HK1263112A1 - Igg subtyping assay for identifying transplantable tissue samples - Google Patents

Description

IgG subtype assay for identification of transplantable tissue samples

Background

The present invention relates generally to the field of organ and tissue transplantation, and more particularly to a procedure for matching donor tissue to a recipient whose body does not reject the tissue if the tissue is transplanted to the recipient.

The transplantation of organs and tissues from a donor to a recipient has become a relatively common procedure. Due to the basic function of the vertebrate immune system (i.e., alerting the body of "non-self" material and helping to eliminate such non-self material), tissue cannot simply be transplanted from the body of one individual of a given species into the body of any other individual of the same species without the possibility of immune complications. If the transplanted tissue is not compatible with the recipient's immune system, the recipient's immune system may react to the transplanted tissue, thereby destroying the transplanted tissue. To avoid such graft rejection, two basic strategies can be employed. First, one or more immune functions of the recipient can be suppressed, thereby reducing or eliminating the immune response to the transplant. Second, grafts and recipients can be screened and matched to reduce the likelihood and/or severity of recipient immune responses to the grafts. The subject matter described herein is primarily directed to this second strategy, although the two strategies are not mutually exclusive-both may be used for the same transplant.

It has long been known that cells in vertebrate tissues express antigens on their surface, and that these antigens can vary significantly between individuals of the same species. For example, human cells display on their surface proteins known as Human Leukocyte Antigens (HLA). For any individual, the HLA displayed by the individual's cells is recognized by the individual's immune system as "self", and the individual's immune system does not (usually) mount an immune response to its own cells. However, the HLA displayed by different individuals can vary widely so that HLA not normally expressed by individual cells, such as HLA of transplanted tissue, can be recognized by the individual's immune system as "non-self" resulting in an immune response against the transplanted tissue. If the immune response initiated against the transplanted tissue is severe enough to induce death of most or all of the transplanted cells or sufficient destruction of the transplanted extracellular material, the transplanted tissue may not exhibit the desired function or property as the cause of the transplant. That is, if the recipient immune response to the transplanted tissue is severe enough, the goal leading to the transplant may not be achieved.

One type of immune response that the recipient's body can generate against the transplanted tissue depends on the binding of antibodies generated by the recipient's body to antigens (e.g., HLA) displayed on the surface of the transplanted tissue. Specific binding between an antibody and its corresponding antigen can catalyze a reaction ("complement fixation") that results in the induction of an effective cytotoxic immune response against the transplanted tissue. Analysis of the recipient immune system components to detect the ability of donor tissues to be "non-self" and to generate cytotoxic responses and selection of donor-recipient pairs to avoid such responses is commonly referred to as "cross-matching" and is widely discussed in the literature (see, e.g., Mulley et al, 2011, neuroproly 16: 125-133).

One known cross-matching technique involves contacting lymphocytes obtained from a donor of potential transplanted tissue with serum obtained from a proposed recipient of the transplanted tissue. Serum includes antibodies that circulate in the recipient's blood. Such binding can be detected if the recipient's serum includes antibodies that specifically bind to antigens present on the donor lymphocytes. In one common detection technique, known as "flow cross-matching", donor lymphocytes are contacted with recipient serum for a period of time, after which unbound antibodies are removed by separating the lymphocytes from the serum and washing the lymphocytes with excess reagents. The lymphocytes are then contacted with a labeling reagent capable of detecting the recipient antibody (e.g., a fluorescent antibody that binds to a human antibody, optionally a specific type of antibody, such as IgM or IgG, or a subtype, such as IgG1, IgG2, IgG3, or IgG4), and unbound labeling reagent is then washed from the lymphocytes. The binding of the recipient antibody to the donor lymphocytes is detected by suspending the lymphocytes in a fluid and passing them through a flow cytometer capable of detecting the labeling agent label. Detection of the label and lymphocytes by flow cytometry (e.g., detection of fluorescence corresponding to fluorescein) indicates that the one or more recipient antibodies recognized by the labeling agent bind to the lymphocytes. Such bound recipient antibodies are commonly referred to as "anti-donor antibodies" (adabs). If multiple flow cross-matching reactions are performed, each using a labeling reagent specific for a different recipient antibody type and/or subtype, a profile of the type and subtype of the ADAb present in the serum of potential recipients can be developed.

