Patent Application: US-16605302-A

Abstract:
non - lethal methods of conditioning a recipient prior to bone marrow transplantation to achieve highly enhanced , stable , long - term hematopoietic chimerism in presence of transient immunosuppression are described . in particular , the administration of non - lethal doses of bone - seeking radiopharmaceuticals such as 153 samarium lexidronam , a radioactive compound linked to a tetraphosphonate group , to target bone marrow cells , are disclosed herein .

Description:
animals . all animal procedures were performed under the supervision and approval of the university of miami institutional animal care and use committee ( iacuc ). mice ( 7 - 8 week old balb / c ( h - 2 d ), c57bl / 6 ( b6 ; h - 2 b ) and c3h / hej ( c3h ; h - 2 k )) were purchased from jackson laboratories ( bar harbor , me .). recipient c57bl / 6 mice were used at 9 - 10 weeks of age . all animals were housed in pathogen - free room in sterile microisolator cages with autoclaved feed and autoclaved acidified water . bone marrow transplantation . balb / c mice , 8 - 9 weeks old , used as donors , were sacrificed on the day of the transplant . bmc were prepared according to a previously published regimen . briefly , after removing femura and tibiae , and cleaning them from muscle tissue and cartilage , bmc were flushed with sterile rpmi - 1640 ( mediatech , inc , herndon , va .) supplemented with 0 . 8 mg / ml gentamycin ( gibco , gaithersburg , md . ), using 23g needle . bmc were filtered through a sterile nylon mesh and counted . fully mch - mismatched c57bl / 6 recipients , 9 - 10 weeks of age , were injected intravenously with either 20 × 10 6 or 100 × 10 6 unmanipulated bmcs resuspended in 0 . 5 and 1 . 0 ml of hbss ( mediatech ) respectively , on either day 7 or 14 . tolerance induction protocol consisted of either 150 or 500 μci of 153 sm - edtmp ( berlex laboratories wayne , n . j . ), i . v ., on day − 7 , and 0 . 5 mg hamster anti - murine cd154 mab ( mr - 1 ), purchased from taconic ( germantown , n . y .) administered intraperitoneally ( i . p .) on days − 1 , 0 , 7 , 14 , 21 and 28 . skin grafting . full - thickness skin donor ( balb / c ) and third party ( c3h / hej ) grafts were transplanted onto the lateral thoracic area of the recipients either the day following bmc - tx , or 4 weeks following the last administration of mr - 1 mab , using techniques described previously . briefly , square , full - thickness skin grafts ( 1 cm 2 ) were prepared from the tail skin of donors . graft beds were prepared on the right ( donor - specific ) and left ( third party ) lateral thoracic wall of recipient mice . grafts were fixed to the beds with 4 sutures of 5 . 0 silk at the corners of the graft and covered with a petroleum jelly - coated gauze and a plaster cast . the grafts were first inspected on the eighth - day following grafting , and every third day thereafter . graft rejection was considered complete when no viable graft tissue was detected by visual inspection . recipient mice were considered to be tolerant when donor - specific skin grafts survived in perfect condition for & lt ; 150 days . immunohemotyping of chimeras . engraftment of donor - derived bmcs was ascertained by flow cytometric analysis ( fcm ) of recipient peripheral blood mononuclear cells ( pbmcs ), splenocytes , thymocytes and bone marrow cells after staining with fitc - conjugated anti - mouse h - 2k b or h - 2k d and cy - chrome - conjugated cd3 monoclonal antibodies ( mabs ) purchased from pharmingen ( san diego , calif .) at multiple time points during the experiment as well as at sacrifice . cells were also assessed for non - specific staining using an ig isotype control ( fitc - conjugated mouse igg 2a and cy - chrome - conjugated rat igg 2b ), and the percentage of cells stained with this ab was subtracted from the values obtained from staining with the specific ab to determine the relative number of positive cells . reconstitution of various cell lineages was assessed using fitc - conjugated anti - mouse h - 2k b or h - 2k d and pe - conjugated anti - mouse cd19 / cd22 in the b cell , pe - conjugated anti - mouse ly - 6g in the granulocyte , and pe - conjugated anti - mouse mac - 3 in the macrophage compartments . recipient animals were first tested 1 week after bmc - tx , every 2 weeks up to 6 weeks , and every 4 weeks thereafter . purified anti - mouse cd16 / cd32 ( fcγ iii / ii ) was used to block non - specific binding to the fc receptors . fcm analyses were preformed using cellquest software on a facscan cytometer purchased from becton dickinson & amp ; co . ( mountain view , calif .). analysis of various t cell receptor families . splenocytes were used to analyze the expression of vb3 + , vb5 + , vb11 + and vb14 + families in the chimeras at the time of sacrifice . for two - color analysis , cells were blocked with purified anti - mouse cd16 / cd32 ( fcγ iii / ii ) ( pharmingen ), and then incubated with fitc - conjugated h - 2k d and pe - conjugated anti - vb3 + , vb5 + , vb11 + or vb14 + ( pharmingen ) for 30 minutes on ice . fitc - conjugated mouse igg2a , pe - conjugated armenian hamster igg , group 2 , mouse igg1 , rat igg2b and rat igm antibodies ( pharmingen ) were used as negative controls . mixed lymphocyte reaction . splenocytes depleted of red blood cells were incubated at 37 ° c . in 5 % co 2 for 3 days in quintuplicate wells containing 2 × 10 5 responders with 2 × 10 5 stimulators treated with mytomicin c ( sigma , st . louis , mo .) in iscove &# 39 ; s tissue culture media ( gibco , gaithersburgh , md .) containing 10 % heat - inactivated fcs , 2 mm l - glutamine ( mediatech ), 25 mm hepes ( mediatech ) and 0 . 05 mm β - mercaptoethanol . responder cells from chimeric mice and stimulator splenocytes , bmcs and keratinocytes were incubated for 3 days in a 96 round - bottom tissue culture plates , and then pulsed with 1 μci [ 3 h ] thymidine ; [ 3 h ] thymidine incorporation was assessed after 8 hours . stimulation indices were calculated by dividing mean counts per minute ( c . p . m .) by responses against self . staining for the presence of anti - donor antibodies . 1 × 10 6 splenocytes , isolated from naïve balb / c donors were incubated with several different dilutions ( 1 : 3 ; 1 : 10 ; 1 : 30 ; 1 : 100 ) of plasma from the chimeric recipients at 4 ° c . for 60 minutes . cells were washed with pbs supplemented with 1 % bsa , 0 . 02 % sodium azide , and then incubated with fitc - conjugated goat anti - mouse igg ( h + l ) ( jackson immunoresearch laboratories , west grove , pa .) and pe - conjugated anti - mouse cd22 for 30 minutes on ice . the cells were then washed with pbs and analyzed on a becton dickinson facscan . plasma from a naïve c57bl / 6 incubated with splenocytes from naïve balb / c donors was used as a baseline . recipient animals ( c57bl / 6 , h - 2 b ) were treated with a single iv dose of 153 sm - edtmp , 150 μci or 500 μci , prior to administration of 20 × 10 6 or 100 × 10 6 allogenic donor bone marrow cells ( bmc ) ( balb / c , h - 2 d ), also administered as a single iv dose . bmc transplantation ( bmc - tx ) was performed on day 7 or 14 following the administration of 153 sm in the presence of transient t lymphocyte co - stimulatory blockade by mr - 1 ( hamster anti - murine cd154 mab ) on days - 1 , 0 , 7 , 14 , 21 and 28 , 0 . 5 mg ip . the lower dose of 153 sm , 150 μci , proved to be as effective as the higher dose , 500 μci . treatment with 153 sm - edtmp resulted in transient myelodepression that occurred one week post administration of the compound and was spontaneously resolved by 4 - 6 weeks post - administration , as shown in fig1 . both the 150 μci and 500 μci doses of 153 sm - edtmp have similar effect on hemolymphopoietic elements . although there is a marked myelodepression , as assessed by a decreased white blood cell counts ( wbc ), administration of 153 sm - edtmp does not have significant effect on red blood cell ( rbc ), hemoglobin ( hb ), and platelet ( plt ) counts . similar data were obtained in animals treated with 153 sm - edtmp and not transplanted with allogeneic bmc ( not shown ). thus , 153 sm - edmp leads to a transient myelodepression of the wbc compartment , which is spontaneously reversible either in the presence or absence of an allogeneic bmc - tx . no dramatic alterations of rbc , plt or hb counts were evident . single administration of bmc resulted in bm engraftment in all recipient animals analyzed . fig2 shows percentages of donor - derived cells in the recipients treated with 100 × 10 6 bmc , anti - cd154 mab , and one of 4 conditioning approaches — 153 sm - edtmp 150 μci , followed by administration of bmc on day 7 ; 153 sm - edtmp 500 μci , followed by administration of bmc on day 7 , 153 sm - edtmp 150 μci , followed by administration of bmc on day 14 ; and 153 sm - edtmp 500 μci , followed by administration of bmc on day 14 . typing of pbl obtained from the recipient animals starting at 2 weeks following the reconstitution with donor - derived bm allogeneic cells , every two weeks up to 6 weeks post - reconstitution , and every 4 weeks afterwards was performed using anti class i h - 2 b - fitc and h - 2 d - fitc . analysis was performed on the