 Of 58 proteins such as heparin, albumin and protamine, amino acids such as, arginine, glycine, and threonine, surfactants such as bile salts, Tween® (detergent) and Pluronic (polyethylene oxide-polypropylene glycol block copolymers), salts such as calcium chloride and sodium phosphate, and lipids such as fatty acids, phospholipids, and bile salts. The ratios are generally between 1:10 and 4:1, carbohydrate to protein, amino acids to protein, protein stabilizer to protein, and salts to protein: between 1:1000 and 1:20, surfactant to protein; and between 1:20 and 4:1, lipids to protein.

In those cases where the enzyme formulation is administered in a polymeric matrix, excipients which modify the solubility of the enzymes such as salts and complexing agents (albumin, protamine) can be used to control the release rate of the protein from a matrix. Agents that enhance degradation of the matrix or release from the matrix can also be incorporated. They can be added to the enzymes, added as a separate phase (i.e., as particulates), or can be codissolved in the polymer phase depending on the compound. In all cases the amount should be between 0.1 and thirty percent (w/w polymer). Types of degradation enhancers include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine and surfactants such as Tween® and Pluronic®.

Pore forming agents that add microstructure to the matrices (i.e., water soluble compounds such as inorganic salts and sugars) are added as particulates. The range should be between one and thirty percent (w/w polymer).

The pH of the formulation is important, especially in regard to safety and comfort during injection, and especially if the preparation is supplied in a liquid formulation. A suitable formulation may contain preservatives, such as sodium benzoate, methylparaben and propylparaben, having a pH of 6.8-8.0 at 25° C. The pH is preferably maintained by a buffer.

Suitable buffering agents include acetate buffers, 2-amino-2-methyl-l-propanol, glycine buffers, phosphate buffers, (tris>hydroxymethyl-aminomethane) (TRIS) buffers, (2->N-morpholino-ethanesulfonic acid) (MES), Bis-Tris, (N->2-acetamido~2-iminodiacetic acid; N->carbarnoylmethyl-irninodiacetic acid) (ADA), (2->(2-amino-2-oxoethyl)amino-ethanesulfonic acid; N->2-acetamido~2-aminoethanesulfonic acid) (ACES), (piperazine-N,N'-bis>2-ethanesulfonic acid; 1,4-piperazinediethanesulfonic acid) (PIPES), (3->N- morpholino~2-hydroxypropanesulfonϊc acid) (MOPSO), Bis-Tris Propane, (N,N-bis>2-hydroxyethyl~2-aminoethanesulfonic acid; 2->bis(2-hydroxyethyl)aminoethanesulfonic acid; 2->bis(2-hydroxyethyl)amino-ethanesulfonic acid (BES), (3->N-morpholino-propanesulfonic acid) (MOPS), (N-tris>hydroxymethyl-methyl-2-aminoethane-sulfonic acid; 2-(>2-hydroxy-l, l-bis(hydroxymethyl)ethyl-amino)ethanesulfonic acid (TES), (N->2-hydroxyethyl)piperazine-N'>2-ethanesulfonic acid) (HEPES), (3->N,N-bis(2-hydroxyethyl)amino— 2-hydroxy-propanesulfonic acid) (DIPSO), (3->N-tris(hydroxymethyl)methylamino— 2-hydrooxypropanesulfonic acid) (TAPSO), (N->2-hydroxyethyl-piperazine-N'->2-hydroxypropanesulfonic acid) (HEPPSO), (POPSO), (N->2-hydroxyethyl)piperazine-N'->3-propasesulfonic acid- (EPPS), triethanolamine (TEA), (N-tris>hydroxymethyl-methylglycine; N->2-hydroxy-l,l-bis(hydroxymethyl)ethyl-glycine) (Tricine), (N,N-bis>2-hydroxyethyl-glycine) (Bicine), (N-tris>hydroxymethyl-methyl-3-aminopropaπesulfonic acid; (>2-dhyroxy-l,l-bis(hydroxymethyl)-ethyl-amino)-l-propanesulfonic acid) (TAPS), (3->(l,l-dimethyl-2-hydroxyethyl)amino~2-hydroxypropanesulfonic acid) (AMPSO), (2->N-cyclohexylamino-ethanesulfonic acid) (CHES), (3->cyclohexylamino— 2-hydroxy- 1-propanesulfonic acid) (CAPSO), 2-amino-2 -methyl- 1-propanol (AMP), and (3->cyclohexylamino~l-propanesulfonic acid) (CAPS), among others. These are available from commercial sources such as Sigma Chemical Co.

EXAMPLES
Example 1: Mast cell (MC) protection against toxicity of sarafotoxin 6b and A tracfaspis e/igaddensis{A.e. v.) venom in MC wild type (WT) and MC deficient mice
The series of graphs in Fig. 1 A-F shows changes in rectal temperatures, levels of sarafotoxins in the peritoneal lavage fluid (at time of death or at 60 min, whichever was sooner) and 24 h survival after i.p. injection of S 6b (1 or [in A only] 10 nmol in 250 μl saline [0.9 % sterile NaCl]) or saline (250 μl) (A-C; in A, survival and sarafotoxin data in the table are only for mice injected with 1 nmol S6b; survival in all other groups was 100%.) or A.e.v. (10 μg in 250 μl PBS) (D-F) into: A+D, Kit+/+ mice, MC-deficient Kitw/Kitw'v mice and Kitw/Kitw'v mice that had been engrafted i.p. with MCs derived in vitro from the bone marrow of JGr+/+ mice (WT BMCMCs^ Kitw/KitW v); B+E, Kit+/+ mice, MC-deficient Kitw/Kitw'v mice and Kitw/KitW v mice that had been engrafted i.p. with ES cell-derived cultured mast cells (ESCMCs) that did (ETA+/-) or did not (ETA-/-) express ETA; and C+F, C57BL/6 mice that had received i.p. injections of vehicle (saline, 300 μl), chymostatin (150 μg in 300 μl of

*- . _ • - •■ * ,~» ,r^o^ . .—,->«-,, ,., 40 of 58 saline) or potato carboxypeptidase inhibitor (PCI, 100 μg in 300 μl of saline) 10 min before injection of S6b or saline. A+D, ***/><0.005 versus corresponding values for Kit+/+ or WT BMCMCs^ Kitw/KitW v mice; B+E, ***p<0-005 versus Kit +/+ mice; +p<0.05, ++p<0.01 or +++p<0.005 versus ETA+/- ESCMCs^ Kitw/Kitw~v mice; C+F, ***p<0.005 versus vehicle; n.s. = not significant (p >0.05).

