# Patent

## Title
Compositions and methods for treating or preventing conditions and diseases associated with Mannheimia haemolytica

## Abstract

Particular aspects show that the signal peptide remains intact on the mature CD18 molecule on ruminant leukocytes rendering these cells susceptible to cytolysis by Lkt. Comparative amino acid sequence analysis of the signal peptide of CD18 of eight ruminants and five non-ruminants revealed that the ruminant CD18 signal peptides contain ‘cleavage-inhibiting’ glutamine (Q), compared to ‘cleavage-conducive’ glycine in non-ruminants, at position −5 relative to the cleavage site. Mutagenesis of Q at position −5 of the bovine CD18 signal peptide to G resulted in the abrogation of Lkt-mediated cytolysis of transfectants expressing bovine CD18 carrying the Q(−5)G mutation. Provided is novel technology to clone cattle and other ruminants expressing CD18 without the signal peptide on their leukocytes, providing ruminants that are less susceptible to M. haemolytica. Methods for treating conditions and/or diseases associated with M. haemolytica (e.g., pneumonic pasteurellosis), comprising administration of polypeptides comprising CD18 signal peptide sequences are also provided.

## Background
FIELD OF THE INVENTION
Aspects of the present invention relate generally to conditions and/or diseases associated with M. haemolytica in ruminants, and more particularly to novel and efficacious compositions and methods for treating or preventing conditions and/or diseases associated with M. haemolytica in ruminants.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the United States national phase, pursuant to 35 U.S.C. 371, of International Patent Application No. PCT/US2010/022932, filed 2 Feb. 2010, which claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 61/149,278 filed 2 Feb. 2009 and entitled COMPOSITIONS AND METHODS FOR TREATING OR PREVENTING CONDITIONS AND DISEASES ASSOCIATED WITH MANNHEIMIA HAEMOLYTICA, all of which are incorporated by reference herein in their entirety.
BACKGROUND
Mannheimia haemolytica (M. haemolytica) is the most significant bacterial pathogen of respiratory disease in cattle, sheep, goats and other ruminants, and causes extensive economic losses world-wide8. M. haemolytica is commonly found in the nasopharynx of healthy ruminants. In conjunction with active viral infection and stress factors, M. haemolytica migrates to the lungs, where it multiplies rapidly, and causes a fibrinonecrotic pleuropneumonia. M. haemolytica produces several virulence factors8. Based on the observation that leukotoxin-deletion mutants (Lkt-deletion mutants) of M. haemolytica cause reduced mortality and much milder lung lesions than the wild-type organisms, Lkt is considered as the most important virulence factor of this organism9-13. Lkt belongs to the family of RTX (repeats in toxins) toxins, and shares extensive homology with the exotoxins produced by other gram-negative bacteria such as Escherichia coli14, Actinobacillus pleuropneumoniae15, and Actinobacillus actinomycetemcomitans16. Cytolytic activity of M. haemolytica Lkt is specific for ruminant leukocytes17-18. Although all the subsets of leukocytes are susceptible to the cytolytic effects of Lkt, PMNs are the most susceptible subset19. PMN-depletion mitigates the lung injury in calves caused by M. haemolytica infection20. Therefore, Lkt-induced PMN lysis and degranulation are the primary causes of acute inflammation and lung injury characteristic of pneumonic pasteurellosis8,13,20.
There is, therefore a pronounced need in the art for novel and efficacious compositions and methods for treating or preventing conditions and/or diseases associated with M. haemolytica (e.g., in mammals, ruminants).
SUMMARY OF EXEMPLARY ASPECTS OF THE INVENTION
In particular aspects of the present invention, studies aimed at mapping the Mannheimia (Pasteurella) haemolytica leukotoxin (Lkt) binding site on its receptor CD18 have unexpectedly shown, as disclosed herein, that the signal peptide of ruminant CD18 remains intact on the mature CD18 molecule on the leukocytes of ruminants and renders these cells susceptible to cytolysis by Lkt.
In additional aspects, comparative analysis of the amino acid (aa) sequence of the signal peptide of CD18 of eight ruminants and five non-ruminants revealed that the signal peptide of CD18 of ruminants contain ‘cleavage-inhibiting’ glutamine (Q), whereas that of non-ruminants contain ‘cleavage-conducive’ glycine (G) at position −5 relative to the cleavage site.
In further aspects, site-directed mutagenesis of Q at position −5 of the signal peptide of bovine CD18 to G resulted in the abrogation of cytolysis of transfectants expressing bovine CD18 carrying the Q(−5)G mutation.
Particular aspects, therefore provide a hitherto unavailable technology to clone cattle and other ruminants expressing CD18 without the signal peptide on their leukocytes, providing ruminants that are less susceptible to pneumonic pasteurellosis that costs millions of dollars world-wide annually.
Particular preferred aspects provide a purified or recombinant ruminant CD18 polypeptide, comprising a ruminant CD18 polypeptide, or portion thereof, having a cleavable signal peptide with a helix-breaking amino acid residue at amino acid position 18 (−5 with respect to signal peptide cleavage site). In certain aspects, the amino acid residue at amino acid position 18 is selected from the group consisting of glycine, proline, arginine, and tyrosine. In further preferred aspects, the ruminant is selected from the group consisting of cattle, bison, buffalo, goat, domestic sheep, big horn sheep, deer, elk, giraffes, yaks, camels, alpacas, llamas, wildebeest, antelope, pronghorn and nilgai. Yet further aspects provide, that the recombinant ruminant CD18 polypeptide is one Q(−5)G CD18 mutant selected from the group consisting of SEQ ID NOS:57, 58, 60, 62, 64, 66, 68, 70 and CD18 signal peptide-comprising portions thereof.
Additional aspects provide an isolated nucleic acid comprising a sequence that encodes the polypeptide of CD18 having a cleavable signal peptide with a helix breaking amino acid residue at amino acid position 18.
Further preferred aspects provide for a recombinant expression vector, comprising a nucleic acid comprising a sequence that encodes a polypeptide comprising a ruminant CD18 polypeptide, or portion thereof, having a cleavable signal peptide with a helix-breaking amino acid residue at amino acid position 18 (−5 with respect to signal peptide cleavage site).
Yet further preferred aspects provide for a recombinant or cloned ruminant cell or ruminant animal, comprising a ruminant cell capable of expressing a polypeptide comprising a ruminant CD18 polypeptide, or portion thereof, having a cleavable signal peptide with a helix-breaking amino acid residue at amino acid position 18 (−5 with respect to signal peptide cleavage site). Additional aspects provide that expression of a polypeptide comprises a ruminant CD18 polypeptide having a cleavable signal peptide comprises expressing from a genomic locus, or from a recombinant expression vector. Further aspects provide that the cell or animal is less susceptible to, or resistant to the effects of M. haemolytica, relative to wild-type control cells.
Additional aspects provide that the cell or animal is that of a ruminant selected from the group consisting of cattle, bison, buffalo, goat, domestic sheep, big horn sheep, deer, elk, giraffes, yaks, camels, alpacas, llamas, wildebeest, antelope, pronghorn and nilgai. Further preferred aspects provide that the cell capable of expressing a polypeptide comprising a ruminant CD18 polypeptide, or portion thereof, having a cleavable signal peptide, there is reduced or no expression of the endogenous wild-type CD18 polypeptide having a non-cleavable signal peptide.
Further preferred aspects provide for a method of providing a recombinant or cloned ruminant cell, comprising introduction into, or engineering within the ruminant cell, a nucleic acid comprising a sequence that encodes a polypeptide comprising a ruminant CD18 polypeptide, or portion thereof, having a cleavable signal peptide with a helix-breaking amino acid residue at amino acid position 18 (−5 with respect to signal peptide cleavage site), wherein the cell is less susceptible to, or resistant to the effects of M. haemolytica, relative to wild-type control cells. Yet further aspects provide for the amino acid residue at amino acid position 18 is selected from the group consisting of glycine, proline, arginine, and tyrosine.
Additional preferred aspects provide for a method where the ruminant is selected from the group consisting of cattle, bison, buffalo, goat, domestic sheep, big horn sheep, deer, elk, giraffes, yaks, camels, alpacas, llamas, wildebeest, antelope, pronghorn and nilgai. Further preferred aspects provide for the recombinant ruminant CD18 polypeptide is one Q(−5)G CD18 mutant selected from the group consisting of SEQ ID NOS:57, 58, 60, 62, 64, 66, 68, 70 and CD18 signal peptide-comprising portions thereof.
Further preferred aspects provide a method of providing a recombinant or cloned ruminant animal, comprising introduction into, or engineering within one or more cells of a ruminant animal, a nucleic acid comprising a sequence that encodes a polypeptide comprising a ruminant CD18 polypeptide, or portion thereof, having a cleavable signal peptide with a helix-breaking amino acid residue at amino acid position 18 (−5 with respect to signal peptide cleavage site), wherein the recombinant or cloned ruminant animal is less susceptible to, or resistant to the effects of M. haemolytica, relative to wild-type control cells. Yet further aspects provide for the use of ruminant stem cells or enucleated ruminant cells.
Additional preferred aspects provide a method where the amino acid residue at amino acid position 18 is selected from the group consisting of glycine, proline, arginine, and tyrosine. Further preferred aspects provide a method where the ruminant is selected from the group consisting of cattle, bison, buffalo, goat, domestic sheep, big horn sheep, deer, elk, giraffes, yaks, camels, alpacas, llamas, wildebeest, antelope, pronghorn and nilgai.
Further preferred aspects provide a method where the recombinant ruminant CD18 polypeptide is one Q(−5)G CD18 mutant selected from the group consisting of SEQ ID NOS:57, 58, 60, 62, 64, 66, 68, 70 and CD18 signal peptide-comprising portions thereof.
Yet further preferred aspects provide a method of treating or preventing conditions and diseases associated with M. haemolytica in ruminants, comprising administering to a ruminant subject in need thereof, an amount of a polypeptide comprising a CD18 signal peptide, or portion thereof, suitable to treat, prevent or otherwise ameliorate a condition or diseases associated with M. haemolytica in the ruminant.
Additional aspects provide a method of treating whereby the polypeptide comprising a CD18 signal peptide, or portion thereof, is a polypeptide comprising from about 13 to about 24 contiguous amino acid residues of the first 24 amino acids of the N-terminus of the native (full-length nascent) CD18 sequence, wherein the polypeptide is suitable to provide for at least one of binding to M. haemolytica leukotoxin (Lkt) and abrogation of Lkt-induced cytolysis. Further aspects provide a method whereby the polypeptide comprises a contiguous portion of the CD18 signal polypeptide beginning at amino acid residue 5. Yet further aspects provide a method whereby the polypeptide comprises residues 5 to 17 of the CD18 signal polypeptide.
Further preferred aspects provide a method whereby the CD18 sequence is selected from the group consisting of cattle, bison, buffalo, goat, domestic sheep, big horn sheep, deer, elk, giraffes, yaks, camels, alpacas, llamas, wildebeest, antelope, pronghorn, nilgai, human, chimp, mouse, rat and pig. Additional preferred aspects provide a method whereby the CD18 sequence is selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 21, 26, 57, 58, 60, 62, 64, 66, 68 and 70.
Certain preferred aspects provide a method whereby the CD 18 signal peptide sequence is selected from the group consisting of cattle, bison, buffalo, goat, domestic sheep, big horn sheep, deer, elk, giraffes, yaks, camels, alpacas, llamas, wildebeest, antelope, pronghorn and nilgai, human, chimp, mouse, rat and pig. Additional preferred aspects provide a method whereby the CD 18 signal peptide sequence is selected from the group consisting of SEQ ID NOS:28, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50, 52 and 54.
Certain aspects further comprise administration of an anti-leukotoxin antibody reagent or epitope-binding portion thereof.
In particular aspects, administering the amount of the polypeptide comprising a CD18 signal peptide, or portion thereof, comprises administration to a ruminant previously vaccinated against M. haemolytica. In certain embodiments, vaccination against M. haemolytica comprises administration of M. haemolytica leukotoxin (Lkt) or a portion thereof.
Additional aspects provide an antibody specific for a CD18 signal peptide, or portion thereof comprising from about 13 to about 24 contiguous amino acid residues of the first 24 amino acids of the N-terminus of the native (full-length nascent) ruminant CD18 sequence, wherein the polypeptide is suitable to provide for at least one of binding to M. haemolytica leukotoxin (Lkt) and abrogation of Lkt-induced cytolysis. In certain embodiments, the polypeptide comprises a contiguous portion of the CD18 signal polypeptide beginning at amino acid residue 5. In certain aspects, the polypeptide comprises residues 5 to 17 of the CD18 signal polypeptide. In particular embodiments, the CD18 sequence is selected from the group consisting of cattle, bison, buffalo, goat, domestic sheep, big horn sheep, deer, elk, giraffes, yaks, camels, alpacas, llamas, wildebeest, antelope, pronghorn, nilgai, human, chimp, mouse, rat and pig. In certain embodiments, the CD18 sequence is selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 21, 26, 57, 58, 60, 62, 64, 66, 68 and 70.
Yet further aspects provide a method of treating for treating or preventing conditions and diseases associated with M. haemolytica in ruminants, comprising administering to a ruminant subject in need thereof, an amount of an antibody specific for a CD18 signal peptide, or portion thereof comprising from about 13 to about 24 contiguous amino acid residues of the first 24 amino acids of the N-terminus of the native (full-length nascent) ruminant CD18 sequence, wherein the polypeptide is suitable to provide for at least one of binding to M. haemolytica leukotoxin (Lkt) and abrogation of Lkt-induced cytolysis, wherein a method of treating for treating or preventing conditions and diseases associated with M. haemolytica in ruminants is provided. In certain aspects, the antibody is according to those anti-ruminant CD18 signal peptide antibodies described herein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C show, according to particular exemplary aspects, that the CD18 signal peptide analog P5 (aa 5-24) inhibits Lkt-induced cytolysis of bovine PMNs. See working EXAMPLE 2 for details.
FIGS. 2A and 2B show, according to particular exemplary aspects, that peptide P5 inhibits Lkt binding to bovine PMNs. See working EXAMPLE 2 for details.
FIGS. 3A-3C show, according to particular exemplary aspects, that N- and C-terminal truncations of peptide P5 identifies the minimal peptide sequence of bovine CD18 bound by Lkt as aa 5-17. See working EXAMPLE 2 for details.
FIG. 4A-4H show, according to particular exemplary aspects, that anti-signal peptide serum binds to membrane CD18 of PMNs of all ruminants tested. See working EXAMPLE 3 for details.
FIGS. 5A-5D show, according to particular exemplary aspects, that the signal peptide of bovine CD18 is not cleaved. See working EXAMPLE 4 for details.
FIGS. 6A-6B show, according to particular exemplary aspects, Signal peptide of CD18 of ruminants contain glutamine at aa position −5 relative to the cleavage site, whereas that of non-ruminants contain glycine. See working EXAMPLE 5 for details.
FIG. 7 shows, according to particular exemplary aspects, that mutation of glutamine at position −5 of the signal peptide of bovine CD18 to glycine abrogates Lkt-induced cytolysis of transfectants expressing CD18 with Q(−5)G mutation. See working EXAMPLE 6 for details.
FIGS. 8A-8C shows, according to particular exemplary aspects, that peptides spanning aa 500-600 of bovine CD18 fail to inhibit Lkt-induced cytolysis of bovine PMNs. See working EXAMPLE 6 for details.
FIG. 9 shows, according to particular exemplary aspects, that co-incubation of peptide P17 with in vitro cultures of M. haemolytica abrogate leukotoxic activity. See working EXAMPLE 7 for details.
FIG. 10 shows mean clinical scores of calves inoculated with M. haemolytica only (Group I), M. haemolytica along with the irrelevant peptide PSC (Group II) and M. haemolytica along with the peptide P17 (Group III) at different time points post-inoculation. At 24 hours post-inoculation, the mean clinical score of Group III was statistically significantly lower than that of other groups (P<0.05). See working EXAMPLE 7 for details.
FIGS. 11A-11B shows, according to particular aspects, representative gross-(A) and histo-(B) pathology of the lungs of calves infected with M. haemolytica with or without peptides. See working EXAMPLE 7 for details.



