Patent application title: Allo and Auto-Reactive T-Cell Epitopes
Inventors:
Stanislaw Joseph Urbaniak (Aberdeen, GB)
Robert Norman Barker (Inverurie, GB)
IPC8 Class: AG01N3353FI
USPC Class:
435 724
Class name: Involving a micro-organism or cell membrane bound antigen or cell membrane bound receptor or cell membrane bound antibody or microbial lysate animal cell leukocyte (e.g., lymphocyte, granulocyte, monocyte, etc.)
Publication date: 2009-03-19
Patent application number: 20090075303
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Patent application title: Allo and Auto-Reactive T-Cell Epitopes
Inventors:
Stanislaw Joseph Urbaniak
Robert Norman Barker
Agents:
LICATA & TYRRELL P.C.
Assignees:
Origin: MARLTON, NJ US
IPC8 Class: AG01N3353FI
USPC Class:
435 724
Abstract:
The present invention relates to a pharmaceutical composition for the
prevention of alloimmunisation of a subject or the immunosuppression of a
response elicited by alloimmunisation of a subject or an autoimmune
haemolytic disease for said composition comprising an immunologically
effective epitope of a rhesus protein or an immunologically active
analogue or derivative thereof.Claims:
1-23. (canceled)
24: A method for determining effect of one or more epitopes from a rhesus protein on a human lymphocyte, in vitro, comprising:(a) stimulating the lymphocyte with one or more epitope/peptide of a rhesus protein;(b) between 4 to 7 days later resuspending the cultures and transferring aliquots into plates prepared in the following manner:(i) coating each well in the plate with monoclonal anticytokine capture antibody;(ii) washing the plate at least once with Hanks Buffered Salt Solution (HBSS);(iii) blocking any non-specific binding using an appropriate solution;(c) incubating the plates with lymphocyte culture for 12-36 hours at 30-40.degree. C. in an atmosphere of substantially 5% CO2 and substantially 95% air;(d) washing the plates at least once with Tween/PBS to remove unbound lymphocytes;(e) introducing an appropriate biotinylated monoclonal detection antibody to each well and incubating for 30-60 minutes at room temperature;(f) washing the plates at least once with Tween/PBS to remove unbound detection antibody;(g) introducing ExtrAvidin-alkaline phosphatase conjugate and incubating for 15-45 minutes;(h) washing the plates at least once with Tween/PBS to remove unbound ExtrAvidin-alkaline phosphatase conjugate;(i) developing the plates with p-nitrophenyl phosphate in 0.05M carbonate alkaline buffer pH 9.6 added to each well; and(j) reading the absorbance at 405 nm.
25: The method according to claim 24 wherein the rhesus protein is selected from the group consisting of RhD, RhC, Rhc, RhE, Rhe and Rh50 protein.
26: The method according to claim 24 wherein the epitope/peptide of a rhesus protein is selected from at lease one of SEQ ID Numbers 2, 5, 6, 11, 12, 14, 28, 29, 31, 38, 39, 44, 47, 50, 51, 66, 75, 77, 78, 79, 81 and 84.
27: The method according to claim 24 wherein the epitope/peptide is artificially synthesized.
28: The method according to claim 24 wherein the monoclonal anti-cytokine capture antibody is specific for IFN-.gamma., IL-10 or TGF-.beta..
Description:
[0001]The present invention relates to the mapping of allo-reactive T-cell
epitopes on the rhesus (RhD and RhCc/Ee) proteins and to the use of such
epitopes to modulate the corresponding immune responses to these
antigens.
[0002]Human blood contains a genetically complex rhesus (Rh) blood group system. For example, humans are either RhD positive or negative and this can lead to problems during transfusions or pregnancy when RhD negative individuals are exposed to RhD positive blood and become immunised to produce anti-D.
[0003]The most important allele in the RhD blood group system is the D antigen. The RhD antigen is carried by the RhD protein which is a transmembrane protein consisting of 417 amino acids with 12 putative transmembrane domains and 6 extracellular loops. A series of peptides have been constructed in the present invention based on the RhD protein each being 15 amino acids (AA) long, and tested in vitro against T-lymphocytes from normal individuals, donors who have been alloimmunised to produce anti-D, and patients with warm type autoimmune haemolytic anaemia.
[0004]The full amino acid sequence of the RhCE polypeptide and the differences in sequence for c, e and D polypeptides is shown in FIG. 1 hereinafter (Reference: The Blood Group Antigen Facts Book, p 94, Editors; M E Reid & C Lomas-Francis, Academic Press London).
[0005]The complexity of the blood system can cause problems during pregnancy when a woman who is RhD negative is carrying a RhD positive fetus, as the woman is at risk of being immunized by the RhD positive blood cells of her own baby. This immunisation can take place during situations when the mother's and baby's blood can become mixed, for example during amniocentesis, antepartum haemorrhage but mainly at parturition.
[0006]Once the mother's immune system has been exposed to RhD positive blood cells, she will produce anti-D antibodies which can cross the placenta and cause Rh haemolytic disease in any subsequent RhD positive pregnancies. Such haemolytic disease can be fatal for the neonate.
[0007]Currently, purified anti-D immunoglobulin is injected whenever a mother is exposed to fetal RhD positive red blood cells which may occur during e.g., amniocentesis, antepartum haemorrhage but mainly at parturition. About 17% of Caucasian women are RhD negative so that most industrialized countries have RhD prevention programmes wherein all RhD negative women receive prophylaxis with anti-D immunoglobulin at delivery or in association with the other high risk events alluded to above. Further in many countries, routine antepartum prophylaxis to minimize the incidence of Rh haemolytic disease is practised.
[0008]There are a number of problems with this approach. In the first place efficacy is never entirely complete since events can be missed or undeclared or a fetal haemorrhage can be larger than the anti-D can neutralize. Secondly, current anti-D immunoglobulin comes from deliberately immunised donors, which puts volunteers, often male (paid or not) at some small risk. In addition it takes at least 12 months to accredit the donors during which time their blood products are not available. For these reasons there is a worldwide shortage of anti-D immunoglobulin. Finally, there are also concerns about the safety of recipients who may be exposed to transfusion transmitted infections such as by inadvertent infection with agents, for example variant Creutzfeld-Jacob Disease (vCJD) for which there is no satisfactory test.
[0009]Other groups that can be at risk from alloimmunisation are those who are regular recipients of bloods products, for example those suffering from haemological malignant disease, sickle cell disease or thalassemia.
[0010]Certain RhD peptides have been found to specifically stimulate the helper T-cells of alloimmunised individuals. Conversely, certain RhD peptides have been found to stimulate the production of immunosuppressive cytokines by helper T-cells. There is furthermore some correlation between the HLA-DR type of allo- and auto-immunised donors and the peptides which stimulate helper T-cell responses.
[0011]An object of the present invention is to provide an effective treatment for subjects that have become alloimmunised or have an autoimmune disease against red blood cells.
[0012]A further objective of the invention is to provide an effective prophylactic to prevent alloimmunisation.
[0013]In a first embodiment of the invention there is provided a pharmaceutical composition for the prevention of alloimmunisation of a subject, said composition comprising an immunologically effective epitope of a rhesus protein or an immunologically active analogue or derivative thereof.
[0014]We have mapped helper T-cell epitopes on the RhD protein. The characterization of a helper epitope that is targeted in most alloimmunised donors and the identification of correlations between HLA-DR type and particular dominant epitopes opens the way for the evaluation of peptide immunotherapy as a novel way to regulate the immune response to RhD and to prevent Rh haemolytic disease and anti-D related transfusion problems.
[0015]Currently, anti-D which is given to pregnant women during significant events in pregnancy may be considered as a passive form of immunotherapy because it has the effect of blocking the effects of immune events on a temporary basis.
[0016]The replacement of passive with active peptide immunotherapy in RhD negative women is an attractive option since safe synthetic tolerogens can be developed and given before pregnancy thus avoiding foetal exposure. Suppression throughout pregnancy would mean that only one injection was necessary, considerably simplifying management of RhD negative women and also it may be possible for the first time to reverse rather than prevent alloimmunisation by administration of tolerogenic peptides to individuals who already have produced anti-D with the objective of "switching-off" the immune response to RhD.
[0017]Tolerogenic peptides to other Rh antigens, as determined by the current invention, would be of equivalent value in preventing, or modifying the production of alloantibodies by the respective antigens, including (but not exclusively) RhC, Rhc, RhE and Rhe; and Rh50 (peptides are shown in Table 4) in autoimmune haemolytic anaemia.
[0018]Accordingly the categories of individual in whom prior immunization would be considered are as follows: -- [0019](1) All women during their child bearing years; and [0020](2) regular recipients of blood products; who might be exposed to blood transfusion for example haemological malignant disease, sickle cell disease and thalassemia.
[0021]Such a pharmaceutical composition can be given to expectant mothers with RhD negative blood and a RhD positive child in this respect, the composition would result in the mother not producing an immune response at any occasion when the fetuses blood comes in contact with her own immune system. In this connection, there is a reduced likelihood that any subsequent baby which is RhD positive would suffer from haemolytic disease.
[0022]The use of synthetic peptides in accordance with the present invention removes concerns about viral infection being transmitted either by anti-D immunoglobulin used for passive immunotherapy or by red blood cells given to volunteer recipients. The time consuming and expensive procedures required to validate accredited donors and donations are also important considerations.
[0023]In addition, by use of these compositions, volunteers who are often RhD negative men, can avoid the usual injection of red blood cells when they are deliberately immunised for the production of anti-D immunoglobulin.
[0024]In a second embodiment of the invention there is provided a pharmaceutical composition for the immunosuppression of a response elicited by alloimmunisation of a subject or an autoimmune haemolytic disease, said composition comprising an immunologically effective epitope of a rhesus protein or an immunologically active analogue or derivative thereof.
[0025]If the immune system of an RhD negative mother has already been in contact with the blood from a RhD positive baby, such a composition can used during subsequent pregnancies with a RhD positive baby to reduce the likelihood of the baby suffering from RhD haemolytic disease.
[0026]In addition, such a composition can be given to patients who have accidentally been given an RhD positive blood transfusion when they are RhD negative. In this connection, the availability of such a composition reduces the need for very large doses of anti-D immunoglobulin for prophylaxis and the likelihood of becoming alloimmunised thereafter.
[0027]Preferably autoimmune disease is idiopathic or secondary autoimmune haemolytic anaemia mediated by `warm-type` autoantibodies. The trigger for this autoimmune disease is unknown and therefore it may occur at anytime and results in the body producing autoantibodies of broad Rh group specificity which attack the bodies own red blood cells.
[0028]Conveniently the rhesus protein is either RhD, RhC, Rhc, RhE or Rhe protein.
[0029]These determine the main Rh-specific antigens found on the surface of a red blood cell.