When various amounts of A.e.v. were administered i.p., WT mice developed significant reductions in body temperature at a dose of 5 μg and death occurred at 50 μg of A.e.\. (data not shown). By contrast, as little as 5 μg of Λ.e.v. induced death in genetically MC-deficient Kitw"sh/Kitw'sh mice. Levels of sarafotoxins in the peritoneal cavity of WT mice were significantly lower than those in the corresponding KitW sh/Kitw~sh mice at 10 μg of A.e.v. and at all higher amounts of A.e.v. tested. Although the i.p. route of injection has been recommended for analyses of the systemic toxicity of snake venoms (A. Rucavado, T.
Escalante, J. M. Gutierrez, supra), many snakebites are to the skin and subcutaneous tissue. We found that MC-deficient mice were also much more susceptible than WT mice to the development of hypothermia and death when A.e.v. (10 μg/mouse) was injected
subcutaneously (data not shown).

A.e.v. contains several toxic compounds, including sarafotoxins 6a, 6b, 6c and 6d, and hemorrhagins, but the most toxic of these is sarafotoxin 6b (S6b) (E. Kochva, Public Health Rev 26, 209 (1998)). When injected with S6b (1 nmol in 250 μl, i.p.), each MC-deficient Kitw/KitW v mouse developed severe hypothermia and died within 1 h whereas the WT Kit+/+ mice developed only a slight reduction in temperature and recovered completely, even when injected with a 10 fold higher amount of S6b (Fig. IA). Injection of unfractionated venom (A.e.v., 10 μg, i.p.) produced very similar results (Fig. ID). Injection of S6b or A.e.v. i.p. also induced extensive degranulation of peritoneal MCs (PMCs; Fig. 6, A, B), a finding which can readily be detected as early as 10 min after i.p. injection of A.e.v., and hardly any sarafotoxins were detected in the peritoneal cavity of Kit+/+ mice at 60 min after injection of S6b, whereas high levels of sarafotoxins were present in the peritoneal cavity of Kitw/Kitw'v mice (Fig. 1 A, D).

Results from injection of sarafotoxin 6b (S6b) and Atractaspis engaddensis venom (A.e.v.) in terms of PMC degranulation are shown, with the results indicating that the compounds induce degranulation of peritoneal mast cells (PMCs) via ETA. (Fig. 6 A-F), Degranulation of PMCs after i.p. injection of S6b (1 nmol in 250 μl saline, (A, C, E), A.e.v.

(10 μg in 100 μl PBS, (B, D, F) or saline or PBS alone (250 μl ["vehicle" in A, BJ) in Kit+/+ mice (A, B), or in Kitw/Kitw'v mice engrafted i.p. with ETA+/- (ETA+/- ESCMCs-* KitW/KitW-v) or ETA-/- (ETA-/- ESCMCs-* Kitw/Kitw'v) ESCMCs (C, D, E, F). Degranulation of PMCs in C57BL/6 mice that received i.p. injections of vehicle (saline, 300 μl), chymostatin (150 μg in 300 μl of saline) or potato carboxypeptidase inhibitor (PCI, 100 μg in 300 μl of saline) 10 min before S6b (E) or A.e.v. (F) injection. Mast cell degranulation is expressed as % of PMCs exhibiting >50% "Extensive", 10-50% "Moderate" or <10% "None" degranulation 60 min after i.p. injection of S6b, A.e.v. or saline. *** p<0.005 versus values for: (A) S6b, (B) A.e.v. (C, D) Kit+/+ or (E, F) vehicle groups; +++ p<0.005 versus values for ETA+/- ESCMCs-* Kitw/Kitw'v mice; n.s. = not significant (p>0.05) versus values for Vehicle-treated mice or for values indicated by double-headed arrows (E+F). n = 12 mice/group (A); n > 10 mice/group (B); n > 5 mice/group (C); n > 7/mice group (D); n = 8 mice/group (E); n = 4 mice/group (F). At 10 min after i.p. injection of A.e.v. (10 μg in 100 μl PBS) or vehicle (PBS) into C57BL/6 mice as in A above, the % of PMCs that exhibited extensive, moderate or no degranulation were 68.8 ± 6.8 % (A.e.v.) versus 7.2 + 0.3 % (PBS), 23.8 + 6.0 % (A.e.v.) versus 19.1 + 2.7 % (PBS), and 7.4 + 1.0 % (A.e.v.) versus 73.7 + 2.9 % (PBS); the differences in the extent of PMC degranulation 10 min after injection of A.e.v. versus PBS was highly significant (p<0.005).

It has also been shown that human cord blood-derived mast cells (hCBMC) express carboxypeptidase A (CPA) and tryptase. To assess whether human mast cells are
theoretically able to degrade venom components, we first had to assess the expression of mast cells proteases within human cord blood-derived mast cells. Both CPA and tryptase are expressed by hCBMC, (data not shown).

Example 2: Administration of Sarafotoxin S6b or A.e. v. to mice with engrafted mast cells
To assess whether the differences in the responses of the Kit+/+ versus Kitw/Kitw~v mice specifically reflected the lack of mast cells in the Kitw/Kitw~v mice, we also administered S6b or A.e.v. to Kitw/Kitw'v mice that had been engrafted with MCs (BMCMCs, bone marrow-derived cultured mast cells) derived from the Kit+/+ mice (WT
BMCMCs-* Kitw/KitW v mice) (M. Maurer et al., J Exp Med 188, 2343 (1998); T. Nakano et al., J Exp Med 162, 1025 (1985)). Upon injection of S6b or A.e.v., MC-engrafted Kitw/Kitw'v mice exhibited very low levels of sarafotoxins in the peritoneal cavity and were protected against both hypothermia and death (Fig. IA, D).