DETAILED DESCRIPTION OF EXEMPLARY ASPECTS OF THE INVENTION
Generally, a nascent membrane protein contains a signal sequence that directs the protein/ribosome to the endoplasmic reticulum (ER) membrane1-3. The signal peptide binds to the signal recognition particle (SRP) which in turn binds to the SRP receptor on the ER membrane and helps in the translocation of the protein into the lumen of the ER. The signal peptide is cleaved from the protein by the ER-resident signal peptidase while it is still growing on the ribosome. Thus the signal peptide is not present on the mature protein that reaches the plasma membrane following post-translational modifications.
As disclosed herein, however, Applicants' mapping the Mannheimia (Pasteurella) haemolytica leukotoxin (Lkt) binding site on its receptor CD18 have led to the unexpected finding that the signal peptide of ruminant CD18 remains intact on the mature CD18 molecule on the leukocytes of ruminants and renders these cells susceptible to cytolysis by Lkt.
Therefore, the signal peptide of ruminant CD18, the β subunit of leukocyte-specific β2-integrins, is an exception to general phenomenon that signal peptides are not present on the mature protein that reaches the plasma membrane. Intriguingly, as disclosed herein, the intact signal peptide of CD18 is responsible for the susceptibility of ruminant leukocytes to Mannheimia (Pasteurella) haemolytica leukotoxin, and the resultant susceptibility of ruminants to severe pneumonia caused by this organism.
Previously, Applicants identified CD18 as the receptor for Lkt on bovine4 and ovine5,6 leukocytes, and mapped the Lkt-binding site to lie between amino acids 1-2917. As disclosed herein, under working EXAMPLE 2, inhibition of Lkt-induced cytolysis of ruminant leukocytes by CD18 peptide analogs revealed that the Lkt-binding site is formed by aa 5-17 of CD18 which, surprisingly, comprise a part of the signal peptide.
As shown herein under working EXAMPLE 3, flow cytometric analysis of ruminant leukocytes with an anti-signal peptide serum indicated the presence of the signal peptide on the mature CD 18 molecules expressed on the cell surface.
As shown herein under working EXAMPLE 4, analysis of the transfectants expressing CD18 containing the ‘FLAG’ epitope at the putative cleavage site confirmed that the signal peptide of CD 18 is not cleaved.
Working EXAMPLE 5 below, discloses a comparative analysis of the amino acid (aa) sequence of the signal peptide of CD18 of eight ruminants and five non-ruminants, and revealed that the signal peptide of CD18 of ruminants contain ‘cleavage-inhibiting’ glutamine (Q), whereas that of non-ruminants contain ‘cleavage-conducive’ glycine (G) at position −5 relative to the cleavage site.
Working EXAMPLE 6 below, discloses that site-directed mutagenesis of Q at position −5 of the signal peptide of bovine CD18 to G resulted in the abrogation of cytolysis of transfectants expressing bovine CD18 carrying the Q(−5)G mutation. According to particular aspects, replacement of ‘cleavage-inhibiting’ Q at −5 position with ‘cleavage-conducive’ G resulted in the cleavage of the signal peptide and the resultant loss of susceptibility of the transfectants to Lkt-induced cytolysis. According to additional aspects, it is possible that abrogation of cytolysis is not due to cleavage of signal peptide, but due to conformational changes caused by the replacement of Q with G. Irrespective of the molecular basis underlying the abrogation of cytolysis, however, the exemplary Q(−5)G mutation presents an exemplary embodiment of a hitherto unavailable technology to, among other things, clone cattle and other ruminants expressing CD18 without the signal peptide on their leukocytes, and thus provide animals that are substantially less susceptible to pneumonic pasteurellosis.
Definitions: “Functional variants” as used herein refers to at least one protein selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70 sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and biologically active variants thereof, where functional or biologically active variants are those proteins that display one or more of the biological activities of at least one protein selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, including but not limited to the activities disclosed herein (e.g., binding to the bacterial toxin Lkt; producing proteins resistant to binding by Lkt)
As used herein, a pharmaceutical or therapeutic effect refers to an effect observed upon administration of an agent intended for the prevention or treatment of a disease or disorder or for amelioration of the symptoms thereof.
As used herein, treatment means any manner in which the symptoms of a condition, disorder or disease or other indication, are ameliorated or otherwise beneficially altered.
As used herein therapeutic effect means an effect resulting from treatment of a subject that alters, typically improves or ameliorates the symptoms of a disease or condition or that cures a disease or condition. A therapeutically effective amount refers to the amount of a composition, molecule or compound which results in a therapeutic effect following administration to a subject.
As used herein, the term “subject” refers to ruminant animals, including mammals, such as cattle.
As used herein, the phrase “associated with” refers to certain biological aspects such as expression of a receptor or signaling by a receptor that occurs in the context of a disease or condition. Such biological aspect may or may not be causative or integral to the disease or condition but merely an aspect of the disease or condition.
As used herein, a biological activity refers to a function of a polypeptide including but not limited to complexation, dimerization, multimerization, receptor-associated kinase activity, receptor-associated protease activity, phosphorylation, dephosphorylation, autophosphorylation, ability to form complexes with other molecules, ligand binding, catalytic or enzymatic activity, activation including auto-activation and activation of other polypeptides, inhibition or modulation of another molecule's function, stimulation or inhibition of signal transduction and/or cellular responses such as cell proliferation, migration, differentiation, and growth, degradation, membrane localization, membrane binding, and oncogenesis. A biological activity can be assessed by assays described herein and by any suitable assays known to those of skill in the art, including, but not limited to in vitro assays, including cell-based assays, in vivo assays, including assays in animal models for particular diseases.
Table 1 contains a brief description and sequence listing including some but not all (e.g., exemplary) of the peptides and polypeptides within the scope of this invention. Therapeutic peptides to be used in the prevention and/or treatment of an infection of M. haemolytica may be a portion of CD18 from ruminants containing the signal sequence. In preferred aspects, the therapeutic contains the amino acid residues of CD18 from ruminants from amino acids 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, and 1 to 10. Additionally, the therapeutic contains the amino acid residues of CD18 from ruminants from amino acids 2 to 25, 2 to 24, 2 to 23, 2 to 22, 2 to 21, 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, and 2 to 10. In further preferred embodiments, the therapeutic contains the amino acid residues of CD18 from ruminants from amino acids 3 to 25, 3 to 24, 3 to 23, 3 to 22, 3 to 21, 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, and 3 to 14, 3 to 13, 3 to 12, 3 to 11, and 3 to 10. In yet further preferred embodiments, the therapeutic contains the amino acid residues of CD18 from ruminants from amino acids 4 to 25, 4 to 24, 4 to 23, 4 to 22, 4 to 21, 4 to 20, 4 to 19, 4 to 18, 4 to 17, 4 to 16, 4 to 15, and 4 to 14, 4 to 13, 4 to 12, 4 to 11, and 4 to 10. In additional, preferred embodiments, the therapeutic contains the amino acid residues of CD18 from ruminants from amino acids 5 to 25, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, and 5 to 14, 5 to 13, 5 to 12, 5 to 11, and 5 to 10.







TABLE 1







Summary of exemplary SEQ ID NOS


and brief descriptions thereof.









