U.S. patent application number 12/583780 was filed with the patent office on 2011-04-07 for anti-viral fusion peptides.
Invention is credited to Kenneth Francis Prendergast.
Application Number | 20110082075 12/583780 |
Document ID | / |
Family ID | 27266087 |
Filed Date | 2011-04-07 |
United States Patent
Application |
20110082075 |
Kind Code |
A1 |
Prendergast; Kenneth
Francis |
April 7, 2011 |
Anti-viral fusion peptides
Abstract
Hybrid peptides capable of binding a blood borne virus to a red
cell, comprising a viral binding peptide component and a malaria
merozoite red cell peptide binding component.
Inventors: |
Prendergast; Kenneth Francis;
(London, GB) |
Family ID: |
27266087 |
Appl. No.: |
12/583780 |
Filed: |
August 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08307742 |
Sep 8, 1994 |
7585508 |
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PCT/GB93/00505 |
Mar 10, 1993 |
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12583780 |
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Current U.S.
Class: |
514/3.8 |
Current CPC
Class: |
C07K 2319/32 20130101;
Y02A 50/58 20180101; Y02A 50/412 20180101; A61P 31/18 20180101;
C07K 14/70514 20130101; Y02A 50/30 20180101; C07K 2319/33
20130101 |
Class at
Publication: |
514/3.8 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61P 31/18 20060101 A61P031/18 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 11, 1992 |
GB |
9205276.0 |
Jul 8, 1992 |
GB |
9214481.5 |
Jul 24, 1992 |
GB |
9215829.4 |
Sep 16, 1992 |
GB |
9219562.7 |
Mar 3, 1993 |
GB |
9304311.5 |
Claims
1. A method of treating a human immunodeficiency virus HIV1 or HIV2
infection wherein a fusion protein is used to treat said infection,
said fusion protein comprising two or more components wherein the
first component comprises the CD4 protein, or a substitution,
deletion, or insertion analog or fragment of the CD4 protein, and
the second component comprises the malaria parasite merozoite
glycophorin binding protein 130 (GBP-130), or a substitution,
deletion, or insertion analog or fragment of the GBP-130 protein,
wherein the CD4 component retains the ability to bind to the HIV
envelope glycoprotein and the GBP-130 component retains the ability
to bind to erythrocytes.
2. A method of preventing the transmission of a human
immunodeficiency virus HIV1 or HIV2 wherein a fusion protein is
used to prevent said transmission, said fusion protein comprising
two or more components wherein the first component comprises the
CD4 protein, or a substitution, deletion, or insertion analog or
fragment of the CD4 protein, and the second component comprises the
malaria parasite merozoite glycophorin binding protein 130
(GBP-130), or a substitution, deletion, or insertion analog or
fragment of the GBP-130 protein, wherein the CD4 component retains
the ability to bind to the HIV envelope glycoprotein and the
GBP-130 component retains the ability to bind to erythrocytes.
Description
FIELD OF INVENTION
[0001] The field of this invention relates to protein molecules in
particular hybrid protein molecules for the prevention of treatment
of human beings afflicted with AIDS and diseases caused by Human
Immuno Deficiency Viruses especially types 1 and 2; and other blood
borne pathogens.
BACKGROUND ART
[0002] The AIDS syndrome is widely believed by the majority of
Scientists (with a few exceptions) to be the end result of
infection with Human Immuno Deficiency Virus type 1 or 2. Despite
available treatment, on diagnosis of AIDS, life expectancy is short
and death occurs usually within two years--death resulting from
overwhelming infection and/or neoplastic diseases provoked by a
profound immuno deficiency. The causative agent of this condition
the HIV virus was proposed by Montagnier L and by Barre-Sinoussi F
et al 1983, Science 220, 868-870 and slightly later by Gallo R et
al 1983, Science 220 865-867.
[0003] The accepted view of most Scientists is that this virus is
necessary at least in part for production of this fatal Immuno
Deficiency Syndrome. One notable exception to this belief is
Deusberg P H who champions the minority view that AIDS is not
caused by the Human Immuno Deficiency Virus and that it is not
transmissible; Deusberg P H Proceedings National Acad Sci USA Vol
88, 1575-1579, February 1991. Regardless of the merits of this
contradictory opinion and academic debate, work continues to find
methods of combating the HIV virus and removing its presence from
infected humans. Since the discovery of the HIV agent in the early
1980's screening of the worlds population has demonstrated that the
HIV/AIDS pandemic is much worse than previously imagined. Evidence
suggests that the impact of the disease will fall on disadvantaged
women and children living in deprived urban areas or in sub-Saharan
Africa or the Third World. It is estimated that 10 million persons,
will contract this condition before the year 2000 Chin J, Lancet
1990; 336: 221-224. Transmission of this virus to newborns from
infected mothers remains a growing problem. At the present time in
Africa, there are at least 2.5 million infected females who have
given birth to an estimated 0.5 million infected infants.
Disadvantaged urban areas in the developed world such as areas of
New York associated with high rates of drug use have also shown
high sero-positivity rates of HIV among neonates ie 2.2% compared
with an overall sero-positivity of 1.25% among neonates in that
city. NOVAK, Journal of America Medical Association 1989; 261:
1745-1750. The social and economic costs of this epidemic are
likely to overburden the resources of health care providers and
remains a growing area of concern. Our understanding of the timing
of maternal to fetal transmission of HIV has been greatly improved
by the study of Ehrnst A et at Lancet, 1981; 338: 203-207. He
demonstrated effectively that the virus is transmitted vertically
at the time of or close to delivery. Despite high viral titre
levels during pregnancy it seems that the mixing of maternal and
fetal blood at time of delivery is the most important mode of
transmission from mother to fetus. In other contexts viral titre
levels are directly related to disease severity; studies by Ho D D
"Quantitation of Human Immuno Deficiency Virus type 1 in the blood
of infected persons." New England. Journal of Medicine 1989;
1621-1625 and the study by Combes R W et at "Plasma Vireama in
Human Immuno Deficiency Infections" New England Journal of Medicine
1989; 321: 1626-1631. Both these studies have demonstrated rising
levels of plasma vireama throughout the course of HIV and have
suggested that high viral titres are associated both with the
degree of immuno suppression, the presence or absence of symptoms
and the likelihood of tranmission to others. It has been shown that
in the early stages of infection, viral titre levels measure 50
virus particles per ml rising to 10-50,000 virus particles per ml
or more at the onset of AIDS.
[0004] In addition to the offspring of infected mothers, other
large groups of the population are at risk or believe themselves to
be at risk from the infection, especially homosexuals, drug
addicts, persons receiving blood transfusions and health care
workers. In the case of health care workers, the risk from a health
care procedure carried out on infected persons is likely to be very
low and is presently estimated to range of 0.3%-0.5% following
needlesticks with an infected needle or surgical instrument.
Operating room personnel frequently exposed to needlestick and
other injuries are especially concerned at the present time. 8This
concern is most marked among Orthopaedic Surgeons who in addition
to needlestick injuries are liable to suffer abrasions or even
transfixion of a digit with steel wires, pins and the like. So
great is the concern that chain-male gloves have been devised to
protect staff from inadvertent needlestick injuries. Disadvantages
are apparent with barrier methods in relation to HIV prevention. In
particular, increased costs--it is known that the dry goods budget
of many hospitals has increased considerably following the onset of
this epidemic. A second disadvantage is the loss of tactile
sensation, making operative procedures more difficult and time
consuming. A further problem is the difficulty in attracting and
keeping staff in high risk areas who report psychological stress on
their part or the part of their spouses, resulting from risk either
real or imagined, particularly following needlesticks. A further
area of concern is the safety of blood transfusions and the blood
supply both in the US and elsewhere. To prevent repeating the
iatrogenic infection of large numbers such as haemophiliacs with
infected pooled factor 8, there has been a growing awareness of the
need to take additional precautions with whole blood and blood
product transfusion. The mainstay of transfusion safety remains
screening, however no amount of screening can guarantee the safety
of a blood transfusion or a blood derived product. Accordingly many
workers have devised different methods to treat blood for donation.
One is directed to the methods of Sieber U.S. Pat. No. 4,915,683
who dislcoses for example photosensatising agents useful to
neutralise viruses and other pathogens in blood for tranfusion.
This approach and other similar methods is non specific however and
requires an irradiating component in addition to a chemical means
of treatment.
[0005] The present disclosure provides a means of improving the
safety of blood products in one step.
[0006] The mechanism of HIV infection involves the binding of viral
GP120 or envelope peptide to the CD4 peptide molecule. The CD4
protein is a 433 amino acid transmembrane glycoprotein which is
found predominantly of the surface of T4 helper cells. It is also
found on the surface of the mega karyocytes, monocytes, macrophages
and other cells.
[0007] The CD4 molecule has three parts; an extracellular part
which is divided into 4 variable domains V1 being responsible for
the binding of HIV GP120; a short transmembrane region and an
intracellular domain.
[0008] The CD4 molecule is part of the Immunoglobulin super family.
That CD4 was the receptor for the virus was shown by Dalglaoise et
at 1984. "The CD4 antigen is an essential component of the receptor
for the AIDS retrovirus" Nature; 212: 763-766. Also Madden P J
Dalglaoise A G McDougall J S et at "The T4 gene encloses the AIDS
virus receptor and is expressed in the immune system and the brain"
Cell: 47, 333-348.
[0009] Maddon P et at Cell: Vol 47, 333-348 have shown that
transformed mouse cells expressing the CD4 molecule possess the
capabilities of binding the HIV virus though lack the ability to
support the HIV infection at a cytoplasmic level. Debate continues
within the art as to the precise binding site of GP120 with the CD4
molecule. Arthus et al Cell: Vol 57, 469-491 May 5, 1989 proposes
that the binding site is contained at least within the first 106
amino acids of the CD4 molecule ie the V1 domain. He bases his
assumption on substitute mutants and binding analysis with soluble
truncated proteins demonstrating a high binding affinity solely
within the first 106 amino acids of the CD4 molecule. Substitution
studies led to the notion that the primary binding site on the CD4
molecule lay between amino acids 41-55.
[0010] Mizukani et at Proc Nat Acad Sci USA 85; 9273 (1988) and
Arthus 1989 "Identification of the residues in human CD4 critical
for the binding of HIV" Cell 57; 469 believes that the folded
structures of the CD4 molecule brings other amino acids into play.
Areas likely to be important in viral CD4 binding are reported to
lie between amino acids 41-47 amino acids 16-49, 31-63 and 74-94 of
the CD4 molecule.
[0011] The remarkable specificity of the virus for the CD4 molecule
has lead to the attempts by many workers to utilise CD4 based
peptides as therapeutic agents. The binding affinity for the GP120
with CD4 lies in the region of 3 nM. Many efforts have been made to
utilise this high specificity.
[0012] In the case of HIV 2, the affinity is 20-25 fold less,
however the binding strength as described is such that even a 25
fold reduction of affinity would not reduce the usefulness of
therapeutic molecules based on CD4 and for that reaon the binding
reaction between GP120 and CD4 remains a logical target for anti
HIV pharmaceuticals.
[0013] However the nature of the molecule means that a linear
peptide will be unlikely to be successful in a therapeutic
context.
[0014] Brodsky et at Immunology Vol 144, 3078-3086 Apr. 15, 1990 No
8 suggests that CD4 based therapeutic molecules should contain
amino acids at least from the first 117 amino acids of the CD4
sequence.
[0015] Moore J P AIDS 1990, 4: 297-3-5 believes that the CD4 GP120
binding reaction is the one step this virus cannot afford to change
by mutation to any appreciable extent. Accordingly, attempts have
been made to use recombinant CD4 peptides as therapeutic
agents.
[0016] Fisher R et al Nature Vol 331, 7 Jan. 1989, "HIV Infection
is blocked in vitro by recombinant soluble CD4" reported the use of
recombinant soluble. CD4 peptides that showed viral blocking
acitivity in vitro. Hussey R et al "A Soluble CD4 protein
selectivity inhibits HIV replications and Syncitium formation"
Nature 331, 7 Jan. 1989, reported encouragingly that Syncitium
formation could be blocked in vito by the same CD4 based soluble
peptides: and Dean et al "Soluble CD4 protein inhibits HIV virus
infection" Nature 331, 7 Jan. 1989; A Traunecker et al "Soluble CD4
molecules neutralise Human Immuno Deficiency Virus type 1", also
Nature Vol 331, 7 Jan. 1989 encouragingly reports that CD4 peptides
may be used as therapeutic agents. The euphoria provided initially
by these experiments was short lived.
[0017] Problems emerged, in particular the rapid clearance of
recombinant soluble CD4 or sCD4 from the blood stream and secondly
the existence of multiple binding sites on the virus. It is known
that the virus contains no less than a 72 GP120 molecules
projecting from its surface. Therefore to fully neutralise a virus
particle it would be necessary to combine recombinant soluble CD4
peptides or peptide based molecules with all 72 of the GP120
molecules the kinetics of this event are unfavourable. The present
disclosure provides means to overcome both of these problems.