It has been observed that ADAb type and subtype can affect the likelihood that transplanted tissue will be rejected by the recipient. For example, Mulley et al recognized that IgG4 would not activate complement, and that adabs detected using donor leukocytes are only potential recipients of the IgG4 subtype may not be likely to activate complement in vivo (i.e., indicating a likely appropriate donor-recipient pairing). Cicciarelli et al reported comparable observations (U.S. patent application publication No. 2010/0261203). Gao et al (2014, am. J. Transplant.14 (7): 1581-1591) recognized that IgG antibodies of the subtypes IgG1 and IgG3, which react with apoptotic cells, are more likely to cause late rejection of transplanted kidneys, and these authors suggested complement-binding capacity attributable to the IgG1 and IgG3 subtypes. However, previous workers examined the binding between antigen coated beads and antibodies in the recipient serum, which assay may be laborious and does not necessarily indicate an interaction between the recipient antibody and the donor cells.

There is a need for improved cross-match assays that can quickly and accurately determine the suitability of a potential tissue graft for implantation into (or onto) an individual recipient. The present disclosure describes such assays.

Summary of the invention

The present invention relates to a method of assessing the transplant compatibility of body tissue of a potential donor with a recipient. The method includes contacting leukocytes obtained from a potential donor with an antibody obtained from a recipient. The binding between potential donor leukocytes and recipient antibodies was assessed for at least IgG subtypes IgG1 and IgG 3. The body tissue is assessed to be compatible with the recipient if substantially no binding between the donor leukocytes and the recipient antibodies of either of the IgG subclasses IgG1 and IgG3 is detected. The invention also relates to a tissue transplantation procedure comprising such an assessment method.

In the assessment method, the binding between donor leukocytes and recipient antibodies is preferably assessed for at least each of the IgG subtypes IgG1, IgG2, IgG3, and IgG4, preferably using differentially labeled antibodies that distinguishably recognize the subtypes, and preferably in a single assay mixture. If desired, the assessment method can further comprise contacting the donor leukocytes with differentially labeled antibodies that distinguishably recognize the donor's B and T cells. After contacting the donor leukocytes with the recipient antibodies, the individual donor leukocytes can be assessed by flow cytometry for the label bound to and/or with the antibodies.

The assessment method is applicable to vertebrate recipients, preferably humans.

Non-specific binding between the recipient antibody and the Fc receptor on the donor leukocyte can be inhibited using known reagents. The donor leukocytes are preferably viable upon contact with the antibody obtained from the recipient.

Brief Description of Drawings

FIG. 1 is a table illustrating the results of the experimental results described in example 5 herein.

Figure 2 is a table illustrating the results of the experimental results described in example 6 herein.

Detailed Description

The present disclosure relates to methods for cross-matching tissue from a donor with a suitable recipient of the tissue. The method involves determining the anti-donor IgG subtype present in a sample containing antibodies obtained from the recipient (e.g., serum prepared from the recipient's blood).

The donor tissue may be tissue that has been collected (and possibly stored and/or frozen) from a living or recently dead vertebrate (e.g., a human donor), or it may be tissue that is still part of the donor candidate. In any of these cases, the individual from whom tissue is or may be collected in the future or from whom tissue is or may be collected at a cadaver is referred to herein as the "donor". Cross-matching methods are typically performed after donor tissue is collected from a donor; however, this method may be performed prior to collecting the tissue to be transplanted (e.g., without collecting the tissue if the recipient is not a suitable recipient), by testing leukocytes obtained from the donor against IgG antibody subtypes in serum obtained from the candidate recipient (i.e., the aforementioned tissue harvest if anti-donor IgG1 or IgG3 antibody subtypes are detected in serum of the candidate recipient). The potential donor leukocytes can be frozen and archived, optionally with or in stored donor tissue.