[0030]In a preferred embodiment an epitope selected from at least one of numbers 2, 5, 6, 6A, 10A, 11, 11A, 12, 12A, 14, 15A, 18A, 28, 29, 31, 38 and 39 hereinbefore set forth.
[0031]The aforementioned are the most common epitopes recognised by T-cells of alloimmunised subjects and those suffering from autoimmune haemolytic anaemia. In autoimmune haemolytic anaemia, the preferred epitopes are 2, 5, 14, 29, 31 and 38. Therefore induced tolerance to such epitopes would stop an immune response being mounted if they appear in the blood of the subject.
[0032]Preferably the epitope is either epitope 12A or 29 since epitope 12A is the most common epitope recognised by alloreactive T-cells, epitope 29 is most commonly recognised in autoimmune haemolytic anaemia.
[0033]Conveniently any of the epitopes or immunoreactive derivatives can be synthesised.
[0034]If the epitope sequences are artificially synthesised microbial contamination is negligible.
[0035]In a third embodiment of the invention there is provided a pharmaceutical composition for the induction of alloimmunisation of a subject, said composition comprising an immunologically effective epitope of a rhesus protein or an immunologically active analogue or derivative thereof disposed in a pharmacologically acceptable vehicle.
[0036]Preferably the rhesus protein is either RhD, RhC, Rhc, RhE or Rhe protein, conveniently an epitope selected from at least one of numbers 2, 5, 6, 6A, 10A, 11, 11A, 12, 12A, 14, 15A, 18A, 28, 29, 31, 38 and 39 hereinbefore set forth.
[0037]Preferably the vehicle is selected such that the composition is in an injectable, oral, rectal, topical or spray-uptake form.
[0038]It is known that mammals may be tolerised to certain stimuli by taking in specific peptide fragments, for example from the nasal mucosa or via the gut. We propose that a good way of abolishing the immune response to RhD in recipient females prior to, during, or after pregnancy is to administer rhesus peptides via the mucosa such as the nasal, buccal, or anal mucosa or transdermally. The peptide fragments in accordance with the present invention will enter via mucosal tissues and effectively tolerise the subject without causing a full blown immune response which may well be the case should the peptide fragments of the present invention reach circulating blood system at the first instance.
[0039]In an injectable form the epitopes can be used to deliberately immunise the subject with an epitope which can for example produce IL-10 or TGF-β which have immunosuppressive effects.
[0040]The outcome of this approach is to develop a "vaccine" using Rh epitopes which will suppress the immune response to Rh proteins.
[0041]In a fourth embodiment of the invention there is provided a tolerising peptide fragment disposed in a pharmacologically effective vehicle, said vehicle being adapted for injection, oral, rectal via a suppository, topical or spray-uptake administration to the subject wherein the tolerising peptide fragment is selected from an epitope of either a RhD, RhC, Rhc, RhE or Rhe protein. Preferably the epitope is selected from at least one of epitope numbers 2, 5, 6, 6A, 10A, 11, 11A, 12, 12A, 14, 15A, 18A, 28, 29, 31, 38 and 39 hereinbefore set forth.
[0042]Thus the pharmaceutically acceptable vehicle may be adapted for transdermal or transmucosal administration or wherein said vehicle may be a formulation with an enteric coating for oral administration.
[0043]In a fifth embodiment of the present invention there is provided a method of tolerizing a subject which comprises administering to said subject a tolerising peptide fragment.
[0044]In a sixth embodiment of the present invention there is provided an epitope from a RhD, RhC, Rhc, RhE or Rhe protein selected from at least one of epitope numbers 2, 5, 6, 6A, 10A, 11, 11A, 12, 12A, 14, 15A, 18A, 28, 29, 31, 38 and 39.
[0045]In a seventh embodiment of the present invention there is provided the use in the manufacture of a medicament for the tolerisation of a patient who may become alloimmunised comprising an epitope selected from a RhD, RhC, Rhc, RhE or Rhe protein or selected from at least one of epitope numbers 2, 5, 6, 6A, 10A, 11, 11A, 12, 12A, 14, 15A, 18A, 28, 29, 31, 38 and 39 disposed in a pharmaceutically acceptable vehicle therefor.
[0046]In an eighth embodiment of the invention there is provided the use in the manufacture of a medicament for the immunosuppression of an alloimmunised patient or a patient with warm-type autoimmune haemolytic anaemia comprising an epitope selected from a RhD, RhC, Rhc, RhE or Rhe protein or selected from at least one of epitope numbers 2, 5, 6, 6A, 10A, 11, 11A, 12, 12A, 14, 15A, 18A, 28, 29, 31, 38 and 39 disposed in a pharmaceutically acceptable vehicle therefor.
[0047]In a ninth embodiment of the invention there is provided a method for determining the effect of an epitope from a rhesus protein on a human lymphocyte, in vitro, comprising the steps of:--
[0048]a) stimulating the lymphocyte with one or more epitope of a rhesus protein;
[0049]b) between 4 and 7 days later resuspending the cultures and transferring aliquots into plates prepared in the following manner;
[0050]c) washing the plate at least once with Hanks Buffered Salt Solution (HBSS);
[0051]d) coating each well in the plate with monoclonal anti-cytokine capture antibody;
[0052]e) blocking any non-specific binding using an appropriate solution;
[0053]f) incubating the plates with the lymphocyte culture for 12-36 hours at 30-40° C. in an atmosphere of substantially 5% CO2 and substantially 95% air;
[0054]g) washing the plates at least once with Tween/PBS;
[0055]h) introducing an appropriate biotinylated monoclonal detection antibody to each well and incubating for 30-60 min at room temperature;
[0056]I) washing the plates at least once with Tween/PBS;
[0057]j) introducing ExtrAvidin-alkaline phosphatase conjugate and incubating for 15-45 mins;
[0058]k) washing the plates again at least once with Tween/PBS;
[0059]l) developing the plates with p-nitrophenyl phosphate in 0.05M carbonate alkaline buffer pH9.6 added to each well; and
[0060]m) reading the absorbance at 405 nm.
[0061]Traditionally, among other techniques, researchers have used a captive assay called ELISPOT to determine the amount of cytokines produced by a cell. This assay produces a colour spot for each cytokine producing cell. A crude calculation based on the number of coloured spots is then used to estimate the amount of cytokines produced. The use of p-nitrophenyl phosphate in the present assay allows the amount of cytokine captured by the antibody in the wall to be established on the basis of the colour change produced which can be measured by the more accurate method of spectrophotometry.
[0062]Accordingly, this method is very sensitive and therefore can identify that a particular RhD protein is capable of stimulating human T-cells to produce potentially immunosuppressive cytokines rather than to proliferate. This is important for the determination of the method of delivery of an epitope. An epitope which leads to T-cell proliferation may be given as a tolerogen through the nasal or mucosal route whereas an epitope which leads to immunosuppressive cytokines may be injected.
[0063]In a tenth embodiment of the present invention there is provided a method for the determination of the propensity of a RhD negative subject to produce anti-D antibodies after exposure to RhD positive blood comprising ascertaining the tissue type of the subject and determining if they are HLA-DRB1*15.
[0064]If the subject has a tissue type of HLA-DRB1*15 they are more likely to raise anti-D antibodies therefore they should be given treatment before being put at risk of exposure to RhD positive red blood cells.
[0065]The invention will now be described, by way of illustration only, with reference to the following examples and the accompanying figures.
[0066]FIG. 1 shows the full amino acid sequence of RhCE polypeptide; differences in the sequence for Rhc, Rhe and RhD polypeptides are also shown (Reference: The Blood Group Antigen Facts Book P94, Editor; M E Reid & C Lomas-Francis, Academic Press London).
[0067]FIG. 2 shows the distribution of stimulatory RhD peptides in donors alloimmunised with RhD antigen from peptides 1 to 42 and 6A to 40A as per Tables 1, 2 and 3; x--RhD peptide added to culture; y--percentage of subjects responding to specific RhD peptides.
[0068]FIG. 3A shows the distribution of stimulating RhD peptides in autoimmune haemolytic anaemia patients; x--RhD peptide stimulus; y--percentage of subjects responding to specific RhD peptides.
[0069]FIG. 3B shows the distribution of stimulating RhD peptides in normal controls; x--RhD peptide stimulus; y--percentage of subjects responding to specific RhD peptides.
[0070]FIG. 4 shows the correlation between Rh epitopes recognised in donors sharing a tissue type. X and Y axes represent the stimulation indices for donors 1 and 2 respectively. Each square represents the response to a peptide. Correlation co-efficient (R)=0.774, p value 9.57E-015
[0071]FIG. 5A shows the response pattern to the induction of TGF-β production of T-cells after incubation with Rh peptides; x--RhD peptide stimulus; y--TGF-β1 secretion (pg/ml). Value D=none.
[0072]FIG. 5B shows the response pattern to the induction of IL-10 production of T-cells after incubation with Rh peptides; x--RhD peptide stimulus; y--IL-10 secretion (ng/ml). Value D=none.
[0073]FIG. 5C shows the response pattern to the induction of IFN-γ production of T-cells after incubation with Rh peptides; x--RhD peptide stimulus; y--IFN-γ secretion (ng/ml). Value D=none.
[0074]FIG. 5D shows the amount of incorporation of 3H-Thymidine into T-cells after incubation with Rh peptides; x--RhD peptide stimulus; y--3H-Thymidine incorporation (mean CPM×10-3±SD) SI=3. Value D=none.
[0075]FIG. 6 shows the inhibition of T-cells that respond to RhD protein by peptides that generate an immunosuppressive cytokine response; x--RhD peptide stimulus; y--3H-Thymidine incorporation (mean CPM×10-3±SD). A--none; B--control (-); C--RhD; D--RhD & 16; E--RhD & 22; F--RhD & 24; G--none; H--PPD; I--PPD & 16; J--PPD & 22; K--PPD & 24.
EXAMPLE 1
[0076]Two complete panels of 68 15-mer peptides, with 5 or 10 amino acid overlaps, were synthesized (Multiple Peptide Service, Cambridge Research Biochemicals, Cheshire, UK and Dept. Of Biochemistry, University of Bristol, UK), corresponding to the sequences of the 30 kD Rh proteins associated with expression of the RhD or RhCc/Ee blood group antigens respectively. The amino acid sequences for each of these proteins were deduced independently from cDNA analyses by 2 laboratories. Since the two polypeptide sequences show 92% homology, 16 of the synthetic peptides were shared between the panels (numbering from the amino terminus, peptides 1-5, 8, 9, 14, 21, 28, 29, 37-39, 41 and 42). In order to ensure purity, each panel was synthesized by fluorenylmethoxycarbonyl chemistry on resin using a base-labile linker, rather than by conventional pin technology, and randomly selected peptides were screened for purity by HPLC and amino acid analysis. The peptides were used to stimulate cultures at 20 μg/ml, although it should be noted that the responses of the cultures had previously been shown to be similar in magnitude and kinetics at peptide concentration between 5-20 μg/ml.