SEQ ID NO









BRIEF DESCRIPTION
Nucleic Acid
Protein





Full Length CD18 Protein




Cattle
SEQ ID NO: 1
SEQ ID NO: 2


Bison
SEQ ID NO: 3
SEQ ID NO: 4


Buffalo
SEQ ID NO: 5
SEQ ID NO: 6


Goat
SEQ ID NO: 7
SEQ ID NO: 8


Domestic Sheep
SEQ ID NO: 9
SEQ ID NO: 10


Wild Sheep
SEQ ID NO: 11
SEQ ID NO: 12


Deer
SEQ ID NO: 13
SEQ ID NO: 14


Elk
SEQ ID NO: 15
SEQ ID NO: 16


Human
SEQ ID NO: 17
SEQ ID NO: 18


Mouse
SEQ ID NO: 19
SEQ ID NO: 20


Rat
SEQ ID NO: 21
SEQ ID NO: 22


Pig
SEQ ID NO: 23
SEQ ID NO: 24


Chimp
SEQ ID NO: 25
SEQ ID NO: 26


Exemplary Mutated CD18


Cattle
SEQ ID NO: 55
SEQ ID NO: 58


Bison
SEQ ID NO: 59
SEQ ID NO: 60


Buffalo
SEQ ID NO: 56
SEQ ID NO: 57


Goat
SEQ ID NO: 61
SEQ ID NO: 62


Domestic Sheep
SEQ ID NO: 63
SEQ ID NO: 64


Wild Sheep
SEQ ID NO: 65
SEQ ID NO: 66


Deer
SEQ ID NO: 67
SEQ ID NO: 68


Elk
SEQ ID NO: 69
SEQ ID NO: 70


Peptide from CD18 Signal


Sequence


Cattle
SEQ ID NO: 27
SEQ ID NO: 28


Bison
SEQ ID NO: 27
SEQ ID NO: 28


Buffalo
SEQ ID NO: 29
SEQ ID NO: 30


Goat
SEQ ID NO: 31
SEQ ID NO: 32


Domestic Sheep
SEQ ID NO: 33
SEQ ID NO: 34


Wild Sheep
SEQ ID NO: 33
SEQ ID NO: 34


Deer
SEQ ID NO: 35
SEQ ID NO: 36


Elk
SEQ ID NO: 37
SEQ ID NO: 38


Human
SEQ ID NO: 39
SEQ ID NO: 40


Chimp
SEQ ID NO: 39
SEQ ID NO: 40


Mouse
SEQ ID NO: 41
SEQ ID NO: 42


Full Length CD18 Protein


Rat
SEQ ID NO: 43
SEQ ID NO: 44


Pig
SEQ ID NO: 45
SEQ ID NO: 46


Exemplary Mutated Peptide


from CD18 Signal Sequence


Human
SEQ ID NO: 47
SEQ ID NO: 48


Chimp
SEQ ID NO: 47
SEQ ID NO: 48


Mouse
SEQ ID NO: 49
SEQ ID NO: 50


Rat
SEQ ID NO: 51
SEQ ID NO: 52


Pig
SEQ ID NO: 53
SEQ ID NO: 54









Variants of at least one protein selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto have utility for aspects of the present invention. Variants can be naturally or non-naturally occurring. Naturally occurring variants (e.g., polymorphisms) are found in humans or other species and comprise amino acid sequences which are substantially identical to the amino acid sequences disclosed herein. Species homologs of the protein can be obtained using subgenomic polynucleotides of the invention, as described below, to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, yeast, or bacteria, identifying cDNAs which encode homologs of the protein, and expressing the cDNAs as is known in the art.
Non-naturally occurring variants which retain substantially the same biological activities as naturally occurring protein variants. Preferably, naturally or non-naturally occurring variants have amino acid sequences which are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenced disclosed herein. More preferably, the molecules are at least 98% or 99% identical. Percent identity is determined using any method known in the art. A non-limiting example is the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1. The Smith-Waterman homology search algorithm is taught in Smith and Waterman, Adv. Appl. Math. 2:482-489, 1981.
As used herein, “amino acid residue” refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are generally in the “L” isomeric form. Residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243:3552-59 (1969) and adopted at 37 C.F.R. §§1.821-1.822, abbreviations for amino acid residues are shown in Table 2:







TABLE 2







Table of Correspondence











SYMBOL













1-Letter
3-Letter
AMINO ACID







Y
Tyr
Tyrosine



G
Gly
Glycine



F
Phe
Phenylalanine



M
Met
Methionine



A
Ala
Alanine



S
Ser
Serine



I
Ile
Isoleucine



L
Leu
Leucine



T
Thr
Threonine



V
Val
Valine



P
Pro
Praline



K
Lys
Lysine



H
His
Histidine



Q
Gln
Glutamine



E
Glu
glutamic acid



Z
Glx
Glu and/or Gln



W
Trp
Tryptophan



R
Arg
Arginine



D
Asp
aspartic acid



N
Asn
Asparagines



B
Asx
Asn and/or Asp



C
Cys
Cysteine



X
Xaa
Unknown or other










It should be noted that all amino acid residue sequences represented herein by a formula have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase “amino acid residue” is defined to include the amino acids listed in the Table of Correspondence and modified and unusual amino acids, such as those referred to in 37 C.F.R. §§1.821-1.822, and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino-terminal group such as NH2 or to a carboxyl-terminal group such as COOH.
Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity can be found using computer programs well known in the art, such as DNASTAR software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
It is reasonable to expect that an isolated 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 will not have a major effect on the biological properties of the resulting variant.
Variants of the at least one protein selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto disclosed herein include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., pegylated molecules). Covalent variants can be prepared by linking functionalities to groups which are found in the amino acid chain or at the N- or C-terminal residue, as is known in the art. Variants also include allelic variants, species variants, and muteins. Truncations or deletions of regions which do not affect functional activity of the proteins are also variants.
A subset of mutants, called muteins, is a group of polypeptides in which neutral amino acids, such as serines, are substituted for cysteine residues which do not participate in disulfide bonds. These mutants may be stable over a broader temperature range than native secreted proteins (see, e.g., Mark et al., U.S. Pat. No. 4,959,314).
Preferably, amino acid changes in the variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
It is reasonable to expect that an isolated 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 will not have a major effect on the biological properties of the resulting secreted protein or polypeptide variant. Properties and functions of the variants are of the same type as a protein comprising the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, and sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, although the properties and functions of variants can differ in degree.
Variants of at least one protein selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., pegylated molecules). The variants also include allelic variants, species variants, and muteins. Truncations or deletions of regions which do not affect functional activity of the proteins are also variants. Covalent variants can be prepared by linking functionalities to groups which are found in the amino acid chain or at the N- or C-terminal residue, as is known in the art.
It will be recognized in the art that some amino acid sequences of the polypeptides of the invention can be varied without significant effect on the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there are critical areas on the protein which determine activity. In general, it is possible to replace residues that form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the protein. The replacement of amino acids can also change the selectivity of binding to cell surface receptors (Ostade et al., Nature 361:266-268, 1993). Thus, the polypeptides of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.
Of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the disclosed protein. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic (see, e.g., Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); and Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).
Amino acids in polypeptides of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as binding to a natural or synthetic binding partner. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).
As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein. Of course, the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of substitutions for any given polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5 or 3.
In addition, pegylation of the inventive polypeptides and/or muteins is expected to provide such improved properties as increased half-life, solubility, and protease resistance. Pegylation is well known in the art.
Fusion Proteins
Fusion proteins comprising proteins or polypeptide fragments of at least one protein selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto can also be constructed. Fusion proteins are useful for generating antibodies against amino acid sequences and for use in various targeting and assay systems. For example, fusion proteins can be used to identify proteins which interact with a polypeptide of the invention or which interfere with its biological function. Physical methods, such as protein affinity chromatography, or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can also be used for this purpose. Such methods are well known in the art and can also be used as drug screens. Fusion proteins comprising a signal sequence can be used.
A fusion protein comprises two protein segments fused together by means of a peptide bond. Amino acid sequences for use in fusion proteins of the invention can be utilize the amino acid sequence shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, and sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or can be prepared from biologically active variants such as those described above. The first protein segment can include of a full-length polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
Other first protein segments can consist of biologically active portions of SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
The second protein segment can be a full-length protein or a polypeptide fragment. Proteins commonly used in fusion protein construction include β-galactosidase, β-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags can be used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
These fusions can be made, for example, by covalently linking two protein segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises a coding region for the protein sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto in proper reading frame with a nucleotide encoding the second protein segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies that supply research labs with tools for experiments, including, for example, Promega Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.), Clontech (Mountain View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).
Pharmaceutical Compositions and Therapeutic Uses
Pharmaceutical compositions of the invention can comprise CD18 signal peptide (or a portion thereof)—comprising polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, and sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto polypeptide-based agents of the claimed invention in a therapeutically effective amount. The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation is determined by routine experimentation and is within the judgment of the clinician. For purposes of the present invention, an effective dose will generally be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto polypeptide constructs in the individual to which it is administered.
A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the subject receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier. Pharmaceutically acceptable salts can also be present in the pharmaceutical composition, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., New Jersey, 1991).
Delivery Methods.
Once formulated, the compositions of the invention can be administered directly to the subject or delivered ex vivo, to cells derived from the subject (e.g., as in ex vivo gene therapy). Direct delivery of the compositions will generally be accomplished by parenteral injection, e.g., subcutaneously, intraperitoneally, intravenously or intramuscularly, myocardial, intratumoral, peritumoral, or to the interstitial space of a tissue. Other modes of administration include oral and pulmonary administration, suppositories, implants, and transdermal applications, needles, and gene guns or hyposprays. Specific oral treatment includes, but is not limited to, the inclusion of the therapeutic in the animal feed. Dosage treatment can be a single dose schedule or a multiple dose schedule.
Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No. WO 93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, APCs, or tumor cells. Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, direct microinjection of the DNA into nuclei, and viral-mediated, such as adenovirus or alphavirus, all well known in the art.
In a preferred embodiments, disorders can be amenable to treatment by administration of a therapeutic agent based on the provided polynucleotide or corresponding polypeptide. The therapeutic agent can be administered in conjunction with one or more other agents including, but not limited to, receptor-specific antibodies and/or chemotherapeutic (e.g., anti-neoplastic agents). Administered “in conjunction” includes administration at the same time, or within 1 day, 12 hours, 6 hours, one hour, or less than one hour, as the other therapeutic agent(s). The compositions may be mixed for co-administration, or may be administered separately by the same or different routes.
The dose and the means of administration of the inventive pharmaceutical compositions are determined based on the specific qualities of the therapeutic composition, the condition, age, and weight of the patient, the progression of the disease, and other relevant factors. For example, administration of polynucleotide therapeutic compositions agents of the invention includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. The therapeutic polynucleotide composition can contain an expression construct comprising a promoter operably linked to a polynucleotide encoding, for example, SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, and sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Various methods can be used to administer the therapeutic composition directly to a specific site in the body. For example, a target tissue is located and the therapeutic composition injected several times in several different locations within the target tissue. Alternatively, arteries which serve a target tissue are identified, and the therapeutic composition injected into such an artery, in order to deliver the composition directly into the target tissue. X-ray imaging is used to assist in certain of the above delivery methods.
Inventive polypeptide-mediated targeted delivery of therapeutic agents to specific tissues can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 mg to about 2 mg, about 5 mg to about 500 mg, and about 20 mg to about 100 mg of DNA can also be used during a gene therapy protocol. Factors such as method of action (e.g., for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy of the subgenomic polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of subgenomic polynucleotides or the same amounts readministered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of, for example, a tumor site, may be required to affect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect.
The therapeutic polynucleotides and polypeptides of the present invention can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.
Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; U.S. Pat. No. 4,777,127; GB Patent No. 2,200,651; EP 0 345 242; and WO 91/02805), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), and adeno-associated virus (AAV) vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.
Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. 264:16985 (1989)); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. 14:2411 (1994), and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:11581-11585.
Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al., Proc. Natl. Acad. Sci. USA 91(24):11581 (1994). Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials or use of ionizing radiation (see, e.g., U.S. Pat. No. 5,206,152 and WO 92/11033). Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun (see, e.g., U.S. Pat. No. 5,149,655); use of ionizing radiation for activating transferred gene (see, e.g., U.S. Pat. No. 5,206,152 and WO 92/11033).)
Recombinant and Cloned Ruminant Animals:
Inventive CD18-related aspects (e.g. polypeptide-mediated treatment, etc) include producing animals that are naturally resistant to the effects of Lkt. This is accomplished by, for example, universally altering the genotype of an animal, wherein, the native CD 18 molecule, which binds to the toxin Lkt, is replaced with the mutant CD 18, which has limited Lkt binding. Universally altering the genotype of animal includes cloning of a given animal having the modified genotype. Additionally, the invention encompasses transgenic animals. Transgenic animals are those that carry a non-native gene that were introduced into the animal using similar techniques as described herein and those well known in the art. Transgenic animals can subsequently be cloned.
Cloned Ruminants:
Cloned fetuses and calves are produced using the chromatin transfer procedure, as described in Kuroiwa et al., (2004) hereby incorporated by reference specifically for its teaching of cloning of cattle. The method consists of sequential application of gene targeting by homologous recombination and rejuvenation of cell lines by production of cloned fetuses. For example, to generate cattle containing a CD18 molecule that had a Q(−5)G mutation (an exemplary mutation which results in limited binding of Lkt to CD18 signal sequence), a male Holstein primary fetal fibroblast line 6594 is transfected with vectors containing the mutated signal sequence to replace the native CD 18 coding region. This fetal cell line containing the mutated CD18 are established at 40-60 days of gestation. Certain fetal cell lines that look promising are recloned to produce calves. To verify that each calf produced contains the mutated CD18 genotype, applicants collect ear biopsies and establish fibroblast cell lines for genotyping (Richt et al., 2007, hereby incorporated by reference to teach analysis of cells and animals post cloning). Genotyping is done by genomic PCR specific to each gene targeting event, followed by sequence analysis. Additionally, applicants verify the calf phenotype by collecting blood samples and isolating PMNs and ensuring that anti-signal peptide serum cannot bind to membrane CD18 of PMNs, as described in FIG. 4 and Example 3.
Gene replacement techniques used in the practice of applicant's invention includes, but is not limited to, the gene replacement techniques described in Kuroiwa et al., (2004) hereby incorporated by reference specifically for its teaching of gene replacement techniques and sequential application of those techniques. For example, the wildtype CD 18 molecule is replaced by the exemplary Q(−5)G mutation in the signal sequence of CD18 that produces a CD18 molecule with a cleavable signal sequence. More specifically two different vectors, each containing specific selection cassettes (e.g., cassettes conferring resistance to neomycin or puromycin) are constructed containing the mutated CD18 molecule Q(−5)G. The mutated CD18 coding sequence is flanked on both the 5′ and 3′ ends with between 1 and 10 kb of native sequences, i.e. sequences that flank the CD18 gene in vivo in cattle. These vectors are then transfected into male Holstein primary fetal fibroblast line 6594.
Additionally, using techniques known in the art, practicing this invention includes gene therapy. Gene therapy or gene introduction encompasses treating an animal in need thereof with a vector that contains a gene sequence encoding the therapeutic to be produced and provided to the animal via the animal's own protein producing mechanisms. According to certain aspects the invention includes expressing nucleic acids sequences encoding CD18 polypeptides (e.g., SEQ ID NOS:2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto include glycosylated forms) by the cell's own machinery.
Antibodies or Antibody Fragments
Agents of the present invention include antibodies and/or antibody fragments, and in particular aspects, they are specific for (directed against): (1) M. haemolytica leukotoxin (Lkt); (2) against Lkt or antigen or portion thereof that is suitable for binding the signal peptide, and optionally wherein the anti-Lkt antibody can also bind Lkt complexed with ruminant CD18 signal peptide; (3) or against a CD18 signal peptide, or portion thereof comprising from about 13 to about 24 contiguous amino acid residues of the first 24 amino acids of the N-terminus of the native (full-length nascent) ruminant CD18 sequence, wherein the polypeptide is suitable to provide for at least one of binding to M. haemolytica leukotoxin (Lkt) and abrogation of Lkt-induced cytolysis; and (4) against signal peptide and able to bind Lkt complexed with ruminant CD18 signal peptide.
Suitable antibodies may be monoclonal, polyclonal or monoclonal antibodies tailored to a specific ruminant species (in analogy with humanized antibodies). Antibodies may be derived by conventional hybridoma based methodology, from antisera isolated from validated protein inoculated animals or through recombinant DNA technology. Alternatively, inventive antibodies or antibody fragments may be identified in vitro by use of one or more of the readily available phage display libraries. Exemplary methods are disclosed herein.
In one exemplary embodiment of the present invention, antibody agents are monoclonal antibodies that may be produced as follows. Target proteins in a baculovirus based system. By this method, target protein cDNAs or epitope-bearing fragments thereof are ligated into a suitable plasmid vector that is subsequently used to transfect Sf9 cells to facilitate protein production. In addition, it may be advantageous to incorporate an epitope tag or other moiety to facilitate affinity purification of the target protein. Clones of Sf9 cells expressing a particular protein are identified, e.g., by enzyme-linked immunosorbant assay (ELISA), lysates are prepared and the target protein purified by affinity chromatography. The purified target protein is, for example, injected intraperitoneally, into BALB/c mice to induce antibody production. It may be advantageous to add an adjuvant, such as Freund's adjuvant, to increase the resulting immune response.
Serum is tested for the production of specific antibodies, and spleen cells from animals having a positive specific antibody titer are used for cell fusions with myeloma cells to generate hybridoma clones. Supernatants derived from hybridoma clones are tested for the presence of monoclonal antibodies having specificity against a particular validated protein or fragments thereof. For a general description of monoclonal antibody methodology, See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988).