The method includes contacting leukocytes (e.g., peripheral blood mononuclear cells) obtained from a donor with antibodies in a sample (e.g., serum) obtained from a potential recipient of donor tissue, and detecting whether antibodies (abs) of multiple IgG subtypes present in the recipient sample bind to the leukocytes of the donor in a single assay after detection. This assessment is preferably performed by flow cytometry using a reagent containing a plurality of differentially labeled antibodies, each of which specifically binds only to certain subtypes (preferably only one subtype) of the IgG antibody. Such differentially labeled abs are described herein as "discriminately recognizing" various subtypes of IgG antibodies. After the excess reagent is separated from the leukocytes, the leukocytes are evaluated by, for example, flow cytometry to detect any markers associated with the leukocytes. From this information, it can be determined which donor-leukocyte binding IgG subtype is present in the recipient serum. If desired, the various donor leukocytes can be distinguished from one another, for example, by known flow cytometry methods, by labeling them with one or more abs that distinguishably recognize (i.e., can be used to distinguish) T and B cells. In the assays described herein, binding of the recipient IgG1 and/or IgG3 antibodies to donor T cells generally indicates a less favorable match than the same binding to donor B cells, although both types of binding indicate a less favorable match than if both types of binding were not present.

The recipient antibodies can be obtained from the recipient's blood, for example, by separating serum (i.e., the cell-free liquid component of the blood) therefrom. The recipient antibody may also or alternatively be used or isolated from any other bodily fluid or wash/lavage fluid known or believed to contain antibodies representative of the patient's body. For example, recipient antibodies may be obtained from pleural aspirate, bronchial lavage, or peritoneal fluid samples. Regardless of how the recipient antibody-containing sample is obtained or prepared, it is important to treat and/or store the IgG antibodies (at least the IgG1 and/or IgG3 subtypes) present in the sample in a manner that retains the ability of the IgG1 and/or IgG3 antibodies therein to bind to the leukocytes of the donor. A preferred sample of recipient antibodies is serum.

If the recipient sample contains donor-leukocyte binding IgG antibodies of the IgG1 or IgG3 subtype, the potential recipient may not be a suitable recipient of the donor tissue and the donor tissue should not be transplanted with the donor. The presence of anti-donor IgG1 or IgG3 subtypes in the recipient sample indicates that graft rejection symptoms may occur if tissue from the donor is transplanted onto the recipient. That is, this indicates that antibody-mediated rejection is more likely than when the recipient's sample does not contain anti-donor IgG1 or IgG3 subtypes. The likelihood of graft rejection symptoms may also be at least approximately correlated with an increased degree of detection of IgG1 and/or IgG 3. Thus, for example, an individual whose leukocytes cause relatively low levels of binding of the recipient IgG1 and/or IgG3 antibodies in the assays described herein would be considered a preferred tissue donor, as compared to another individual whose leukocytes cause significantly higher levels of binding of the recipient IgG1 and/or IgG3 antibodies in the assays. Furthermore, given the greater propensity of IgG3 to induce complement binding, detecting a given level of binding of the recipient-IgG 3 to donor leukocytes (e.g., twice above normal background levels) is considered an indication of a less favorable donor match than detection of the same level of binding of the recipient-IgG 1 to donor leukocytes. Typically, detection of binding of recipient-IgG 3 or-IgG 1 to donor leukocytes is about 2-fold higher than the basal (i.e., normal background) level of such binding, which is considered to indicate an unfavorable donor match (i.e., an increased likelihood of graft rejection symptoms), with higher detection binding generally indicating a more unfavorable donor match.

If the recipient's sample does not contain donor-leukocyte binding IgG antibodies of the IgG1 or IgG3 subtype, the potential recipient may be a suitable recipient of the donor tissue, and the donor tissue may be transplanted onto the recipient with the reasonable expectation that it will not be rejected (at least through physiological processes involving complement fixation).

Thus, the methods described herein can be used to determine whether an individual recipient can be expected to reject tissue obtained from a different individual donor (different, preferably the same species). If the recipient's sample includes donor-leukocyte binding antibodies of the IgG1 or IgG3 subtype, the recipient may be expected to reject the tissue obtained from the donor, and if the donor sample does not include donor-leukocyte binding antibodies of the IgG1 or IgG3 subtype, it may be expected not to reject the tissue (even if the donor sample includes donor-leukocyte binding antibodies of the IgG2 and/or IgG4 subtype).

In contrast to existing methods, the described methods not only identify potentially successful tissue grafts (which can be identified using previous methods that rely on the detection of donor-leukocyte binding antibodies to undifferentiated subtypes of IgG), but also identify potentially successful tissue grafts that were not identifiable using existing methods.