Thus, MCs can reduce levels of sarafotoxins in the peritoneal cavity after i.p. injection of S6b or A.e.v. in vivo and can limit the systemic toxicity and death induced by either S6b or unfractioned A.e.v.

Example 3: A. engadcfensis venom activates mast cells via the ETA receptor on MCs, causing degranulation of proteases
S6b, like ET-I, activates the ETA receptor, which is expressed by MCs (M. Maurer et al., supra). Therefore, we generated mice that contained, as their only MC populations, MCs that did or did not express ETA. Since ETA-/- mice are not viable, we generated MCs in vitro from ETA+A or ETA7- embryonic stem (ES) cells (M. Tsai et al., Proc Natl Acad Sci USA 91, 9186 (2000)), and then adoptively transferred these ES cell-derived cultured MCs (ESCMCs) into Kitw/Kitw'v mice (Fig. IB, E). Engraftment of Kitw/Kitw'v mice with ETA+/- or ETA-/-ESCMCs results in equal numbers of MCs in the peritoneum (M. Maurer et al., supra).
However, each ETA-/- ΕSCMCs-^ Kitw/Kitw'v mouse developed severe hypothermia
(comparable to that in Kitw/KitW v mice) and died within 75 min of i.p. injection of either S6b or A.e.v. (Fig. IB, E). By contrast, the responses to S6b or A.e.v. in ETA+/- ESCMCs -> Kitw/Kitw'v mice were indistinguishable from those of the WT mice (Fig. IB, E). Both ETA+/- ESCMCS-* Kitw/KitW v mice and Kit+/+ mice exhibited extensive PMC degranulation (Fig. S3C, D), as well as almost complete elimination of sarafotoxin from the peritoneal cavity (Fig. IB, E). However, the ETA-/- ΕSCMCs-^ Kitw/Kitw'v mice exhibited significantly lower levels of MC degranulation (Fig. S3C, D) and much higher levels of sarafotoxins (Fig. IB, E).

Example 4: Carboxypeptidase A (CPA), but not chymase or mMCP-4, regulates the toxicity of A. efigadde/isfswΑova.
Taken together, the above examples indicate that A.e.v.-induced toxicity can be diminished by MCs and that this most likely reflects the ability of MCs to directly or indirectly reduce levels of sarafotoxins, such as S6b, the most toxic component of the venom.

To assess which MC-associated mediators might contribute to this effect, we tested inhibitors of two likely candidates, chymase (M. Maurer et al., supra) and carboxypeptidase A (K. P.

Metsarinne et al., A rterioscler Thromb Vase Biol 22, 268 (2002)) (i.e., chymostatin and potato carboxypeptidase inhibitor [PCI], respectively). WT C57BL/6 mice pre-treated with

PCI i.p. developed severe hypothermia and died within 1 h of S6b or A.e.v. injection (Fig.

1C, F). The extent of PMC degranulation in PCI-pre-treated WT mice did not differ from that in the vehicle- or chymostatin-pre-treated WT mice (Fig. S3E, F). However, sarafotoxin
a- CMnβΛκn5mτ io 43 of 58 levels in the peritoneum of PCI-pre- treated WT mice were much higher than those observed in the vehicle- or chymostatin-pre-treated WT mice (Fig. 1C, F), although not quite as high as those in MC-deficient mice injected with S6b or A.e.v. (Fig. IA, D). By contrast, i.p. pre-treatment with chymostatin resulted in only a slight, albeit statistically significant, drop in body temperature as compared to responses in the vehicle-treated WT mice, and only a slight impairment in the reduction in peritoneal levels of sarafotoxins (Fig. 1C, F). PCI also markedly inhibited the ability of mouse peritoneal cells to degrade S 6b ex vivo, whereas chymostatin had little or no effect (Fig. 7). Further experiments have shown that Human cord blood-derived mast cells (hCBMC) can reduce the toxicity of A. engaddensis venom. Co-incubation of A. engaddensis venom with Ca-Ionophore (A23187)-activated hCBMCs ex vivo limits the toxicity of the venom. CBMCs had to be activated by A23187 since these cells were shown to be unresponsive to the venom, most likely because the ETA receptor is not sufficiently expressed by immature mast cells. (Data not shown).

Figure 7 shows evidence that carboxypeptidase A is more important than chymase in mediating mast cell-cell dependent degradation of sarafotoxin 6b (S6b) ex vivo. The results of Fig. 7 were obtained in an experiment in which S6b (1 nmol in 300 μl) was incubated ex vivo for 30 min with vehicle alone (DMEM), or with vehicle containing peritoneal lavage cells (PLCs) from C57BL/6 mice (1 x 106 total cells, containing 2 x 104 PMCs) which were co-incubated with vehicle alone, chymostatin (100 μM) or PCI (400 μg/ml). Remaining levels of S6b were then measured by ELISA; * p<0.05 versus incubation with vehicle; + p<0.05 versus incubation with PLCs + vehicle; #p<0.05 versus incubation with PLC + chymostatin, by Mann- Whitney U test, 2-tailed; n.s. = not significant (p>0.05) for values indicated by brackets. Data are the mean + s.e.m. calculated from the average of duplicate values derived from the results of three separate experiments (n = 3/group).

These results were unexpected, in that our previous pharmacological experiments suggested an important role for MC-associated chymase in the degradation of ET-I (M.
Maurer et al., Nature 432, 512 (2004)). Therefore, to complement our pharmacological inhibitor studies, we also used a genetic approach to characterize the role of chymase versus CPA in limiting the toxicity of A.e.v. The only protease with chymotryptic activity in mouse PMCs is mouse mast cell protease-4 (mMCP-4) (E. Tchougounova, G. Pejler, M. Abrink, J Exp Med 198, 423 (2003)). In mMCP4-/- mice and the respective WT control mice, i.p.
injection of S6b or A.e.\. induced comparable degrees of mild hypothermia, similar levels of intra-peritoneal sarafotoxins, and similar levels of PMC degranulation; moreover, all of the mice survived (Fig. 8). Similar results were obtained when WT and mMCP4-/- mice were injected i.p. with ET-I (Fig. 96A-C). These observations, and the results of our
pharmacological and in vitro studies (Fig. 9D-H), are in accord with results of work with rat MCs in vitro (K. P. Metsarinne et al., supra) in indicating that CPA is more effective than chymase in degrading ET-I in vitro (Fig. 9G,H) as well as in reducing both ET-I levels and ET-I -mediated toxicity in vivo (Fig. 9D,E).