[0077]The control antigens Mycobacterium tuberculosis purified protein derivative (PPD) (Statens Seruminstut, Denmark) and keyhole limpet hemocyanin (KLH) (Calbiochem-Behring, La Jolla, Calif., USA) were dialysed extensively against phosphate buffered saline pH 7.4 (PBS) and filter sterilized before addition to cultures at 50 μg/ml, PPD, but not KLH, readily provokes recall T-cell responses in vitro, since most UK citizens have been immunised with BCG. Concanavalin A (Con A) was obtained from Sigma, Poole, Dorset, UK, and used to stimulate cultures at 10 μg/ml.
[0078]Antibodies
[0079]FITC- or phycoerythrin-conjugated in Abs against human CD3, CD19, CD45 or CD14 were obtained from Dako UK Ltd. Blocking mAbs specific for HLA-DP, -DQ, or -DR supplied by Becton Dickinson (Oxford, UK) were dialysed thoroughly against PBS before addition to cultures at the previously determined optimum concentration of 2.5 μg/ml.
[0080]Isolation of Peripheral Blood Mononuclear Cells and T-Cells
[0081]Peripheral blood mononuclear cells (PBMC) from donors or patients were separated from fresh blood samples using Ficoll-Hypaque. The donors and patients had become alloimmunised with RhD positive blood either through pregnancy, a blood transfusion or through immunization with the relevant blood.
[0082]The viability of PBMC was greater than 90% in all experiments, as judged by trypan blue exclusion. T-cells were isolated from PBMC by passage through glass bead affinity columns coated with human IgG/sheep anti-human IgG immune complexes. Flow cytometry (Becton Dickinson FACScan) demonstrated that typical preparations contained more than 95% T-cells.
[0083]Cell Proliferation Assays
[0084]PBMC were cultured in 100 μl volumes in microtitre plates at a concentration of 1.25×106 cells/ml in an Alpha Modification of Eagle's Medium (ICN Flow, Bucks UK) supplemented with 5% autologous serum, 4 mM L-Glutamine (Gibco, Paisley, UK), 100 U/ml sodium benzylpenicillin G (Sigma), 100 μg/ml streptomycin sulphate (Sigma), 5×10-5M 2-mercaptoethanol (Sigma) and 20 mM HEPES pH7.2 (Sigma). All plates were incubated at 37° C. in a humidified atmosphere of 5% CO2/95% air. The cell proliferation in cultures was estimated from the incorporation of 3H-Thymidine in triplicate wells 5 days after stimulation with antigen as described previously. Proliferation results are presented either as the mean CPM+/-SD of the triplicate samples, or as a stimulation index (SI), expressing the ratio of mean CPM in stimulated versus unstimulated control cultures. An S1>3 with CPM>1000 is interpreted as representing a positive response.
[0085]Activation Assay
[0086]The aforementioned experiments were designed to minimise the response by quiescent or naive T-cells that can recognise RhD protein, but which are not activated by immunisation. To validate the experiments, the T-cells proliferated in the aforementioned experiment were tested using a modification of the method set out in European Journal of Immunology (1994) 24: 1578-1582 to identify if they had been activated in vivo. In this connection, the T-cells were screened to ascertain if they were from the subset bearing CD45RO which is a marker of previous activation or "memory", rather than from the subset bearing CD45RA which is the marker of quiescent or "naive" T-cells.
[0087]As shown in FIG. 2 various peptide fragments have been selected in accordance with their particular peptide sequences. These are given in Tables 1, 2 and 3 which follow and the results achieved by means of the foregoing example are shown in FIG. 2.
[0088]Accordingly we have shown that helper T-cells from all donors deliberately immunised against RhD can be stimulated in vitro by RhD peptides.
[0089]Further there is a variation between alloimmune donors in the T-cell response profile to the RhD peptides. Despite these variations, RhD peptides Nos. 2, 6, 6A, 10A, 11, 11A, 12, 12A, 15A, 18A, 28 and 39 are most commonly targeted and a proliferative response was elicited by peptide 12A in 70% of donors. However significantly related profiles are found in donors sharing HLA-DR alleles. It is predicted that alloreactive T-cell epitopes on the RhD protein would comprise sequences that are foreign to RhD-negative individuals, and would thus not be carried on the related RhCc/Ee protein that is expressed on the erythrocytes of such individuals. With the exception of peptide 28, all of the fragments identified are sequences that fulfil this prediction. It is therefore considered that such peptides, or derived sequences, could be used to stimulate either T-cell response or tolerance in vivo as desired, depending on the route of administration and/or the dose and formulation of the preparation.
[0090]The T-cells which were proliferated were in fact drawn from those that have been previously activated. This is important because it is these cells which will drive anti-D antibody production in RhD-negative donors immunised with RhD.
[0091]It follows that the characterisation of the putative helper T-cell epitopes we have identified is a key step in the development of safe immunogens for anti-immunoglobulin donors and opens the way to the evaluation of peptide immunotherapy as a novel approach to the prevention of haemolytic disease inter alia in neonates.
[0092]These experiments can be carried out using other rhesus proteins, such as RhC, Rhc, RhE or Rhe protein.
[0093]The aforementioned experiments were repeated using blood from subjects suffering from autoimmune haemolytic anaemia. It was therefore established that the T-cells of the subjects exhibited a proliferative response to peptides 2, 5, 14, 29, 31 and 38 (see FIG. 3) and 65% of patients responded to peptide 29. The results also showed a correlation between patients suffering from autoimmune haemolytic anaemia and having tissue type HLA-DR15. With the exception of peptide 31 all of the peptides are shared in common between the RhD and RhCe/Ee proteins.
EXAMPLE 2
[0094]The HLA class II tissue type of the donors tested in Example 1 was ascertained by standard SSP-PCR methods. This was carried out because the molecules that determine tissue type select and bind antigenic peptide fragments for display to T-cells therefore they are important in this investigation.
[0095]The techniques described in Barker et al (1997) Blood 90:2701-2715 were used to determine that the HLA-D loci was more important than either the HLA-DP or HLA-DQ in the presentation of Rh D peptide fragments that stimulate T-cells in vitro.
[0096]A significant proportion of Rh D-negative donors selected for responsiveness to Rh D carry the HLA-DRB1*15 gene (56% versus approx. 29% in a control population). Thus carrying this tissue type is associated with an increase risk of producing anti-D antibodies after exposure to RhD positive erythrocytes, and there is smaller variation in HLA-DR tissue type among responders than in the general population. It has also been shown that the patterns of RhD peptides that elicit T-cell proliferation are significantly related in Rh D-negative donors who share the same HLA-DR type (see FIGS. 3A and 3B).
[0097]For warm-type autoimmune haemolytic anaemia there is also an association with HLA DR15 with 65% of patients carrying this HLA type.
[0098]Nevertheless, a statistical analysis of all the data shows that the effect of HLA-DR type on the identity of the peptides recognised is relatively weak. In other words, many of the RhD peptides stimulate T-cells regardless of tissue type.
[0099]These analyses demonstrate that the selection of RhD peptide fragments for immunisation/tolerisation regimes may not be dependent on prior tissue typing of recipients, an important practical consideration for the clinical application of this approach.
EXAMPLE 3
[0100]Cultured T-cells are stimulated with each of the epitopes given in Tables 1 to 3 and after 5 days the responding cells were transferred to a flat-bottomed microtitre plates (96-well Nunc-Immuno Maxisorp) coated with 50 μl per well of monoclonal anti-cytokine capture antibody diluted in 0.05M alkaline carbonate coating buffer pH 9.6. Unbound capture antibody was removed by two washes with HBSS and non-specific binding potential blocked by incubation with 200 μl per well of phosphate buffered saline, pH 7.4 (PBS containing 3% BSA).
[0101]Five days after stimulation, lymphocyte cultures were mixed to resuspend the cells and duplicate 100 μl aliquots were transferred into wells coated with the respective capture antibody specific for IFN-γ and or IL-10 or TGF-β. The plates coated with capture antibodies and layered by lymphocytes were then incubated for a further 24 hours at 37° C. in a humidified atmosphere of 5% CO2 and 95% air. After this incubation the PBMC were removed by four washes with 0.2% Tween/PBS. One hundred microlitre aliquots of the appropriate biotinylated monoclonal detection antibody in 0.2% BSA/PBS were then added to the wells and incubated at room temperature for 45 minutes. After six washes with 0.5% Tween/PBS, 100 μl of 1:100,000 ExtrAvidin-alkaline phosphatase conjugate (Sigma) was then added to each of the wells and incubated at room temperature for 30 minutes. The ExtrAvidin conjugate was removed by eight washes with 0.2% Tween/PBS, and the plates developed using 100 μl per well of p-nitrophenyl phosphate (Sigma) 1.0 mg/ml in 0.05M carbonate alkaline buffer pH 9.6. The absorbence of 405 nm was then measured using a Multiscan plate reader (Labsystems Basingstoke UK).
[0102]Cytokine secretion was measured by interpolation from a standard curve generated by incubating duplicate wells with doubling dilutions of recombinant human IFN-γ or IL-10 or TGF-β (Pharmingen). The standard curves were modelled by a smoothed cubic spline function applied to the absorbence reading and the cytokine concentrations after a quasilogarithmic transformation, where:
quasiloge(z)=loge[z+ [z2+1]).
[0103]This method is very sensitive and therefore can identify that a particular RhD peptide is capable of stimulating human T-cells to produce potentially immunosuppressive cytokines rather than to proliferate.
[0104]From FIGS. 5A and 5B it can be seen that epitopes 10, 16, 22, 24 and 34 induce IL-10 and/or TGF-β production by human T-cells. IL-10 and TGF-β molecules are known to have immunosuppressive properties. In preliminary experiments RhD peptides that induce IL-10 have been shown to inhibit T-cell proliferation in response to the entire RhD protein in vitro. Accordingly, prior administration of RhD peptides that elicit T-cell IL-10 or TGF-β production can be used to prevent RhD negative individuals from responding to RhD. It is also possible to inhibit established responses. This novel approach to manipulating the immune system has other application, including treatment of warm-type autoimmune haemolytic anaemia, in which the Rh proteins are important targets. The identification of peptides with similar properties derived from other antigens could also lead to therapy for a wide range of autoimmune diseases where the antigens/proteins are identified.
[0105]No IL-4 production was detected in any culture. In FIG. 5C it can be seen that epitopes 5, 21 and 27 stimulate IFN-γ secretion. FIG. 5D shows the level of incorporation of 3H-Thymidine into the T-cells after stimulation with the RhD peptides.
[0106]From FIG. 6 it can be seen that the addition of such peptides to T-cell cultures specifically blocks the proliferative response to the RhD protein, but not to a control antigen PPD. This result is very important since it raises the possibility that these peptides may also be able to inhibit damaging responses in vivo if given to patients, whilst not suppressing the rest of the immune system.