The method has the following advantages: multiple evaluations (e.g., detection of IgG1 and detection of IgG3) can be performed in a single reaction mixture using a single aliquot of the sample. Since all multiple assessments are performed using the same sample, the difficulties arising from multiple assays relying on multiple corresponding controls are avoided.

The present methods have the additional advantage that they are performed using intact donor cells rather than synthetic beads linked to donor antigens. Donor cells (e.g., live PBMCs) can be expected to closely mimic antigen presentation by donor tissue; in contrast, synthetic beads with donor antigens can present donor antigens significantly differently in the same way as donor tissue presents the same antigens. Thus, the methods described herein can be expected to more accurately predict the recipient's response to the donor tissue.

The methods described herein are used to match a human recipient to a human donor of a tissue. However, it is also understood that the same method, using only routine modifications, can be used to match individual recipients of substantially any vertebrate species with donors of tissue of the same species. Likewise, the same method, by routine modification, can be used to match an individual recipient of a first vertebrate species with tissue obtained from a donor of a second, different vertebrate species (although it is recognized that other assays of recipient appropriateness may be performed for inter-species tissue transplantation).

Examples

The subject matter of the present disclosure is now described with reference to the following examples. These embodiments are provided for illustrative purposes only, and the subject matter is not limited to these embodiments, but includes all variations that are apparent in light of the teachings provided herein.

Example 1

Isolation of PBMC from donor blood

Peripheral blood mononuclear cells (PBMCs, which include lymphocytes and monocytes) were isolated from donor whole blood collected in citrate dextrose (ACD) vacuum tubes. The donor blood was transferred to a 15 ml conical tube and mixed by inversion with 2 ml of methylcellulose (1% solution, weight/volume; Sigma-Aldrich, St. Louis, Mo., USA). The tube was rotated in an incubator at 37 degrees celsius for 15 minutes and allowed to stand at 37 degrees celsius for an additional 30 minutes to separate the plasma layer containing the enriched lymphocytes from the red blood cell layer. The plasma layer was carefully transferred to a new 15 ml conical tube and treated with dulbecco phosphate buffered saline (DPBS, Lonza, walker, MD, USA) at a rate of 1: 1 (plasma: DPBS) and mixed by inversion.

The diluted plasma was covered with Lymphocyte Separation Medium (LSM) using a pasteur pipette, so that the diluted plasma: the ratio of LSM is 1: 1. the tubes were centrifuged at 400 xg for 25 minutes at 22 degrees celsius in a swinging bucket rotor using slow deceleration. The clear white band at the interface containing PBMCs was transferred to a new 15 ml conical tube and diluted with DPBS to a final volume of 10 ml. The tubes were centrifuged at 260 Xg for 10 min at 22 ℃ to pellet PBMC. The supernatant containing platelets was discarded, leaving a PBMC-rich pellet. The pellet was carefully resuspended in 2 ml DPBS and the volume ratio 1: 2 dilution of trypan blue cells were counted in a hemocytometer.

IgG subtype determination

IgG subtype analysis was performed using a high sensitivity flow cytometry cross-matching (FCXM) platform, as described below. Briefly, PBMC isolated from donor blood were aliquoted into separate 5ml polystyrene round bottom tubes at a concentration of 5 × 10⁵Individual cells/tube. Cells were washed with DPBS and pelleted by centrifugation at 800 × g for 5 minutes at 22 ℃. Fc receptors on cell surfaces were blocked using Fc receptor blocker reagent (obtained from Innovex Biosciences, Richmond, CA, USA) for 10 minutes at room temperature (approximately 20 degrees celsius). Blocking reagent was removed by washing the cells twice with DPBS and then incubating with patient (i.e., recipient) serum for 30 minutes at 4 degrees celsius. Donor cells were also incubated with negative and positive control serum samples. Positive control sera were prepared by pooling sera from 5 highly sensitized patients (cPRA values greater than 98% for each patient). The positive sera reacted with PBMC cells from all donors, yielding consistently positive FCXM results. Negative control sera (obtained from Gemini Bio-Products, WestSacramento, Calif., USA) were collected from healthy male donors of serotype AB in FDA approved facilities in the United states. The material is defibrinated from source plasma AB. All donor units were tested for viral markers and found to be non-reactive. The serum produced consistently negative FCXM results.