[0018] Further discouragement was provided by Eric Daar et al, Proc
Nat Acad Sci USA Vol 87, 6574-6578. "High concentrations of
recombinant SCD4 are required to neutralise primary immuno
deficiency type 1 isolates." This report based on the treatment of
45 patients shows that soluble CD4 peptides failed to consistently
reduce HIV titres and further suggests a reason for the failure
being lower GP120-sCD4 affinities than being reported in vitro.
Daar suggests that mutant strains possessing lower CD4 affinity
than the strains used in laboratory testing may be responsible for
the failure of the agents.
[0019] The present disclosure proposes that clinical disappointment
with the T4 agents, such as disclosed in PCT WO89/019140 relates
more to the short half life of the peptides in plasma and to the
fact that 72 binding sites exist on a single virus, rather than due
to lack of affinity between the agent and the virus itself. So
great is the affinity between CD4 molecule and the GP120 molecule
that even affinities 1000 fold less than those seen in vitro should
be pharmaceutically effective.
[0020] Despite the failure of unmodified soluble CD4 as a HIV
therapy, other workers have advanced alternatives in the hope of
utilising the GP120-CD4 interaction. Examples of some of these
alternatives are fusion peptides where a CD4 component is fused to
another peptide having a different function.
[0021] Capon D J et at disclosed the use of CD4 fused to the FC
component of the human Igh antibodies. The objective being to
mobilise the compliment system and thereby destroy the virus
particle by the compliment cascade. Capon refers to these new
molecules as immuno adhesives. A possible disadvantage associated
with this approach is the necessity of a fully functional
compliment system, unlikely to the case in advanced HIV infections.
See Capon et al "Designing CD4 Immunoadhesins for AIDS Therapy"
Nature Vol 337.
[0022] The molecular machines of the present disclosure do not
require a functional immune system and may be effective in cases
where no immune function exists.
[0023] At the present time the main stay of treatment relies of the
substance Azidothymine or AZT, this susbtance acts on the HIV
enzyme reverse transcriptase.
[0024] An increasing number of patients and earlier in their
illness are receiving treatment with AZT, however tolerance of this
drug can be a problem. AZT is known to be lymphotoxic and is
additionally associated with substantial anaemia. Haemato toxicity
appears to be major limiting factor in the use of AZT in the
treatment of ARC and AIDS Richman et al "The toxicity of the
Azidothymine", New England of Medicine, 317; p 192-197, 1987. So
profound is the anaemia provoked by this agent that many patients
require frequent blood transfusions or treatment with Epogen (R), a
novel genetically engineered erythropotein. The molecular machines
of the present disclosure may be used in combination with AZT or
other treatments and will augment the effectiveness of blood
transfusions given to patients receiving AZT who are suffering from
severe anaemia. Other agents acting on the reverse transscriptase
such as DDC or DDI suffer high side effect profiles also and are
toxic compounds in their own right. Equally, agents acting at other
sites such as glycosylation are known to exhibit severe toxic
effects on the host.
[0025] The agents of the present disclosures are anticipitated to
have lower toxic effects on the host than existing treatment
modalities.
[0026] The agents may be used alone or in combination with existing
treatments.
[0027] So great is the side effect problem of anti-AIDS
therapeutics many feel it is likely that no one agent will be
successful in combating this disease. One is directed to Fisher
World Patent WO 89/11860 for a discussion of combination
therapy.
[0028] More recently Taylor R P in Proceedings National Acad
Science 88, p 3305-3309 refocused our attention on the work of
Nelson published in 1950's who some years ago proposed an imune
function for the red cells. Red cells (erythrocytes) are often
considered to have only oxygen carrying and acid base balancing
functions in the blood stream. According to Nelson red cells have
additional immune functions. In particular Nelson proposed that
opsonised antigens ie antigens whose surfaces were bound to
compliment derived peptides could be fused to the compliment 0.1
receptor molecule (CR1) on the red cell surface. By fusing a virus
or bacterium to the red cell surface in this way the virus or
antigen becomes bound to the red cell surface in the manner of a
barnacle bound to the surface of a ship.
[0029] A virus immobilised in this way would be harmless and be
carried around the circulation on the red cell surface to await
final phagocytosis and digestion at the end of the red cells life
(usually 120 days from formation). It is thought that many antigens
and pathogens are cleared from circulation by this mechanism rather
than by direct phagocytosis and the like so frequently discussed in
immunology texts. Accordingly, Taylor R P proposed a technique to
utilise this long forgotten mechanism. He suggested the use of
hybrid antibodies capable of binding both to the HIV virus and to
the compliment 1 receptor molecule of the red cell surface. The
adherence of virus particles to the red cell surface membrane in
this way would clear the blood stream of free virus particles and
free GP120. Thereby attempting to achieve Nelson's proposition that
one day red blood cells could be harnessed to rid mankind of
infection.
[0030] The peptide machines of the present disclosure provide
totally different mechanisms to achieve Nelson's dream ie to bring
about the binding of HIV virus particles to the red cell surface
membrane.
[0031] The agents of the present disclosure carry the additional
advantage in that antibodies to the compliment peptides and red
cell surface CR1 are not a feature. This would be an advantage in
that the risk of destabilising an already unbalanced immune system
is not expected to be a feature of the molecules of the present
disclosure.
[0032] The Third World or underdeveloped world is no stranger to
epidemics and has prior to the development of AIDS and HIV been
ravaged by infectious disease; the most serious on a global scale
is probably malaria. Various forms of importance to humans are
Plasmodium Falciparum, Plasmodium Vivax, Plasmodium Ovale,
Plasmodium Malariae. This serious parasite infests red blood cells.
The life cycle is complex and consists in part of a short life
cycle in the salivary gland of mosquitoes. Following innoculation
into a human being, the malaria organism migrates to the liver and
to the red blood cell. It multiplies forming further merozoites.
Merozoites may then proceed to infect other red blood cells, or may
find their way into another mosquito where they replicate in the
mosquito salivary gland and later proceed to infect another human
being.
[0033] The course of malaria is a variable one and may be
characterised by a short acute illness which can bring death in a
matter of hours; or a longer more chronic illness associated with
debility and anaemia. Other forms of malaria infect animals such as
plasmodium Knowlesi which is known to infect the Rhesus monkey; and
many other zooanotic forms.
[0034] The preferred location for the malaria parasite is within
the red cells of the infected host and for much of its life span it
lives intracellularly protected from the host immune system.
[0035] Merozoites are thought to spend but a brief period free in
the circulation. Accordingly, considerable research efforts have
been expended to discover the means whereby the merozoite forms of
the parasite gains attachment and gains entry into the human red
cell.
[0036] Margaret E Perkins Journal Experimental Medicine 160
September 1984, 788-798 is responsible for the formulation of a
relationship between the Plasmodium Falciparum glycophorin binding
molecule and glycophorin A and B two silao glycoproteins found on
the surface of red cells.
[0037] More laterally, Holt and Perkins et al American Journal
Tropical Medicine Hygiene 1989, p 245-251 disclose species and
stain variations of Plasmodium Falciparum wherein some strains of
the organism exhibit preference for sialo glycoproteins
glycophorins A, B and C; and also demonstrated was the varying
requirements for the N-acetyl-neuramic acid residues (NeuNac).
[0038] The glycophorin A molecule is a highly glycolated peptide.
Pasvol has suggested that glycophorin binding peptide binds to the
region of glycophorin close to the lipid bi-layer. Pasvol G et al
"Inhibition of Malaria Parasite Invasion by Monoclonical antibodies
against glycophorin A correlates with a reduction in red cell
membrane deformality." Blood 74; No 5, October 1985, 1836-1843.
Debate continues within the literature as to the requirement for
sialic acid on the glycophorin molecule to effect invasion by
merozoites.
[0039] Some strains of malaria are totally dependent on normally
sialated glycophorin A to gain entry into the red cell, whereas
other strains seem to be independent of sialic acid. See Mitchell
et al 67, No 5 May 1986 1519-1521. Perkins and Roco in Journal of
Immunology, 88, Vol 141, 3190-3196 No 9, again stress the
importance of sialic acid where normally silated glycophorin is
necessary to achieve successful binding of merozoite peptides, in
particular to pf 200.
[0040] For several years a peptide called the glycophorin binding
protein was believed to be the primary peptide responsible for
binding merozoites to erythrocytes. A gene coding for GBP was
isloated by M Ravetch and Kochan J and disclosed in Science Vol
227, p 1593-1596, 29 Mar. 1985 and incorportated herein fully by
reference.
[0041] GBP 130 is characterised by a tandem repeated sequence
believed to be the site of erythrocyte binding, "A tandem repeated
sequence determines the binding domatin for an erythrocyte receptor
binding protein of plasmodium falciparum". Kochan J, Perkins M and
Ravetch J Cell, Vol 44, 689-696 March 1986. See FIG. 2 p 691 which
also discloses the full sequences and genetic code of the GBP 130
molecule and is incorporated fully herein by reference.
[0042] Other workers have challenged the supremacy of the GBP 130
as the primary binding molecule of the malaria merozoite, Orlandi
P; Kim Lee Sim et al Molecular and Biochemical Parasitology, 40
(1990) 285-294 "Characterisation of the 175 kilodalton erythrocyte
binding antigen of plasmodium flaciparum" suggested a different
peptide, the EBA 175 molecule, as being responsible for merozoite
binding or at least playing some role therein. The EBA 175 molecule
like the GBP 130 molecule has an affinity for the red blood cell
surface and binds thereto.
[0043] It is known that the EBA 175 molecule has an affinity for
oligosaccharides which are found on the surface of the red cell
molecule. However a problem arises in that the EBA 175 molecule
does not bind effectively with the malaria merozoite parasite.
Therefore it is thought that the EBA 175 serves a function as a
bridge. This disclosure proposes an alternative mechanism in that
the EBA 175 molecule is responsible for bringing the merozoite
close to the erythrocyte membrane surface, thereafter GBP 130 drags
the merozoite closer still by binding with the base of the
glycophorin A peptide; thus bringing the lipid bilayer of the
malaria parasite into approximation with the lipid bilayer of the
red cell membrane and thereby allowing the incorportation of the
parasite into the erythrocyte itself. This disclosure suggests the
merozoite is winched into the RBC cytoplasm.
[0044] The genetic sequence and the peptide sequence of EBA 175 was
disclosed by Kim Lee Sim, Orlandi P et al "Primary structure of the
175 K plasmodium falciparum erythrocyte binding antigen and
identification of a peptide which elicits antibodies that inhibit
malaria merozoite invasion" See J Cell Biology Vol 111 (1990) p
1877-1884 FIG. 2 of P 1880 for the sequence of amino acids and DNA
sequence.
[0045] To further complicate the picture Dagmar Nolte et al
described two close relatives of the glycophorin binding peptide
130 molecules which they call GBPH or glycophorin binding peptide
homologues. These molecules like the GBP molecule display several
tandem repeat sequences and a high affinity for the erythrocyte
surface membrane surface peptides. It has been proposed by Nolte
and co-workers that it is the GBPH molecule and not the GBP
molecule that is responsible for erythrocyte binding of the
parasite in that the GBP molecule is released as an immunogenic
decoy to distract the immune system from the real binding peptide
the GBPH.
[0046] The nucleotide sequence and amino acid sequence of one form
of the peptide GPBH is dislcosed by Dagmar Nolte et al in the
Journal of Molecular and Biochemical Parasitology 49, (1991) p
253-264 see FIG. 2 of p 257 incorporated herein fully by reference.
See also FIG. 3 of p 258 the same journal and paper which lists a
comparison between GBP 130 and GBPH. Binding and entry of
merozoites into RBC's probably involves several peptides or several
alternatives as fail safes for the organism.
[0047] The picture is further complicated by other research notably
by Peterson Gregory who proposes PMMSA (Pre major merozoite surface
antigen) as being responsible for erythrocyte binding either in
this state or following fragmentation into smaller fragments. The
genetic sequence and the peptide sequence of the PMMSA molecule is
given in the Journal of Molecular and Biochemical Parasitology 27
(1988) 291-302 see FIGS. 3 of P 294 and 295 Peterson G et al, and
is incorporated fully herein by reference.
[0048] Erythrocyte binding using different peptides and surface
molecules is exhibited by other species of the malaria parasite in
particular the Plasmodium Vivax organism. This organism can infect
only persons expressing the Duffy marker. The Duffy antigen is a
red cell surface marker and is one of many blood group markers and
carried by a percentage of the population. Persons not expressing
Duffy antigens are therefore immune from infection by Plasmodium
Vivax. The Plasmodium Vivax merozoite expresses a Duffy binding
receptor molecule the P. vivax Duffy receptor, cloned and sequenced
by Xiangdang Fang and disclosed in Molecular and Biochemical
Parasitology 44 (1991) p 125-132 see especially FIG. 1 of p 127 for
the genetic sequence and amino acid sequence. Similar to Plasmodium
Vivax is Pladmodium Knowlesi which also uses the Duffy antigen.
This organism parasitises Rhesus monkeys. Also in the same Journal,
same figure, same page is listed the genetic sequence of Plasmodium
Knowlesi Duffy receptor molecule which may find a use in the
machines of the present disclosure.