Figure 9 presents evidence that carboxypeptidase A is more important than mMCP4 in limiting endothelin-1 (ET-l)-induced toxicity. Since S6b and ET-I are highly homologous (T. Nakano et al., J Exp Med 162, 1025 (1985)), we also assessed whether CPA might be the dominant mast cell-associated enzyme involved in the degradation of ET-I in vivo. I.p.
injection of 1.2 nmol ET-I (in 300 μl saline) did not result in statistically significant differences of (A) body temperature, (9B) intra-peritoneal ET-I levels (measured at 60 min), or (9C) levels of PMC degranulation between WT and mMCP4-/- mice. Pre-treatment of ET-1 -injected WT mice with potato carboxypeptidase inhibitor (PCI, 100 μg in 300 μl of saline) resulted in severe hypothermia, failure to reduce levels of ET-I in the peritoneal cavity

(measured at time of death or at 60 min, whichever was sooner), and death (9D, E), without influencing the extent of PMC degranulation induced by ET-I (9F). Although the effects were much less dramatic than those observed in PCI pre-treated mice, chymostatin (150 μg in 300 μl of saline) pre-treated mice injected i.p. with either S6b (Fig. 1C) or ET-I (9D) also exhibited significant hypothermia compared to values in the corresponding vehicle-treated (saline, 300 μl) mice. By contrast, no significant differences were observed in the body temperature responses of mMCP4-/- versus WT mice to either S6b (Fig. 8A) or ET-I (A). We suspect that the in vivo effects of chymostatin may well reflect the ability of this compound to diminish CPA activity. Indeed, we find that chymostatin can reduce the activity of CPA by up to 40% in vitro (9G), as assessed by comparing the effects of vehicle (Veh), chymostatin (Ch y, 120 μg) or potato carboxypeptidase inhibitor (PCI, 150 μg) added to lysates from 3 x 106 C57BL/6 peritoneal cells containing 1 x 105 peritoneal MCs (total volume 150 μl) to change the absorbance of a chromogenic substrate specific for CPA (R. D. Theakston, D. A. Warrell, E. Griffiths, supra). PCI, and to a much lesser albeit significant extent chymostatin, also prevented in part the degradation of ET-I in vitro (9H). ET-I (1 nmol in 250 μl) was incubated ex vivo for 30 min with vehicle alone (DMEM), or with vehicle containing peritoneal lavage cells (PLCs) from C57BL/6 mice (1 x 106 total cells, containing 2 x 104 PMCs) which were co-incubated with vehicle alone, chymostatin (100 μM) or PCI (400 μg/ml). Remaining levels of ET-I were then measured by ELISA. * p<0.05 versus incubation with vehicle; + /?<0.05 versus incubation with PLCs + vehicle; #ρ<0.05 versus incubation with PLC + chymostatin, by Mann- Whitney U test, 2-tailed. Data are the mean 4- s.e.m. calculated from the average of duplicate values derived from the results of three separate batches of cells, each derived from different mice (n = 3/group). (9C, F) Percentage of PMCs exhibiting >50% "Extensive", 10-50% "Moderate" or <10% "None" degranulation 60 min after injection of ET-I. * p<0.01, *** /?<0.005 versus vehicle; n.s. = not significant (p<0.05) versus values for WT (9C) or Vehicle-treated (9F) mice or for values indicated by bracket (9B); t = all mice dead, n = 8 (C57BL/6) or 6 (mMCP4-/-) mice/group (9 A-C); n = 4 (vehicle) or 5 (chymostatin and PCI) mice/group (9D-G); n > 3/group.
We also used a lentivirus-based shRNA approach to generate mice that contained as their only MC populations either normal MCs or MCs with significantly decreased CPA activity (>75% reduction, Fig. 2A). KitW sh/KitW sh mice that had been engrafted with either control (i.e., empty vector) MCs or CPA shRNA MCs exhibited the same numbers of PMCs (Fig. 2B), similar levels of PMC degranulation in response to S6b or A.e.v. (Fig. 2C) and similar numbers of MCs in the mesentery (Fig. 2D, and data not shown). However, upon injection of S6b or Ae.v. i.p., the CPA shRNA MC-engrafted Kitw'sh/Kitw'sfl mice developed severe . hypothermia, retained high concentrations of sarafotoxins in the peritoneal cavity and died within 1 h (Fig. 2E, F), findings very similar to those observed in either MC-deficient Kitw~ Sh/Kitw-Sh (Fig. 2E, F) or Kitw/Kitw'v (Fig. IA, D) mice, or in C57BL/6 mice which had been pre-treated with PCI (Fig. 1C, F).