After incubation, cells were washed with washing buffer containing DPBS and 1% fetal bovine serum (FBS, Gemini Bio-products, WestSacramento, Calif., USA). Cells were resuspended in wash buffer and transferred to individual tubes containing lyophilized custom mixtures of differentially labeled antibodies that specifically recognize various IgG subclasses (obtained from BDLyotube, BD Pharmingen, San Jose, CA, USA). Incubation of donor cells with the lyophilized antibody mixture was performed in the dark for 20 minutes, and then the cells were washed with a washing buffer to remove excess antibody.

Specific anti-HLA IgG subtype antibodies that bind to cells are detected using the multicolor flow cytometry detection methods described herein. The level of IgG subtype in the patient serum was compared to the level observed in the negative control serum. Fold changes in IgG subtype levels in patient sera relative to negative control sera were calculated. Levels of IgG subtype in the patient serum were 2-fold higher than in the negative control serum, indicating a positive for this subtype.

Example 2

Protocol for identifying IgG subtypes by flow cytometry

The following description is similar to the procedure described in example 1 with some minor variations.

PBMCs were isolated from whole blood or frozen PBMCs were used.

Washed twice with phosphate buffered saline (PBS, ph7.4) and viability counted.

Centrifuge at 1500rpm for 5 minutes.

The supernatant was discarded and the cell pellet was resuspended at every 10 th⁷0.3ml of FcR blocker per cell (InnovexCat # NB 309).

Incubate at room temperature (about 20 degrees celsius) for 10 minutes.

Washed twice with excess PBS.

The cells were treated with 10⁷The individual cells/ml were resuspended in staining buffer (i.e., phosphate buffered saline, pH7.4, containing 5% v/v fetal bovine serum).

100 microliters per test (10 microliters per test)⁶One) cell suspension was aliquoted into each 12x75mm tube.

To each tube was added 20 microliters of pure serum.

Incubate at 4 ℃ for 30 minutes.

Wash with 2.5ml staining buffer.

The supernatant was discarded.

Resuspended in 100. mu.l staining buffer and the contents transferred to lyotube.

Incubate at room temperature for 20 minutes.

Wash with 2.5ml staining buffer.

The supernatant was discarded and resuspended in 0.4ml of staining buffer.

Samples were taken and the amount of individual IgG subtypes was measured.

Example 3

Use of IgG typing in Positive flow-cell count Cross-matched (FCXM) Heart transplantation

Positive FCXM is often a barrier to heart transplantation due to the risk of hyperacute rejection and antibody-mediated rejection (AMR). Although highly sensitive to the presence of donor-specific antibodies, FXCM does not determine whether these antibodies bind complement.

Case reporting: 61 year old African American men with coronary artery disease after myocardial infarction and coronary artery bypass graft developed heart failure due to ischemic cardiomyopathy with Ejection Fraction (EF) of 15-20%. Despite drug treatment and implantation of cardiac resynchronization therapy devices, he implanted a cardiochaperone II Left Ventricular Assist Device (LVAD) as a bridge to the transplantation due to progressive symptoms. The course of disease following LVAD is complicated by recurrent gastrointestinal bleeding that requires multiple transfusions and infections at the exit site of the driveline. He upgraded to the 1A state, but failed to obtain donors due to high Population Reactive Antibodies (PRAs) (class I69%, class II 3%). Desensitization was attempted using plasmapheresis, intravenous immunoglobulin, rituximab, mycophenolate mofetil, and bortezomib, but there was no significant response.

Suitable donors were identified that had negative complement-dependent cytotoxicity results for the expected cross-matching type. However, FCXM was strongly positive for both T and B cells (median channel shift: T cells 362/50, B cells 359/100). Recipient serum analysis was performed using custom antibodies recognizing different IgG subtypes. Of the 4 IgG subtypes, only subtypes 2 and 4 were identified in the recipient serum. Since only IgG subtypes 1 and 3 are known to be complement-bound, transplantation is considered safe.

Cardiac transplantation was performed and the recipients received an induced immunosuppressive therapy ("inducibility") using basiliximab and a regimen immunosuppressive using prednisone, tacrolimus and mycophenolate mofetil. He had normal graft function immediately after surgery, without hyperacute rejection. Repeated echoes taken 12 days after transplantation showed normal biventricular function. He performed 14 protocol endocardial myocardial biopsies within 12 months with no apparent cell rejection. Staining of all C4d and CD68 samples showed no evidence of antibody-mediated rejection. He continued to maintain a good migration function EF 70% on the first annual visit. The regadenoson stress test performed at this time showed no evidence of ischemia.