[0049] When developing therapeutic agents directed against the
malaria parasite itself then it is the teachings of the art to
identify the precise molecule responsible for merozoite binding in
the clinical context. However, this disclosure teaches that where
malaria peptides are to be employed as erythrocyte binding agents
more generally then it is not important to identify the precise
peptide the malaria organism uses to effect invasion, rather any
malaria peptide capable of binding to an erythrocyte surface
membrane may have a therapeutic use for the purposes of the
machines of the present disclosure and also segments of such a
peptide.
[0050] It is disclosed that the machines of the present invention
consists of hybrid peptides formed by the fusion of malaria derived
peptide sequences sufficient to exhibit erythrocyte membrane
affinity fused to a CD4 derived peptide sequence sufficient to
demonstrate affinity for the HIV virus. The restriction map and
peptide sequence of the CD4 molecule are disclosed by Madden et al
Proceedings National Acad Science USA 84; 1987' p 1955-1959 see
especially FIGS. 1 and 2 incorporated herein fully by
reference.
[0051] Also Madden et al Cell Vol 42, P 93-104 August 1985
especially FIG. 6 of P 97.
[0052] It will be appreciated that the CD4 peptide components will
not be restricted to segments of the naturally occurring CD4
molecule. Also, the malaria derived peptide fragments may be any
that are capable of binding to the red-cell membrane and one is
especially directed to those referenced herein before.
BRIEF SUMMARY
[0053] The present disclosure provides novel hybrid or fusion
peptides having a minimum of two different peptide components
possessing different functionality. One component, preferably the
CD4 molecule or segment or derivative thereof which, is capable of
binding to an HIV virus is fused or joined to a malaria derived
peptide or derivative or fragment thereof which is capable of
binding to a red cell membrane. The fusion peptide will in one
aspect bind the free HIV virus to a red cell producing possible
therapeutics benefits of: [0054] a) reduced free virus in
circulation; [0055] b) reduced free GP120 in circulation; [0056] c)
reduced symptoms and infectivity, both generally and prior to
delivery; [0057] d) augmented therapeutic usefulness of blood
tranfusions; [0058] e) greater safety of blood tranfusions; [0059]
f) immune augmentation by novel vaccine effects; [0060] g)
usefulness as prophylaxis, eye drops for theatre staff;
preparations, creams, lotions and foams for contraceptive use;
preparations for exposed cuts or abrasions; [0061] h) simpler safer
and economic viral titre measurements; [0062] i) preparation for
cleaning surgical instruments.
[0063] It will be appreciated that more than the minimum two
components may be used to form the fusion peptides, mixed in any
combination to produce more complex molecular machines without
departing from the scope and spirit of the invention.
eg
##STR00001##
or where A denotes a malaria fragment and B denotes a CD4 derived
fragment and `m` and `n` are natural numbers, the disclosure
contemplates in some aspects the hybrid molecules such as:-- [0064]
Am Bn; (ABA)n [0065] (AB) n; (BAB) m and the like which also fall
with the scope and spirit of the present disclosure.
DESCRIPTION OF DRAWINGS
[0066] The FIGS. 1(a) and 1(b) are provided on Drawings sheet 1 of
1.
[0067] FIG. 1(a) depicts free HIV virus particles drawn to and
immobilised on a red cell (RBC).
[0068] FIG. 1(b) depicts the formation of a novel vaccine within
the host blood stream utilising HIV components (live virus) derived
from the host rather than provided from external sources as would
be usual; for vaccines.
DETAILED DISCLOSURE AND EXEMPLARY EMBODIMENTS
[0069] The molecular machines of the present disclosure are
prepared by the conjugation of at least two separate components
into a single larger molecular entity or conjugate peptide. A
minimum of one HIV binding component is joined to a minimum of one
RBC peptide. A minimum of one HIV binding component is joined to a
minimum of one RBC binding component. The components may be joined
directly or indirectly to a junctional sequence or cross
linkers.
[0070] The simplest form of a molecular machine will consist of a
HIV binding peptide and an RBC binding peptide fused by peptide
bonds giving the following structure:
TABLE-US-00001 HIV Binding RBC Binding Peptide Peptide Middle of
molecule & site of fusion not critical
[0071] The functional areas of the machines are preferably situated
towards either end. Therefore the middle of the molecule and the
site of fusion is not critical.
[0072] It will be appreciated that the two components may be fused
directly or an intervening segment or peptide sequence or linker be
interposed between them. Such a segment could be any peptide
sequence such as: [0073] a) the junctional areas of the CD4
molecule; [0074] b) the Fc segment of an immunoglobulin; [0075] c)
or any macromolecule or cross linker or part thereof.
[0076] The interposed segment may or may not retain functionally of
its own but must not interfere with the function of HIV binding and
RBC binding. The preferred HIV binding component is a CD4 peptide
sequence all or part thereof retaining HIV binding. However, it
will be apparent to the skilled artisan that a CD4 therapeutic
agent such as CD4-Fc retaining a funtional HIV binding moiety would
also provide a suitable HIV binding component. In that event, the
Fc fragment would become a spacer or peptide cross linker between
the CD4 segment and the RBC binding peptide.
[0077] The alternative becomes:
TABLE-US-00002 CD4 SPACER RBC BINDING PEPTIDE PEPTIDE CROSS
LINKER
[0078] It will be appreciated that the use of a CD4-Fc peptide or
any CD4-P where P is a non specific peptide sequence, in place of a
simple CD4 peptide segment falls, within the scope and spirit of
this invention regardless of whether the middle component retains
functionality or not. One is directed to WO 89/02922 Genetech
Denial J Capon for a description of immunoadehesions comprising eg
CD4-Fc fusion peptides which may be used in place of CD4; and is
incorporated fully herein by reference.
[0079] The envisaged mode of action of the therapeutic machines of
the present disclosure is that one component of the molecular
machines, the CD4 derived component will bind with the HIV virus
GP120 molecule or free GP120 whilst the merozoite derived peptide
segment will bind with the erythrocyte membrane surface. Either
glycophorin A, B, C or to the Duffy antigen where a Duffy receptor
peptide is used.
[0080] The outcome of this binding will be to immobilise the virus
particle onto the surface of the red cell much like a barnacle to
the surface of a ship, and thereby neutralise it.
[0081] Thereafter the virus will be carried around with the red
cell harmlessly to await destruction by mechanisms such as
phagocytosis.
[0082] It is likely that the peptides will have a long half life in
vivo. It is known that merozoites have a predilection for young red
cells rather than mature red cells. Therefore the problem of short
half life seen with previous CD4 based therapies will be eliminated
to a certain extent. The effect of this viral binding will be to
sweep the circulation clear of free virus particles, reducing the
victims infectiveness to others. The peptides of the present
disclosure will find uses as molecules to be injected prior to
surgery, molecules to be injected prior to delivery, molecules to
be mixed with blood transfusions (whole blood) to be given to AIDS
victims thereby to improve the therapeutic effectiveness of
tranfusions and molecules to reduce the high viral titre levels
believed to be associated with symptomatic AIDS and molecules for
prophylaxis generally.
[0083] A further benefit of the machines of the present disclosure
is the possibility of incorporation of the virus into the red cell
cytoplasm. It will be appreciated that although this step is not
essential to the present disclosure, immobilisation being
sufficient, nevertheless were a virus to attempt infection of a red
cell such infection resulting in the dismantling of the virus outer
coat and the incorporation of the virus RNA into the red cell
cytoplasm this would be a completely wasted exercise from the viral
point of view. Red cells lack a nucleus and a virus once
incorporated into a red cell cytoplasm is immobilised and
neutralised forever.
[0084] It is envisaged that normal dosage levels of a peptides of
the present disclosure will range from 0.01 mg/kg to 10 mg/kg. The
agents of the present disclosure may also find a use in diagnostic
testing and viral titre quantification.
[0085] The molecular machines of the present disclosure may act
beneficially in further ways.
[0086] The GP120 binding component (CD4 derived sequence) may bind
the molecular machines to infected cells expressing GP120.
Expression of GP120 by infected cells occurs prior to viral
shedding
[0087] The resultant complex,
##STR00002##
may mobilise an immune reaction against the infected cell killing
the infected cell thus preventing viral release.
[0088] Further were this immune response to be in the form of
cellular immunity or continue in the absence of the molecular
machines of the present disclosure, then the said machines would
constitute a long awaited vaccine or immuno modulator against
HIV.
[0089] It is known for instance that cellular immunity is highly
desirable in combating HIV infection. It is known that HIV victims
with enhanced cellular immunity live longer and remain symptom free
for long periods.
[0090] The generation of a cellular immune response has been found
to follow where an infection antigen (virus) has been ingested by a
macrophage APC (antigen presenting cell). Live viruses are
processed differently to non infectious particles by APC's. Live
viral antigens are digested in the cytoplasm of the APC
(cytoplasmic pathway) and digested particles presented on the APC
surface in association with class 1 MHC markers provoke a cellular
immune response (Whitton J L Oldstone B A Class 1 MHC can present
endogenous peptide to cytotoxic lymphocytes J Exp Med 1989 170:
1051-56). Accordingly the degradation of HIV (live virus) coated
RBC's in the spleen or reticulo endothelial system could encourage
a cellular immune response. By capturing endogenous live virus and
by binding the said live virus to a cell destined for destruction
by phogocytosis, the molecular machines of this invention provide a
vaccine of a particularly novel variety in that: [0091] 1) the
vaccine as administered contains no components derived from the
pathogen target of the vaccine (no HIV component). eg rGP120;
[0092] 2) the vaccine obtains its pathogen derived component direct
from the host and modifies host produced live virus; [0093] 3) the
red cell surface is incorporated into the final immunogenic
construct.
[0094] It should be noted that the art teaches that vaccines should
usually contain material derived from the target pathogen. This
disclosure teaches vaccines which do not provide target
pathogen-derived material in that the material derived from the
target pathogen of the vaccine is provided later in the host. The
precise mechanism for the generation of cellular immunity is not
understood. It is a further unproven postulate of this disclosure
that cellular immunity generally proceeds from the processing of
pathogens bound to erythrocytes when the said-pathogens are
engulfed with their red cell symbiot in the liver, bone marrow or
spleen. Possible proof of this hypothesis lie in the following
facts: [0095] 1) the fact that splenectomy leads to a defect in
cellular immunity; [0096] 2) the fact the splenectomy is beneficial
in hypoimmune states such as ITP idiopathic thrombocytopaenic
purpurpae. [0097] 3) The fact that cellular immunity is a delayed
phenomenon which would be consistent in that the spleen must wait
for RBC's with bound antigens to `age` sufficiently before
engulfing them.
[0098] The precise role of the spleen in the `immune` system is not
clearly understood. However, although it is useful to speculate on
the above hypothesis they are disclosed herein as notions worthy of
further study, and not binding to the disclosure.
Examples of Clinical Mode of Use of Molecular Machines
Example 1 of Clinical Mode of Use
[0099] The molecular machines of the present disclosure in one
aspect are prepared and packaged in lypholised form and stored in
refridgerated conditions. The lypholised fusion peptides are
reconstituted by means of injectable solvents such as injectable
preparations of normal saline or dextrose 5% or the like together
with buffers and other stabilising ingredients incorporated as
necessary.
[0100] The reconstituted molecular machines (fusion peptides) are
administered to patients by either the Direct or Indirect
method.
[0101] Direct method involves the intravenous or even intra
muscular administration of the reconstituted injectable solution of
the molecular machines of amounts usually not exceeding 1,000 mg of
agent per ml. The agents may be infused slowly by means of an
intravenous drip or when safe to do so or if necessary by bolus
injection.
[0102] The slower administration rates provide more even mixing of
agent with red cells therefore labelling many more red cells.
[0103] A disadvantage with targetting large numbers of red cells is
the risk of haemolysis. But the advantage is longer half life of
the agent. Bolus administration or rapid administration reduces
haemolysis because fewer red cells are carrying the agents, however
those fewer red cells will have a shorter plasma half life.
[0104] Indirect methods of administration envisage the mixing of
the reconstituted agents with red cells outisde the body. The
agents may be added to blood transfusion units all or part prior to
administration or the agents may be mixed with a quantity of the
patients own blood. Ten mls of the patients own blood is mixed with
citrated buffer and thereafter with the reconstituted molecular
machines. A degree of mechanical agitation is provided. The treated
blood is now reintroduced into a suitable vein, slowly at first to
observe for untoward reactions.
[0105] The procedures above may be repeated until side effects such
as haemolysis or idiosyncratic reactions prevent their further use.
It is envisaged that the agent will be administered as courses of
treatment on a 3 to 4 monthly basis.
[0106] It will be appreciated that the agents are not necessarily
restricted to lypholised forms. It has recently been shown that
following liquid storage recombinant peptides perform better than
freeze dried preparations, in some cases this was demonstrated for
IFN see Pearlamn and Tue Nguyen J Pharm Phamacol 1992, 44 (suppl
1): 178-185.
Example 2
[0107] The agents may be formulated in slow release forms allowing
the gradual leaching of active peptide molecular machines over a
time period. Examples of such methods are the incorporation of the
agents into biodegradable polymers. Such polymers could be injected
as subdermal `rice grains` or form part of an intrauterine
contraceptive device.