In addition to the baculovirus expression system, other suitable bacterial or yeast expression systems may be employed for the expression of a particular target protein or polypeptides thereof. As with the baculovirus system, it may be advantageous to utilize one of the commercially available affinity tags to facilitate purification prior to inoculation of the animals. Thus, the target protein cDNA or fragment thereof may be isolated by, e.g., agarose gel purification and ligated in frame with a suitable tag protein such as 6-His, glutathione-S-transferase (GST) or other such readily available affinity tag. See, e.g., Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press pp. 160-161 (ed. Glick, B. R. and Pasternak, J. J. 1998).
In additional embodiments of the present invention, antibody agents are ruminantized anti-target protein monoclonal antibodies. The phrase “ruminantized antibody” refers to an antibody derived from a non-ruminant antibody—typically a mouse monoclonal antibody. Alternatively, a ruminantized antibody may be derived from a chimeric antibody that retains or substantially retains the antigen-binding properties of the parental, non-ruminant, antibody but which exhibits diminished immunogenicity as compared to the parental antibody when administered to ruminant. The phrase “chimeric antibody,” as used herein, refers to an antibody containing sequence derived from two different antibodies (see, e.g., U.S. Pat. No. 4,816,567) which typically originate from different species. Most typically, chimeric antibodies comprise human and murine antibody fragments, generally bovine constant and mouse variable regions.
Because ruminantized antibodies are far less immunogenic in ruminant than the parental mouse monoclonal antibodies, they can be used for the treatment of ruminant with far less risk of anaphylaxis. Thus, these antibodies may be preferred in therapeutic applications that involve in vivo administration to a ruminant such as, e.g., use as radiation sensitizers for the treatment of neoplastic disease or use in methods to reduce the side effects of, e.g., cancer therapy.
Ruminantized antibodies may be achieved by a variety of methods including, for example: (1) grafting the non-ruminant complementarity determining regions (CDRs) onto a ruminant framework and constant region (a process referred to in the art as “humanizing”), or, alternatively, (2) transplanting the entire non-ruminant variable domains, but “cloaking” them with a ruminant-like surface by replacement of surface residues (a process referred to in the art as “veneering”). In the present invention, ruminantized antibodies will include both “ruminantized” and “veneered” antibodies. These methods, in the context of humanized antibodies, are disclosed in, e.g., Jones et al., Nature (1986) 321:522-525; Morrison et al., Proc. Natl. Acad. Sci., U.S.A., (1984) 81:6851-6855; Morrison and 01, Adv. Immunol. (1988) 44:65-92; Verhoeyer et al., Science (1988) 239:1534-1536; Padlan, Molec. Immun. (1991) 28:489-498; Padlan, Molec. Immunol. (1994) 31(3):169-217; and Kettleborough, C. A. et al., Protein Eng. (1991) 4:773-83 each of which is incorporated herein by reference.
The phrase “complementarity determining region” refers to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. See, e.g., Chothia et al., J. Mol. Biol. (1987) 196:901-917; Kabat et al., U.S. Dept. of Health and Human Services NIH Publication No. 91-3242 (1991). The phrase “constant region” refers to the portion of the antibody molecule that confers effector functions. In the present invention, mouse constant regions are substituted by human constant regions. The constant regions of the subject humanized antibodies are derived from human immunoglobulins. The heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu.
One method of ruminantized antibodies comprises aligning the non-ruminant heavy and light chain sequences to ruminant heavy and light chain sequences, selecting and replacing the non-ruminant framework with a ruminant framework based on such alignment, molecular modeling to predict the conformation of the ruminantized sequence and comparing to the conformation of the parent antibody. This process is followed by repeated back mutation of residues in the CDR region which disturb the structure of the CDRs until the predicted conformation of the ruminantized sequence model closely approximates the conformation of the non-ruminant CDRs of the parent non-ruminant antibody. Such ruminantized antibodies may be further derivatized to facilitate uptake and clearance, e.g., via recpetors in analogy with the use of Ashwell receptors (see, e.g., U.S. Pat. Nos. 5,530,101 and 5,585,089, both incorporated herein by reference.
Ruminantized antibodies to a particular target protein can also be produced using transgenic animals that are engineered to contain ruminant immunoglobulin loci. In analogy with humanized antibodies for example, WO 98/24893 discloses transgenic animals having a human Ig locus wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of endogenous heavy and light chain loci. WO 91/10741 also discloses transgenic non-primate mammalian hosts capable of mounting an immune response to an immunogen, wherein the antibodies have primate constant and/or variable regions, and wherein the endogenous immunoglobulin-encoding loci are substituted or inactivated. WO 96/30498 discloses the use of the Cre/Lox system to modify the immunoglobulin locus in a mammal, such as to replace all or a portion of the constant or variable region to form a modified antibody molecule. WO 94/02602 discloses non-human mammalian hosts having inactivated endogenous Ig loci and functional human Ig loci. U.S. Pat. No. 5,939,598 discloses methods of making transgenic mice in which the mice lack endogenous heavy claims, and express an exogenous immunoglobulin locus comprising one or more xenogeneic constant regions.
Using a transgenic animal described above, an immune response can be produced to a selected antigenic molecule (e.g., target protein or fragment thereof), and antibody-producing cells can be removed from the animal and used to produce hybridomas that secrete ruminant monoclonal antibodies. Immunization protocols, adjuvants, and the like are known in the art, and are used in immunization of, for example, a transgenic mouse as described in WO 96/33735. This publication discloses monoclonal antibodies against a variety of antigenic molecules including IL-6, IL-8, TNFα, human CD4, L-selectin, gp39, and tetanus toxin. The monoclonal antibodies can be tested for the ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein. WO 96/33735 discloses that monoclonal antibodies against IL-8, derived from immune cells of transgenic mice immunized with IL-8, blocked IL-8-induced functions of neutrophils. Human monoclonal antibodies with specificity for the antigen used to immunize transgenic animals are also disclosed in WO 96/34096.
For purposes of the present invention, target polypeptides and variants thereof are used to immunize an animal or transgenic animal as described above. Monoclonal antibodies are made using methods known in the art, and the specificity of the antibodies is tested using isolated target polypeptides. The suitability of the antibodies for clinical use is tested by, for example, exposing HCMV-infected cells to the antibodies and measuring cell growth and/or phenotypic changes. Ruminant monoclonal antibodies specific for a particular validated protein, or for a variant or fragment thereof can be tested for their ability to inhibit, for example, cell migration. Such antibodies would be suitable for pre-clinical and clinical trials as pharmaceutical agents for preventing or controlling virus or bacterial-mediated effects, conditions or diseases.
It will be appreciated that alternative target protein inhibitor antibodies may be readily obtained by other methods commonly known in the art. One exemplary methodology for identifying antibodies having a high specificity for a particular validated protein is the phage display technology.
Phage display libraries for the production of high-affinity antibodies are described in, for example, Hoogenboom, H. R. et al., Immunotechnology (1998) 4(1):1-20; Hoogenboom, H. R., Trends Biotechnol. (1997) 15:62-70 and McGuinness, B. et al., Nature Bio. Technol. (1996) 14:1149-1154 each of which is incorporated herein by reference. Among the advantages of the phage display technology is the ability to isolate antibodies of ruminant origin that cannot otherwise be easily isolated by conventional hybridoma technology. Furthermore, phage display antibodies may be isolated in vitro without relying on an animal's immune system.
Antibody phage display libraries may be accomplished, for example, by the method of McCafferty et al., Nature (1990) 348:552-554 which is incorporated herein by reference. In short, the coding sequence of the antibody variable region is fused to the amino terminus of a phage minor coat protein (pIII). Expression of the antibody variable region-pIII fusion construct results in the antibody's “display” on the phage surface with the corresponding genetic material encompassed within the phage particle.
A target protein, or fragment thereof suitable for screening a phage library may be obtained by, for example, expression in baculovirus Sf9 cells as described, supra. Alternatively, the target protein coding region may be PCR amplified using primers specific to the desired region of the validated protein. As discussed above, the target protein may be expressed in E. coli or yeast as a fusion with one of the commercially available affinity tags.
The resulting fusion protein may then be adsorbed to a solid matrix, e.g., a tissue culture plate or bead. Phage expressing antibodies having the desired anti-target protein binding properties may subsequently be isolated by successive panning, in the case of a solid matrix, or by affinity adsorption to a validated protein antigen column. Phage having the desired target protein inhibitory activities may be reintroduced into bacteria by infection and propagated by standard methods known to those skilled in the art. See Hoogenboom, H. R., Trends Biotechnol., supra for a review of methods for screening for positive antibody-pIII phage.
Vaccination of Ruminants
Agents of the present invention include compositions that elicit a specific immune response in a ruminant in need thereof. According to certain embodiments, the elicitation of the specific immune response treats, reduces the likelihood, and/or limits a M. haemolytica infection and/or the symptoms thereof. In particular aspects, these compositions that elicit a specific immune response are: (1) M. haemolytica leukotoxin (Lkt); (2) Lkt or antigen or portion thereof that is suitable for binding the signal peptide, and optionally wherein the immune response elicited can also recognize Lkt complexed with ruminant CD18 signal peptide; (3) a CD18 signal peptide, or portion thereof comprising from about 13 to about 24 contiguous amino acid residues of the first 24 amino acids of the N-terminus of the native (full-length nascent) ruminant CD 18 sequence; and/or (4) signal peptide, wherein the elicited immune response is capable of binding to Lkt complexed with ruminant CD18 signal peptide.
According to certain embodiments, a composition that elicits a specific immune response is a vaccine. The terms “vaccine” “vaccination” and “vaccinating” mean the inoculation of a substance or composition (a vaccine) into the body of the subject for the purpose of producing immunity against a disease that is for the purpose of treating or preventing a disease. Accordingly, vaccination may be therapeutic or prophylactic. By therapeutic vaccination is meant the administration of a vaccine to a subject already suffering from a M. haemolytica infection, typically for the purpose of heightening or broadening the immune response to thereby halt, impede or reverse the progression of the disease.
Vaccination in accordance with the invention may provide protective immunity against a M. haemolytica infection to the subject being vaccinated. That is, the component(s) of the vaccine may elicit a protective immune response in the subject, for example by inducing the production of autoantibodies, innate immunity or adaptive immunity against the component(s). As used herein, the term “protective immunity” refers to the ability of a molecule or composition administered to a subject to elicit an appropriate immune response in the subject and thereby provide protection to the subject from the development or progression of a M. haemolytica infection.
The efficacy of compositions that elicit a specific immune response for use in accordance with the invention may be enhanced by the use of one or more adjuvants. Adjuvants capable of enhancing the delivery or protective or therapeutic efficacy of vaccines (for example by boosting the immune response produced) are well known to those skilled in the art.
Compositions that elicit a specific immune response may be prepared according to methods which are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant. For administration in accordance with the present invention, a suitable vaccine may be formulated in a pharmaceutically acceptable carrier according to the mode and route of administration to be used. The carriers, diluents and adjuvants must be “acceptable” in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Typically a sterile water or isotonic formulation is employed. For example, a suitable isotonic solution is phosphate buffered saline or Ringer's solution.
Those skilled in the art will appreciate that the methods and vaccinations contemplated by the present invention may be carried out in conjunction with other therapies or preventative measures for the treatment or prevention of M. haemolytica infections or symptoms associated with such diseases. For such combination therapies, each component of the combination therapy may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired effect. Alternatively, the components may be formulated together in a single dosage unit as a combination product.
Example 1
Materials and Methods
Inhibition of Lkt binding to, and cytolysis of target cells by the peptide analogs of CD18. Flow cytometric analysis of Lkt binding to target cells, and MTT dye-reduction cytotoxicity assay for detection of Lkt-induced cytolysis have been previously described by Applicants4,7. Detection of inhibition of Lkt binding to, and Lkt-induced cytolysis of, target cells by peptides in the disclosed studies were performed essentially as described, with the obvious exception that Lkt was pre-incubated with the peptides before incubation with the target cells.
Peptides. The nested set of 20-mer peptides spanning aa 1-291 of bovine CD18, and the N- and C-terminally truncated versions of the Lkt-binding minimal peptide were synthesized at Sigma-Genosys. An irrelevant peptide (20-mer) derived from major surface protein 1 (MSP1) of Anaplasma marginale was used as the negative control.
Cloning and expression of bovine CD18 carrying the mutation Q to G. The bovine cDNA for CD 1828 was previously subcloned into the mammalian expression vector pCI-neo to yield pMD14. To produce the Q(−5)G mutation in bovine CD18, site-directed mutagenesis was performed using the GeneTailorTM site-directed mutagenesis system (Invitrogen). CD18 sequence after the point mutation was checked by DNA sequencing. Transfection of P815 cells with Lipofectamine TM 2000 was carried out according to the supplier's recommendations.
Cloning and Expression of Bovine CD18 Carrying the ‘FLAG’ Epitope at the Cleavage Site.
The GeneTailorTM site-directed mutagenesis system (Invitrogen) was used to insert the ‘FLAG’ epitope (DYKDDDDK; SEQ ID NO:75) into the vector pMD1 carrying bovine CD18 cDNA, at the signal peptide cleavage site (between aa 22 and 23). The insertion was carried out in two steps (12 bp at a time) according to the manufacturer's instructions. The insertion of ‘FLAG’ epitope into CD18 was confirmed by DNA sequencing. The vector carrying the ‘FLAG’-tagged CD18 was transfected into P815 cells with Lipofectamine TM 2000 according to the manufacturer's protocol.
Statistical Analysis. One-way ANOVA was employed to determine whether the differences in % inhibition caused by the different peptides are statistically significant.
Preparation of Lkt. Production of Lkt from M. Haemolytica A1 has been Previously described by Applicants29. The undiluted toxin preparation contained 640 Units of toxin per ml. All experiments were performed with the same batch of toxin aliquoted and frozen at −20° C.
Cell lines and antibodies. The cell lines P815 (murine mastocytoma), and BL3 (bovine lymphoma3), were propagated in complete Dulbecco's minimum Eagle's medium or RPMI 1640, respectively, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 20 ug/ml of gentamicin (complete medium). The transfectant 2B2, expressing full-length bovine CD18 on the cell surface, was previously developed in Applicants' laboratory by transfecting P815 with cDNA for bovine CD184. The transfectants were selected and propagated in the complete DMEM together with 500 ug/ml of Geneticin (Invitrogen). PMNs were isolated from peripheral blood by density gradient centrifugation using Ficoll-Paque (Amersham Pharmacia Biotech.), followed by hypotonic lysis of the erythrocyte pellet, as previously described30. Anti-bovine CD18 monoclonal antibody (mAb) BAQ30A was obtained from Washington State University Monoclonal Antibody Center. The Lkt-non-neutralizing mAb MM605 (IgG2a) was previously developed in Applicants' laboratory29. FITC-conjugated MM-605 was used in flow cytometry to detect Lkt-binding7.
Peptides.
A nested set of 20-mer peptides spanning aa 1-291 of bovine CD18 was synthesized with either 6 or 15 aa overlap. Once the peptide which inhibits Lkt-induced cytotoxicity was identified, another set of peptides were synthesized with N-terminal truncation by dropping one aa at a time while keeping the C-terminal aa constant. Once the N-terminal aa of the minimal peptide was identified, another set of peptides were synthesized with C-terminal truncation by dropping one aa at a time while keeping the N-terminal aa constant. Peptides were purchased from Sigma Genosys. All the peptides were referred to by the sequence number of their first amino acid. An irrelevant peptide (20-mer) derived from major surface protein 1 (MSP1) of Anaplasma marginale was used as the negative control. All the peptides were resuspended in dimethysulfoxide (ATCC) at a concentration of 1 mg/ml, aliquoted and stored at −20° C.
Detection of Inhibition of Lkt-Induced Cytolysis of Target Cells by the Peptide Analogs of CD18.
The MTT [3-(4,5-dimethylthiazoyl-2-YI]-2,5-diphenyl tetrazolium bromide; Sigma] dye reduction cytotoxicity assay for detection of Lkt-induced cytolysis of target cells has been previously described by Applicants30. This assay measures the ability of the endoplasmic reticulum-resident enzymes in viable cells to convert a tetrazolium dye into a purple formazan precipitate, which is later dissolved in acid isopropanol. The optical density (OD) of the end product, representing the intensity of the purple color which developed, is directly proportional to the viability of the cells. The percent cytotoxicity was calculated as follows:

% cytotoxicity=[1−(OD of toxin-treated cells/OD of toxin-untreated cells)]×100.

Inhibition of Lkt-induced cytolysis of target cells by the peptide analogs was detected by the MTT assay with the obvious exception that Lkt was pre-incubated with the peptides before incubation with the target cells. Lkt was used at the dilution that causes 50% cytolysis of target cells. The percent inhibition of cytolysis was calculated as follows:

% Inhibition of cytolysis=[(1−% cytolysis in the presence of peptide)/% cytolysis in the absence of peptide]×100.

Flow cytometric analysis of inhibition of Lkt binding to target cells. Flow cytometric analysis of Lkt binding to target cells has been previously described by Applicants. Inhibition of Lkt binding to target cells by the peptides in the presently disclosed studies was performed essentially as previously described by Applicants with the obvious exception that Lkt was pre-incubated with the peptides before incubation with the target cells.
Flow Cytometric Analysis of the Cell Surface Expression of CD18 on Transfectant.
Transfectants were examined for the cell surface expression of CD 18 using anti-CD 18 MAb by flow cytometry, as previously described4.
Example 2
Peptide Analogs of Bovine CD18 Inhibited Lkt Binding to, and Cytolysis of, Ruminant PMNS
Applicants previously mapped the Lkt binding site on bovine CD 18 to lie between amino acids (aa)1-2917. In this EXAMPLE, inhibition of Lkt-induced cytolysis of target cells by a nested set of 20-mer peptides spanning aa 1-291 of bovine CD18 was used to determine the Lkt binding site on bovine CD 18. Lkt-induced cytolysis of bovine PMNs was strongly inhibited by two peptides, P1 and P5, containing aa 1-20 and 5-24, respectively, at a concentration of 5 ug/50 ul (FIG. 1a).
Specifically FIG. 1a shows, according to particular exemplary aspects, that the CD18 signal peptide analog P5 (aa 5-24) inhibits Lkt-induced cytolysis of bovine PMNs. Inhibition of Lkt-induced cytolysis of bovine PMNs by a nested set of peptides (20-mers) spanning the aa 1-291 was tested by the MTT dye-reduction cytotoxicity assay. All data are expressed as mean±s.d. (n=3). a. Inhibition of cytolysis of bovine PMNs by Lkt. The peptides are designated by the sequence number of their first amino acid. Since the most pronounced aa sequence difference in the CD 18 of ruminants and non-ruminants was observed in the N-terminal region, the point of origin of first 5 peptides was staggered by 5 aa, and the rest of the peptides were staggered by 14 aa. A 20-mer peptide derived from major surface protein 1 of Anaplasma marginale (MSP-1) was used as the negative control. The peptides were used at a concentration of 5 μg/50 μl. % Inhibition was calculated as described in the Methods. b. Determination of the concentration of P1 and P5 that gives 50% inhibition of Lkt-induced cytolysis of bovine PMNs. The peptides P1 and P5 were used in the inhibition assay at concentrations ranging from 3.1 ug/ml to 100 ug/ml. c. Specificity of inhibition of Lkt-induced cytolysis of bovine PMNs by the peptide P5. Two peptides containing the same aa as the peptide P5, but in a randomly scrambled sequence, were used in the cytotoxicity assay along with P5.
Comparison of the concentration of peptides P1 and P5 which causes 50% inhibition of Lkt-induced cytolysis of bovine PMNs revealed the potency of peptide P5 to be higher than that of peptide P1 (12 ug/ml versus 26 ug/ml; FIG. 1b). Similar results were obtained with PMNs of other ruminants (goats, domestic sheep, wild sheep, and deer) as well (data not shown). Two other peptides containing the same as P5, but in a randomly scrambled sequence, failed to inhibit Lkt-induced cytolysis of target cells indicating that the inhibition of Lkt-induced cytolysis of bovine PMNs by the peptide P5 is specific (FIG. 1c).
Flow cytometric analysis confirmed the inhibition of Lkt binding to bovine PMNs by peptide P5 (FIG. 2).
Specifically, FIG. 2 shows, according to particular exemplary aspects, that peptide P5 inhibits Lkt binding to bovine PMNs. Binding of Lkt to bovine PMNs was tested by flow cytometry, in the absence of any peptide (a & b; black histogram), or in the presence of peptide P5 (a; gray histogram), or an irrelevant peptide (b; gray histogram) by flow cytometry. Shaded histogram represents the background fluorescence given by the cells. The x and y axis show the fluorescence intensity and the number of cells, respectively. Results of one representative experiment out of three are shown.
Inhibition of Lkt-induced cytolysis of target cells by shorter versions of peptides derived from P5 by N- and C-terminal truncations identified aa 5-17 of ruminant CD18 as the sequence that serves as the receptor for Lkt. Hence the peptide made up of aa 5-17 is the minimal peptide analog of ruminant CD18 that effectively inhibits Lkt-induced cytolysis of ruminant PMNs and other leukocyte subsets (FIG. 3).
Specifically, FIG. 3 shows, according to particular exemplary aspects, that N- and C-terminal truncations of peptide P5 identifies the minimal peptide sequence of bovine CD18 bound by Lkt as aa 5-17. Inhibition of Lkt-induced cytolysis of BL-3 cells by N-terminally truncated or C-terminally truncated versions of peptide P5 were determined by the MTT dye-reduction cytotoxicity assay. Peptides were used at concentrations ranging from 1.56 to 100 um/ml. All data are expressed as mean±s.d. (n=3). a. Inhibition of Lkt-induced cytolysis of BL-3 cells by peptides with N-terminal truncation. The peptides used were: P5 (aa 5-24); P6 (aa 6-24); P7 (aa 7-24); P8 (aa 8-24); P9 (aa 9-24); P10 (aa 10-24); P11 (aa 11-24); P12 (aa 12-24); P6 (aa 6-24); P6 (aa 6-24). b. Inhibition of Lkt-induced cytolysis of BL-3 cells by peptides with C-terminal truncation. Results of the inhibition assay in a above, identified the N-terminal aa of the minimal peptide sequence as aa #5. Therefore, the C-terminally truncated versions of peptide P5 were synthesized with aa #5 as the N-terminal aa. The peptides used were: P24 (aa 5-24); P23 (aa 5-23); P22 (aa 5-22); P21 (aa 5-21); P20 (aa 5-20); P19 (aa 5-19); P18 (aa 5-18); P17 (aa 5-17); P16 (aa 5-16). Results of the inhibition assay in a and b above, identified the minimal peptide sequence of bovine CD18 bound by leukotoxin as aa 5-17. c. Comparison of inhibition given by the peptide P5 (aa 5-24) and the minimal peptide P17 (aa 5-17).
Example 3
The Amino Acid Sequence of the Bovine CD18 Peptide Analog that Inhibits Lkt-Induced Cytolysis was Shown to be from the Signal Sequence of CD18
The amino acids 5-17 constitute the bulk of the predicted signal sequence (amino acids 1-22) of CD18. As appreciated in the relevant art, paradigm dictates that signal peptides of plasma membrane proteins are cleaved by the signal peptidase in the ER1-3. Therefore Applicants checked for the presence of the signal peptide on mature cell surface CD 18.
As shown in FIG. 4, flow cytometric analysis with an anti-serum specific for the signal peptide of bovine CD18 indicated that the CD18 molecules are on the surface of ruminant PMNs and, indeed, have the signal peptide intact.
Specifically, FIG. 4 shows, according to particular exemplary aspects, that anti-signal peptide serum binds to membrane CD18 of PMNs of all ruminants tested. PMNs from the indicated animals were tested by flow cytometry for the binding of a chicken anti-serum (1/1000 dilution) developed against a synthetic peptide spanning aa 5-19 of bovine CD18 signal peptide. Serum from un-immunized chicken, and MDBK cells, were used as the negative controls for the serum and cells, respectively. The different panels show the results of flow cytometry with PMNs of (a) cattle; (b) goats; (c) domestic sheep; (d) wild sheep; (e) deer; (f) Jurkat cells (human T-lymphoma); (g) RAW264.A cells (mouse macrophage); (h) HEK-293 (human embryonic kidney). Shaded histogram: cells only; silver histogram: cells treated with serum from un-immunized chicken; gray histogram: cells treated with anti-serum against signal peptide of bovine CD 18. Results of one representative experiment out of three are shown.
Example 4
The Signal Peptide of Ruminant CD18 was Shown not to be Cleaved from the Mature Protein
To confirm the fact that the signal peptide of bovine CD18 is not cleaved, Applicants introduced a minigene encoding the ‘FLAG’ epitope21 at the putative signal peptide cleavage site (between aa 22 and 23). Transfectants stably expressing bovine CD18 containing the ‘FLAG’ epitope between aa 22 and 23 were tested with two monoclonal antibodies (MAbs), M1 and M2, specific for the ‘FLAG’ epitope. M1 recognizes the free N-terminal end of ‘FLAG’, while M2 recognizes ‘FLAG’ irrespective of its sequence context21.
As shown in FIG. 5, the MAb M2, but not M1, bound to the transfectants expressing ‘FLAG’-containing CD18 confirming that the signal peptide of bovine CD18 is indeed not cleaved.
Specifically, FIG. 5 shows, according to particular exemplary aspects, that the signal peptide of bovine CD18 is not cleaved. a. Introduction of the ‘FLAG’ epitope at the signal peptide cleavage site and its detection by two monoclonal antibodies. The ‘FLAG’ epitope (DYKDDDDK) introduced at the signal peptide cleavage site (between aa 22 and 23) can be detected by two anti-‘FLAG’ epitope antibodies M1 and M2. M1 recognizes the free N-terminal end of ‘FLAG’ (if cleavage occurs), while M2 recognizes ‘FLAG’ irrespective of its sequence context. b. Flow cytometric analysis of bovine CD18 expression. The untransfected parent cells P815 (shaded histogram), or P815 cells transfected with either bovine CD18 (2B2; black histogram) or bovine CD18 containing the ‘FLAG’ epitope at the cleavage site (BFL; gray histogram), were tested for expression of bovine CD18 by flow cytometric analysis with an anti-bovine CD18 monoclonal antibody. C.-d. Flow cytometric analysis of expression of the ‘FLAG’ epitope. The transfectants 2B2 and BFL were tested for the expression of the ‘FLAG’ epitope with the monoclonal antibody M2 (c.) and M1 (d.). Results of one representative experiment out of three are shown.
Example 5
The Signal Peptide of CD18 of Non-Ruminants was Shown to Contain ‘Cleavage-Conducive’ Glycine, Whereas that of Ruminants was Shown to Contain ‘Cleavage-Inhibiting’ Glutamine at Position −5 Relative to the Cleavage Site
Applicants' finding that a sequence (aa 5-17) within the signal peptide of ruminant CD18 serve as the receptor for M. haemolytica Lkt, and the fact that the cytolytic activity of Lkt is absolutely specific for ruminant leukocytes, prompted examination of the signal peptide of CD18 of ruminants and non-ruminants. The amino acid sequences of CD18 of eight ruminants and five non-ruminants were compared (FIG. 6).
Specifically, FIG. 6 shows, according to particular exemplary aspects, that the signal peptide of CD18 of ruminants contain glutamine at aa position −5 relative to the cleavage site, whereas that of non-ruminants contain glycine. a. The “−3,−1, rule” for cleavage of signal peptides (Von Hejne22). b. Comparison of signal peptide sequences of ruminants and non-ruminants (GenBank Accession #: cattle: M81233; bison: EU553919; buffalo: AY842449; goat: AY452481; domestic sheep: DQ470837; wild sheep: DQ104444; deer: EU623794; elk: EU553918; human: NM0002211; chimpanzee: NM001034122; mouse: X14951; rat: NM001037780; pig: U13941). DS, BHS and chimp denote domestic sheep, wild sheep (bighorn sheep) and chimpanzee, respectively. Arrow indicates the signal peptide cleavage site.
The predicted signal sequence of both the ruminant and non-ruminant CD18 contains 22 aa. The “−3−1 rule” of Von Hejne22 for signal peptide cleavage calls for the presence of amino acids with small uncharged amino acids at position −1 and −3 relative to the cleavage site. Both ruminant and non-ruminant CD18 signal peptides conform to this rule. The amino acid residue at position −5 could also determine whether the signal peptide gets cleaved or not23. Helix-breaking residues glycine and proline are conducive for signal peptide cleavage22. Arginine is also conducive to signal peptide cleavage22,23. Glutamine on the other hand has been shown to inhibit cleavage of signal peptide23. Astonishingly, CD18 of all five non-ruminants examined contained the ‘cleavage-conducive’ glycine (humans, mice, rats, and chimpanzees) or arginine (pigs), while CD18 of all eight ruminants examined contained ‘cleavage-inhibiting’ glutamine.
Example 6
Transfectants Expressing Bovine CD18 Containing the Mutation of Glutamine at −5 Position to Glycine were Shown to be not Susceptible to M. Haemolytica Lkt-Induced Cytolysis
The observation that the signal peptide of CD18 of ruminants (Lkt-susceptible) contains Q at −5 position whereas that of non-ruminants (Lkt-non-susceptible) contains G raised the question as to whether site-directed mutagenesis of Q to G [Q(−5)G] would result in the abrogation of Lkt-induced cytolysis of transfectants expressing Q(−5)G mutation in the signal peptide of CD18. Indeed, as disclosed herein, that is precisely what was found. The Q(−5)G mutation in the signal peptide of bovine CD18 abrogated Lkt-induced cytolysis of the transfectants expressing the mutated CD18 (FIG. 7).
Specifically, FIG. 7 shows, according to particular exemplary aspects, that mutation of glutamine at position −5 of the signal peptide of bovine CD18 to glycine abrogates Lkt-induced cytolysis of transfectants expressing CD18 with Q(−5)G mutation. Transfectants expressing bovine CD18 (2B2), those expressing bovine CD18 containing the Q(−5)G mutation (BQG), and the parent cells (P815) were tested for susceptibility to Lkt-induced cytolysis by the MTT dye-reduction cytotoxicity assay. All data are expressed as mean±s.d. (n=3).
In summary, particular aspects disclosed herein demonstrate for the first time that the aa 5-17 within the signal peptide of ruminant CD18 serve as the receptor for M. haemolytica Lkt, and that the failure of the signal peptide to be cleaved from mature CD18 molecules renders the ruminant leukocytes susceptible to Lkt.
Dileepan et al.24,25 has previously reported that Lkt binding site lies within aa 500-600, more precisely between aa 541-581 of bovine CD18. The present results indicate that this conclusion is erroneous for various reasons.
First, two different sets of synthetic peptides spanning aa 500-600 failed to inhibit Lkt-induced cytolysis of bovine PMNs (FIG. 8). Specifically, FIG. 8 shows, according to particular exemplary aspects, that peptides spanning aa 500-600 of bovine CD18 fail to inhibit Lkt-induced cytolysis of bovine PMNs. a. Nested sets of peptides spanning aa 500-600. One set (#1) of peptides (20-mer) spanning aa 500-600 were synthesized and tested in the inhibition assay. Since none of the peptides inhibited Lkt-induced cytolysis of bovine PMNs, another set (#2) of peptides with points of origin different from that of the first set were synthesized. b & c. Inhibition of Lkt-induced cytolysis of bovine PMNs by the nested set of peptides was tested by the MTT dye-reduction cytotoxicity assay. All data are expressed as mean±s.d. (n=3).
Second, synthetic peptides containing the signal sequence aa 5-17 completely inhibited Lkt-induced cytolysis of PMNs of bovine (FIG. 1) and other ruminants. If Lkt bound to CD18 between aa 541 and 581, one would expect to see cytolysis of target cells when Lkt is incubated with a synthetic peptide representing the signal sequence (aa 5-17). Third, Applicants; transfectants expressing CD18 containing the Q(−5)G mutation in the signal peptide are not lysed by Lkt although the aa 500 to 600 are intact in the CD18. According to particular aspects, the failure of Dileepan et al24,25 to identify aa 5-17 in the signal peptide as the Lkt binding region was likely due to the fact that their transductants were developed with K562 cells. According to further aspects, K562 cells transfected with bovine CD18 do not express CD18 with intact signal peptide (Applicants' unpublished observations). Therefore, according to additional aspects, K562 cells likely carry a signal peptidase that cleaves the signal peptide in spite of the presence of Q at position −5. K562 is a poorly characterized cell-line, and conflicting reports regarding the lineage of this cell-line can be found in the literature26,27. Applicants' studies indicate that the findings of Dileepan et al.24,25 are unique to bovine CD18 transductants developed with K562 cells, and do not reflect the molecular events occurring in ruminant leukocytes.
According to particular aspects, the replacement of ‘cleavage-inhibiting’ Q at −5 position with ‘cleavage-conducive’ G likely results in the cleavage of the signal peptide and the resultant loss of susceptibility of the transfectants to Lkt-induced cytolysis. According to additional aspects, abrogation of cytolysis is not due to cleavage of signal peptide, but rather due to conformational changes caused by the replacement of Q with G. According to further aspects, both effects are involved. Irrespective of the molecular basis or mechanism underlying the abrogation of cytolysis, the exemplary Q(−5)G mutation embodiment provides for a hitherto unavailable technology to, among other things, clone cattle and other ruminants expressing CD18 without the signal peptide on their leukocytes, and hence provide animals less susceptible to pneumonic pasteurellosis, and which will save millions of dollars annually with world-wide benefit.
Example 7
Endobronchial Inoculation of a Peptide Analog of CD18 was an Effective Inhibitor of M. Haemolytica-Caused Pneumonia in a Calf Challenge Model
Overview.
In this Example, a study was conducted that confirmed the ability of the peptide spanning amino acids 5-17 of bovine CD18 to inhibit or mitigate the disease caused by M. haemolytica, in a calf challenge model.
Leukotoxin (Lkt) produced by Mannheimia haemolytica is the major virulence factor of this organism. Lkt-induced cytolysis and degranulation of alveolar macrophages and polymorphonuclear leukocytes is responsible for the acute inflammation and lung injury characteristic of pneumonia caused by M. haemolytica. Applicants identified a peptide analog of CD18 (P17, spanning amino acids 5-17) that effectively inhibited Lkt-induced cytolysis of ruminant leukocytes in in vitro cytotoxicity assays (Shanthalingam and Srikumaran, 2009). The objective of this study was to determine the ability of this peptide to inhibit or mitigate lung lesions in a calf challenge model of M. haemolytica. Three groups of four calves each were endobronchially inoculated with logarithmic phase cultures of M. haemolytica (5×109 CFU per 10 ml of culture medium) alone (Group I), or along with a control peptide (Group II), or with the CD18 peptide analog P17 (Group III). Animals were observed for clinical signs at different time points, euthanized at 90 hours post-inoculation, and necropsied. The total clinical disease scores for Group III calves were lower than those for group I and II at all time points except 48 hours. This difference was statistically significant (P<0.05) at 24 hours post-inoculation. All the calves presented gross pulmonary lesions consistent with fibrinonecrotic pneumonia characteristic of M. haemolytica infection.
The difference in percent volume of lungs exhibiting gross pneumonic lesions among the three groups was not statistically significant (P=0.9). However, M. haemolytica isolated from the lungs of Group III calves was 100- to 1000-fold less than those isolated from the calves in Group I and Group II. This difference, expressed as CFU of M. haemolytica per g of lung tissue, was statistically significant (P<0.001) indicating that the CD18 peptide analog reduced leukotoxic activity in the lungs enabling more effective bacterial clearance by the phagocytes.
In particular aspects, prolonging the presence and activity of the CD18 peptide analog in the lungs using a nanoparticle delivery system such as crystallized dextran microspheres enhances its protective ability.
MATERIALS AND METHODS
Preparation of M. Haemolytica Inoculum for Endobronchial Challenge.
M. Haemolytica serotype-1 strain SH789, isolated from the pneumonic lung of a calf, was streaked on blood agar plate and incubated overnight at 37° C. The following day few colonies were transferred to 3 ml of pre-warmed brain heart infusion (BHI) broth and incubated for 3 hours at 37° C. with constant shaking (200 cycles/minute). Two BHI agar plates were ‘lawned’ with this bacterial culture using sterile cotton swabs and incubated overnight at 37° C. The following day (day of inoculation) M. haemolytica was harvested from the BHI agar plates and transferred to 40 ml of pre-warmed BHI broth in a 250 ml flask, and incubated for 2.5 hours at 37° C. with constant shaking to obtain cultures in the logarithmic phase of growth. The culture was centrifuged at 6000×g at 20° C. for 30 minutes and the pellet was washed once with RPMI 1640 (without phenol red) medium. The bacterial pellet was re-suspended in 4 ml of RPMI 1640 (without phenol red), and 1 ml of this culture was added to 50 ml of pre-warmed RPMI 1640 (without phenol red) containing L-glutamine (1 ml L-glutamine/100 ml RPMI) in a 250 ml flask. Bacteria were incubated for 3 hours at 37° C. with constant shaking to obtain logarithmic phase culture and the optical density (OD) was measured. The culture was appropriately diluted to obtain a concentration of 1×109 CFU of M. haemolytica per ml. Five ml of this preparation per calf was used for endobronchial challenge. The bacterial concentration was confirmed the following day by culturing diluted aliquots of the inoculum on BHI agar and counting the resulting colonies.
Peptides.
The peptide (P17) containing amino acids 5-17 of bovine CD18 (NH2-RPQLLLLAGLLAL-OH) (SEQ ID NO.:71), and the peptide (PSC) containing the same amino acids as peptide P17 but in a randomly scrambled sequence (NH2-LRALLPLQLLAGL-OH) (SEQ ID NO.:72), were synthesized at Neopeptide (Cambridge, Mass.). Both peptides were re-suspended in dimethysulfoxide (ATCC) at a concentration of 20 mg/ml and stored at −20° C. until used. Based on the results of in vitro neutralization of Lkt by peptide P17, each calf was endobronchially inoculated with 2 mg of peptide in 5 ml of RPMI mixed with 5×109 CFU of M. haemolytica in 5 ml of RPMI.
In Vitro Neutralization of Lkt.
Five ml aliquots of M. haemolytica containing 1×109 CFU/ml of RPMI 1640 were mixed with 5 ml aliquots of the peptide (P17 or PSC) at a concentration of 5, 4, or 2 mg per 5 ml of RPMI 1640 (without phenol red), and incubated for 4-5 hours at 37° C. with constant shaking. The bacteria were removed from the culture by centrifugation (13, 500×g for 20 min at 4° C.), and the supernatant fluid was filter-sterilized and stored at −20° C. until tested by the cytotoxicity assay for leukotoxic activity.
Detection of Lkt-Induced Cytolysis of Target Cells.
The MTT [3-(4,5-dimethylthiazoyl-2-YI)-2,5-diphenyl tetrazolium bromide; Sigma] dye reduction cytotoxicity assay for detection of Lkt-induced cytolysis of target cells has been previously described by us (Gentry and Srikumaran, 1991). This assay measures the ability of the ER-resident enzymes in viable cells to convert a tetrazolium dye into a purple formazan precipitate, which is later dissolved in acid isopropanol. The optical density (OD) of the end product, representing the intensity of the purple color developed, is directly proportional to the viability of the cells. Briefly, the target cells were re-suspended in colorless RPMI 1640 (without phenol red) at a concentration of 5×106 cells ml−1, and seeded into 96 well round bottom microtiter plates (50 ul/well) containing the serially diluted Lkt in duplicates and incubated at 37° C. for 1 hour. Cells were centrifuged at 600×g for 5 min following incubation, and the supernatant fluid was discarded. The cell pellets were re-suspended in 100 ul of colorless RPMI 1640 and 20 ul of 0.5% MTT dye were added to each well. After 1 hour of incubation at 37° C., the plates were centrifuged at 600×g for 5 min and the supernatant fluid was removed. The formazan precipitate was thoroughly dissolved in 100 ul acid isopropanol and the OD of the samples was measured using an ELISA reader at 540 nm. The percent cytotoxicity was calculated as follows: % cytotoxicity=[1−(OD of toxin-treated cells/OD of toxin-untreated cells)]×100.
Detection of Inhibition of Lkt-Induced Cytolysis of Target Cells by the Lkt-Neutralizing Abs in Serum.
For Lkt neutralization, 50 ul of toxin preparation at a 50% toxicity end point titer of 40 Units/ml was incubated with 50 ul of serum (2 fold dilutions starting at 1:20) at 4° C. for 1 hour. Bovine lymphoma cells (BL3; 5×106/ml) were added, and the MTT assay was performed as described above. The percent inhibition of cytolysis was calculated as follows: % Inhibition of cytolysis=[1−(% cytolysis in the presence of serum/% cytolysis in the absence of serum)]×100.
Animal Inoculation.
All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Washington State University before the onset of the study. Twelve Holstein calves were randomly assigned to three experimental groups. Calves were matched for age when assigned into three groups. Prior to inoculation, serum samples and pharyngeal and nasal swabs were collected from all the calves. Group I calves received endobronchial injections of 5×109 CFU of M. haemolytica in 10 ml of RPMI. Group II calves received 5×109 CFU of M. haemolytica and 2 mg of peptide PSC in 10 ml of RPMI. Group III calves received 5×109 CFU of M. haemolytica and 2 mg of peptide P17 in 10 ml of RPMI. The inoculum was flushed down with an additional 10 ml of RPMI in all calves. Clinical disease in each calf was scored at different time points post-inoculation. Calves were humanely euthanized 90 hours post-inoculation, and the percent volume of lungs exhibiting gross pulmonary pathology was calculated using morphometric methods.
Scoring of Clinical Disease.
Physical examination of each calf was performed immediately prior to experimental infection and at 6, 18, 24, 42, 48, 66, 72, and 90 hours post-inoculation. Signs of clinical disease were allocated points according to the scoring system followed by Malazdrewich et al (2003; Table 3).