Described in this example is the case of heart transplantation with IgG typing despite the presence of positive FCXM. The lack of complement-bound IgG subtypes 1 and 3 in the recipient serum resulted in successful heart transplantation, no rejection after 1 year, and normal graft function.

Example 4

Method for preparing reagents suitable for simultaneous flow cytometry identification of four IgG subtypes

1. Fluorescently labeled individual anti-IgG 1, 2, 3, 4 antibodies (Ab) were purchased from different companies and clones of these antibodies were determined.

2. Each clone was evaluated as appropriate for the experiment (binding of a second anti-IgG antibody to a first antibody linked to HLA antigen (Ag) on PBMC).

3. The amount of each secondary anti-IgG Ab that needs to be included in the reagent is determined. This requires that each anti-IgG Ab be tested separately.

4. Experiments were performed with a combination of 4 anti-IgG antibodies (groups of 2, 3 or all 4 together) and determined whether there was any steric hindrance preventing the second antibody from binding to the first antibody that bound to HLA Ag on PBMCs. If so, one or more different abs are selected in place of at least one sterically hindered Ab.

5. The reagent used to block the Fc receptor was selected as ready for use. The currently used reagent is an "Fc receptor blocker", which is commercially available, as described above, and works as intended. Others are known in the art.

6. "standard beads" labeled with individual IgG subtypes were obtained and used as internal experimental standards for each experiment run.

7. Normalization of the reagents was performed using standard beads and freshly isolated PBMCs. Normalization of the reagents required multiple experiments with the same concentration of anti-IgG antibody, using the same amount of standard beads, but PBMCs isolated from various donors, as described above. This indicates that for each experiment performed, the results are consistent with standard beads, and that the assay can detect the level of IgG subclasses bound to PBMCs isolated from various donors.

8. The above-described normalization experiments were validated using freshly isolated PBMCs from multiple donors (e.g., 20 or more donors).

Each of these steps is preferably a document so that details can be checked by an authority if necessary or desired.

Example 5

IgG subtype specific flow cross-matching assays can increase the number of successful transplants in sensitized renal recipients

Positive flow cytometric cross-matching (FXCM) is considered a contraindication for successful kidney transplantation. Standard FXCM does not distinguish between various subtypes of immunoglobulin molecules (IgG1, IgG2, IgG3, and IgG 4). Only the IgG1 and IgG3 subtypes are able to maximize complement activation. IgG2 and IgG4 were relatively benign. We present preliminary results of our study that evaluated a new FCXM test that specifically detected and quantified IgG subtypes responsible for positive cross-matching.

The method used in the experiment described in this example will now be described. Pre-transplant sera from 7 recipients and blood samples from their respective donors were evaluated. IgG subtype analysis was performed using FCXM. PBMCs isolated from donor samples were incubated with patient and control sera. The cells were then incubated in a lyophilized custom antibody mixture that specifically recognized the various IgG subtypes bound to the cells, which were then subjected to FCXM analysis. All sera were tested for C1q (complement activation).

The results of these experiments are as follows. Standard FCXM was positive in most cases studied (5/7), so transplantation was not usually performed based on these results alone. However, using the IgG subtype analysis described herein, we were able to determine that the positive cross-match results were attributable to the presence of non-complement-binding IgG2 or IgG4 antibodies, and that transplantation was possible. IgG typing and C1q results were almost identical. All cases showed that the presence of non-complement activating antibodies was responsible for positive FCXM (except CF). CFL showed the presence of IgG3 antibody with negative C1 q; possibly as a result of denaturing the antibody. No clinical rejection episodes were observed in any of the cases or no dialysis requirements were observed for any of the recipients within the first week. These results are summarized in the table shown in fig. 1. In fig. 1, "Cr." refers to serum creatine determination.

The experiments described in this example demonstrate that we have developed an IgG subtype FCXM assay that has the ability to identify the presence of complement activating antibodies. This assay has been shown to be highly accurate in detecting IgG subtypes that give rise to positive flow cross-matching. The use of this assay may result in successful transplantation even in the presence of positive FCXM in highly sensitized recipients.