[0108] Intrauterine contraceptive devices (IUD'S) impregnated with
the agents of the present disclosure would slowly leak the agents
towards the cervix. Cerival erosions are known to be an area of
major importance in women's vulnerability to AIDS. Indeed the
heavier menses associated with IUD's would leach more of the
peptides from the polymer and by targetting menstrual blood or on
their own provide a useful degree of protection against the virus.
Such a use would be very helpful in the Third World.
[0109] For a useful reference on polymer delivery systems see Kost
J and Langer R Tibech April 1992 Vol 10, 127-131 incorportated
fully herein by reference.
Example 3
[0110] The novel peptide agents of the present disclosure may by
incorporated into creams and gels or foams for use as topical
preparations. Such compositions may be used in conjunction with:
[0111] a) barrier contraceptives; [0112] b) in place of existing
lubricating agents or with lubricating agents generally
[0113] The said creams, gels or foams may also be used to dress
wounds, cuts or open sores or as contraceptive agents or in
conjunction with contraceptives especially barrier methods. In the
case of HIV positive patients such sores would become reduced
sources of infection for others.
[0114] In the case of medical personnel such creams, gels or foams
and the like could render abrasions or cuts more protected against
contamination by free virus from patients. Such a mode of use as a
prophylactic may be especially helpful for theatre personnel.
Example 4
[0115] The agents of the present disclosure are formulated into
drops, especially eyedrops. During surgical operations eye splashes
are a frequent occurrence. Theatre staff although strongly warned
to use eye protection often fail to do so for reasons of
discomfort, haste or carelessness. Existing eyewash preaprations do
not contain specific anti-viral components. The present agents are
formulated as surgical eyedrops or eye washes useful as emergency
decontaminants of splashed eyes during operative procedures.
Examples of Clinical Use as Testing Agents
The Liquid Phase Use of Malaria Erythrocyte Binding Peptides Fused
to CD4 in HIV Tests and Viral Titre Quantification
[0116] A measured volume of blood or fluid to be assayed is mixed
with a known quantity of molecular machines; usually 1 ml of sample
blood with 20 ug of CD4-merozoite fusion peptides. following a
period of incubation, agitating the sample so as to bring red cells
into contact with the virus, the sample is further incubated with a
fluorescent labelled anti HIV GP120, anti GP160 or anti GP41
antibody or cocktail thereof. Separation is carried out by
centrifugation or filtration. After adequate washing of cells by
appropriate buffer, cell fluorescence is measured in a cell sorter.
The advantages of such a liquid phase HIV test are: [0117] 1) high
specificity owing to the concentration of antigen in one small spot
on a red cell, helping fluorescent contrast [0118] 2) ease of
automation [0119] 3) quantification of viraemia. Up to now HIV
viraemia has been difficult to quanity. Cheaper methods of viral
quanitification will assist in new drug development.
Examples of Methods of Manufacture
[0120] The fusion peptides of the present disclostire may be
manufactured by a wide variety of manufacturing methods and
strategies; which may be applied in various combinations.
[0121] Both the number and variety of suitable methods of
manufacture are increasing rapidly at the present time and are
widely known and practiced by persons skilled in the art.
Accordingly in the interests of brevity reference will be made to
some but by no means to all of the manufacturing methods suited to
the present disclosure.
[0122] The various manufacturing methods may be combined in
different combinations to form a final strategy. In one strategy
the fusion peptides may be manufactured by means of the separate
manufacture of components such as components A and B and subsequent
fusion of A and B invitro using various means eg by chemical
linkers. Peptide sequences even greater than 150 amino aicds long
may be manufactured entirely synthetically invitro. Accordingly
where component A or B of the fusion peptide is less than 150 amino
acids in length this component may be made synthetically as a
single component. Where component A or B is very long component A
or B may be manufactured synthetically in smaller fragments to be
bonded chemically there after invitro, the said bonding to be
preferably peptide bonds and achieved by methods such as segment
condensation or by other methods known to the art.
[0123] The two peptide components A and B may also be fused using
chemical linkers or by disulphide bonds as disclosed by Chan et al
1977, J Bio Chem 252; 1515-1522. Miskimins et at 1979 Bio Chemi
Biophys Ref Comune 1991; 143-151 discloses a fusion peptide formed
using cystamine as the cross linking agent. Cawley et at 1980 Cell
22, 563-70 discloses the use or hetrobifunctional cross linking
agents to form a hybrid molecule consisting of RICIN A joined to
epiderminal groth factor EGF. Other useful strategies for fusing
peptide sequences employ linking groups such as the chelating agent
DTPA. To facilitate the fusion of peptide sequences by means of
chemical linking groups it may be helpful to fix one or both
peptide sequences onto a solid substrate such as resin beads or
polyacrylamide gel or cross linked polystyrine or other anchoring
mority.
[0124] The recent art continues to provide new techniques for
applying the Merrifield synthesis of peptides on a scaled up basis
useful for the large scale manufacture ie of peptides such
techniques are termed "scale up of solid phase peptide synthesis".
Merrifield synthesis may be very suited to the tandem repeat
segements of malaria derived peptide components. In the interests
of brevity a hill account of peptide synthesis will not be given
which techniques are widely known and practised by the skilled
artisans but reference will be made to some more interesting or
recent developments in peptide manufacture.
[0125] A recent development involves the use of a new anchoring
moiety involving the bondying of (HYCRAM) (R) (Orpegen GMBh,
Czernyring 22, D-6900 Heidelberg FRG) a
4-hydroxy-crotonoyl-amidomethyl group bonded to an
aminomethyl-polyacrylamide gel via spacer molecules such as a
B-alanaine or sarcosine or the like. Fmoc-amino acids can be linked
to the HYCRAM (R) by esterification. Also any Boc-amino acid or any
Ddz-amino acid 3,5-di methoxy phenyl-2-propyl-2-oxycarbanyl-amino
acid salt may be bonded to the HYCRAM (R) anchor.
[0126] Synthesis then proceeds using the Boc-/benzyl, the
Ddz-/t-butyl or the Fmoc-/t-butyl protocols as usual.
[0127] Detachment of the peptide from the HYCRAM (R) support
employs palladium tatra-kis (triphynyl-phosphane) a catalyst in a
suitable solvent such as 50% (v/v) dimethylsuphoxide with dimethyl
formamide; N-methylpyrrolidine, tetrahydrofuran and water. Oxygen
tetrahydrofuran must be excluded. Acceptor molecules, morpholine,
dimedine or N,N'-di methylbarbiturate may be added to take up the
allylic group.
[0128] The Ddz-/t-butyl amino acid protections are easier to cleave
using with 1-5% (v/v) trifluoroacetic acid in dichloromethane a
process taking 10 to 30 minutes or by means of the more
environmental friendly acetic acid or dioxane containing 1% (w/v)
HCL gas. The other useful protocol is the Fmoc-/t-butyl strategy.
Cleavage of F moc can be achieved using 20-50% (v/v)
piperidine/dimethyl formamide. Deprotection can be monitored in
both cases photometrically. The activation of Boc-; Fmoc-; or
Ddz-amino acid derivatives may employ the inexpensive (DCC)
diclohexylcarbodi-imide. Pre activation using HOBT
(N-hydroxybenzotriazole) can be employed to form symmetric
anyhydrides of protected amino acids or their esters. Other
activating agents are the Castro Reagent or BOP;
Benzotriazole-1-yl-oxy-tris(dimethyl amino) phosphonium hexa
fluorophosphate; one is directed to CASTRO B et al (1957)
Tetrahedron Lett 14, 1219; and TBTU the Knorr reagent,
Benzotriazole-1-yl-oxy-1,1, 3, 3-tetramethyluronium
tetrafluoroborate one is directed to Knorr R et al (1989)
Tetrahedron Lett 30, 1927. Fragment condensation can be achieved
using the BOP or the TBTU reagent with HOBT in excess. Protected
peptides may also be in excess, however solvents and excesses can
often be recycled.
[0129] It will be appreciated that by blocking incomplete fragment
condensations shorter by-products can be discriminated from the
desired polypeptide. Using this system high purity polypeptides can
be produced.
[0130] Monitoring of the production process will usually involve
U.V. absorption techniques.
[0131] A typical production process involves either the separate
synthesis of peptide sequences by their expression in suitable
hosts, and their subsequent purification and fusion; or chemical
synthesis such as on a solid substrate for example by the
sequential addition of amino acid residues or peptide fragments
which are protected, the protection of the amino acid residues as
required and the subsequent reacting of the peptide chains with
linking agents before removing the peptide chains from the said
solid substrates and the final purification by the various means is
such as reverese phase chromatography; or any combination of the
above.
[0132] In the case of some of the exemplary embodiments it may be
convenient to manufacture the fusion peptides by means of a fused
gene. A fused gene is a genetic sequence which codes for both
components of the hybrid component molecule. One is directed to
Murphy U.S. Pat. No. 4,675,382 for a detailed disclosure of the use
of fused genes in the manufacture of hybrid peptides having the
components MSH or Melanocyte Stimulating Hormone fused to dipcheria
A toxins.
[0133] Alternatively peptide fragments may be manufactured by DNA
cloning and expression in suitable hosts and recovery with
subsequent condensation in vitro.
[0134] Generally cloned sequences useful for the production of
fusion peptides will have the transmembrane domain and the
cytoplasmic domain sequences removed.
[0135] For a useful general description of DNA cloning and modular
hydridization technology, one is directed to Mainiatis et al
Molecular cloning, A Laboratory Manual, Cold Spring Harbour
lanoratory (See Second Edition 1989); and to Horvath et al An
Automated DNA synthesiser employing Deoxynucleoide 3
phorphoramidites, methods in enzymology 154: 313-326, 1987. One is
also directed to "Principles of Gene Manipulation an Introduction
to Genetic Engineering" Old R W and Primrose. S B Ed 4; Blackwell
Scientific Publications ISBN 0-632-02608-1 incorporated fully herin
by reference. DNA may be made by the chemical synthesis of DNA
polymer fragments using phosphotriester, phosphite or
phosphoramidite chemistry. For a description of solid phase
techniques one is directed to Chemical and Enzymatic Synthesis of
Gene Fragments--A Laboratory manual ed H G Gassen and L Lang,
Verlag Chemiee, Weinheim 1982; and Gait M J et al Nucleic Acids
Research 1982, 10, 6243; Spoat B S et al Tetrehedron Letters 1980,
21, 719; Matteuci M D et al J of American Chemical Society 1981,
103, 3185; Sinha N D et al Nucleic Acids Research 1984, 12, 4539;
Adams S P et al J American Chemical Society. 1983, 195; 661; and
Matthes H W D et al Embro Journal 1984, 3, 801; and are
incorporated herein fully by reference.
[0136] Reverse transceptase techniques may also be used to generate
a complimentary c DNA strand by means of the reverse transciption
of malaria parasite derived mRNA. Kits are available for this
purpose.
[0137] The DNA fragments may be ligated by either blunt-ended or
staggered-ended termini using restriction enzymes; digestion;
filling in as required; and treatment with alkali and phosphatase
for protection and subsequent ligation with suitable ligases.
[0138] Appropriate leader sequences may be chosen from the many
available.
[0139] The cloning of the DNA sequences of the hybrid peptides of
this invention may take place in prokaryotes such as E coli for
example K12 strain 294 or E coli B or E coli X1776 by way of non
limiting examples or by means of the polymerase chain reaction.
[0140] Subseqent expression of the hybrid peptides may take place
in any host cell, but preferably mammalian host cells. Other useful
cells are fungi, yeasts, insects and prokaryotes. Signals suited to
the chosen host cell are chosen as appropriate, in the case of
prokaryotes one can chose from a large group including alkaline
phosphatase and the like.
[0141] Where prokaryotes are used to express the hybrid peptides,
then they are transformed by an expression vector usually a plasmid
such as PBR322 into which the DNA encoding the fusion peptide or
fragment has been ligated such a plasmid will also feature suitable
marker sequences, promoters and Shine-Dalgarno sequences may be
chosen as appropriate.
[0142] A prokaryote host such as E coli may be transformed by
treatment using a solution of CaCl.sub.2 as described by Cohen et
al PNAS 1973, 69; 2110 or by treatment with a solution comprising a
mixture of RbCl, Mncl.sub.2, potassium acetate and glycerol and
then subsequently with 3-(N-morpholino)-propene-sulphonic acid and
RbCl and glycerol.
[0143] One is directed to "DNA Cloning" Vol II D M Glover ed, IRL
Press Ltd 1985 for a description of transforming techniques.
[0144] Alternatively where the chosen host is a yeast such as
Saccharomyces cerevisiae the plasmid YRp7 as the expression vector
may be used. One is directed to Stinchcomb et al Nature 282, 39,
(1979), Kingsman et al Gene 7; 141 (1979); Tschemper et al, Gene
10; 175 (1980).
[0145] On some occasions difficulties were encountered in cloning
segments of the plasmodium falciparum DNA resulting from the very
rich A T DNA sequences which cause problems when cloned in E coli
organisms.
[0146] Using yeasts such as saccharoyces cerevisiae large segements
such as 230 kilobases may be cloned successfully using yeast
artificial chromosomes such as pYAC-4; Tony Triglia and David Kemp,
Molecular and Biochemical Parasitology, 44, 1991, 207-212. As has
mentioned herein before the cloning of soluble of T4 peptides has
already been achieved and represents little difficulty. One is
directed to PCT Patent Application WO 89/11860 and to U.S. Pat. No.