TABLE-US-00001 TABLE 1 PEPTIDE NUMBER PEPTIDE SEQUENCE RESIDUES RhCE (R2 cE) 1 SSKYPRSVRRCLPLW 2-16 2 CLPLWALTLEAALIL 12-26 3 AALILLFYFFTHYDA 22-36 4 THYDASLEDQKGLVA 32-46 5 KGLVASYQVGQDLTV 42-56 6 QDLTVMAALGLGFLT 52-66 7 LGFLTSNFRRHSWSS 62-76 8 HSWSSVAFNLFMLAL 72-86 9 FMLALGVQWAILLDG 82-96 10 ILLDGFLSQFPPGKV 92-106 11 PPGKVVITLFSIRLA 102-116 12 SIRLATMSAMSVLIS 112-126 13 SVLISAGAVLGKVNL 122-136 14 GKVNLAQLVVMVLVE 132-146 15 MVLVEVTALGTLRMV 142-156 16 TLRMVISNIFNTDYH 152-166 17 NTDYHMNLRHFYVFA 162-176 18 FYVFAAYFGLTVAWC 172-186 19 TVAWCLPKPLPKGTE 182-196 20 PKGTEDNDQRATIPS 192-206 21 ATIPSLSAMLGALFL 202-216 22 GALFLWMFWPSVNSP 212-226 23 SVNSPLLRSPIQRKN 222-236 24 IQRKNAMFNTYYALA 232-246 25 YYALAVSVVTAISGS 242-256 26 AISGSSLAHPQRKIS 252-266 27 QRKISMTYVHSAVLA 262-276 28 SAVLAGGVAVGTSCH 272-286 29 GTSCHLIPSPWLAMV 282-296 30 WLAMVLGLVAGLISI 292-306 31 GLISIGGAKCLPVCC 302-316 32 LPVCCNRVLGIHHIS 312-326 33 IHHISVMHSIFSLLG 322-336 34 FSLLGLLGEITYIVL 332-346 35 TYIVLLVLHTVWNGN 342-356 36 VWNGNGMIGFQVLLS 352-366 37 QVLLSIGELSLAIVI 362-376 38 LAIVIALTSGLLTGL 372-386 39 LLTGLLLNLKIWKAP 382-396 40 IWKAPHVAKYFDDQV 392-406 41 FDDQVFWKFPHLAVG 402-416 42 DDQVFWKFPHLAVGF 403-417
TABLE-US-00002 TABLE 2 PEPTIDE NUMBER PEPTIDE SEQUENCE RESIDUES RhCE (R1 Ce) 1 (C) SSKYPRSVRRCLPLC 2-16 2 (C) CLPLCALTLEAALIL 12-26 22 (e) GALFLWMFWPSVNSA 212-226 23 (e) SVNSALLRSPIQRKN 222-236 RhD 6 (also C) QDLTVMAAIGLGFLT 52-66 7 (also C) LGFLTSSFRRHSWSS 62-76 10 (also C) ILLDGFLSQFPSGKV 92-106 11 (also C) PSGKVVITLFSIRLA 102-116 12 SIRLATMSALSVLIS 112-126 13 SVLISVDAVLGKVNL 122-136 15 MVLVEVTALGNLRMV 142-156 16 NLRMVISNIFNTDYH 152-166 17 NTDYHMNMMHIYVFA 162-176 18 IYVFAAYFGLSVAWC 172-186 19 SVAWCLPKPLPEGTE 182-196 20 PEGTEDKDQTATIPS 192-206 22 GALFLWIFWPSFNSA 212-226 23 SFNSALLRSPIERKN 222-236 24 IERKNAVFNTYYAVA 232-246 25 YYAVAVSVVTAISGS 242-256 26 AISGSSLAHPQGKIS 252-266 27 QGKISKTYVHSAVLA 262-276 30 WLAMVLGLVAGLISV 292-306 31 GLISVGGAKYLPGCC 302-316 32 LPGCCNRVLGIPHSS 312-326 33 IPHSSINGYNFSLLG 322-336 34 FSLLGLLGEIIYIVL 332-346 35 IYIVLLVLDTVGAGN 342-356 36 VGAGNGMIGFQVLLS 352-366 40 IWKAPHEAKYFDDQV 392-406
TABLE-US-00003 TABLE 3 PEPTIDE NUMBER PEPTIDE SEQUENCE RESIDUES RhCE (R1 Ce) 1A (C) RSVRRCLPLCALTLE 7-21 22A (e) WMFWPSVNSALLRSP 217-231 RhD 6A (also C) MAAIGLGFLTSSFRR 57-71 7A (also C) SSFRRHSWSSVAFNL 67-81 10A (also C) FLSQFPSGKVVITLF 97-111 11A (also C) VITLFSIRLATMSAL 107-121 12A TMSALSVLISVDAVL 117-131 13A VDAVLGKVNLAQLVV 127-141 15A VTALGNLRMVISNIF 147-161 16A ISNIFNTDYHMNMMH 157-171 17A MNMMHIYVFAAYFGL 167-181 18A AYFGLSVAWCLPKPL 177-191 19A LPKPLPEGTEDKDQT 187-201 20A DKDQTATIPSLSAML 197-211 21A LSAMLGALFLWIFWP 207-221 22A WIFWPSFNSALLRSP 217-231 23A LLRSPIERKNAVFNT 227-241 24A AVFNTYYAVAVSVVT 237-251 26A SLAHPQGKISKTYVH 257-271 27A KTYVKSAVLAGGVAV 267-281 30A LGLVAGLISVGGAKY 297-311 31A GGAKYLPGCCNRVLG 307-321 32A NRVLGIPHSSIMGYN 317-331 33A IMGYNFSLLGLLGEI 327-341 34A LLGEIIYIVLLVLDT 337-351 35A LVLDTVGAGNGMIGF 347-361 39A LLNLKIWKAPHEAKY 387-401 40A HEAKYFDDQVFWKFP 397-411
TABLE-US-00004 TABLE 4 PEPTIDE NUMBER PEPTIDE SEQUENCE RESIDUES Rh50 GP 1 MRFTFPLMAIVLEIA 1-15 2 VLEIAMIVLFGLFVE 11-25 3 GLFVEYETDQTVLEQ 21-35 4 TVLEQLNITKPTDMG 31-45 5 PTDMGIFFELYPLFQ 41-55 6 YPLFQDVHVMIFVGF 51-65 7 IFVGFGFLMTFLKKY 61-75 8 FLKKYGFSSVGINLL 71-85 9 GINLLVAALGLQWGT 81-95 10 LQWGTIVQGILQSQG 91-105 11 LQSQGQKFNIGIKNM 101-115 12 GIKNMINADFSAATV 111-125 13 SAATVLISFGAVLGK 121-135 14 AVLGKTSPTQMLIMT 131-145 15 MLIMTILEIVFFAHN 141-155 16 FFAHNEYLVSEIFKA 151-165 17 EIFKASDIGASMTIH 161-175 18 SMTIHAFGAYFGLAV 171-185 19 FGLAVAGILYRSGLR 181-195 20 RSGLRKGHENEESAY 191-205 21 EESAYYSDLFAMIGT 201-215 22 AMIGTLFLWMFWPSF 211-225 23 FWPSFNSAIAEPGDK 221-235 24 EPGDKQCRAIVDTYF 231-245 25 VDTYFSLAACVLTAF 241-255 26 VLTAFAFSSLVEHRG 251-265 27 VEHRGKLNMVHIQNA 261-275 28 HIQNATLAGGVAVGT 271-285 29 VAVGTCADMAIHPFG 281-295 30 IHPFGSMIIGSIAGM 291-305 31 SIAGMVSVLGYKFLT 301-315 32 YKFLTPLFTTKLRIH 311-325 33 KLRIHDTCGVHNLHG 321-335 34 HNLHGLPGVVGGLAG 331-345 35 GGLAGIVAVAMGASN 341-355 36 MGASNTSMAMQAAAL 351-365 37 QAAALGSSIGTAVVG 361-375 38 TAVVGGLMTGLILKL 371-385 39 LILKLPLWGQPSDQN 381-395 40 PSDQNCYDDSVYWKV 391-405 41 NCYDDSVYWKVPKTR 395-409 Other Peptides BR SKYPNCAYKTTQANKH AV2 TIPEQSFQGSPSADT AV4 TVKADFEFSSAPAPD AV6 TVEERQQFGELPVSE P23 ELKIISRCQVCMKKRH HA PKYVKQNTLKLAT
Sequence CWU
1
152115PRTHomo sapiensResidues 2-16 1Ser Ser Lys Tyr Pro Arg Ser Val Arg
Arg Cys Leu Pro Leu Trp 1 5 10
15215PRTHomo sapiensResidues 12-26 2Cys Leu Pro Leu Trp Ala Leu Thr
Leu Glu Ala Ala Leu Ile Leu 1 5 10
15315PRTHomo sapiensRhCE (R2 CE) Residues 22-36 3Ala Ala Leu
Ile Leu Leu Phe Tyr Phe Phe Thr His Tyr Asp Ala 1 5
10 15415PRTHomo sapiensRhCE (R2 CE) Residues
32-46 4Thr His Tyr Asp Ala Ser Leu Glu Asp Gln Lys Gly Leu Val Ala 1
5 10 15515PRTHomo sapiensRhCE
(R2 CE) Residues 42-56 5Lys Gly Leu Val Ala Ser Tyr Gln Val Gly Gln Asp
Leu Thr Val 1 5 10
15615PRTHomo sapiensRhCE (R2 CE) Residues 52-66 6Gln Asp Leu Thr Val Met
Ala Ala Leu Gly Leu Gly Phe Leu Thr 1 5
10 15715PRTHomo sapiensRhCE (R2 CE) Residues 62-76 7Leu
Gly Phe Leu Thr Ser Asn Phe Arg Arg His Ser Trp Ser Ser 1
5 10 15815PRTHomo sapiensRhCE (R2 CE)
Residues 72-86 8His Ser Trp Ser Ser Val Ala Phe Asn Leu Phe Met Leu Ala
Leu 1 5 10 15915PRTHomo
sapiensRhCE (R2 CE) Residues 82-96 9Phe Met Leu Ala Leu Gly Val Gln Trp
Ala Ile Leu Leu Asp Gly 1 5 10
151015PRTHomo sapiensRhCE (R2 CE) Residues 92-106 10Ile Leu Leu Asp
Gly Phe Leu Ser Gln Phe Pro Pro Gly Lys Val 1 5
10 151115PRTHomo sapiensRhCE (R2 CE) Residues
102-116 11Pro Pro Gly Lys Val Val Ile Thr Leu Phe Ser Ile Arg Leu Ala 1
5 10 151215PRTHomo
sapiensRhCE (R2 CE) Residues 112-126 12Ser Ile Arg Leu Ala Thr Met Ser
Ala Met Ser Val Leu Ile Ser 1 5 10
151315PRTHomo sapiensRhCE (R2 CE) Residues 122-136 13Ser Val
Leu Ile Ser Ala Gly Ala Val Leu Gly Lys Val Asn Leu 1 5
10 151415PRTHomo sapiensRhCE (R2 CE)
Residues 132-146 14Gly Lys Val Asn Leu Ala Gln Leu Val Val Met Val Leu
Val Glu 1 5 10
151515PRTHomo sapiensRhCE (R2 CE) Residues 142-156 15Met Val Leu Val Glu
Val Thr Ala Leu Gly Thr Leu Arg Met Val 1 5
10 151615PRTHomo sapiensRhCE (R2 CE) Residues 152-166
16Thr Leu Arg Met Val Ile Ser Asn Ile Phe Asn Thr Asp Tyr His 1
5 10 151715PRTHomo sapiensRhCE (R2
CE) Residues 162-176 17Asn Thr Asp Tyr His Met Asn Leu Arg His Phe Tyr