Figure 2 A-F shows that the knock-down of carboxypeptidase A (CPA) using shRNA in mast cells (MCs) renders mice susceptible to A. engaddensis venom (Λ.e.v.)- and sarafatoxin 6b (Sόb)-induced morbidity and mortality. Fig. 2A shows CPA activity in peritoneal cells from C57BL/6 or Kitw'sh/Kitw'sh mice engrafted with BMCMCs that were infected witfi CPA shRNA or empty vector. *p<0.05 versus C57BL/6 or empty vector control. Fig. 2 B-D, Kitw'sh/Kitw'sh mice engrafted with BMCMCs infected with CPA shRNA or empty vector (control) exhibit: normalization of peritoneal mast cell (PMC) numbers (B), similar PMC responsiveness to S6b and A.e.v. (C), and the appearance of MCs in the mesentery (D; arrows = MCs, scale bars = 100 μm). In C, ***p<0.005 versus C57BL/6; n.s. = not significant (p >0.05) versus values for C57BL/6 mice (B) or for the comparisons indicated by brackets (B+C). C,E+F, Extent of PMC degranulation (C) and Fig. 2 (E+F) changes in rectal temperature, levels of sarafotoxins in the peritoneal lavage fluid (at time of death or at 60 min, whichever was sooner) and 24 h survival in C57BL/6 mice or Kitw'sh/Kitw" sh mice that were engrafted i.p. with empty vector (empty vector MCs-> Kitw~sl'/Kitw~sh) or CPA shRNA (CPA shRNA MCs-> Kitw'sh/Kitw-sb) expressing BMCMCs and then were injected with 1 nmol S6b (E) or 10 μg A.e.v. (F) 4 weeks later. In E+F, **p <0.01 or
* * *p<0.005 versus C57BL/6 or empty vector MCs -> KitW-sh/KitW-sh mice.

Example 5: Mast cells protect against Rattlesnake and Copperhead venoms, which do not contain S6b
To assess whether MCs can reduce the toxicity of snake venoms which do not contain S6b or other ET-like peptides, we injected mice with venoms from the Western Diamondback Rattlesnake (Crotalus atrox, 150 μg) or the Southern Copperhead {Agkistrodon contortrix contortrix, 75 μg), species representatives of pit vipers (Crotalidae), which account for the great majority of snake envenomations in North America (D. McNamee, Lancet 357, 1680 (2001)). MC-deficient Kitw/Kitw'v mice injected with Crotalus atrox venom (C.α.v.) or Agkistrodon contortrix contortrix venom (A.c.c.v.) i.p. exhibited significantly lower body temperatures than WT mice and all of them died within 24 h. By contrast, most of the Kit+/+ mice survived (survival was 87% in Cα.v.-injected mice and 100% in A. c.c. v.-injected mice; Fig. 3A, C) and these mice appeared to recover fully, with body temperatures returning to normal by 1-3 d. Moreover, the selective engraftment of Kitw/KitW v mice with WT BMCMCs resulted in levels of protection against hypothermia and death, which were statistically indistinguishable from those in the WT mice (Fig. 3A, C). Very similar results were obtained when these experiments were repeated using C57BL/6, Kitw'sh/Kitw'sh and WT MC-engrafted Kitw'sh/Kitw'sh mice (data not shown). In WT (C57BL/6) mice injected with venom i.p., PCI pre-treatment significantly worsened C.a.v.~ or A.c.c.v.-induced hypothermia and mortality (Fig. 3B, D). However, pre-treatment of C.a.\.- or Ac.c.v.-injected mice with PCI did not result in levels of hypothermia and/or death rates (Fig. 3B, D) that were quite as striking as those observed in MC-deficient mice, especially in mice injected with C.a.\. One possible explanation for these results is that MC-derived enzymes other than CPA (and/or additional MC-derived mediators) may also have some protective effects against C. atrox or A.c.
contortrix venoms, and/or in counteracting the pathology, which they induce.

Fig. 3 illustrates the protective effect of MCs in a series of graphs that show changes in rectal temperature nd 24 h survival after i.p. injection of Agkistrodon contortrix contortrix (Southern Copperhead) venom (A.c.c. venom, 150 μg in 250 μl PBS [A, BJ) or Crotalus atrox (Western Diamondback Rattlesnake) venom (C. a. venom, 75 μg in 250 μl [C, D]) into:

KIt+/+ mice, MC-deficient Kitw/Kitw'v mice and Kitw/Kitw'v mice that had been reconstituted i.p. with BMCMCs from KH+I+ mice (WT BMCMCs-* Kitw/Kitw'v) (A, C) or into C57BL/6 mice that received i.p. injections of vehicle (saline, 300 μl), chymostatin (150 μg in 300 μl of saline) or potato carboxypeptidase inhibitor (PCI, 100 μg in 300 μl of saline) 10 min before i.p. injection of snake venoms (B, D). Mice, which survived for 3 h, all appeared healthy and returned to baseline body temperature within 1-3 d (body temperatures at 1-3 d were measured in surviving mice in one of the 3 experiments whose data were pooled to give the depicted 24 h survival and up to 3 d body temperature data). A, C, *p<0.05 or ***p<0.005 versus Kit+/+ or WT BMCMCs-* Kitw/KitW v mice; B, D, *p<0.05 or ***p< 0.005 versus vehicle.

We couldn't assess PMC degranulation in vivo in these experiments because of the excessive bleeding that had occurred within 60 min of venom injection (or by the time of death) in all of the venom-injected mice. However, we detected extensive PMC degranulation when PMCs were exposed to either type of venom ex vivo and in C57BL/6 mice which had been exposed to either type of venom for only 10 min after i.p. injection of the venom in vivo (data not shown).

Example 6: Mast cells limit the size of hemorrhagic lesions
We also found that MCs diminished the extent of the hemorrhagic lesions that developed upon the injection of C.a.v. into the skin (Fig. 4).

Figure 4 illustrates the results of experiments in which C.a.v. (50 μg in 100 μl) was injected s.c. in the shaved back skin of age-matched female WT mice, Kitw'sh/Kitw'sh mice, or Kitw'sb/Kitw-Sh mice that had been engrafted i.d., 6 weeks earlier, with 2xlO6 BMCMCs (WT BMCMCs-> Kitw'sh/KitW sh) and the size of the hemorrhagic lesions induced was measured 2 h later, by averaging the lengths of the largest diameter and the diameter perpendicular to that. n =13 (WT and Kitw-sh/Kitw-sh) or 5 (WT BMCMCs-* KitW sh/KitW sh). * /xθ.05; *** p<0.005 for the comparisons indicated by brackets.

Thus, mast cells not only have effects that can reduce the systemic toxicity and mortality associated with the i.p. injection of this venom, but also can diminish the local pathology induced when such venom is injected subcutaneously.