094,322 filed Sep. 4, 1987 and to U.S. Pat. No. 141,649 filed
January 1987 and the PCT WO 89/01940 filed Sep. 1, 1988 which are
incorporated here fully by reference.
[0147] A wide choice of promoters is available for use in yeast
cell expression systems and include by way on non limiting examples
3-phosphoglycerate kinase and one is directed to Hitzman et al J
Biol Chem 255; 2073 (1980); also enolase,
glyceraldehyde-3-phosphate dehydroginase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, gluocse-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triose phosphate
isomerase, phospho glucose isomerase ad glucokinaise being
glycolytic enzymes and one is directed to Hess et al J Adv Enzyme
Reg 7; 149 (1968) and to Holland Biochemistry 17: 4900; 91978.
[0148] Other promoters suitable for yeast expression systems
include the promoter regions for alcohol deyhydrognase 2, 100
cytocrome C, acid phosphatase, also mettallothioneins.
glycoraldehyde-3-phosphate dehydrogenase and others one is directed
to Hitzman R et al European patent publication No 73, 657A.
[0149] Where insect cells such as Lepidoptera cells are the chosen
expression host a suitable vector would be Baculovirus. Such a
system would contain the target peptide encoding sequence linked to
a baculovirus promoter within a shuttle vector with sufficient
baculovirus DNA flanking the target peptide encoding sequence to
permit recombination.
[0150] One is directed to Summers et al TAES Bull (Texas
Agricultural Experiemental Station Bulletin) NR 1555 May 1987.
[0151] One is also directed to SmithKlein (WO/US/89/05550).
[0152] Insect larvae can also be used to produce transformed insect
cells, particularly Heliothis virescens caterpillars and one is
direced to PCT/WO/88/02030 Miller at al.
[0153] Where plant cells are the chosen host expression cells the
cowpea plant provides a suitable expression system. One is directed
to the system developed by the Agriculture Genetics Company of
Cambridge UK employing techniques involving the use of cowpea
mosaic virus (CPMV).
[0154] Where mammalian cells are the chosen hosts for expression,
these cells may be grown invitro in tissue culture or suitable
bioreactors or in vivo in animals.
[0155] Vectors useful for mammalian cells host systems involve the
use of DNA derived from animal viruses such as SV40 virus;
retroviruses such as RSV, MMTV, MOMLV, baculovirus, Vaccinovirus,
Adenovirus, polyoma or bovine papilloma virus.
[0156] Promoters suitable for mammalian cells systems may be chosen
from the many available. One is directed to Friers et al Nature
273; 113 (1987) and Greenway P J et al Gene 18; 353-360 (1982) and
Okayamah Mol. Cell. Biol 3; 280; 1; 1983 by way of example.
[0157] Additionally suitable enhancers may be chosen from the many
available. One is directed to Laimins L et al PNAS 78; 993 (1981)
and Lusky M L et al Mol Cell Bio 3; 1108 (1983) and Banerji J L et
al Cell 33; 729 (1983) and Osbourne T F et al Mol Cell Bio 4; 1293
(1984).
[0158] For a description of some available selection techniques one
is directed to Southern et al J Molec Appl Genet. 1; 327 (1982) and
to Mulligan et al Science 209; 1422 (1980) and to Sugden et al Mol
Cell Biol 5; 410-413 (1985).
[0159] Some techniques useful for the introduction of the
expression vector in to the host cell involve protoplast fusion,
calcium phosphate precipitation, electroporation and other
techniques as mentioned herein before.
[0160] The expression of the fusion peptide or fragment by the host
may be confirmed by using an anti CD4 antibody such as a
complimentary probe. One is directed to Dalgleish et al Nature 312;
763-766 (1984), Klatzman et al Immunol Today 7; 291-297 1986;
McDougal et al J Immunol 153 135; 3151-3162 1985 and McDougal et al
J Immunol 137 2937-2944 (1986) and to verification experiments
mentioned herein after.
[0161] Mammalian host cells suitable for the expression of
fragments of, or in some cases the entire fusion peptides may now
be chosen from the large number now available such as VERO or
CHO-K1, the hybridoma SP2\0-AG14 of the myloma cell line P3x63Sgh
or their P3xSgh derivatives also J558L, cells.
[0162] Other suitable eukaryotic host cells include COS cells;
human embryonic kidney cells; mouse plasmacytoma cells; mouse:
sertoli cells; baby hamster kidney cells 9BHK cells); Chinese
hamster ovary cells--DKFR(CHO); monkey kidney cells; African green
monkey cells; human cervical carcinoma cells (HELA cells); human
liver cells, and mouse mammary tumour cells by way of non limiting
examples.
[0163] It will be appreciated that the chosen host cells will
preferably express the minimum levels proteases within their
cytoplasm. It will also be appreciated that amino acid sequence
variations of the peptide sequences involved may be insertions,
substitutional or deletional variations involving single amino acid
residues or peptide fragments; combinations of or multiples of such
variations, or strain or species variation.
[0164] The purpose of such variations may be to increase the
affinity of the components, to improve stability, to reduce the
cost of preparation or to increase the half life, or to lesson the
severity of side effects such as atopic reactions. The final form
of the molecular machines of the present disclosure may involve any
combination of substitutions, delations or insertion of amino acid
sequences, provided the binding ability of the epitopes or peptide
sequences are retained. It will be appreciated that also included
in the present disclosure are glycosylation variations ie variants
completely unglycolated, variants having glycosylated amino acids
other than those glycosylated in the natural peptides or variants
having a greater number of amino acid residues glycosylated or
fewer glycosylated residues or residues glycosylated by
oligosaccharides other than those oligosaccharides usually
associated with the said sequences.
[0165] Following their expression by the host cell, the peptides
are recovered and purified by means known to the ordinary skilled
artisan.
[0166] Such methods may include acid extraction, ethanol
precipitation using amonium sulphate, anion or cation exchange
chromatography, phosphocellulose chromatography, amino, affinity
chromatography, hydroxyappetite chromatography and reverse phase
chromatography.
[0167] Generally purification and processing involves four stages:
[0168] 1) extraction of peptides from host cells; [0169] 2) initial
purification; [0170] 3) final purification; [0171] 4) product
polishing.
[0172] Extraction may be accomplished using sonication techniques;
or solid shear techniques--on a small scale.
[0173] Lysozyme based techniques are expensive. More usually in the
case of bacterial hosts homogenisers or liquid shear techniques are
employed.
[0174] Other useful techniques involve osmotic shock, freezing and
thawing or alkali homogenisation.
[0175] In the case where a product is expressed as an inclusion
body cell paste may be solublised by solvents such as
8M--guanidinium.
[0176] Centrifugation provides the removal cellular debris, and
continuous flow centrifuges are preferred for large scale
operations. As an alternative to centrafugation cross flow
filtration using flat or tubular membranes and high shear forces
may provide a useful alternative to centrifugation.
[0177] Initial purification involves mainly the removal of excess
water and product concentration.
[0178] Precipitation using amonium sulphate, organic solvents,
polyethylene glycol or other polymers can be used to acomplish this
step; with the addition of some variety of chromatography usually
absorptive chromotagraphy techniques employing ion exchange,
hydrophobic or bioaffinity interactions; followed by washing and
desorption; chromatography is not restricted to columns by may take
place in membranes or even in spirally wound cartridges.
[0179] In cases where peptide fragments have been expressed as
inclusion bodies an additional step of `refolding` is required.
Because of low yields after refolding inclusion body production is
often uneconomical. However two approaches are practised to refold
peptides into the natural or desired three dimensional state; the
empirical approach and the rationalist approach.
[0180] In the empirical approach multiple solvents are applied and
an optimum strategy is determined using phase diagrams as disclosed
by Ahmed and Biglow 1979 J Mol. Biol. 131; 6097-6017
[0181] The rationalist approach seeks to produce conditions
favouring the native state while at the same time keeping
intermediates in solution.
[0182] A problem may arise in connection with disulphide bridges
which may form in non-native configurations. A way around this
problem is to oxidise disulphides under denaturing conditions,
using gel filtration remove covalent aggregates and thereafter
dilute the product in a non denaturing buffer. The peptide which
collapses into an amorphous tangle often rearranges itself into the
native form.
[0183] Highly resolving chromatography is the preferred technique
for final purification. For large scale applications columns are
preferred and techniques such as gel filtration, ion exchange,
hydrophobic interaction or affinity chromatography may be used
alone or in combination as dictated by economies of scale.
[0184] Gel filtration is best suited to small batch volumes and
suffers from the disadvantage of slow speed.
[0185] Ion exchange chromtography techniques are very useful in
early purification stages and can deal with large volumes at great
speed, producing yields of high resolution.
[0186] Hydrophobic interaction chromatography provides both high
resolution and high speed even at large batch volumes.
[0187] Bio affinity chromatography produces the highest resolution,
at high speed, but batch size may require curtailment. This
technique is an ideal late stage technique.
[0188] The increasing availability of monoclonal antibodies at
lower prices has led to greater use of bio affinity chromatographic
techniques.
[0189] A wide choice of chromatographic matrices is now available
on the market suitable for large scale use.
[0190] Particle size is decreasing from the 90 um of traditional
gels to approximately 40 um for newer gels such as Sepharose HR(R);
Sephacryl HR(R); Superdex (R) (Pharmcia-LKB) or Fractogel (R)
(Toso-Haas).
[0191] Other polymeric particles are Superose (R) (Pharmacia-LKB)
or the TSK-PW (R) varieties manufactured by (Toso Haas,
Philadelphia USA), providing very low particle size.
[0192] It will be appreciated that the process of chromatography
involves the choice and development of a strategy continaing one or
more steps ie high resolution by single step or a multistep
procedure the final choice to be determined by: [0193] 1) the
chosen peptide production process which determines the form of the
starting materials to be purified [0194] 2) cost
[0195] Usually the first chromatography column will involve large
diameter packings circa 100 um which are often chosen so that they
can by resanitised by sodium hydroxide to reduce costs. Low
resolution steps to be followed by higher reoslution steps until
the desired product purity is obtained.
[0196] A typical strategy might involve: [0197] Step One
Hydrophobic interaction chromatography at low ionic strength.
Purpose to absorb proteinases. Target peptides not absorbed and
collected. Alternatively use proteinase inhibitors. [0198] Step Two
Rerun through hlc column adding salts to bind the target peptide to
the hlc column. The objective is volume reduction. Alternatively
employ ultra filtration as described. Use a step gradient to elute
the target peptide. [0199] Stage Three A step gradient to a low
ionic stength buffer will remove remaining salts. Alternatively
employ polyethyleneimine precipitation, centrifugation and
diafiltration.
[0200] In the case of therapeutic peptides such as the molecular
machines of this disclosure where administration to a human is
considered then the step of produt polishing is vital.
[0201] Product polishing involves the removal of polymers of the
product and other pyrogens.
[0202] Techniques for product polishing involve additional gel
filtration with or without a buffer exchange step; treatment with
alyhdrogel (aluminim hydroxide) or treatment with specific
lecithins or anion exchanges.
[0203] Following their extraction and purification the hybrid
peptides may be combined with carriers, binders, preservatives,
antioxidants, antimcrobial agents and the like to formulate a
pharmaceutical composition; and one is directed to the British and
American Pharmacopoea incorporated herein by reference. It is
anticipated that intravenous administration of injectable
compositions will be a principle route of administration of the
hybrid peptides. However other compositions for administration
include microphores, lyosomes or sustained release formulations,
replantable or microcapsular sustained release matrices; intra
muscular administration may also be used.
[0204] The hybrid peptides may also be components of capsules,
tablets, creams, gels, lotions, drops, patches or may be
administered where appropriate by any other of the known means of
administration of a drug to a human where appropriate.
[0205] Hydro phobic solvents may be used for intravenous or
parentral administration in some circumstances. Examples of such
solvents are propylene glycol, polythylene glycol, vegetable oils,
organic esters.
[0206] Aqueous carriers which are preferred are by way of example
water for injection, emulsions or suspensions, saline or buffers,
alcoholic aqueous solutions, sodium chloride solutions, ringers
lactate or dextrose.
[0207] The manufacturing process only finishes with the secure
packaging of the peptides in a suitable vacuum or atmosphere in
suitable containers usually vials of glass or polymers with
suitable stoppers. The said glass or polymer vials are then housed
in a sturdy housing of cardboard or polymer after appropriate
sealing. The cardboard housing will ideally be further wrapped in a
clear polymer such as cellulose and provided with a seal to
disclose tampering. All relevant information such as product name,
date of manufacture, batch number and date of expriy and the like
should be clearly visible and printed on the vials and housing as
required by law and good manufacturing practice. The products
should also be accompanied by leaflets, indicating material facts
relevant to their use such as dosage, side effects, contra
indications and the like it will be appreciated that the
manufacturing process should take place in an appropriately
designed facility, the floors, walls and ceilings of which should
be finished to a high standard. Product flow must be arranged to
reduce risk of mix up. Water systems employed should meet the US
Pharmacopia specifications for pure water and be of injection
quality in final purification stages.
[0208] Compressed air and nitrogen must be filtered through 0.2 u
inch filters and be free of oil.