Val Phe Ala 1 5 10
151815PRTHomo sapiensRhCE (R2 CE) Residues 172-186 18Phe Tyr Val Phe Ala
Ala Tyr Phe Gly Leu Thr Val Ala Trp Cys 1 5
10 151915PRTHomo sapiensRhCE (R2 CE) Residues 182-196
19Thr Val Ala Trp Cys Leu Pro Lys Pro Leu Pro Lys Gly Thr Glu 1
5 10 152015PRTHomo sapiensRhCE (R2
CE) Residues 192-206 20Pro Lys Gly Thr Glu Asp Asn Asp Gln Arg Ala Thr
Ile Pro Ser 1 5 10
152115PRTHomo sapiensRhCE (R2 CE) Residues 202-216 21Ala Thr Ile Pro Ser
Leu Ser Ala Met Leu Gly Ala Leu Phe Leu 1 5
10 152215PRTHomo sapiensRhCE (R2 CE) Residues 212-226
22Gly Ala Leu Phe Leu Trp Met Phe Trp Pro Ser Val Asn Ser Pro 1
5 10 152315PRTHomo sapiensRhCE (R2
CE) Residues 222-236 23Ser Val Asn Ser Pro Leu Leu Arg Ser Pro Ile Gln
Arg Lys Asn 1 5 10
152415PRTHomo sapiensRhCE (R2 CE) Residues 232-246 24Ile Gln Arg Lys Asn
Ala Met Phe Asn Thr Tyr Tyr Ala Leu Ala 1 5
10 152515PRTHomo sapiensRhCE (R2 CE) Residues 242-256
25Tyr Tyr Ala Leu Ala Val Ser Val Val Thr Ala Ile Ser Gly Ser 1
5 10 152615PRTHomo sapiensRhCE (R2
CE) Residues 252-266 26Ala Ile Ser Gly Ser Ser Leu Ala His Pro Gln Arg
Lys Ile Ser 1 5 10
152715PRTHomo sapiensRhCE (R2 CE) Residues 262-276 27Gln Arg Lys Ile Ser
Met Thr Tyr Val His Ser Ala Val Leu Ala 1 5
10 152815PRTHomo sapiensRhCE (R2 CE) Residues 272-286
28Ser Ala Val Leu Ala Gly Gly Val Ala Val Gly Thr Ser Cys His 1
5 10 152915PRTHomo sapiensRhCE (R2
CE) Residues 282-296 29Gly Thr Ser Cys His Leu Ile Pro Ser Pro Trp Leu
Ala Met Val 1 5 10
153015PRTHomo sapiensRhCE (R2 CE) Residues 292-306 30Trp Leu Ala Met Val
Leu Gly Leu Val Ala Gly Leu Ile Ser Ile 1 5
10 153115PRTHomo sapiensRhCE (R2 CE) Residues 302-316
31Gly Leu Ile Ser Ile Gly Gly Ala Lys Cys Leu Pro Val Cys Cys 1
5 10 153215PRTHomo sapiensRhCE (R2
CE) Residues 312-326 32Leu Pro Val Cys Cys Asn Arg Val Leu Gly Ile His
His Ile Ser 1 5 10
153315PRTHomo sapiensRhCE (R2 CE) Residues 322-336 33Ile His His Ile Ser
Val Met His Ser Ile Phe Ser Leu Leu Gly 1 5
10 153415PRTHomo sapiensRhCE (R2 CE) Residues 332-346
34Phe Ser Leu Leu Gly Leu Leu Gly Glu Ile Thr Tyr Ile Val Leu 1
5 10 153515PRTHomo sapiensRhCE (R2
CE) Residues 342-356 35Thr Tyr Ile Val Leu Leu Val Leu His Thr Val Trp
Asn Gly Asn 1 5 10
153615PRTHomo sapiensRhCE (R2 CE) Residues 352-366 36Val Trp Asn Gly Asn
Gly Met Ile Gly Phe Gln Val Leu Leu Ser 1 5
10 153715PRTHomo sapiensRhCE (R2 CE) Residues 362-376
37Gln Val Leu Leu Ser Ile Gly Glu Leu Ser Leu Ala Ile Val Ile 1
5 10 153815PRTHomo sapiensRhCE (R2
CE) Residues 372-386 38Leu Ala Ile Val Ile Ala Leu Thr Ser Gly Leu Leu
Thr Gly Leu 1 5 10
153915PRTHomo sapiensRhCE (R2 CE) Residues 382-396 39Leu Leu Thr Gly Leu
Leu Leu Asn Leu Lys Ile Trp Lys Ala Pro 1 5
10 154015PRTHomo sapiensRhCE (R2 CE) Residues 392-406
40Ile Trp Lys Ala Pro His Val Ala Lys Tyr Phe Asp Asp Gln Val 1
5 10 154115PRTHomo sapiensRhCE (R2
cE) Residues 111-125 41Phe Asp Asp Gln Val Phe Trp Lys Phe Pro His Leu
Ala Val Gly 1 5 10
154215PRTHomo sapiensRhCE (R2 cE) Residues 403-417 42Asp Asp Gln Val Phe
Trp Lys Phe Pro His Leu Ala Val Gly Phe 1 5
10 154315PRTHomo sapiensRhCE (R1 Ce) Residues 2-16
43Ser Ser Lys Tyr Pro Arg Ser Val Arg Arg Cys Leu Pro Leu Cys 1
5 10 154415PRTHomo sapiensRhCE (R1
Ce) Residues 12-26 44Cys Leu Pro Leu Cys Ala Leu Thr Leu Glu Ala Ala Leu
Ile Leu 1 5 10
154515PRTHomo sapiensRhCE (R1 Ce) Residues 212-226 45Gly Ala Leu Phe Leu
Trp Met Phe Trp Pro Ser Val Asn Ser Ala 1 5
10 154615PRTHomo sapiensRhCE (R1 Ce) Residues 222-236
46Ser Val Asn Ser Ala Leu Leu Arg Ser Pro Ile Gln Arg Lys Asn 1
5 10 154715PRTHomo sapiensRhD
Residues 52-66 47Gln Asp Leu Thr Val Met Ala Ala Ile Gly Leu Gly Phe Leu
Thr 1 5 10 154815PRTHomo
sapiensRhD Residues 62-76 48Leu Gly Phe Leu Thr Ser Ser Phe Arg Arg His
Ser Trp Ser Ser 1 5 10
154915PRTHomo sapiensRhD Residues 92-106 49Ile Leu Leu Asp Gly Phe Leu
Ser Gln Phe Pro Ser Gly Lys Val 1 5 10
155015PRTHomo sapiensRhD Residues 102-116 50Pro Ser Gly Lys
Val Val Ile Thr Leu Phe Ser Ile Arg Leu Ala 1 5
10 155115PRTHomo sapiensRhD Residues 112-126 51Ser
Ile Arg Leu Ala Thr Met Ser Ala Leu Ser Val Leu Ile Ser 1
5 10 155215PRTHomo sapiensRhD Residues
122-136 52Ser Val Leu Ile Ser Val Asp Ala Val Leu Gly Lys Val Asn Leu 1
5 10 155315PRTHomo
sapiensRhD Residues 142-156 53Met Val Leu Val Glu Val Thr Ala Leu Gly Asn
Leu Arg Met Val 1 5 10
155415PRTHomo sapiensRhD Residues 152-166 54Asn Leu Arg Met Val Ile Ser
Asn Ile Phe Asn Thr Asp Tyr His 1 5 10
155515PRTHomo sapiensRhD Residues 162-176 55Asn Thr Asp Tyr
His Met Asn Met Met His Ile Tyr Val Phe Ala 1 5
10 155615PRTHomo sapiensRhD Residues 172-186 56Ile
Tyr Val Phe Ala Ala Tyr Phe Gly Leu Ser Val Ala Trp Cys 1
5 10 155715PRTHomo sapiensRhD Residues
182-196 57Ser Val Ala Trp Cys Leu Pro Lys Pro Leu Pro Glu Gly Thr Glu 1
5 10 155815PRTHomo
sapiensRhD Residues 192-206 58Pro Glu Gly Thr Glu Asp Lys Asp Gln Thr Ala
Thr Ile Pro Ser 1 5 10
155915PRTHomo sapiensRhD Residues 212-226 59Gly Ala Leu Phe Leu Trp Ile
Phe Trp Pro Ser Phe Asn Ser Ala 1 5 10
156015PRTHomo sapiensRhD Residues 222-236 60Ser Phe Asn Ser
Ala Leu Leu Arg Ser Pro Ile Glu Arg Lys Asn 1 5
10 156115PRTHomo sapiensRhD Residues 232-246 61Ile
Glu Arg Lys Asn Ala Val Phe Asn Thr Tyr Tyr Ala Val Ala 1
5 10 156215PRTHomo sapiensRhD Residues
242-256 62Tyr Tyr Ala Val Ala Val Ser Val Val Thr Ala Ile Ser Gly Ser 1
5 10 156315PRTHomo
sapiensRhD Residues 252-266 63Ala Ile Ser Gly Ser Ser Leu Ala His Pro Gln
Gly Lys Ile Ser 1 5 10
156415PRTHomo sapiensRhD Residues 262-276 64Gln Gly Lys Ile Ser Lys Thr
Tyr Val His Ser Ala Val Leu Ala 1 5 10
156515PRTHomo sapiensRhD Residues 292-306 65Trp Leu Ala Met
Val Leu Gly Leu Val Ala Gly Leu Ile Ser Val 1 5
10 156615PRTHomo sapiensRhD Residues 302-316 66Gly
Leu Ile Ser Val Gly Gly Ala Lys Tyr Leu Pro Gly Cys Cys 1
5 10 156715PRTHomo sapiensRhD Residues
312-326 67Leu Pro Gly Cys Cys Asn Arg Val Leu Gly Ile Pro His Ser Ser 1
5 10 156815PRTHomo
sapiensRhD Residues 322-336 68Ile Pro His Ser Ser Ile Met Gly Tyr Asn Phe
Ser Leu Leu Gly 1 5 10
156915PRTHomo sapiensRhD Residues 332-346 69Phe Ser Leu Leu Gly Leu Leu
Gly Glu Ile Ile Tyr Ile Val Leu 1 5 10
157015PRTHomo sapiensRhD Residues 342-356 70Ile Tyr Ile Val
Leu Leu Val Leu Asp Thr Val Gly Ala Gly Asn 1 5
10 157115PRTHomo sapiensRhD Residues 352-366 71Val