Example 6

Discrimination of IgG subtypes using a novel flow cross-matching assay to increase successful transplantation in sensitized cardiac and renal recipients

The transplantation procedure does not want to transplant in the presence of positive flow cross-matching. Standard flow cytometric cross-matching (FCXM) does not distinguish between the various subtypes of immunoglobulin molecules (IgG1, IgG2, IgG3, and IgG 4). We developed a new FCXM assay that was able to specifically detect and quantify the amount of complement-activated (IgG1 and IgG3) and non-complement-activated (IgG2 and IgG4) IgG antibodies. We used this assay to provide preliminary results in two separate heart and kidney transplantation models. We demonstrate that successful transplantation can be achieved in the presence of positive cross-matching using this assay, which is capable of distinguishing between various IgG subtypes.

The method used in the experiment described in this example will now be described. Pre-transplant sera from 7 heart recipients (including those described in example 3) and 7 kidney recipients and blood samples from their respective donors were used in this study. IgG subtype analysis was performed using FCXM. PBMCs isolated from donor samples were incubated with patient and control sera. The cells were then incubated in a lyophilized custom antibody mixture that specifically recognized the various IgG subtypes bound to the cells, which were then subjected to FCXM analysis. All sera were tested for C1 q.

The results obtained for the renal recipient patients are described in example 5 and figure 1.

The results obtained for the cardiac recipient patients are as follows. Most of the heart transplant cases studied (6/7) had a positive cross-match and therefore were not usually transplanted. However, using the IgG subtype analysis described herein, we were able to determine that the positive cross-match results were attributable to the presence of non-complement-binding IgG2 or IgG4 antibodies, and that transplantation was possible. In most cases, the C1q results are consistent with the cross-matching results. Two cases (JO, MP) were positive for C1 q; possibly due to the prozone effect of HLA-specific IgM antibodies. All cases had positive 30-and 90-day survival rates after transplantation with no primary graft dysfunction or >2R rejection (using the international heart lung transplantation society for care of heart transplant recipients terminology). Antibody-mediated rejection was documented and both cases (JF, RP) receiving induction therapy continued to have normal graft function. These results are summarized in the table shown in fig. 2.

The experiments described in this example and example 5 demonstrate that the IgG subtype assay described herein is highly accurate for detecting IgG subtypes that cause positive flow cross-matching. These results demonstrate for the first time that the assay promotes safe transplantation even in the presence of standard positive flow cross-matching, which is generally contraindicated. Clinical implementation of our IgG subtype assays can have a large impact on increasing the number of successful transplants, particularly in sensitized recipients.

The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

Although the subject matter has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations may be devised by those skilled in the art without departing from the true spirit and scope of the subject matter described herein. The appended claims are to encompass all such embodiments and equivalents.

Claims (30)

1. A method of transplanting tissue obtained from a vertebrate donor to a recipient, the method comprising:

contacting leukocytes obtained from a donor with an antibody obtained from a recipient,

assessing binding between donor leukocytes and recipient antibodies of at least IgG subclasses IgG1 and IgG3, and

the donor tissue is transplanted into the recipient if substantially no binding is detected between the donor leukocytes and the recipient antibody of either of IgG subclasses IgG1 and IgG 3.

2. The method of claim 1, comprising assessing binding between the donor leukocytes and recipient antibodies of at least IgG subtypes IgG1, IgG2, IgG3, and IgG 4.

3. The method of claim 2, wherein the assessment of binding between donor leukocytes and recipient antibodies of each of the IgG subtypes IgG1, IgG2, IgG3, and IgG4 is performed in a single assay.

4. The method of claim 1, wherein the assessment of binding between the donor leukocytes and the antibody is performed by contacting the recipient antibody with a differentially labeled antibody that specifically recognizes antibodies of the IgG1 and IgG3 subclasses.

5. The method of claim 1, wherein the assessment of binding between the donor leukocytes and the antibody is performed by contacting the recipient antibody with a differentially labeled antibody that distinguishably recognizes antibodies of the subclasses IgG1, IgG2, IgG3 and IgG 4.

6. The method of claim 5, further comprising contacting the donor leukocytes with differentially labeled antibodies that distinguishably recognize the donor's B and T cells.

7. The method of claim 1, wherein the individual donor leukocytes are assessed by flow cytometry after contacting the donor leukocytes with the recipient antibody.