[0209] Final purification stages should ideally take place under
laminate flow hoods or equivalent in suitably clean rooms.
[0210] In short the manufacture of the peptides must take place in
conditions meeting GMP standards.
[0211] Validation and quality control are ongoing necessary steps
to ensure public safety and product quality.
[0212] The disclosure will be illustrated by means of exemplary
embodiments which are non limiting.
[0213] In the exemplary embodiments that follow a number of
assumptions are made which will be appreciated by the reader, these
assumptions to being common knowledge to persons skilled in the
art.
1) It is generally considered easier by all arts from DNA cloning
to woodworking to delete ie shorten an object than to add to it.
Therefore peptide sequences as useful formulae longer than
necessary will be provided by way of illustration; truncation or
deletions to be carried out as desired. 2) Any substitutions and
any modifications of the wild type CD4 or indeed the malaria
erythrocyte binding molecules will fall within the scope and the
spirit of the invention. Avi Ashkanazi et al Proceedings national
Acadamey of Science USA Vol 87, p 7150-71 54 September 1990 reports
substitution experiements where alanine residues were substituted
for naturally occurring amino acid residues within the CD4
molecule. The results demonstrated that single alanine substitution
made little difference in most cases to the ability of CD4 segments
to bind to the HIV GP120. In some cases either no binding
whatsoever was found or greatly improved binding was found more
particularly the substitutions G4OA and D63A; of special interest
are the double substitution G4OA and D63A which exhibited a 2.3
fold increase in affinity and the triple mutant Q40A D63A Q89L
which exhibited a four fold affinity increase. 3) It is common
knowledge that species variation or strain variations of peptides
exist in nature. Accordingly it will be appreciated tha peptide
segments derived from proteins of any strain of the malaria
organism, filling the same role fall within the scope and spirit of
the invention if used for the same purpose and in the `same way` as
the molecular machines of the invention.
Exemplary Embodiment Number 1
[0214] Exemplary embodiment number 1 is directed to a molecular
machine or hybrid protein which comprises a peptide sequence
derived from the CD4 molecule or fragment thereof which possesses
the capability of binding to HIV GP120, fused to a peptide sequence
or fragment thereof derived from the merozoite glycophorin binding
peptide GBP130.
[0215] The peptide sequence of the CD4 molecule providing derived
fragments useful for the preparation for the first component was
disclosed by Paul Jay Maddon et al in Cell, Vol 42, 93-104, August
1985. See FIG. 6 page 97; incorporated fully herin by
reference.
[0216] The second component peptide of the hybrid peptide of the
present embodiment may be derived from the peptide sequence of
GBP130 glycophorin binding peptide 130 as disclosed by Jarema
Kochan et al in Cell Vol 44, 689-696 Mar. 14, 1986. See FIG. 2 page
691 and incorporated fully herein by reference.
[0217] In this example the peptide sequence consisting of 1-371 of
the CD4 molecule is fused at the C terminal end to the N terminal
end of a peptide sequence derived from the GBP130 molecule amino
acid residues 201-774 of the GBP molecule or a fragment
thereof.
##STR00003##
[0218] In this example the preferred fusion is by peptide bonds, it
will also be appreciated that the site of fusion is illustrative
but not limiting. Fusion may take place at other amino acid
residues of both the above peptide components. The example will be
further described by a useful formula of
##STR00004##
to avoid any possible confusion
[0219] Therefore the formula of
##STR00005##
in would aspect would be,
TABLE-US-00003 +1 10 20 N Terminal gln gly asn lys val val leu gly
lys lys gly asp thr val glu leu thr cys thr ala ser gln lys lys ser
ile gln phe his trp lys asn ser asn gln ile lys ile leu gly asn gln
gly ser phe leu thr lys gly pro ser lys leu asn asp arg ala asp ser
arg arg ser leu trp asp gln gly asn phe pro leu ile ile lys asn leu
lys ile glu asp ser asp thr tyr ile cys glu val glu asp gln lys glu
glu val gln leu leu val phe gly leu thr ala asn ser asp thr his leu
leu gln gly gln ser leu thr leu thr leu glu ser pro pro gly ser ser
pro ser val gln cys arg ser pro arg gly lys asn ile gln gly gly lys
thr leu ser val ser gln leu glu leu gln asp ser gly thr trp thr cys
thr val leu gln asn gln lys lys val glu phe lys ile asp ile val val
leu ala phe gln lys ala ser ser ile val tyr lys lys glu gly glu gln
val glu phe ser phe pro leu ala phe thr val glu lys leu thr gly ser
gly glu leu trp trp gln ala glu arg ala ser ser ser lys ser trp ile
thr phe asp leu lys asn lys glu val ser val lys arg val thr gln asp
pro lys leu gln met gly lys lys leu pro leu his leu thr leu pro gln
ala leu pro gln tyr ala gly ser gly asn leu thr leu ala leu glu ala
lys thr gly lys leu his gln glu val asn leu val val met arg ala thr
gln leu gln lys asn leu thr cys glu val trp gly pro thr ser pro lys
leu met leu ser leu lys leu glu asn lys glu ala lys val ser lys arg
glu lys ala val trp val leu asn pro glu ala gly met trp gln cys leu
leu ser asp ser gly gln val leu leu glu ser asn ile lys val leu pro
thr trp ser thr pro val ser gln lys pro ser thr ser thr arg ser asn
asn glu val lys ile arg ala ala ser asn gln glu thr leu thr ser ala
asp pro glu gly gln ile met arg glu tyr ala ala asp pro glu tyr arg
lys his leu glu ile phe tyr lys ile leu thr asn thr asp pro asn asp
glu val glu arg arg asn ala asp asn lys glu asp leu thr ser ala asp
pro glu gly gln ile met arg glu tyr ala ser asp pro glu tyr arg lys
his leu glu ile phe tyr lys ile leu thr asn thr asp pro asn asp asp
val glu arg arg asn ala asp asn lys glu asp leu thr ser ala asp pro
glu gly gln ile met arg glu tyr ala ala asp pro glu tyr arg lys his
leu glu val phe his lys ile leu thr asn thr asp pro asn asp glu val
glu arg arg asn ala asp asn lys glu asp leu thr ser ala asp pro glu
gly gln ile met arg glu tyr ala ala asp pro glu tyr arg lys his leu
glu ile phe his lys ile leu thr asn thr asp pro asn asp glu val glu
arg arg asn ala asp asn lys glu asp leu thr ser ala asp pro glu gly
gln ile met arg glu tyr ala ala asp pro glu tyr arg lys his leu glu
val phe his lys ile leu thr asn thr asp pro asn asp glu val glu arg
arg asn ala asp asn lys glu leu thr ser ser asp pro glu gly gln ile
met arg glu tyr ala ala asp pro glu tyr arg lys his leu glu ile phe
his lys ile leu thr asn thr asp pro asn asp glu val glu arg arg asn
ala asp asn lys glu asp leu thr ser ala asp pro glu gly gln ile met
arg glu tyr ala ala asp pro glu tyr arg lys his leu glu ile phe tyr
lys ile leu thr asn thr asp pro asn asp glu val glu arg arg asn ala
asp asn lys glu asp leu thr ser ala asp pro glu gly gln ile met arg
glu tyr ala ala asp pro glu his leu glu ile phe his lys ile leu thr
asn thr asp pro asn asp glu val glu arg arg asn ala asp asn lys glu
glu leu thr ser ser asp pro glu gly gln ile met arg glu tyr ala ala
asp pro glu tyr arg lys his leu glu ile phe tyr lys ile leu thr asn
thr asp pro asn asp glu val glu arg arg asn ala asp asn lys glu asp
leu thr ser ala asp pro glu gly gln ile met arg glu tyr ala ser asp
pro glu tyr arg lys his leu glu ile phe tyr lys ile leu thr asn thr
asp pro asn asp asp val glu arg arg asn ala asp asn lys glu asp leu
thr ser ala asp pro gly glu gln ile met arg glu tyr ala ala asp pro
glu tyr arg lys his leu glu ile phe his lys ile leu thr asn thr asp
pro pro asn asp glu val glu arg gl:n asn ala asp asn asn glu ala -
C TERMINAL,
which formula describes the embodiment but is intended to be non
limiting.
[0220] This exemplary embodiment is also directed to a protein gene
which comprises a DNA sequence which codes for the CD4 molecule or
fragment thereof which binds HIV GP120 fused to a DNA sequence
which encodes the glyphorin 130 or fragment thereof which possesses
the capability of binding to red cells.
[0221] DNA sequences suitable for forming the cloned DNA of the
fusion peptide may be found in the disclosure of Paul Jay Maddon
Cell, Vol 42 93-104, August 1985 see FIG. 6 page 97. Some or all of
the DNA sequence coding for the CD4 molecule as disclosed may be
fused to the DNA sequence conditioning for the DNA glyphorin
binding peptide. It may be preferable to include the leader
sequence of the CD4 molecule. It may also be preferable to delete
the transmembrane and cytoplasmic domains. A DNA sequence encoding
the glycophorin peptide 130 and suitable for the fabrication for
the cloned genes useful for the preparation of the hybrid peptides
of this disclosure may be found in Jarema Kochan et at Cell Vol 4
689-696 Mar. 14, 1986 FIG. 2 page 691. Suitable sites of fusion
might be in the region of site 733 ie Leu AAA or in the vicinity of
site 736 ie vat, GTT or in the vicinity of Site 739 ie Ser TCT in
the case of GBP130; and in the region of site 1257 for the CD4
derived sequence.
##STR00006##
[0222] By way of further illustration the CD4 DNA sequence is
restricted at the BspM1 site position 514 and fused to some or all
of the DNA sequence coding for GBP130 as disclosed above or
restricted at other convenient sites
##STR00007##
[0223] The DNA sequence for the CD4 component is also obtained by
restricting the DNA sequence encoding for the CD4 peptide component
as previously disclosed at the Pvu11 site, position 691 and joined
to the cloned DNA encoding the glycophorin binding peptide 130
fragment as previously described or smaller fragments thereof,
restricted as desired.
##STR00008##
[0224] Fusion will be carried out by suitable ligases and gene
fragments, even those gene fragments encoding additional amino
acids or sequences of amino acids may be inserted to enable the
ligation provided the inserted segments do not interfere with the
functionality of either the CD4 derived peptide or the GBP130
derived peptide segment.
[0225] It is also envisaged that the hybrid peptide will have 2
components; the first being a CD4 derived peptide sequence
comprising amino acids 1-177 ie +1 gln-177 val fused to a
glyophorin binding protein (GBP) 130 derived peptide sequence such
as Leu 226-Ala 774 or Thr 227 Ala 274, fusion to take place at Leu
226 or Thr 227 by way of example fusion to consist of a peptide
bond formed between CD4 amino acid 177 and the glycophrin binding
peptide 130 amino acid 226 or 227 fused at the C terminal end of
the CD4 derived peptide and the N terminal end of the GBP130
peptide sequence. The said sequences and the enumeration of the
said sequences to be found in the sources mentioned herein
before.
[0226] This variation of the embodiment contains only the tandem
repeat sequence of the GBP-130 molecule.
##STR00009##
[0227] In this exemplary embodiment it is also envisaged that a
peptide sequence comprising the first 117 amino acids of the CD4
molecule or fragment thereof are fused C terminally to a peptide
sequence of the glycophorin binding peptide GBP130. A suitable site
of fusion would be Ala 370 N terminally to provide an amino acid
lead sequence to be followed by the tandem repeat sequence
beginning with Leu 376; the sequence terminating at Asp 425. It is
appreciated that smaller sections of such a tandem sequence could
be used as can smaller sections of the CD4 peptide sequence. The
example is intended to be illustrative rather than limiting.
##STR00010##
[0228] In all the preceeding variations of exemplary embodiment
number 1 the CD4 component peptide may be fused at the N terminal
end to the C terminal end of the GBP130 derived peptide sequence.
It was shown by P Yeh et al PNAS March 1992 P1904-1908 that
functionality is not lost when CD4 is fused N terminally.
Exemplary Embodiment Number 2
[0229] This exemplary embodiment is directed to a molecular machine
or hydrbid peptide characterised by the fusion of some or all of
the amino acid peptide sequence, amino acid residues 1-371 of the
CD4 molecule fused at the C terminal end to a peptide sequence
derived from the glycophorin binding peptide homologue molecule.
Amino acid resides 70-427 or segments thereof of the glyophorin
binding peptide homolgue(s). The amino acid sequence of the CD4
molecule has been disclosed by Paul Jay Maddon, the reference is
given in Exemplary Embodiment No. 1. The glycophorin binding
peptide homologue amino acid sequence is disclosed by Dagmar Nolte
et al in Molecular and Biochem Parasitology 49, (1991) page 253-264
FIG. 2 page 257, incorporated fully herein by reference.
##STR00011##
[0230] It may avoid confusion to describe the exemplary embodiment
in one aspect by a useful formula which is intended to be non
limiting.