Gly Ala Gly Asn Gly Met Ile Gly Phe Gln Val Leu Leu Ser 1
5 10 157215PRTHomo sapiensRhD Residues
392-406 72Ile Trp Lys Ala Pro His Glu Ala Lys Tyr Phe Asp Asp Gln Val 1
5 10 157315PRTHomo
sapiensRhCE (R1 Ce) Residues 7-21 73Arg Ser Val Arg Arg Cys Leu Pro Leu
Cys Ala Leu Thr Leu Glu 1 5 10
157415PRTHomo sapiensRhCE (R1 Ce) Residues 217-231 74Trp Met Phe
Trp Pro Ser Val Asn Ser Ala Leu Leu Arg Ser Pro 1 5
10 157515PRTHomo sapiensRhD Residues 57-71
75Met Ala Ala Ile Gly Leu Gly Phe Leu Thr Ser Ser Phe Arg Arg 1
5 10 157615PRTHomo sapiensRhD
Residues 67-81 76Ser Ser Phe Arg Arg His Ser Trp Ser Ser Val Ala Phe Asn
Leu 1 5 10 157715PRTHomo
sapiensRhD Residues 97-111 77Phe Leu Ser Gln Phe Pro Ser Gly Lys Val Val
Ile Thr Leu Phe 1 5 10
157815PRTHomo sapiensRhD Residues 107-121 78Val Ile Thr Leu Phe Ser Ile
Arg Leu Ala Thr Met Ser Ala Leu 1 5 10
157915PRTHomo sapiensRhD Residues 117-131 79Thr Met Ser Ala
Leu Ser Val Leu Ile Ser Val Asp Ala Val Leu 1 5
10 158015PRTHomo sapiensRhD Residues 127-141 80Val
Asp Ala Val Leu Gly Lys Val Asn Leu Ala Gln Leu Val Val 1
5 10 158115PRTHomo sapiensRhD Residues
147-161 81Val Thr Ala Leu Gly Asn Leu Arg Met Val Ile Ser Asn Ile Phe 1
5 10 158215PRTHomo
sapiensRhD Residues 157-171 82Ile Ser Asn Ile Phe Asn Thr Asp Tyr His Met
Asn Met Met His 1 5 10
158315PRTHomo sapiensRhD Residues 167-181 83Met Asn Met Met His Ile Tyr
Val Phe Ala Ala Tyr Phe Gly Leu 1 5 10
158415PRTHomo sapiensRhD Residues 177-191 84Ala Tyr Phe Gly
Leu Ser Val Ala Trp Cys Leu Pro Lys Pro Leu 1 5
10 158515PRTHomo sapiensRhD Residues 187-201 85Leu
Pro Lys Pro Leu Pro Glu Gly Thr Glu Asp Lys Asp Gln Thr 1
5 10 158615PRTHomo sapiensRhD Residues
197-211 86Asp Lys Asp Gln Thr Ala Thr Ile Pro Ser Leu Ser Ala Met Leu 1
5 10 158715PRTHomo
sapiensRhD Residues 207-221 87Leu Ser Ala Met Leu Gly Ala Leu Phe Leu Trp
Ile Phe Trp Pro 1 5 10
158815PRTHomo sapiensRhD Residues 217-231 88Trp Ile Phe Trp Pro Ser Phe
Asn Ser Ala Leu Leu Arg Ser Pro 1 5 10
158915PRTHomo sapiensRhD Residues 227-241 89Leu Leu Arg Ser
Pro Ile Glu Arg Lys Asn Ala Val Phe Asn Thr 1 5
10 159015PRTHomo sapiensRhD Residues 237-251 90Ala
Val Phe Asn Thr Tyr Tyr Ala Val Ala Val Ser Val Val Thr 1
5 10 159115PRTHomo sapiensRhD Residues
257-271 91Ser Leu Ala His Pro Gln Gly Lys Ile Ser Lys Thr Tyr Val His 1
5 10 159215PRTHomo
sapiensRhD Residues 267-281 92Lys Thr Tyr Val His Ser Ala Val Leu Ala Gly
Gly Val Ala Val 1 5 10
159315PRTHomo sapiensRhD Residues 297-311 93Leu Gly Leu Val Ala Gly Leu
Ile Ser Val Gly Gly Ala Lys Tyr 1 5 10
159415PRTHomo sapiensRhD Residues 307-321 94Gly Gly Ala Lys
Tyr Leu Pro Gly Cys Cys Asn Arg Val Leu Gly 1 5
10 159515PRTHomo sapiensRhD Residues 317-331 95Asn
Arg Val Leu Gly Ile Pro His Ser Ser Ile Met Gly Tyr Asn 1
5 10 159615PRTHomo sapiensRhD Residues
327-341 96Ile Met Gly Tyr Asn Phe Ser Leu Leu Gly Leu Leu Gly Glu Ile 1
5 10 159715PRTHomo
sapiensRhD Residues 337-351 97Leu Leu Gly Glu Ile Ile Tyr Ile Val Leu Leu
Val Leu Asp Thr 1 5 10
159815PRTHomo sapiensRhD Residues 347-361 98Leu Val Leu Asp Thr Val Gly
Ala Gly Asn Gly Met Ile Gly Phe 1 5 10
159915PRTHomo sapiensRhD Residues 387-401 99Leu Leu Asn Leu
Lys Ile Trp Lys Ala Pro His Glu Ala Lys Tyr 1 5
10 1510015PRTHomo sapiensRhD Residues 397-411
100His Glu Ala Lys Tyr Phe Asp Asp Gln Val Phe Trp Lys Phe Pro 1
5 10 1510115PRTHomo sapiensRh50
GP Residues 1-15 101Met Arg Phe Thr Phe Pro Leu Met Ala Ile Val Leu Glu
Ile Ala 1 5 10
1510215PRTHomo sapiensRh50 GP Residues 11-25 102Val Leu Glu Ile Ala Met
Ile Val Leu Phe Gly Leu Phe Val Glu 1 5
10 1510315PRTHomo sapiensRh50 GP Residues 21-35 103Gly
Leu Phe Val Glu Tyr Glu Thr Asp Gln Thr Val Leu Glu Gln 1
5 10 1510415PRTHomo sapiensRh50 GP
Residues 31-45 104Thr Val Leu Glu Gln Leu Asn Ile Thr Lys Pro Thr Asp Met
Gly 1 5 10
1510515PRTHomo sapiensRh50 GP Residues 41-55 105Pro Thr Asp Met Gly Ile
Phe Phe Glu Leu Tyr Pro Leu Phe Gln 1 5
10 1510615PRTHomo sapiensRh50 GP Residues 51-65 106Tyr
Pro Leu Phe Gln Asp Val His Val Met Ile Phe Val Gly Phe 1
5 10 1510715PRTHomo sapiensRh50 GP
Residues 61-75 107Ile Phe Val Gly Phe Gly Phe Leu Met Thr Phe Leu Lys Lys
Tyr 1 5 10
1510815PRTHomo sapiensRh50 GP Residues 71-85 108Phe Leu Lys Lys Tyr Gly
Phe Ser Ser Val Gly Ile Asn Leu Leu 1 5
10 1510915PRTHomo sapiensRh50 GP Residues 81-95 109Gly
Ile Asn Leu Leu Val Ala Ala Leu Gly Leu Gln Trp Gly Thr 1
5 10 1511015PRTHomo sapiensRh50 GP
Residues 91-105 110Leu Gln Trp Gly Thr Ile Val Gln Gly Ile Leu Gln Ser
Gln Gly 1 5 10
1511115PRTHomo sapiensRh50 GP Residues 101-115 111Leu Gln Ser Gln Gly Gln
Lys Phe Asn Ile Gly Ile Lys Asn Met 1 5
10 1511215PRTHomo sapiensRh50 GP Residues 111-125 112Gly
Ile Lys Asn Met Ile Asn Ala Asp Phe Ser Ala Ala Thr Val 1
5 10 1511315PRTHomo sapiensRh50 GP
Residues 121-135 113Ser Ala Ala Thr Val Leu Ile Ser Phe Gly Ala Val Leu
Gly Lys 1 5 10
1511415PRTHomo sapiensRh50 GP Residues 131-145 114Ala Val Leu Gly Lys Thr
Ser Pro Thr Gln Met Leu Ile Met Thr 1 5
10 1511515PRTHomo sapiensRh50 GP Residues 141-155 115Met
Leu Ile Met Thr Ile Leu Glu Ile Val Phe Phe Ala His Asn 1
5 10 1511615PRTHomo sapiensRh50 GP
Residues 151-165 116Phe Phe Ala His Asn Glu Tyr Leu Val Ser Glu Ile Phe
Lys Ala 1 5 10
1511715PRTHomo sapiensRh50 GP Residues 161-175 117Glu Ile Phe Lys Ala Ser
Asp Ile Gly Ala Ser Met Thr Ile His 1 5
10 1511815PRTHomo sapiensRh50 GP Residues 171-185 118Ser
Met Thr Ile His Ala Phe Gly Ala Tyr Phe Gly Leu Ala Val 1
5 10 1511915PRTHomo sapiensRh50 GP
Residues 181-195 119Phe Gly Leu Ala Val Ala Gly Ile Leu Tyr Arg Ser Gly
Leu Arg 1 5 10
1512015PRTHomo sapiensRh50 GP Residues 191-205 120Arg Ser Gly Leu Arg Lys
Gly His Glu Asn Glu Glu Ser Ala Tyr 1 5
10 1512115PRTHomo sapiensRh50 GP Residues 201-215 121Glu
Glu Ser Ala Tyr Tyr Ser Asp Leu Phe Ala Met Ile Gly Thr 1
5 10 1512215PRTHomo sapiensRh50 GP
Residues 211-225 122Ala Met Ile Gly Thr Leu Phe Leu Trp Met Phe Trp Pro
Ser Phe 1 5 10
1512315PRTHomo sapiensRh50 GP Residues 221-235 123Phe Trp Pro Ser Phe Asn
Ser Ala Ile Ala Glu Pro Gly Asp Lys 1 5
10 1512415PRTHomo sapiensRh50 GP Residues 231-245 124Glu
Pro Gly Asp Lys Gln Cys Arg Ala Ile Val Asp Thr Tyr Phe 1
5 10 1512515PRTHomo sapiensRh50 GP
Residues 241-255 125Val Asp Thr Tyr Phe Ser Leu Ala Ala Cys Val Leu Thr
Ala Phe 1 5 10