Example 7: Mast cells protect against honeybee and brown recluse spider venom
In addition to reptiles, stinging insects and spiders can account for significant venom-associated morbidity, and mortality (R. S. Vetter, P. K. Visscher, S. Camazine, West J Med 170, 223 (1999); J. O. Schmidt, Toxicon 33, 917 (1995); R. G. Jones, R. L. Corteling, G. Bhogal, J. Landon, Am J Trop Med Hyg 61, (1999)) (Furbee et ah, (2006) Clin Lab Med, 26:211-226).

For example, death can be induced in some mouse strains by a single sting of worker bees of either the European (Apis mellifera mellifera) or "Africanized" (Apis m. scutellata) honeybee (J. O. Schmidt, Toxicon 33, 917 (1995)). It has been estimated that - 150-1200 stings can deliver the LD50 of honeybee venom in humans (R. S. Vetter, P. K. Visscher, S. Camazine, West J Med 170, 223 (1999)), and the introduction of the "Africanized" honeybee into North America has resulted in an increased incidence of human deaths due to multiple honeybee stings (R. S. Vetter, P. K. Visscher, S. Camazine, supra; R. G. Jones, R. L.
Corteling, G. Bhogal, J. Landon, supra).

Although it is well-known that honeybee venom contains compounds which can induce MC degranulation (M. R. Ziai, S. Russek, H. C. Wang, B. Beer, A. J. Blume, J Pharm Pharmacol 42, 457 (1990)), it had been thought that this contributed to the pathology associated with such stings (M. C. Calixto, K. M. Triches, J. B. Calixto, Inflamm Res 52, 132 (2003)). However, we found that MCs can confer significant protection against the hypothermia and death induced by the s.c. injection of Apis mellifera venom (Fig. 5A). While mice in all groups appeared to exhibit the same initial response to such injections, namely, intense scratching of the injection sites, all of the MC-deficient Kitw'sh/Kitw'sh mice, but none of the WT mice or the WT BMCMCs-engrafted Kitw-sh/Kitw-sH mice, in addition to
developing profound hypothermia, also developed gross hematuria (Fig. 5A).
Intradermal injection of brown recluse spider (Loxosceles reclusά) venom (L.r.v) at a concentration equivalent to a single spider bite resulted in severe drop in body temperature in MC-deficient Kitw/Kitw'v mice while WT mice (Kit+/+) exhibited significantly reduced hypothermia. More importantly, all of the Kii1 /Kitw'v mice, but none of the WT mice or the WT BMCMCs-engrafted Kitw/Kitw'v mice died, showing that MCs can also confer significant protection against death induced by the i.d. injection of Loxosceles reclusa venom (Fig. 5B).

Fig. 5 plots changes in rectal temperature and 24 h survival and occurrence of gross hematuria after injection of (A) Apis mellifera venom (A.m.-v.) or (B) Loxosceles reclusa venom (X.r.v.). A. A.m.v. was injected s.c. at 5 different sites (3 injections distributed over the length of the back skin and 2 into the belly skin, each containing 100 μg Λ.m.v. in 50 μl PBS) into: WT mice, mast cell-deficient Kitw-sh/Kitw-sh mice or Kitw'sh/Kitw-sh mice that had been engrafted i.d., 6 weeks earlier, with 1.5xlO6 BMCMCs into each of the 5 injection areas (WT
The amount of venom per injection (i.e., 100 μg) roughly reflects the amount that can be delivered by one bee sting (J. O. Schmidt, Toxicon 23, 9X1 (1995)). All of the WT or WT BMCMCs^ Kitw'sh/Kitw^h mice appeared healthy and their body temperatures returned to baseline within 2 d. B. L.r.v. was injected i.d. into the back skin (2.5 μg in 50μl PBS) into: WT mice, mast cell-deficient Kitw/Kitw'v mice or Kitw/Kitw'y mice that had been engrafted i.d., 6 weeks earlier, with 2.OxIO6 BMCMCs into an area of

1.5cm x 1.5cm (WT BMCMCs-* KitW sh/KitWsh). The amount of venom injected (i.e., 2.5 μg) roughly reflects the amount that can be delivered by one spider bite. *** p<0.005 versus either C57BL/6 or WT BMCMCs-* KitWsh/KitWsh mice (A); * p<0.05 vs. KU+/+ (B).

Example 8: Figure 8. Carboxypeptidase A is more important than mMCP4 in limiting endothelin-l (ET-l)-induced toxicity.
Since S6b and ET-I are highly homologous (Y. Kloog et al., Science 242, 268 (1988)), we also assessed whether CPA might be the dominant mast cell-associated enzyme involved in the degradation of ET-I in vivo. Lp. injection of 1.2 nmol ET-I (in 300 μl saline) did not result in statistically significant differences of (A) body temperature, (B) intra-peritoneal ET-I levels (at time of death or at 60 min, whichever was sooner), or (C) levels of PMC degranulation between WT and mMCP4-/- mice. Pre-treatment of ET-I -injected WT mice with potato carboxypeptidase inhibitor (PCI, 100 μg in 300 μl of saline) resulted in severe hypothermia, failure to degrade ET-I, and death (D, E), without influencing the extent of PMC degranulation induced by ET-I (F). Although the effects were much less dramatic than those observed in PCI pre- treated mice, chymostatin (150 μg in 300 μl of saline) pre-treated mice injected i.p. with either S6b (Fig. 1C) or ET-I (D) also exhibited significant hypothermia compared to values in the corresponding vehicle-treated (saline, 300 μl) mice. By contrast, no significant differences were observed in the body temperature responses of mMCP4-/- versus WT mice to either S6b (Fig. 7A) or ET-I (A). We suspect that the in vivo effects of chymostatin may well reflect the ability of this compound to diminish CPA activity. Indeed, we find that chymostatin can reduce the activity of CPA by up to 40% (G) in vitro, as assessed by comparing the effects of incubation with vehicle (Veh), chymostatin (Chy) or potato carboxypeptidase inhibitor (PCI) on the ability of peritoneal cells from C57BL/6 mice to change the absorbance of a chromogenic substrate specific for CPA. (C, F) Percentage of PMCs exhibiting >50% "Extensive", 10-50% "Moderate" or <10% "None" degranulation 60 min after injection of ET-I. * p<0.0l, *** p<0.005 versus vehicle; n.s. = not significant; t = all mice dead, n = 8 (C57BL/6) or 6 (mMCP4-/-) mice / group (A-C); n = 4 (vehicle) or 5 (chymostatin and PCI) mice / group (D-F); n > 3 / group.