8. The method of claim 1, wherein the individual donor leukocytes are assessed by flow cytometry after contacting the donor leukocytes with a recipient antibody and after contacting the recipient antibody with a differentially labeled antibody that discriminately recognizes antibodies of the subclasses IgG1, IgG2, IgG3, and IgG 4.

9. The method of claim 1, wherein the individual donor leukocytes are assessed by flow cytometry after contacting the donor leukocytes with the recipient antibodies, after contacting the recipient antibodies with differentially labeled antibodies that distinguishably recognize antibodies of the subclasses IgG1, IgG2, IgG3, and IgG4, and after contacting the donor leukocytes with differentially labeled antibodies that distinguishably recognize B and T cells of the donor.

10. The method of claim 1, wherein the recipient is a vertebrate.

11. The method of claim 10, wherein the recipient is the same species as the donor.

12. The method of claim 11, wherein the recipient is a human.

13. The method of claim 1, wherein non-specific binding between the recipient antibody and the Fc receptor on the donor leukocyte is inhibited.

14. The method of claim 13, wherein non-specific binding is inhibited by contacting the donor leukocytes with a peptide that inhibits binding between the antibody and the surface Fc receptor prior to contacting the donor leukocytes with the recipient antibody.

15. The method of claim 1, wherein the donor leukocytes are viable upon contact with the recipient antibody.

16. A method of assessing transplant compatibility of body tissue of a potential donor with a recipient, the method comprising:

contacting leukocytes obtained from a potential donor with an antibody obtained from a recipient,

assessing binding of potential donor leukocytes to recipient antibodies of at least the IgG subclasses IgG1 and IgG3,

the body tissue is assessed to be transplant compatible with the recipient if substantially no binding is detected between the donor leukocytes and the recipient antibody of either of the IgG subclasses IgG1 and IgG 3.

17. The method of claim 16, comprising assessing binding between the donor leukocytes and recipient antibodies of at least IgG subtypes IgG1, IgG2, IgG3, and IgG 4.

18. The method of claim 17, wherein the assessment of binding between donor leukocytes and recipient antibodies for each of the IgG subtypes IgG1, IgG2, IgG3, and IgG4 is performed in a single assay.

19. The method of claim 16, wherein the assessment of binding between the donor leukocytes and the antibody is performed by contacting the recipient antibody with a differentially labeled antibody that specifically recognizes antibodies of the IgG1 and IgG3 subclasses.

20. The method of claim 16, wherein the assessment of binding between the donor leukocytes and the antibody is performed by contacting the recipient antibody with a differentially labeled antibody that distinguishably recognizes antibodies of the subclasses IgG1, IgG2, IgG3 and IgG 4.

21. The method of claim 20, further comprising contacting the donor leukocytes with differentially labeled antibodies that distinguishably recognize the donor's B and T cells.

22. The method of claim 16, wherein the individual donor leukocytes are assessed by flow cytometry after contacting the donor leukocytes with the recipient antibody.

23. The method of claim 16, wherein the individual donor leukocytes are assessed by flow cytometry after contacting the donor leukocytes with the recipient antibody and after contacting the recipient antibody with a differentially labeled antibody that discriminately recognizes antibodies of the subclasses IgG1, IgG2, IgG3, and IgG 4.

24. The method of claim 16, wherein the individual donor leukocytes are assessed by flow cytometry after contacting the donor leukocytes with the recipient antibodies, after contacting the recipient antibodies with differentially labeled antibodies that distinguishably recognize antibodies of the subclasses IgG1, IgG2, IgG3, and IgG4, and after contacting the donor leukocytes with differentially labeled antibodies that distinguishably recognize B and T cells of the donor.

25. The method of claim 16, wherein the recipient is a vertebrate.

26. The method of claim 25, wherein the recipient is the same species as the donor.

27. The method of claim 26, wherein the recipient is a human.

28. The method of claim 16, wherein non-specific binding between the recipient antibody and the Fc receptor on the donor leukocyte is inhibited.

29. The method of claim 28, wherein non-specific binding is inhibited by contacting the donor leukocytes with a peptide that inhibits binding between the antibody and the surface Fc receptor prior to contacting the donor leukocytes with the recipient antibody.

30. The method of claim 1, wherein the donor leukocytes are viable upon contact with an antibody obtained from the recipient.