[0231] Therefore the formula of
##STR00012##
in one aspect is;
TABLE-US-00004 N Terminal Q G N K V V L G K K G D T V E L T C T A S
Q K K S I Q F H W K N S N Q I K I L G N Q G S F L T K G P S K L N D
R A D S R R S L W D Q G N F P L I I K N L K I E D S D T Y I C E V E
D Q K E E V Q L L V F G L T A N S D T H L L Q G Q S L T L T L E S P
P G S S P S V Q C R S P R G K N I Q G G K T L S V S Q L E L Q D S G
T W T C T V L Q N Q K K V E F K I D I V V L A F Q K A S S I V Y K K
E G E Q V E F S F P L A F T V E K L T G S G E L W W Q A E R A S S S
K S W I T F D L K N K E V S V K R V T Q D P K L Q M G K K L P L H L
T L P Q A L P Q Y A G S G N L T L A L E A K T G K L H Q E V N L V V
M R A T Q L Q K N L T C E V W G P T S P K L M L S L K L E N K E A K
V S K R E K A V W V L N P E A G M W Q C L L S D S G Q V L L E S N I
K V L P T W S T P V S Q Y K Q A A D Y S F R E S R V L A E G K S T S
K K N A K T A L R K T K Q T T L T S A D P E G Q I M K A W A A D P E
Y R K H L N V L Y Q I L N N T D P N D E L E T S A D P E G Q I M K A
Y A A D P E Y R K H L N V L Y Q I L N N T D P N D E V E S S A D P E
G Q I M K A Y A A D P E Y R K H V N V L Y Q I L N N T D P N D E L E
T S A D P E G Q I M K A Y A A D P E Y R K H V N V L Y Q I L N H T D
S S E V E T S A D P E G Q I M K A Y A A D P E Y R K H V N V L Y Q I
L N H T D S S E V E T S A D P E G Q I M K A Y A A D P E Y R K H V N
V L Y Q I L N N T D P N D E L E T S A D P E G Q I M K A Y A A D P E
Y R K H V N V L Y Q I L N N T D P N D E L E T S A D P E G Q I M K A
Y A A D P E Y R K H V N V L Y Q I L N N T D P N D E S S - C
Terminal;
which formula is but one possible variation of the many apparent to
the skilled artisan.
[0232] This example is also directed to a molecular machine or
fusion peptide which is characterised by the fusion of CD4 peptide
sequence to a glycophorin binding peptide homologue sequence. The
CD4 derived sequence comprises some or all of the amino acids from
number 1-177 of the CD4 molecule. The sequence numbering and source
of CD4 peptide sequence has been referenced herein before, see
exemplary embodiment No. 1.
[0233] The above CD4 peptide sequence to be fused C terminally to
the N terminal of glycophorin binding homologue peptide sequence
amino acid residues 70-427 or some or part thereof. The source of
glycophorin binding peptide homologue being the sequence disclosed
by Dagmar Nolte (full reference given above).
##STR00013##
[0234] This example is also directed to a molecular machine or
hybrid peptide formed by the fusion of an amino acid peptide
sequence derived from the CD4 molecule amino acid residues 1-371 or
fragment thereof fused C terminally to a peptide sequence derived
from the glycophorin binding peptide homologue fused N terminally
at amino acid 109; the sequence comprising amino acids 109-427 some
or part thereof. The sequencing of CD4 and glycophorin peptide
binding homologue as already referenced.
##STR00014##
[0235] This example is also directed to a molecular machine or
hybrid peptide which comprises the fusion of an amino sequence
derived from the CD4 molecule the sequence as previously disclosed
and consists of amino acid residues 1-177 or fragments thereof
fused C terminally to a tandem repeat sequence(s) derived from the
glycophorin peptide binding homologue molecule as disclosed herein
before. Such a sequence may consist of amino acid residues 230-268.
It will be appreciated that an even shorter fragment could be used.
It will be also appreciated that one or more of the tandem repeat
sequences could be included. It will be further appreciated that
the tandem repeat sequence could be shortened to an even smaller
fragment containing only the RBC binding epitopes; and that this
smaller fragment could be repeated.
[0236] It will be still further appreciated that related
glycophorin binding peptide homologue molecules whose sequences are
known but not as yet disclosed in the public domain could provide
peptide sequences--tandem repeat units--fulfilling the functions of
GBP130 or the GBPH sequences as disclosed above "in the same way"
without departing from the scope and spirit of the invention.
##STR00015##
[0237] This example is further directed to a fused gene coding for
a hybrid peptide comprising a amino acid sequence derived from the
CD4 molecule joined to an amino acid sequence derived from the
glycophorin binding peptide homologue molecule. A typical fused
gene might consist of;
##STR00016##
[0238] Where X is any or no intervening sequence, X being
inspecified any non limiting.
[0239] Ligation to take place using ligases and additional linker
segments by conventional means, known to the skilled artisan.
[0240] This example is further directed to a fusion gene consisting
of a DNA sequence encoding a peptide derived from the CD4 molecule
truncated at the PvuII site 691 fused to a single tandem repeat
encoding DNA sequence derived from glycophorin binding peptide
homologue gene as disclosed by Nolte as referenced herin before or
part thereof.
##STR00017##
[0241] Where X is any intervening sequence X being, inspecified and
non limiting. Suitable linkers and joining segments to be provided
as desired.
[0242] All the preceding exemplary embodiments may be fused C
terminally of CD4 segment to the N terminal of the GBPH
component.
Exemplary Embodiment Number 3
[0243] This example is directed to a molecular machine or hybrid
peptide formed by the fusion of a peptide sequence derived from the
CD4 molecule or fragment thereof; the amino acid resides 1-371
fused at their C terminal end to a peptide sequence derived from
the EBA 175 antigen otherwise known as the erythrocyte binding
antigen 175; amino acid residues 20-1435 all or part thereof. In
this example some or all ie fragments of the component peptide
sequences may be used to fabricate the hybrid peptide. The sequence
of amino acids and DNA sequence of the gene encoding EBA 175 was
disclosed by B Kim Lee Sim et al in the Journal Cell Biology Vol
111, 1990 p 1877-1884, FIG. 2, p 1880; incorporated fully herein by
reference. The peptide sequence for CD4 being already referenced
herein before.
##STR00018##
[0244] To avoid any possible confusion it may be helpfyl to
describe the embodiment by a useful formula which is intended as
non limiting.
[0245] CD4 is truncated at 371 and EBA-175 truncated at 19 ie
excluding the hydrophobic sequence. CD4 after Maddon. EBA-175 after
Kim Lee Sim J Cell Biology Vol 111 1990, 1880
[0246] Therefore the molecular machines
##STR00019##
in one aspect would be:
TABLE-US-00005 N Terminal QGNKVVLGKKGDTVELTCTASQKKSIQFHWKNSN
QIKILGNQGSFLTKGPSKLNDRADSRRSLWDOGNFPLIIKNLKIE
DSDTYICEVEDOKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPP
GSSPSVQCRSPRGKNIQGGKTLSVSQLELGDSGTWTCTVLQNQKK
VEFKIDIVVLAFQKASSIVYKKEGEQVEFSFPLAQFTVEKLTGSG
ELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLH
LTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNL
TCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCL
LSDSGQVLLESNIKVLPTWSTPVKARNEYDIKENEKFLDVYKEKF
NELDKKKYGNVQKTDKKIFTFIENKLDILNNSKFNKRWKSYGTPD
NIDKNMSLINKHNNEEMFNNNYQSFLSTSSLIKQNKYVPINAVRV
SRILSFLDSRINNGRNTSSNNEVLSNCREKRKGMKWDCKKKNDRS
NYVCIPDRRIQLCIVNLSIIKTYTKETMKDHFIEASKKESQLLLK
KNDNKYNSKFCNDLKNSFLDYGHLAMGNDMDFGGYSTKAENKIQE
VFKGAHGEISEHKIKNFRKEWWNEFREKLWEAMLSEHKNNINNCK
NIPQEELQITQWIKEWHGEFLLERDNRSKLPKSKCKNNTLYEACE
KECIDPCMKYRDWIIRSKFEWHTLSKEYETQKVPKENAENYLIKI
SENKNDAKVSLLLNNCDAEYSKYCDCKHTTTLVKSVLNGNDNTIK
EKREHIDLDDFSKFGCDKNSVDTNTKVWECKNPYILSTKDVCVPP
RRQELCLGNIDRIYDKNLLMIKEHILAIAIYESRILKRKYKNKDD
KEVCKIINKTFADIRDIIGGTDYWNDLSNRKLVGKINTNSKYVHR
NKKNDKLFRDEWWKVIKKDVWNVISWVFKDKTVCKEDDIENIPQF
FRWFSEWGDDYCQDKTKMIETLKVECKEKPCEDDNCKSKCNSYKE
WISKKKEEYNKGAKQYQEYQKGNNYKMYSEFKSIKPEVYLKKYSE
KCSNLNFEDEFKEELHSDYKNKCTMCPEVKDVPISIIRNNEQTSQ
EAVPEENTEIAHRTETPSISEGPKGNEQKERDDDSLSKISVSPEN
SRPETDAKDTSNLLKLKGDVDISMPKAVIGSSPNDNINVTEQGDN
ISGVNSKPLSDDVRPDKKELEDQNSDESEETVVNHISKSPSINNG
DDSGSGSATVSESSSNTGLSIDDDRNGDTFVRTQDTANTEDVIRK
ENADKDEDEKGADEERHSTSESLSSPEEKMLTDNEGGNSLNHEEV
KEHTSNSDNVQQSGGIVNMNVEKELKDTLENPSSSLDEGKAHEEL
SEPNLSSDQDMSNTPGPLDNTSEETTERISNNEYKVNEREDERTL
TKEYEDIVLKSHMNRESDDGELYDENSDLSTVNDESEDAEAKMKG
NDTSEMSHNSSQHIESDQQKNDMKTVGDLGTTHVGINEISVPVTG
EIDEKLRESKESKIHKAEEERLSHTDIHKINPEDRNSNTLHLKDI
RNEENERHLTNQNINISQERDLQKHGFHTMNNLHGDGVSERSQIN
HSHHGNRQDRGGNSGNVLNMRSNNNNFNNIPSRYNLYDKKLDLDL
YENRNDSTTKELIKKLAEINKCENEISVKYCDHMIHEEIPLKTCT
KEKTRNLCCAVSDYCMSYFTYDSEEYYNCTKREFDDPSYTCFRKE
AFSSMIFKFLITNKIYYYFYTYKTAKVTIKKINFSLIFFFFFSF- C Terminal,
which is intended to be non limiting, viarations of which will be
apparent to the skilled artisan.
[0247] This example is also directed to a molecular machine or
fusion peptide comprising the fusion of a peptide sequence derived
from CD4 consisting of the first 177 amino acid residues or
fragment peptide sequence thereof; amino acid 1-177 fused at the C
terminal end to the N terminal end of a peptide sequence derived
from EBA 175' amino acid 1062-1103. This particular peptide
sequence is also named EBA peptide 4 and has the ability to inhibit
the binding of EBA 175 to red blood cells. Also included would be
fused genes formed by the fusion of DNA sequences encoding the CD4
molecule fused to Sequences encoding the erythrocyte binding
antigen 175 or its derived epeptide EBA peptide 4. The DNA
sequences of the EBA peptide number 4 and EBA 175 molecules may be
obtained from the Journal of Cell Biology Vol 111 1990, p 1880 in
the article by J Kim Lee Sim et al in the same journal.
##STR00020##
[0248] It is also envisaged that the CD4 component may be fused at
the N terminal end to the C terminal end of any of the preceding
examples.
Exemplary Embodiments Using P. Vivax Duffy
Exemplary Embodiment Number 4
[0249] In this embodiment the P Vivax Duffy Receptor molecule is
joined to the CD4 receptor molecule both truncated at their
transmembrane extrecellular domain regions.
##STR00021##
[0250] Obviously further deletions and substitutions may be made to
this molecule without departing from its intended function, the
listing of all possible deletions and substitutions obvious to an
artisan skilled in the art would be tedious and is excluded in the
interests of brevity.
[0251] It is to be noted that the amino acid sequence of CD4 as
presented in this embodiment is the same as for Maddon P N A Sci
1987 but in reverse order.
[0252] It is also to be noted that the CD4 peptide segment may be
fused at the N terminal end to the C terminal of the P Vivax Duffy
peptide, or at the C terminal end of the CD4 molecule to the N
terminal of the P Vivax Duffy Receptor; by peptide bonds. This
embodiment is also directed to a molecular machine or hybrid
peptide comprosing the fusion or joining together of two amino acid
sequences; one derived from the CD4 molecule and consisting some of
a fragment of amino acid or peptide sequence derived from
plasmodium Vivax Duffy Receptor molecule or fragment thereof; amino
acid sequence 23-1051 joined at the C terminal ends which will
require that the component sequences be expressed separately in
separate hosts and fused subsequently. Many strategies are
available for joining two peptide sequences together at both C
terminal ends. One strategy envisages terminating both peptide
sequences with `cys` or incorporating cys near each C terminal end
and fusion of the two sequences by means of sulphidryl bonds. Other
strategies involve the use of joining sequences containing cys
facilitating sulphidryl bonds linking the joining segment to the
component peptide sequences. The genetic sequence and amino acid
sequence of Plasmodium Vivax Duffy Receptor is disclosed by
Xiangdang Fang et al in Molecular and Biochimical Parasitology 44,
(1991) 125-132 one is directed to FIG. 1 of p 127.