Example 8: Figure S6. Cra/a/us a/rox(C.a.\.) atoάAgά/strodon con tort r/x con /ortrix venom (A.c.c.v.) induce degranulation of mouse peritoneal mast cells ex v/'vσ.
Peritoneal lavage cells from C57BL/6 mice were incubated for 2 h with 3H-5-hydroxytryptamine (3H-serotoniπ) and stimulated for 15 min. Results are mean + s.e.m. (n = 4) of values pooled from 2 independent experiments that gave similar results. * p<0.05, *** /?<0.005 versus vehicle-treated cells; n.s. = not significant.

Example 9: Mast cells limit the size of hemorrhagic lesions induced by Crofa/us atrσx venom (Cla.\.) in the skin.
Ca. v. (50 ug in 100 μl) was injected s.c. in the shaved back skin of age-matched female WT mice, KitW sh/Kitw-sh mice, or Kitw-sh/Kitw-sh mice that had been engrafted i.d., 6 weeks earlier, with 2xlO6 BMCMCs (WT BMCMCs^ Kitw-Sh/Kitw-Sh) and the size of the hemorrhagic lesions induced was determined 2 h later, by averaging the lengths of the largest diameter and the diameter perpendicular to that, n =13 (WT and Kitw~sh/Kitw'sh) or 5 (WT BMCMCs^> Kitw-Sh/Kitw-Sh), * /?<0.05, *** /?<0.005.

Example 10: Mast cells limit the toxicity of Ap/s /ne////era (honey bee) venom (A./n.v.).
was injected s.c. at 5 different sites (3 injections distributed over the length of the back skin and 2 into the belly skin, each containing 100 μg A.m.v. in 50 μl PBS) into: WT mice, mast cell-deficient Kitw'sh/Kitw"sh mice or Kitw'sh/Kitw"sh mice that had been engrafted i.d., 6 weeks earlier, with 1.5xlO6 BMCMCs into each of the 5 injection areas (WT
BMCMCs -*Kitw'sh/Kitw'sh). The amount of venom per injection (i.e., 100 μg) roughly reflects the amount that can be delivered by one bee sting (J. O. Schmidt, supra). All of the WT or WT BMCMCs-> Kitw'sh/Kitw'sh mice appeared healthy and their body temperatures returned to baseline within 2 d. B. L.r.v. was injected i.d. into the back skin (2.5 μg in 50μl PBS) into: WT mice, mast cell-deficient Kitw/KitW v mice or Kitw/KitW v mice that had been engrafted i.d., 6 weeks earlier, with 2.0xl06 BMCMCs into an area of 1.5cm x 1.5cm (WT
BMCMCs-* Kitw'sh/Kitw'sh). The amount of venom injected (i.e., 2.5 μg) roughly reflects the amount that can be delivered by one spider bite. ***/?<0.005 versus either C57BL/6 or WT BMCMCs-* Kitw-Sh/Kitw-Sh mice (A); * p<0.05 vs. Kit+/+ (B).

We have also shown that human cord blood-derived mast cells (hCBMC) can reduce the toxicity of honeybee venom. Co-incubation of honeybee venom with hCBMCs ex vivo results in almost complete detoxification of the venom (data not shown).

Example 11: Degradation of Sarafotoxin 6b (S6b) by different proteases
Referring now to Figure 10, various commercially available proteases or vehicle alone were incubated for 60 min with 1 nmol S6b (total volume 250 μl) and remaining S6b concentrations were then measured using ELISA and expressed as % of S6b + vehicle incubation.

Concentrations of proteases were chosen according to the manufacturer's suggestions and are shown in the graph. The ELISA signal decreased if the candidate protease cleaved the S6b because the antibody, a monoclonal capture antibody against Endothelin (with 100% cross-reactivity to Sarafotoxin) only detects the complete 21 amino acid peptide, and not a cleaved and thus smaller peptide. For the detection of S6b a commercial Endothelin EIA kit which shows 100% cross-reactivity to Sarafotoxins (from Cayman Chemicals) was used according to the manual provided by the manufacturer. Proteases were purchased from Elastin Products Company, Owensville, MO (carboxypeptidase A, carboxypeptidase B, chymotrypsin, chymase, tryptase) or from Sigma (Papain). CPA, CPB and chymotrypsin were purified from human pancreas. Chymase was purified from human skin mast cells. Tryptase was purified from human lung mast cells. Papain was purified from Carica papaya.

All proteases tested, except for tryptase, degraded the S 6b. Most effective were CPA

(carboxypeptidase A), chymotrypsin and papain. Carboxypeptidase B and chymase were also effective.

Example 12: Both human carboxypeptidase A and human tryptase limit the toxicity of honeybee venom
As shown in Figure 12, both carboxypeptidase A and tryptase limit the toxicity of honeybee venom. Apis mellifera venom (500 μg/ml in PBS) was co-incubated with human purified lung tryptase (1 μg/ml), human purified pancreas CPA (1 μg/ml) or vehicle (PBS) at 37°C. After 60 minutes 100 μl were injected i.p. into MC-deficient Kitw'sh/Kitw'sh mice.
Control mice were injected with the same concentrations of proteases alone, n = 3 mice / group, *** = p<0.005 vs. Vehicle co-incubated venom. Results are summarized below:
These results show that co-incubation of honeybee venom with human CPA or human tryptase can significantly detoxify the venom. Injection of tryptase and carboxypeptidase alone does not (CPA) or only slightly (tryptase) affect body temperature of the injected mice. Additionally, mass spectroscopy data has been obtained which shows the degradation of bee venom components by human tryptase, CPA and chymase. As described in Miller et aL, "Cloning and characterization of complementary DNA for human tryptase.," J Clin Invest. 1989, Oct;84(4) : 1188-95, human mast cell tryptase human tryptase consists of a 244-amino acid catalytic portion and a 30-amino acid leader sequence. The protein contains a typical His, Asp Ser catalytic triad and regions of amino acid sequence that are highly conserved in serine proteases, in general, were conserved in tryptase. For example, the catalytic portion of human tryptase had an 84% amino acid sequence similarity with that of dog tryptase. Thus, sequence variation can be tolerated in the tryptase embodiment of the present invention, as discussed above in connection with other sequences.