lymphoid gate , and the values were normalized to 100 %. cd3 + t lymphocytes of donor origin were also present , suggesting mixed chimerism of the lymphoid lineage as well . in fig3 is shown the percentage of donor - derived cells in the recipients treated with 20 × 10 6 bmc , anti - cd154 mab , and one of the 4 conditioning approaches : 153 sm - edtmp 150 μci , followed by administration of bmc on day 7 ; 153 sm - edtmp 500 μci , followed by administration of bmc on day 7 ; 153 sm - edtmp 150 μci , followed by administration of bmc on day 14 ; and 153 sm - edtmp 500 μci , followed by administration of bmc on day 14 . typing of pbl obtained from the recipient animals starting at 2 weeks following the reconstitution with donor - derived bm allogeneic cells , every two weeks up to 6 weeks post - reconstitution , and every 4 weeks afterwards was performed using anti class i h - 2 b - fitc and h - 2 d - fitc . analysis was performed on the lymphoid gate , and the values were normalized to 100 %. cd3 + t lymphocytes of donor origin were also present , suggesting mixed chimerism of the lymphoid lineage as well . therefore , administration of 153 sm - edmp in the presence of costimulatory blockade leads to long - lasting hematopoietic chimerism in the recipients of allogeneic bmc . the dose of 153 sm - edmp ( 150 μci vs . 500 μci ) and the timing of bmc - tx relative to 153 sm - edmp administration do not grossly influence the results . bmc dose , on the other hand , directly correlates with the levels of chimerism achieved . as shown in fig4 the percentage of donor - derived cells in the control animals treated with 100 × 10 6 bmc and one of the 4 conditioning approaches was assessed . the conditioning regimens were 153 sm - edtmp 150 μci , followed by administration of bmc on day 7 ; 153 sm - edtmp 500 μci , followed by administration of bmc on day 7 ; 153 sm - edtmp 150 μci , followed by administration of bmc on day 14 ; and 153 sm - edtmp 500 μci , followed by administration of bmc on day 14 . this fourth regimen differs from the previous , since no anti - cd154 mab to induce costimulatory blockade was used . typing of pbl obtained from the recipient animals starting at 2 weeks following the reconstitution with donor - derived bmc allogeneic cells , every two weeks up to 6 weeks post - reconstitution , and every 4 weeks afterwards was performed using anti class i h - 2 b - fitc and h - 2 d - fitc . analysis was performed on the lymphoid gate , and the values were normalized to 100 %. [ 0045 ] fig5 shows the percent of donor - derived cells in the control animals treated with 20 × 10 6 bmc , and one of the 4 conditioning approaches : 153 sm - edtmp 150 μci , followed by administration of bmc on day 7 ; 153 sm - edtmp 500 μci , followed by administration of bmc on day 7 ; 153 sm - edtmp 150 μci , followed by administration of bmc on day 14 ; and 153 sm - edtmp 500 μci , followed by administration of bmc on day 14 ( this regimen differs from the previous , since no anti - cd154 mab to induce costimulatory blockade was used ). typing of pbl obtained from the recipient animals starting at 2 weeks following the reconstitution with donor - derived bmc allogeneic cells , every two weeks up to 6 weeks post - reconstitution , and every 4 weeks following that was performed using anti class i h - 2 b - fitc and h - 2 d - fitc . analysis was performed on the lymphoid gate , and the values were normalized to 100 %. thus , the data from fig4 - 5 show that in the absence of co - stimulatory blockade , 153 sm - edmp administration followed by bmc - tx only leads to transient chimerism , regardless of the dose of bmc ( 20 × 10 6 or 100 × 10 6 ). the percentage of donor - derived cells in the control animals treated with 20 × 10 6 bmc or 100 × 10 6 bmc along with anti - cd154 mab ( in the absence of 153 sm - edtmp treatment ) is shown in fig6 . typing of pbl obtained from the recipient animals starting at 2 weeks following the reconstitution with donor - derived bmc allogeneic cells , every two weeks up to 6 weeks post - reconstitution , and every 4 weeks following that was performed using anti class i h - 2 b - fitc and h - 2 d - fitc . analysis was performed on the lymphoid gate , and the values to were normalized to 100 %. the results indicate that treatment with bmc - tx and co - stimulatory blockade without administration of 153 sm - edmp , leads to transient chimerism when a low dose ( 20 × 10 6 ) bmc is administered and to low level , stable chimerism when 100 × 10 6 bmc are administered . [ 0048 ] fig7 shows a two - color flow cytometric analysis of the proportion of donor - derived