TABLE 3







Evaluation and scoring of clinical signs










Clinical signs
Clinical score







Body temperature >103.5° F.
2



Inappetance
1



Lethargy/depression
1



Marked weakness/recumbency
2



Moribund state
3



Cough
1



Nasal discharge
1



Respiratory rate >60 breaths/min
1



Dyspnea
2



Abnormal breath sounds on auscultation
1










Serotyping.

M. haemolytica isolated from pharyngeal and nasal swabs prior to inoculation and from lung tissue at necropsy, were typed using anti-serotype A1 serum (kindly provided by Dr. Robert Briggs, National Animal Disease Center, Ames, Iowa). One milliliter of fresh culture was centrifuged at 6800×g for 3 minutes and the pellet was re-suspended in 100 μl of Hanks' balanced salt solution (HBSS) medium containing 0.25% of bovine serum albumin (BSA). Twenty-five μl of culture was then placed on the agglutination plate. Anti-A1 specific serum was diluted in HBSS medium containing 0.25% of BSA and 25 μl of the diluted serum at 1/16 dilution was added to the bacteria and mixed by gentle rocking of the plate. The reference A1 strain and culture media were used as the positive and negative controls, respectively.
Quantitation of Gross Pulmonary Pathology.
On necropsy lungs were removed and gross pneumonic lesion development in each lobe of lung was observed. The entire lung from each animal was sliced at 1 cm thickness and the total and pneumonic lesion areas were traced onto transparent acetate sheets. The traced portions were scanned into ImgaeJ, NIH Image (National Institute of Health; Nethesda, Md.), which was used to measure areas representing both the total serial section and the gross pneumonic lesions within it. Measured areas for each serial section were used to calculate the volume of each lung and the grossly pneumonic regions within it using Simpson's rule: V=(1⁄3) h [(A0+An)+4(A1+A3+ . . . +An-1)+2(A2+A4+ . . . +An-2)] where V is the total or pneumonic lung volume, h is the thickness of each slice in centimeters, and A, A0, A1, A2, An represent measured lung or pneumonic lesion areas for lung slices 0, 1, 2, n. These values were then used to calculate the percent volume of the lung exhibiting gross pulmonary pathology in each calf.
Lung tissues from representative gross lesions in each calf were collected for histopathological evaluation. Tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin using standard techniques. After routine processing, 5 μm tissue sections were stained with hematoxylin and eosin and used for subjective, non-quantitative histopathological examination.
Re-Isolation of M. haemolytica from Pneumonic Lungs.
Using aseptic techniques, fresh lung samples and pharyngeal and nasal swabs were obtained for isolation and characterization of bacteria including M. haemolytica and other Pasteurella species. Serotyping of M. haemolytica isolates were performed using anti-serotype A1 specific sera.
Bacteriological Examination.
Small samples of tissue (1 g) were obtained from affected regions from the same lobe. The samples were homogenized into 3 mls of RPMI 1640 and diluted 10-fold (1×10−1 to 1×10−6). Ten μl aliquots of each dilution were applied to BHI agar plates on 5 spots and incubated at 37° C. overnight and viable counts were determined. Representative colonies were checked for M. haemolytica by colony PCR. Primers used were as follows: forward 5′-AGAGGCCAATCTGCAAACCTC-3′ (SEQ ID NO.:73) and reverse 5′-GTTCGTATTGCCCAACGCCG-3′ (SEQ ID NO.:74). Counts were expressed as CFU of M. haemolytica/g of lung tissue.
Statistical Analysis.
Clinical scores and the percent volume of the lung exhibiting gross pneumonic lesions were expressed as the mean±SEM. Clinical scores between the groups were compared using one-way analysis of variance tests (ANOVA). Pneumonic lung scores and the quantity of M. haemolytica/g of lung tissue of all three groups were also compared using one-way ANOVA. Differences were considered significant at a value of P<0.05.
Results
Co-Incubation of Peptide P17 with In Vitro Cultures of M. haemolytica Abrogates Leukotoxic Activity.
Before proceeding with the in vivo experiments, the inhibitory effect of the peptide P17 on the leukotoxic activity of in vitro cultures of M. haemolytica was determined. M. haemolytica cultures were incubated with 2, 4, or 5 mg of peptide P17 or the control peptide PSC, and the leukotoxic activity in the culture supernatant fluids was determined by the dye-reduction cytotoxicity assay. The supernatant fluids from M. haemolytica cultures incubated with the peptide P17 did not exhibit significant leukotoxic activity whereas those from cultures incubated with the control peptides had significant leukotoxic activity (FIG. 9). The difference in % cytotoxicity exhibited by the supernatant fluids from the cultures incubated with the peptide P17 and PSC was statistically significant (P<0.001). Inhibition of cytotoxicity did not decrease when the quantity of peptide P17 was reduced from 5 to 4 and 2 mg, indicating that the peptide P17 can inhibit the Lkt-induced cytolysis of target cells even at 2 mg (per 5×109 CFU of M. haemolytica).
Pre-Inoculation Status of Calves (Nasopharyngeal Flora and Anti-Lkt Antibodies).
The results of bacterial isolation are summarized in Table 4. Ten out of 12 calves carried M. haemolytica in their pharynx whereas only 4 calves carried it in the nasal cavity. None of the isolates belonged to serotype 1, the serotype of M. haemolytica used for inoculation. The unavailability of antisera specific for all known serotypes of M. haemolytica prevented us from identifying the precise serotype of these bacteria. Pasteurella multocida and Bibersternia trehalosi were isolated from two and three calves, respectively. All the calves used in the experiment had low titers of Lkt-neutralizing antibodies as revealed by the cytotoxicity inhibition assay. The titers ranged from 1/20 to 1/320.