##STR00022##
[0253] It may be helpful to decribe the molecular machines by a
useful formula of a amino acids, the useful formula is intended to
be non limiting.
[0254] Therefore a useful formula
##STR00023##
Where J is a joining segment or cross linker in one aspect would
be:
TABLE-US-00006 K D D F S I T L I N Y H E G K K Y L I I L K R K L E
K A N N R D V C N F F L H F S Q V N N V L L E R T I E T L L E C K N
E Y V K G E N G Y K L A K G H H C V E E D N L E R W L Q G T N E R R
S E E N I K Y K Y G V T E L K I K Y A Q M N G K R S S R I L K E S I
Y G A H N F G G N S Y M E G K D G G D K T G E E K D G E H K T D S K
T D N G K G A N N L V M L D Y E T S S N G Q P A G T L D N V L E F V
T G H E G N S R K N S S N G G N P Y D I D H K K T I S S A I I N H A
F L Q N T V M K N C N Y K R K R R E R D W D C N T K K D V C I P D R
R Y Q L C M K E L T N L V N N T D T N F H R D I T F R K L Y L K R K
L I Y D A A V E G D L L L K L N N Y R Y N K D F C K D I R W S L G D
F G D I I M G T D M E G I G Y S K V V E N N L R S I F G T D E K A Q
Q R R K Q W W N E S K A Q I W T A M M Y S V K K R L K G N F I W I C
K L N V A V N I E P Q I Y R W I R E W G R D Y V S E L P T E V Q K L
K E K C D G K I N Y T D K K V C K V P P C Q N A C K S Y D Q W E T R
K K N Q W D V L S N K F I S V K N A E K V Q T A G I V T P Y D I L K
Q E L D E F N E V A F E N E I N K R D G A Y I E L C V C S V E E A K
K N T Q E V V T N V D N A A K S Q A T N S N P I S Q P V D S S K A E
K V P G D S T H G N V N S G Q D S S T T G K A V T G D G Q N G N Q T
P A E S D V Q R S D I A E S V S A K N V D P Q K S V S K R S D D T A
S V T G I A E A G K E N L G A S N S R P S E S T V E A N S P G D D T
V N S A S I P V V S G E N P L V T P Y N G L R H S K D N S D S D G P
A E S M A N P D S N S K G E T G K G Q D N D M A K A T K D S S N S S
D G T S S A T G D T T D A V D R E I N K G V P E D R D K T V G S K D
G G G E D N S A N K D A A T V V G E D R I R E N S A G G S T N D R S
K N D T E K N G A S T P D S K Q S E D A T A L S K T E S L E S T E S
G D R T T N D T T N S L E N K N G G K E K D L Q K H D F K S N D T P
N E E P N S D Q T T D A E G H D R D S I K N D K A E R R K H M N K D
T F T K N T N S H H L N S N N N L S N G K L D I K E Y K Y R D V K A
T R E D I I L M S S V R K C N N N I S L E Y C N S V E D K I S S N T
C S R E K S K N L C C S I S D F C L N Y F D V Y S Y E Y L S C M K K
E F E D P S Y K C F T K G G F K -COOH CROSS LINKER COOH V P T S E T
P terminal OR JOINING SEQUENCE insert cys in Duffy insert cys in
CD4 and insert cys in and insert cys in J sequence J J sequence L V
K I N S E L L V Q G S D S L L C Q W M G A E P N L V W V A K E R K S
V K A E K N E L K L S L M L K P S T P G W V E C T L N K Q L Q T A R
M V V L N V E Q H L K G T K A E L A L T N N G S G A Y Q P L A Q P L
T L H L P L K K G M Q L K P D Q T V R K V S V E K N K L D F T I W S
K S S S A R E A Q W W L E G S G T L K E V T F A L P F S F E V Q E G
E K K Y V I S S A K Q F A L V V I D I K F E V K K Q N Q L V T C T W
T G S D Q L E L Q S V S L T K G G Q I N K G R P S R C Q V S P S S G
P P S E L T L T L S Q G Q L L H T D S N A T L G F V L L Q V E E K Q
D E V E C I Y T D S D E I K L N K I I L P F N G Q D W L S R R S D A
R D N L K S P G K T L F S G Q N G L I K I Q N S N K W H F Q I S K K
Q S A T C L E V T D G K K G L V V K N G Q NH.sub.2 Terminal;
the numbering of this useful formula is after that of the
referenced sourced peptides. It will be appreciated that the
skilled artisan could provide countless variations on the above
without departing from the scope and spirit of the disclosure.
Exemplary Embodiment Number 5
[0255] This example is directed to a molecular machine or hybrid
peptide formed by the fusion of a peptide sequence derived from the
P Knowlesi Duffy Receptor binding molecule as disclosed by
Xiangdong Fang in Molecular and Biochemical Parasitology 44, (1991)
125-132 see FIG. 1 P 127. The choice of peptide fragment is non
critical provided it contains erythrocyte binding ability and the
method of joining to the CD4 component is also non critical,
examples of typical methods of joining peptides are given in the
preceding examples.
##STR00024##
[0256] The fusion may also be by peptide bonds N terminal of CD4
component to C terminal of P Knowlesi or N terminal of P Knowlesi
component to C terminal of CD4 component
Exemplary Embodiment Number 6
[0257] This example is directed to a molecular machine or hybrid
peptide formed by the fusion of a peptide sequence derived from the
expression of the PMMSA gene or precursor to the major merozoite
surface antigen genes as disclosed by M Gregory Peterson in
Molecular and Biochemical Parasitology, 27 (1988) P291-302. One is
directed to p 294 and p 295 FIG. 3.
[0258] In this example an amino acid sequence derived from the
expression of nucleic acid residues 177-5169 all or part fused at
the C terminal end to the C terminal end of the CD4 derived peptide
sequence such as formed from the expression of nucleic acid
residues 114-1260 or fragment thereof.
[0259] The fusion at the C terminal end being accomplished by
suitable joining sequences chemical ligands or sulphidryl bonds or
other means.
[0260] It will also be envisaged that cys residues be added as
required as described in exemplary embodiment number 14; the site
of insertion is non critical providing binding function is
maintained.
##STR00025##
[0261] Alternatively a fused gene joining the CD4 component to the
PMMSA component N terminally to C terminally by peptide bonds may
be utilised to provide alternative variations apparent to the
skilled artisan.
Exemplary Embodiment Number 7
[0262] Exemplary embodiment number 7 contemplates joining sequences
suitable for joining two peptide fragments at the C terminal ends.
The joining sequences could be a straight peptide chain which will
have cys amino acid residues added to form sulphidryl bonds with
cys residues also inserted into the peptide sequences to be joined.
Other methods of ligation are also envisaged. A suitable straight
segment, but by no means the only suitable straight segment may be
found in the joining chain amino acid resodues 95-112 inclusive of
the CD4 molecule; the sequence disclosed by Paul Jay Maddon and
already referenced herein before. `Cys` residues may be inserted at
any site for example between amino acid 97 and 98 and between 107
and 108 to name but two possible sites.
[0263] The CD4 molecule is part of the immunoglobulin
superfamily.
[0264] The immunoglobulin superfamily provide many examples of
straight and bent chains with cys already inserted for the purposes
of such bonding.
[0265] One is free to choose from any of these peptide segments
without departing from the scope and spirit of the invention as
disclosed.
[0266] One is also free to use other straight or bent peptide
sequences.
TABLE-US-00007 NH.sub.2
val.gln.leu.leu.val.phe.gly.leu.thr.ala.asn.asn.ser.asp.thr.his.-
leu.leu.gln. C Term insert insert cys cys
Exemplary Embodiment Number 8
[0267] Exemplary embodiment number 8 illustrates how a joining
sequence J can be used to join an EBA175 fragment with a GBP130
fragment and both to be joined to a CD4 derived peptide or
variations already listed. The J peptide sequence may be similar to
that already provided or have additional cys residues added.
##STR00026##
[0268] It will be appreciated that branched chains such as the one
illustrated or variations thereof, anyone of which functioning in
the same way would be apparent to the skilled artisan and fall
within the scope and spirit of the disclosure.
Exemplary Embodiment Number 9
[0269] This example envisages molecular machined formed by the
fusion of ZOOANOTIC MEROZOITE derived peptide fragments whose
function in nature is to bind with non human red blood cells, fused
to CD4 segments in the manner already suggested. [0270] Useful
sources of peptides are [0271] Plasmodium Chabaudi [0272]
Plasmodium Yeolii [0273] Plasmodium Berghei [0274] Plasmodium
Gallinacium [0275] Plasmodium Cyanomogli
[0276] The molecular machine so produced would be [0277] (ZOONOSIS)
[0278] Animal parasite
TABLE-US-00008 [0278] merozite derived CD4 RBC binding peptide
[0279] The amino acid sequences of such peptides are to be found in
the Protein Data Bases together with restriction maps, in the
public domain, available on payment; of a fee and incorporated
fully herin by reference. Such novel molecular machines find a use
in HIV testing
METHOD OF USE OF EXEMPLARY EMBODIMENT 9
[0280] A sample of human blood to be tested is mixed with a known
quantity of animal blood treated with the molecular machines of
example 9.
[0281] The sample is agitated and thereafter the animal RBC removed
selectively by anti-animal RBC antibodies bound to argarose or
polyacrylamide gels or the like. Fluorescent labelled anti HIV
antibody is then employed demonstrate virus particles adherent to
the animal red cell. Quantification may then take place by
automated systems in the usual way.
Experimental Verification
Experiment 1A: Immunological Characterisation Using Antibodies
Directed Against CD4
[0282] Purified samples of the molecular machines in amounts
ranging from 0.1 ug to 1 ug are used to coat the walls of ELISA
plates and are thereafter incubated with preparations of OKT4A mAb
(Ortho Diagnostics); the activity of the OKT4A are first altered by
incubation with varying amounts of soluble CD4 preparations. A
labelled anti-mouse serum is then used to assess the amount of
bound mouse antibody.
Experiment 1 B: Immunological Characterisation Using Antibodies
Directed Against the Malaria Derived Component
[0283] Purified samples of the molecular machines in amounts
ranging from 0.1 ug to 1 ug are used to coat the walls of ELISA
plates and are thereafter incubated with monoclonal antibodies
directed against the erythrocyte binding peptide component. Binding
is revealed by goat anti mouse antibodies the said goat antibodies
to be labelled by conventional means.
Experiment 2: the Binding of the Disclosed Molecular Machines to
Red Cell Surfaces.
[0284] A buffered solution containing 100,000 human red cells is
incubated with 20 ug of fusion peptides. The washed red cells are
thereafter incubated with 1.times.10.sup.11 M of OKT4A (Ortho
Diagnostics). The RBC's are washed again with appropriate isotonic
buffer and incubated with peroxidase labelled goat anti mouse IGG
serum (BIOYSIS). Further washing and the addition of
3,3',5,5'-tetramethylbenzidine permits assessment of binding by the
measurement of absorbance at 600 nm.
Experiment 3: the Binding Kinetics of RBC: and the Disclosed
Molecular Machines
[0285] A known quantity of a purified sample of fusion peptide 0.1
ug to 1 ug in increments of .times.2 are incubated with 100,000
RBC's in isotonic buffer solution. 125-I labelled OKT4A or
otherwise labelled OKT4A are added at set times 1 min, 2 min, 4, 8,
16, 30 min and 1 hour; the red cells arecentrifuged through Percoll
2 minutes after the addition of 125-1 labelled OKT4A. Accordingly
both the optimum ratio of RBC to molecular machines can be
calculated together with the binding time to saturation of each
incremental dose of fusion peptides.
Experiment 4: the Binding of Live Virus to Red Cells Treated with
the Disclosed Fusion Peptides
[0286] 10 ml of whole blood drawn from HIV Elisa and WB negative
blood donors is centrifuged after mixing with Alserer's solution
and rediluted to haematocrits of 25%, 35%, 45%, 55% and 65%, 75%
this corresponding to the range of haematocrits seen in gross
anaemia with dilution, to packed cells. The whole blood samples are
incubated with amounts of fusion peptides ranging from 0.1 ug in
increasing increments of .times.2 to 10 ug. Thereafter the samples
are incubated after mixing with 1 ml of suspension of cultured
HIV-1 Bru particles having viral dose levels ranging from 50 to
50,000 viral particles per ml. Centrifugation is carried out after
60 minutes. The supernatant and red cell are separately assayed for
bound virus using techniques already described, or by conventional
means.
Experiment 5: In Vitro Demonstration of Clinical Utility
[0287] 50 ml samples of blood are obtained from HIV positive and
AIDS patients and divided into of 10 ml after appropriate buffering
and anticoagulation. One 10 ml sample from each patient is assayed
for viral titre levels by conventional means. The remaining 4
samples of 10 ml of blood from each patient are treated by
molecular machines of exemplary embodiments 1,2,3,4,5,6 and 8 in
amounts ranging from 0.1 ug to 1 mg and thereafter assayed for
viral titre levels by conventional means. Additional smaples are
centrifuged and the packed cells rediluted and assayed for viral
titre levels separate from the plasma also assayed.
Sequence CWU 1
1
* * * * *