CONCLUSION
Our data show that MCs and proteases therefrom can substantially reduce the morbidity and mortality induced in mice by the venoms of three different snakes. Moreover, the activation of MC degranulation by snake venom components appears to represent a critical early step in eliciting MC-dependent protection against the toxicity associated with that venom. It is possible that MCs can enhance resistance to the venoms of other poisonous snakes as well, as many snake venoms can activate MCs (A. Weisel-Eichler, F. Libersat, J Comp Physiol A Neuroethol Sens Neural Behav Physiol 190, 683 (2004)). Indeed, evidence was recently reported that the venom system in snakes and lizards derives from a single early origin in squamate evolution (-200 million years ago) and that such venoms share some similar or identical toxins, including nerve growth factor, crotamine and B -type natriuretic peptide; in each case the respective reptile toxin (A. C. Mancin et al., Biochem MoI Biol Int 42, 1171 (1997)) or a highly homologous mammalian peptide or protein has been shown to induce MC degranulation (F. L. Pearce, H. L. Thompson, J Physiol 372, 379 (1986); H. Yoshida et al., Regul Pept 61, 45 (1996)).

Moreover, a variety of venoms derived from several animal species other than reptiles and honeybees also have been shown to activate MCs, including those from the platypus (Ornithorhynchus anatinus), scorpions (Scorpionida) (A. Weisel-Eichler, F. Libersat, supra), ants (Formicidae) (N. K. Lind, Toxicon 20, 831 (1982)) and the Portuguese man-of-war (Physalia spp.) (S. M. Cormier, J Exp Zool 218, 117 (1981)). It will be of interest to assess whether mast cells might enhance resistance to the venoms of these species, as well as to those of the honeybee and the three snake species tested.

The hypothesis that MCs might reduce the toxicity of snake venom was proposed by R. D. Higginbotham in 1965, based on morphological observations of mast cell degranulation at sites of s.c. injection of Russell's Viper (Daboia russellii) venom and other lines of evidence (R. D. Higginbotham, / Immunol 95, 867 (1965)). For example, the mast cell-associated proteoglycan, heparin, which is highly anionic, can bind to certain cationic components of Russell's Viper venom and can reduce the toxicity of the venom when admixed with it prior to its injection into mice (R. D. Higginbotham, supra). However, the MC deficiency of Kitw/Kitw'v mice had not yet been reported (Y. Kitamura, S. Go, K.
Hatanaka, Blood 52, 447 (1978)) at the time of Higginbotham' s study, so his hypothesis that mast cells could confer protection against snake venom-induced toxicity could not be tested directly. Moreover, many toxic components of snake venoms are not highly cationic (C. Valiente, E. Moreno, A. Sittenfeld, B. Lomonte, J. M. Gutierrez, Toxicon 30, 815 (1992); W. K. You, Y. J. Jang, K. H. Chung, O. H. Jeon, D. S. Kim, Biochem Biophys Res Commun 339, 964 (2006)) and therefore would not be expected to bind strongly to heparin.

We have shown herein that the protease component of MCs can enhance the resistance of mice to the morbidity and mortality induced by the venoms of various species probably in large part by undergoing degranulation and releasing CPA and other MC cytoplasmic granule-associated enzymes, which can degrade important toxic components of the venom. MCs also can enhance the resistance of mice to the toxicity of honeybee and brown recluse spider venom. Moreover, it is possible that mast cell functions other than the secretion of CPA and proteases also may contribute to the ability of these cells to reduce the morbidity and mortality associated with certain venoms. For example, MC-dependent enhancement of local vascular permeability would favor extravasation of plasma inhibitors of venom metalloproteases and other toxins (J. E. Biardi, D. C. Chien, R. G. Coss, J Chem Ecol 32, 137 (2006)).

In summary, we have identified a heretofore-unproven role for MCs: enhancing innate host resistance to the toxicity of certain animal venoms. Our observations also provide a new perspective on the presence, within MCs, of prominent cytoplasmic granules, which contain a large amount and, in some species, a large diversity, of proteases and peptidases (C. Huang, A. SaIi, R. L. Stevens, J Clin Immunol 18, 169 (1998); G. H. Caughey, MoI Immunol 38, 1353 (2002)). It is likely that mast cell proteases and peptidases can have beneficial roles in many settings, not only in host defense (E. Tchougounova, G. Pejler, M. Abrink, J Exp Med 198, 423 (2003); C. Huang, A. SaIi, R. L. Stevens, / Clin Immunol 18, 169 (1998); J. Mallen-St Clair, C. T. Pham, S. A. Villalta, G. H. Caughey, P. J. Wolters, J Clin Invest 113, 628 (2004)). However, we speculate that the storage in MC cytoplasmic granules of large amounts of proteases and peptidases, which can be released to the exterior very rapidly upon suitable MC activation, reflects, at least in part, the selective pressure of the exposure of animals to diverse exogenous toxins contained in vertebrate and invertebrate venoms, as well as the advantage of being able to degrade and thereby control the toxicity of potent endogenous molecules such as ET-I (M. Maurer et al., supra).

The present examples, methods, procedures, specific compounds and molecules are meant to exemplify and illustrate the invention and should in no way be seen as limiting the scope of the invention, which is provisionally defined by the appended claims. Any patents or publications mentioned in this specification are indicative of levels of those skilled in the art to which the patent pertains and are intended to convey details of the invention which may not be explicitly set out but would be understood by workers in the field. Such patens or publications are hereby incorporated by reference to the same extent as if each was specifically and individually incorporated by reference and for the purpose of describing and enabling the method or material referred to.