1512615PRTHomo sapiensRh50 GP Residues 251-265 126Val Leu Thr Ala Phe Ala
Phe Ser Ser Leu Val Glu His Arg Gly 1 5
10 1512715PRTHomo sapiensRh50 GP Residues 261-275 127Val
Glu His Arg Gly Lys Leu Asn Met Val His Ile Gln Asn Ala 1
5 10 1512815PRTHomo sapiensRh50 GP
Residues 271-285 128His Ile Gln Asn Ala Thr Leu Ala Gly Gly Val Ala Val
Gly Thr 1 5 10
1512915PRTHomo sapiensRh50 GP Residues 281-295 129Val Ala Val Gly Thr Cys
Ala Asp Met Ala Ile His Pro Phe Gly 1 5
10 1513015PRTHomo sapiensRh50 GP Residues 291-305 130Ile
His Pro Phe Gly Ser Met Ile Ile Gly Ser Ile Ala Gly Met 1
5 10 1513115PRTHomo sapiensRh50 GP
Residues 301-315 131Ser Ile Ala Gly Met Val Ser Val Leu Gly Tyr Lys Phe
Leu Thr 1 5 10
1513215PRTHomo sapiensRh50 GP Residues 311-325 132Tyr Lys Phe Leu Thr Pro
Leu Phe Thr Thr Lys Leu Arg Ile His 1 5
10 1513315PRTHomo sapiensRh50 GP Residues 321-335 133Lys
Leu Arg Ile His Asp Thr Cys Gly Val His Asn Leu His Gly 1
5 10 1513415PRTHomo sapiensRh50 GP
Residues 331-345 134His Asn Leu His Gly Leu Pro Gly Val Val Gly Gly Leu
Ala Gly 1 5 10
1513515PRTHomo sapiensRh50 GP Residues 341-355 135Gly Gly Leu Ala Gly Ile
Val Ala Val Ala Met Gly Ala Ser Asn 1 5
10 1513615PRTHomo sapiensRh50 GP Residues 351-365 136Met
Gly Ala Ser Asn Thr Ser Met Ala Met Gln Ala Ala Ala Leu 1
5 10 1513715PRTHomo sapiensRh50 GP
Residues 361-375 137Gln Ala Ala Ala Leu Gly Ser Ser Ile Gly Thr Ala Val
Val Gly 1 5 10
1513815PRTHomo sapiensRh50 GP Residues 371-385 138Thr Ala Val Val Gly Gly
Leu Met Thr Gly Leu Ile Leu Lys Leu 1 5
10 1513915PRTHomo sapiensRh50 GP Residues 381-395 139Leu
Ile Leu Lys Leu Pro Leu Trp Gly Gln Pro Ser Asp Gln Asn 1
5 10 1514015PRTHomo sapiensRh50 GP
Residues 391-405 140Pro Ser Asp Gln Asn Cys Tyr Asp Asp Ser Val Tyr Trp
Lys Val 1 5 10
1514115PRTHomo sapiensRh50 GP Residues 395-409 141Asn Cys Tyr Asp Asp Ser
Val Tyr Trp Lys Val Pro Lys Thr Arg 1 5
10 1514216PRTHomo sapiensBR 142Ser Lys Tyr Pro Asn Cys
Ala Tyr Lys Thr Thr Gln Ala Asn Lys His 1 5
10 1514315PRTHomo sapiensAV2 143Thr Ile Pro Glu Gln
Ser Phe Gln Gly Ser Pro Ser Ala Asp Thr 1 5
10 1514415PRTHomo sapiensAV4 144Thr Val Lys Ala Asp
Phe Glu Phe Ser Ser Ala Pro Ala Pro Asp 1 5
10 1514515PRTHomo sapiensAV6 145Thr Val Glu Glu Arg
Gln Gln Phe Gly Glu Leu Pro Val Ser Glu 1 5
10 1514616PRTHomo sapiensP23 146Glu Leu Lys Ile Ile
Ser Arg Cys Gln Val Cys Met Lys Lys Arg His 1 5
10 1514713PRTHomo sapiensHA 147Pro Lys Tyr Val Lys
Gln Asn Thr Leu Lys Leu Ala Thr 1 5
10148417PRTHomo sapiensRhCE Residues 111-125 148Met Ser Ser Lys Tyr Pro
Arg Ser Val Arg Arg Cys Leu Pro Leu Cys 1 5
10 15Ala Leu Thr Leu Glu Ala Ala Leu Ile Leu Leu Phe
Tyr Phe Phe Thr 20 25 30His
Tyr Asp Ala Ser Leu Glu Asp Gln Lys Gly Leu Val Ala Ser Tyr 35
40 45Gln Val Gly Gln Asp Leu Thr Val Met
Ala Ala Ile Gly Leu Gly Phe 50 55
60Leu Thr Ser Ser Phe Arg Arg His Ser Trp Ser Ser Val Ala Phe Asn 65
70 75 80Leu Phe Met Leu Ala
Leu Gly Val Gln Trp Ala Ile Leu Leu Asp Gly 85
90 95Phe Leu Ser Gln Phe Pro Ser Gly Lys Val Val
Ile Thr Leu Phe Ser 100 105
110Ile Arg Leu Ala Thr Met Ser Ala Met Ser Val Leu Ile Ser Ala Gly
115 120 125Ala Val Leu Gly Lys Val Asn
Leu Ala Gln Leu Val Val Met Val Leu 130 135
140Val Glu Val Thr Ala Leu Gly Thr Leu Arg Met Val Ile Ser Asn
Ile145 150 155 160Phe Asn
Thr Asp Tyr His Met Asn Leu Arg His Phe Tyr Val Phe Ala
165 170 175Ala Tyr Phe Gly Leu Thr Val
Ala Trp Cys Leu Pro Lys Pro Leu Pro 180 185
190Lys Gly Thr Glu Asp Asn Asp Gln Arg Ala Thr Ile Pro Ser
Leu Ser 195 200 205Ala Met Leu Gly
Ala Leu Phe Leu Trp Met Phe Trp Pro Ser Val Asn 210
215 220Ser Pro Leu Leu Arg Ser Pro Ile Gln Arg Lys Asn
Ala Met Phe Asn225 230 235
240Thr Tyr Tyr Ala Leu Ala Val Ser Val Val Thr Ala Ile Ser Gly Ser
245 250 255Ser Leu Ala His Pro
Gln Arg Lys Ile Ser Met Thr Tyr Val His Ser 260
265 270Ala Val Leu Ala Gly Gly Val Ala Val Gly Thr Ser
Cys His Leu Ile 275 280 285Pro Ser
Pro Trp Leu Ala Met Val Leu Gly Leu Val Ala Gly Leu Ile 290
295 300Ser Ile Gly Gly Ala Lys Cys Leu Pro Val Cys
Cys Asn Arg Val Leu305 310 315
320Gly Ile His His Ile Ser Val Met His Ser Ile Phe Ser Leu Leu Gly
325 330 335Leu Leu Gly Glu
Ile Thr Tyr Ile Val Leu Leu Val Leu His Thr Val 340
345 350Trp Asn Gly Asn Gly Met Ile Gly Phe Gln Val
Leu Leu Ser Ile Gly 355 360 365Glu
Leu Ser Leu Ala Ile Val Ile Ala Leu Thr Ser Gly Leu Leu Thr 370
375 380Gly Leu Leu Leu Asn Leu Lys Ile Trp Lys
Ala Pro His Val Ala Lys385 390 395
400Tyr Phe Asp Asp Gln Val Phe Trp Lys Phe Pro His Leu Ala Val
Gly 405 410
415Phe149417PRTHomo sapiensRhCe Residues 121-135 149Met Ser Ser Lys Tyr
Pro Arg Ser Val Arg Arg Cys Leu Pro Leu Cys 1 5
10 15Ala Leu Thr Leu Glu Ala Ala Leu Ile Leu Leu
Phe Tyr Phe Phe Thr 20 25
30His Tyr Asp Ala Ser Leu Glu Asp Gln Lys Gly Leu Val Ala Ser Tyr
35 40 45Gln Val Gly Gln Asp Leu Thr Val
Met Ala Ala Ile Gly Leu Gly Phe 50 55
60Leu Thr Ser Ser Phe Arg Arg His Ser Trp Ser Ser Val Ala Phe Asn 65
70 75 80Leu Phe Met Leu
Ala Leu Gly Val Gln Trp Ala Ile Leu Leu Asp Gly 85
90 95Phe Leu Ser Gln Phe Pro Ser Gly Lys Val
Val Ile Thr Leu Phe Ser 100 105
110Ile Arg Leu Ala Thr Met Ser Ala Met Ser Val Leu Ile Ser Ala Gly
115 120 125Ala Val Leu Gly Lys Val Asn
Leu Ala Gln Leu Val Val Met Val Leu 130 135
140Val Glu Val Thr Ala Leu Gly Thr Leu Arg Met Val Ile Ser Asn
Ile145 150 155 160Phe Asn
Thr Asp Tyr His Met Asn Leu Arg His Phe Tyr Val Phe Ala
165 170 175Ala Tyr Phe Gly Leu Thr Val
Ala Trp Cys Leu Pro Lys Pro Leu Pro 180 185
190Lys Gly Thr Glu Asp Asn Asp Gln Arg Ala Thr Ile Pro Ser
Leu Ser 195 200 205Ala Met Leu Gly
Ala Leu Phe Leu Trp Met Phe Trp Pro Ser Val Asn 210
215 220Ser Ala Leu Leu Arg Ser Pro Ile Gln Arg Lys Asn
Ala Met Phe Asn225 230 235
240Thr Tyr Tyr Ala Leu Ala Val Ser Val Val Thr Ala Ile Ser Gly Ser
245 250 255Ser Leu Ala His Pro
Gln Arg Lys Ile Ser Met Thr Tyr Val His Ser 260
265 270Ala Val Leu Ala Gly Gly Val Ala Val Gly Thr Ser
Cys His Leu Ile 275 280 285Pro Ser
Pro Trp Leu Ala Met Val Leu Gly Leu Val Ala Gly Leu Ile 290
295 300Ser Ile Gly Gly Ala Lys Cys Leu Pro Val Cys
Cys Asn Arg Val Leu305 310 315
320Gly Ile His His Ile Ser Val Met His Ser Ile Phe Ser Leu Leu Gly
325 330 335Leu Leu Gly Glu
Ile Thr Tyr Ile Val Leu Leu Val Leu His Thr Val 340
345 350Trp Asn Gly Asn Gly Met Ile Gly Phe Gln Val
Leu Leu Ser Ile Gly 355 360 365Glu
Leu Ser Leu Ala Ile Val Ile Ala Leu Thr Ser Gly Leu Leu Thr 370