lymphoid ( b cells ), nk , and myeloid ( granulocytes ) lineages in representative mixed chimeras prepared using a non - lethal conditioning regiment of 20 × 10 6 bmc , 153 sm - edtmp , and anti - cd154 mab ( upper panels ) as well as 20 × 10 6 bmc and anti - cd154 mab ( lower panels ). analysis was performed using class i h - 2 d - fitc and either cd22 ( b cells ), nk , or gran1 ( granulocytes ), all pe . analysis was performed on the lymphoid gate , and the values were normalized to 100 %. in fig8 is shown a two - color flow cytometric analysis of the proportion of donor - derived lymphoid ( b cells ), nk , and myeloid ( granulocytes ) lineages in representative mixed chimeras prepared using a non - lethal conditioning regiment of 100 × 10 6 bmc , 153 sm - edtmp , and anti - cd154 mab ( upper panels ) as well as 100 × 10 6 bmc and anti - cd154 mab ( lower panels ). analysis was preformed using class i h - 2 d - fitc and either cd22 ( b cells ), nk , or gran1 ( granulocytes ), all pe . analysis was performed on the lymphoid gate , and the values were normalized to 100 %. as is evident from the data presented in fig7 and 8 , long - term , stable multilineage chimerism is achieved in the group treated with a combination of bmc - tx , 153 sm - edmp , and anti - cd154 mab . the survival of full thickness tail - derived skin grafts placed on the recipients treated with 20 × 10 6 bmc , 153 sm - edtmp , and anti - cd154 mab , or indicated control groups is shown in fig9 . grafts were prepared 30 days following the last administration of anti - cd154 mab in the treated animals . two different donor strain combinations , balb / c ( h - 2 d ) and c3h / j ( h - 2 k ) were used . each recipient received skin grafts from both strains : donor - type , balb / c ( h - 2 d ), as well as third - party , c3h / j ( h - 2 k ). third party grafts were rejected within the same time frame as were donor - specific grafts placed on naive recipients . grafts were followed for a minimum of 128 days and were considered rejected when viable tissue was no longer detected at the transplant site . therefore , tolerance to donor - specific skin grafts is obtained when animals receive a low dose of bmc ( 20 × 10 6 ), only if 153 sm - edmp is part of the treatment , while co - stimulation alone ( along with bmc ) is not sufficient to achieve the same result . the survival of full thickness tail - derived skin grafts placed on the recipients treated with 100 × 10 6 bmc , 153 sm - edtmp , and anti - cd154 mab , or indicated control groups is depicted graphically in fig1 . grafts were prepared 30 days following the last administration of anti - cd154 mab in the treated animals . two different donor strain combinations , balb / c ( h - 2 d ) and c3h / j ( h - 2 k ) were used . each recipient received skin grafts from both strains : donor - type , balb / c ( h - 2 d ), as well as third - party , c3h / j ( h - 2 k ). third party grafts were rejected within the same time frame as were donor - specific grafts placed on naïve recipients . grafts were followed for a minimum of 128 days and were considered rejected when viable tissue was no longer detected at the transplant site . thus , when a high dose of bmc is given ( 100 × 10 6 ), the enhancing effect of 153 sm - edmp administration is still visible on chimerism levels , that are reproducibly higher , but lost on graft survival since co - stimulatory blockade only (+ bmc - tx ) appears similarly efficacious . radionuclide complexes between lanthanides and bone specific carriers may be formulated into any pharmaceutically acceptable dosage form , including liquids , emulsions , suspensions and the like . liquid solutions for injection are particularly preferred . pharmaceutical compositions of the complexes for use according to the invention may also contain suitable diluents , excipients , buffers , stabilizers and carriers . sterile water or sterile isotonic saline solutions are particularly preferred . while the invention has been illustrated via the preferred embodiments described above , it will be understood that the invention may be practiced employing various modifications evident to those skilled in the art without departing from the spirit and scope of the invention as generally described herein , and as further set forth by the appended claims . 1 . saba n , flaig t . bone marrow transplantation for nonmalignant diseases . j hematother stem cell res . 2002 ( 2 ): 377 - 87 . 2 . furst d e . stem cell transplantation for autoimmune disease : progress and problems . curr opin rheumatol . 2002 ; 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