TABLE 4







Bacteria isolated from calves pre-inoculation and at necropsy.













M. haemolytica
P. multocida
B. trehalosi














Animal #
Region
Before
After
Before
After
Before
After





16
P
(+)
(+)
(−)
(−)
(−)
(−)



N
(−)
(+)
(−)
(−)
(−)
(−)


21
P
(+)
(+)
(−)
(−)
(−)
(−)



N
(−)
(+)
(−)
(−)
(−)
(−)


25
P
(+)
(+)
(−)
(−)
(−)
(−)



N
(−)
(−)
(−)
(+)
(−)
(−)


26
P
(−)
(−)
(−)
(−)
(−)
(−)



N
(−)
(−)
(−)
(+)
(−)
(−)


29
P
(+)
(+)
(−)
(−)
(−)
(−)



N
(+)
(−)
(−)
(+)
(−)
(−)


36
P
(+)
(+)
(−)
Past
(−)
(−)



N
(−)
(−)
(+)
(+)
(−)
(−)


52
P
(+)
(−)
(+)
(+)
(−)
(+)



N
(−)
(−)
(−)
(+)
(−)
(−)


68
P
(+)
(−)
(−)
(+)
(−)
(−)



N
(−)
(−)
(−)
(+)
(−)
(−)


86
P
(+)
(−)
(−)
(−)
(−)
(−)



N
(−)
(−)
(−)
(+)
(−)
(−)


146 
P
(+)
(−)
(−)
(−)
(+)
(−)



N
(+)
(+)
(−)
(−)
(−)
(−)


168 
P
(+)
(+)
(−)
(−)
(+)
(+)



N
(+)
(+)
(−)
(−)
(−)
(−)


170 
P
(−)
(−)
(−)
(−)
(+)
(+)



N
(+)
(+)
(−)
(+)
(−)
(−)





Before: before the challenge;


After: at necropsy;


P: Pharynx;


N: Nasal cavity;


(+): Present;


(−): Absent;


Past: Pasteurella species






Clinical Disease Scores.

Physical examination of each calf was conducted immediately prior to experimental infection and 6, 18, 24, 42, 48, 66, 72, and 90 hours post-infection. All calves were clinically normal (clinical score=0) pre-inoculation. Within 6 hours of infection, all calves developed clinical symptoms of disease. Rectal temperature increased to 105-106° F. within 6 hours post-inoculation and returned to baseline 24 hours post-inoculation as previously reported by other workers (Corrigan et al., 2007). All calves in Groups I and II had nasal discharge throughout the study period, but the Group III calves had nasal discharge only up to 24 hours post-inoculation. The clinical scores for nasal discharge of Group III calves were statistically significantly different from those of Group I and II (P<0.05) of calves inoculated with peptide P17. The total observational disease scores for Group III calves (peptide P17) were lower than those for group I and II at all time points except 48 hours. This difference was statistically significant (P<0.05) at 24 hours post-inoculation.
Gross Lesions.
All the calves presented gross pulmonary lesions consistent with fibrinonecrotic pneumonia characteristic of M haemolytica-caused pneumonia. The pulmonary lesions in all calves were qualitatively similar but differed in severity and extent. Affected lung tissue exhibited consolidation, congestion, and prominent interlobular septae due to fibrin deposition. These lesions were mainly present in the right lung and to a limited extent in the left lung (FIG. 11A). The percent volume of the lungs exhibiting gross pneumonic lesions, as determined by morphometic techniques (Malazdrewich, et al., 2004) are shown in Table 5. The difference in the percent volume of lungs exhibiting gross pneumonic lesions among the three groups was not statistically significant (P=0.9).
Histopathological examination of pulmonary tissues from the calves revealed that the lesions observed in the calves were characteristic of pneumonia caused by M. haemolytica. Interlobular septa were markedly widened by fibrin and fribrous tissue. Within lobules, discreet foci of parenchymal necrosis were outlined by dense bands of degenerate neutrophils, often with streaming nuclei Coat cells'). Within necrotic foci, alveolar walls were lysed and alveolar spaces were filled with fibrin, red blood cells and nuclear debris. In some calves large colonies of coccobacilli were present within affected areas. Parenchyma adjacent to necrotic foci was either collapsed or filled with fibrin and macrophages (FIG. 11B).







TABLE 5







Gross pneumonic lesions expressed as a % of total lung volume












Group
Animal #
Age (months)
Lesion (%)

Mean (%)















I
25
4
16.62





16
4
14.03



52
6
3.07
 {close oversize brace} 
10.03 ± 2.85



146
3
8.38


II
29
4
24.57



21
4
4.07



68
6
10.70
 {close oversize brace} 
12.09 ± 4.39



168
3
9.01


III
36
4
15.67



26
4
2.7



86
6
13.22
 {close oversize brace} 
10.53 ± 3.02



170
3
8.51









Re-Isolation of M. haemolytica Serotype 1 from Pneumonic Lungs of Calves.

Pure cultures of bacteria (M. haemolytica) were recovered from the pneumonic lungs of all calves. All isolates were identified as M. haemolytica by PCR, and confirmed as serotype 1, by serotyping analysis. All heart blood cultures were negative for M. haemolytica, indicating that the infection was confined to the respiratory tract. M. haemolytica were isolated from pharynx and nasal cavity of most of the calves (Table 2). All the isolates from pharynx belonged to serotype 1 but the isolates from nasal cavity were not. Isolation of M. haemolytica from the lungs revealed that the calves in Group III carried approximately 100- to 1000-fold less organisms in the lungs than the calves in Group I and Group II (Table 6). This difference, expressed as CFU of M. haemolytica per g of lung tissue, was statistically significant (P<0.001).







TABLE 6







Number of M. haemolytica (CFU per gram of lung tissue)


isolated from the lungs of calves at necropsy











Group I
Group II
Group III















2.38 × 106
1.89 × 107
5.29 × 103



1.50 × 105
6.73 × 105
4.92 × 102



2.68 × 105
6.50 × 107
5.70 × 104



 2.9 × 107
1.87 × 107
8.00 × 102


Mean
7.9 ± 7.0 × 106
2.58 ± 1.374 × 107
1.589 ± 1.374 × 104









Example Discussion
M. haemolytica Lkt-induced cytolysis and degranulation of macrophages and PMNs is responsible for the acute inflammation and lung injury that is characteristic of pneumonia caused by this organism. According to certain embodiments, abrogation of Lkt-induced cytolysis prevents or mitigates the lung lesion. Applicants have shown that a peptide representing the amino acid sequence of Lkt-binding site on its receptor, CD18, effectively inhibits the Lkt-induced cytolysis of target cells (Shanthalingam and Srikumaran, 2009). Applicants have confirmed that this peptide analog of CD18 abrogates the leukotoxic activity of in vitro cultures of M. haemolytica (FIG. 9) which prompted us to test the efficacy of this peptide in a calf challenge model.
M. haemolytica serotype 1 was the obvious choice for this study since it is the serotype that predominantly causes pneumonia in cattle although other serotypes such as 2, 4 and 7 commonly inhabits the nasopharynx of healthy cattle (Frank and Smith 1983, Frank, 1988; Gonzalez and Maheswaran, 1993). All the pharyngeal isolates obtained from the calves pre-inoculation belonged to serotypes other than serotype 1 allowed us to track the inoculated M. haemolytica by serotyping. The presence of Lkt-neutralizing antibodies in the serum indicates that the calves had these antibodies in the epithelial lining fluid which, according to certain embodiments, had an effect on the bacterial clearance. The calves were matched for Lkt-neutralizing antibody titers. According to additional aspects, colostrum-deprived calves are used to eliminate any possible effects due to Lkt-neutralizing antibodies.
All the calves developed high rectal temperature within 6 hours of inoculation which dropped to normal levels in 24 hours. LPS represents 10 to 25% of the dry weight of M. haemolytica bacteria (Keiss et al., 1964) and it forms high-molecular-weight aggregates with Lkt (Li and Clinckenbeard, 1999). Since LPS stimulates alveolar macrophages to produce TNFα and interleukin-8, leading to inflammation, it is likely that some of the effects that we observed were LPS related. The total observational disease scores for Group III calves were lower than those for group I and II at all time points beyond 6 hours. This difference was statistically significant (P<0.05) at 24 hours post-inoculation. According to certain embodiments, the peptide is absorbed from the surface of the respiratory epithelium by 24 hours post-inoculation, which results in the loss of protective effect of the peptide. According to additional aspects, use of peptides absorbed to solid particles which slowly release the peptide provides for prolonging the protective effect of the peptides which mitigate the disease.
The peptide P17 strongly inhibited Lkt-induced cytolysis of bovine PMNs in in vitro assays. It is surprising that the gross pneumonic lesions in calves inoculated with this peptide and M. haemolytica (Group III) were no less than those in calves inoculated with the control peptide PSC and M. haemolytica (Group II), or M. haemolytica alone (Group I). According to particular aspects, the ability of the peptide to mitigate lung lesions could be enhanced by: (1) increasing the quantity of the peptide; (2) decreasing absorption of the peptide from the lung epithelial surface; or (3) protecting the peptide from proteolytic degradation. For example, by use of peptides adsorbed on solid particles which slowly release the peptide is likely to prolong the protective effect of the peptides which could be expected to mitigate the lung lesions (Freiberg and Zhu, 2004; Schroder and Stahl, 1984; Schroder, 1985). Although the lung lesions were similar in extent in all three groups, it is possible that the animals in Group III would have recovered from the disease if they were not euthanized at 90 hours post-inoculation, as we did in this study. This scenario is supported by the finding that the number of M. haemolytica recovered from the lungs of Group III animals was 100 to 1000 times less than that recovered from the lungs of animals in Group I and II (Table 6). The significantly lower number of bacteria isolated from the lungs of Group III animals is likely due to the presence of relatively larger number of functional phagocytes in the lungs which were protected from Lkt by the peptides. In contrast, the animals in Groups I and II would have had relatively smaller number of phagocytes in the lungs because of their cytolysis by Lkt.
Molecules such as proteins and peptides are often marginally stable and consequently could be easily damaged or degraded (Tibbetts et al., 2000). In vivo degradation when exposed to enzymes results in short biological half-lives (Tibetts, 2000). According to particular aspects, prolonging the presence of peptides in the lungs extends protection of the phagocytes from the Lkt, resulting in more effective clearance of bacteria from the lungs which in turn prevents or mitigates lung lesions.
In particular aspects, nanoparticle delivery systems are used to improve protein/peptide stability and provide sustained release. Adsorbing the peptides to solid particles such as dextran represents a method of prolonging the presence and activity of the peptides in the lungs. Dextran, under certain controlled conditions, aggregates into porous microspheres, forming crystallized dextran microspheres that can absorb/adsorb peptides, drugs and biologicals, protecting them against degradation and prolonging their release. Such dextran microspheres are available which are biodegradable, biocompatible, non-toxic, non-immunogenic and are removed from the body by normal physiological routes. These characteristics are uniquely advantageous for peptide delivery. According to particular aspects, crystallized dextran microspheres are used for the delivery of peptides and prolong the protective effects of the peptide.
References cited for Examples 1-6, and incorporated herein by reference for their relevant teachings as referred to herein:

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References cited for Example 7, and incorporated herein by reference for their relevant teachings as referred to herein:

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## Claims

1. A ruminant cell that is genetically engineered to contain a nucleic acid sequence encoding a ruminant CD18 protein having a cleavable signal peptide with a helix-breaking amino acid residue at an amino acid positioned 5 residues upstream of the signal peptide cleavage site.

2. The ruminant cell of claim 1, wherein the cell is less susceptible to, or resistant to the effects of M. haemolytica, relative to wild-type control cells.

3. The ruminant cell of claim 1, wherein the cell is that of a ruminant selected from the group consisting of cattle, bison, buffalo, goat, domestic sheep, big horn sheep, deer, elk, giraffes, yaks, camels, alpacas, llamas, wildebeest, antelope, pronghorn and nilgai.

4. The ruminant cell of claim 1, wherein, said cell does not express endogenous wild-type CD18 polypeptide having a non-cleavable signal peptide.

5. The ruminant cell of claim 1, wherein said helix breaking amino acid is selected from the group consisting of G, P, and R.