375 380Gly Leu Leu Leu Asn Leu Lys Ile Trp Lys
Ala Pro His Val Ala Lys385 390 395
400Tyr Phe Asp Asp Gln Val Phe Trp Lys Phe Pro His Leu Ala Val
Gly 405 410
415Phe150417PRTHomo sapiensRhcE Residues 131-145 150Met Ser Ser Lys Tyr
Pro Arg Ser Val Arg Arg Cys Leu Pro Leu Trp 1 5
10 15Ala Leu Thr Leu Glu Ala Ala Leu Ile Leu Leu
Phe Tyr Phe Phe Thr 20 25
30His Tyr Asp Ala Ser Leu Glu Asp Gln Lys Gly Leu Val Ala Ser Tyr
35 40 45Gln Val Gly Gln Asp Leu Thr Val
Met Ala Ala Leu Gly Leu Gly Phe 50 55
60Leu Thr Ser Asn Phe Arg Arg His Ser Trp Ser Ser Val Ala Phe Asn 65
70 75 80Leu Phe Met Leu
Ala Leu Gly Val Gln Trp Ala Ile Leu Leu Asp Gly 85
90 95Phe Leu Ser Gln Phe Pro Pro Gly Lys Val
Val Ile Thr Leu Phe Ser 100 105
110Ile Arg Leu Ala Thr Met Ser Ala Met Ser Val Leu Ile Ser Ala Gly
115 120 125Ala Val Leu Gly Lys Val Asn
Leu Ala Gln Leu Val Val Met Val Leu 130 135
140Val Glu Val Thr Ala Leu Gly Thr Leu Arg Met Val Ile Ser Asn
Ile145 150 155 160Phe Asn
Thr Asp Tyr His Met Asn Leu Arg His Phe Tyr Val Phe Ala
165 170 175Ala Tyr Phe Gly Leu Thr Val
Ala Trp Cys Leu Pro Lys Pro Leu Pro 180 185
190Lys Gly Thr Glu Asp Asn Asp Gln Arg Ala Thr Ile Pro Ser
Leu Ser 195 200 205Ala Met Leu Gly
Ala Leu Phe Leu Trp Met Phe Trp Pro Ser Val Asn 210
215 220Ser Pro Leu Leu Arg Ser Pro Ile Gln Arg Lys Asn
Ala Met Phe Asn225 230 235
240Thr Tyr Tyr Ala Leu Ala Val Ser Val Val Thr Ala Ile Ser Gly Ser
245 250 255Ser Leu Ala His Pro
Gln Arg Lys Ile Ser Met Thr Tyr Val His Ser 260
265 270Ala Val Leu Ala Gly Gly Val Ala Val Gly Thr Ser
Cys His Leu Ile 275 280 285Pro Ser
Pro Trp Leu Ala Met Val Leu Gly Leu Val Ala Gly Leu Ile 290
295 300Ser Ile Gly Gly Ala Lys Cys Leu Pro Val Cys
Cys Asn Arg Val Leu305 310 315
320Gly Ile His His Ile Ser Val Met His Ser Ile Phe Ser Leu Leu Gly
325 330 335Leu Leu Gly Glu
Ile Thr Tyr Ile Val Leu Leu Val Leu His Thr Val 340
345 350Trp Asn Gly Asn Gly Met Ile Gly Phe Gln Val
Leu Leu Ser Ile Gly 355 360 365Glu
Leu Ser Leu Ala Ile Val Ile Ala Leu Thr Ser Gly Leu Leu Thr 370
375 380Gly Leu Leu Leu Asn Leu Lys Ile Trp Lys
Ala Pro His Val Ala Lys385 390 395
400Tyr Phe Asp Asp Gln Val Phe Trp Lys Phe Pro His Leu Ala Val
Gly 405 410
415Phe151417PRTHomo sapiensRhD Residues 141-155 151Met Ser Ser Lys Tyr
Pro Arg Ser Val Arg Arg Cys Leu Pro Leu Trp 1 5
10 15Ala Leu Thr Leu Glu Ala Ala Leu Ile Leu Leu
Phe Tyr Phe Phe Thr 20 25
30His Tyr Asp Ala Ser Leu Glu Asp Gln Lys Gly Leu Val Ala Ser Tyr
35 40 45Gln Val Gly Gln Asp Leu Thr Val
Met Ala Ala Ile Gly Leu Gly Phe 50 55
60Leu Thr Ser Ser Phe Arg Arg His Ser Trp Ser Ser Val Ala Phe Asn 65
70 75 80Leu Phe Met Leu
Ala Leu Gly Val Gln Trp Ala Ile Leu Leu Asp Gly 85
90 95Phe Leu Ser Gln Phe Pro Ser Gly Lys Val
Val Ile Thr Leu Phe Ser 100 105
110Ile Arg Leu Ala Thr Met Ser Ala Leu Ser Val Leu Ile Ser Val Asp
115 120 125Ala Val Leu Gly Lys Val Asn
Leu Ala Gln Leu Val Val Met Val Leu 130 135
140Val Glu Val Thr Ala Leu Gly Asn Leu Arg Met Val Ile Ser Asn
Ile145 150 155 160Phe Asn
Thr Asp Tyr His Met Asn Met Met His Ile Tyr Val Phe Ala
165 170 175Ala Tyr Phe Gly Leu Ser Val
Ala Trp Cys Leu Pro Lys Pro Leu Pro 180 185
190Glu Gly Thr Glu Asp Asn Asp Gln Thr Ala Thr Ile Pro Ser
Leu Ser 195 200 205Ala Met Leu Gly
Ala Leu Phe Leu Trp Ile Phe Trp Pro Ser Phe Asn 210
215 220Ser Ala Leu Leu Arg Ser Pro Ile Glu Arg Lys Asn
Ala Val Phe Asn225 230 235
240Thr Tyr Tyr Ala Val Ala Val Ser Val Val Thr Ala Ile Ser Gly Ser
245 250 255Ser Leu Ala His Pro
Gln Gly Lys Ile Ser Lys Thr Tyr Val His Ser 260
265 270Ala Val Leu Ala Gly Gly Val Ala Val Gly Thr Ser
Cys His Leu Ile 275 280 285Pro Ser
Pro Trp Leu Ala Met Val Leu Gly Leu Val Ala Gly Leu Ile 290
295 300Ser Val Gly Gly Ala Lys Tyr Leu Pro Gly Cys
Cys Asn Arg Val Leu305 310 315
320Gly Ile Pro His Ser Ser Ile Met Gly Tyr Asn Phe Ser Leu Leu Gly
325 330 335Leu Leu Gly Glu
Ile Ile Tyr Ile Val Leu Leu Val Leu Asp Thr Val 340
345 350Gly Ala Gly Asn Gly Met Ile Gly Phe Gln Val
Leu Leu Ser Ile Gly 355 360 365Glu
Leu Ser Leu Ala Ile Val Ile Ala Leu Thr Ser Gly Leu Leu Thr 370
375 380Gly Leu Leu Leu Asn Leu Lys Ile Trp Lys
Ala Pro His Glu Ala Lys385 390 395
400Tyr Phe Asp Asp Gln Val Phe Trp Lys Phe Pro His Leu Ala Val
Gly 405 410
415Phe152417PRTHomo sapiensRhCe Residues 151-165 152Met Ser Ser Lys Tyr
Pro Arg Ser Val Arg Arg Cys Leu Pro Leu Trp 1 5
10 15Ala Leu Thr Leu Glu Ala Ala Leu Ile Leu Leu
Phe Tyr Phe Phe Thr 20 25
30His Tyr Asp Ala Ser Leu Glu Asp Gln Lys Gly Leu Val Ala Ser Tyr
35 40 45Gln Val Gly Gln Asp Leu Thr Val
Met Ala Ala Leu Gly Leu Gly Phe 50 55
60Leu Thr Ser Asn Phe Arg Arg His Ser Trp Ser Ser Val Ala Phe Asn 65
70 75 80Leu Phe Met Leu
Ala Leu Gly Val Gln Trp Ala Ile Leu Leu Asp Gly 85
90 95Phe Leu Ser Gln Phe Pro Pro Gly Lys Val
Val Ile Thr Leu Phe Ser 100 105
110Ile Arg Leu Ala Thr Met Ser Ala Met Ser Val Leu Ile Ser Ala Gly
115 120 125Ala Val Leu Gly Lys Val Asn
Leu Ala Gln Leu Val Val Met Val Leu 130 135
140Val Glu Val Thr Ala Leu Gly Thr Leu Arg Met Val Ile Ser Asn
Ile145 150 155 160Phe Asn
Thr Asp Tyr His Met Asn Leu Arg His Phe Tyr Val Phe Ala
165 170 175Ala Tyr Phe Gly Leu Thr Val
Ala Trp Cys Leu Pro Lys Pro Leu Pro 180 185
190Lys Gly Thr Glu Asp Asn Asp Gln Arg Ala Thr Ile Pro Ser
Leu Ser 195 200 205Ala Met Leu Gly
Ala Leu Phe Leu Trp Met Phe Trp Pro Ser Val Asn 210
215 220Ser Ala Leu Leu Arg Ser Pro Ile Gln Arg Lys Asn
Ala Met Phe Asn225 230 235
240Thr Tyr Tyr Ala Leu Ala Val Ser Val Val Thr Ala Ile Ser Gly Ser
245 250 255Ser Leu Ala His Pro
Gln Arg Lys Ile Ser Met Thr Tyr Val His Ser 260
265 270Ala Val Leu Ala Gly Gly Val Ala Val Gly Thr Ser
Cys His Leu Ile 275 280 285Pro Ser
Pro Trp Leu Ala Met Val Leu Gly Leu Val Ala Gly Leu Ile 290
295 300Ser Ile Gly Gly Ala Lys Cys Leu Pro Val Cys
Cys Asn Arg Val Leu305 310 315
320Gly Ile His His Ile Ser Val Met His Ser Ile Phe Ser Leu Leu Gly
325 330 335Leu Leu Gly Glu
Ile Thr Tyr Ile Val Leu Leu Val Leu His Thr Val 340
345 350Trp Asn Gly Asn Gly Met Ile Gly Phe Gln Val
Leu Leu Ser Ile Gly 355 360 365Glu
Leu Ser Leu Ala Ile Val Ile Ala Leu Thr Ser Gly Leu Leu Thr 370
375 380Gly Leu Leu Leu Asn Leu Lys Ile Trp Lys
Ala Pro His Val Ala Lys385 390 395
400Tyr Phe Asp Asp Gln Val Phe Trp Lys Phe Pro His Leu Ala Val
Gly 405 410 415Phe
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