U.S. patent application number 15/884017 was filed with the patent office on 2018-08-02 for non-genotoxic conditioning regimen for stem cell transplantation.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Akanksha Chhabra, Benson M. George, Judith A. Shizuru, Irving L. Weissman.
Application Number | 20180214524 15/884017 |
Document ID | / |
Family ID | 62977017 |
Filed Date | 2018-08-02 |
United States Patent
Application |
20180214524 |
Kind Code |
A1 |
Weissman; Irving L. ; et
al. |
August 2, 2018 |
NON-GENOTOXIC CONDITIONING REGIMEN FOR STEM CELL
TRANSPLANTATION
Abstract
The present invention provides a clinically applicable method of
stem cell transplantation that facilitates engraftment and
reconstitutes immunocompetence of the recipient without requiring
radiotherapy or chemotherapy, and without development of GVHD or
graft rejection.
Inventors: |
Weissman; Irving L.;
(Stanford, CA) ; Shizuru; Judith A.; (Palo Alto,
CA) ; Chhabra; Akanksha; (San Francisco, CA) ;
George; Benson M.; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Stanford |
CA |
US |
|
|
Family ID: |
62977017 |
Appl. No.: |
15/884017 |
Filed: |
January 30, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62452218 |
Jan 30, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/39541 20130101;
C07K 16/2875 20130101; A61K 39/001 20130101; C07K 16/2803 20130101;
A61P 37/06 20180101; A61K 2039/505 20130101; C07K 16/2866 20130101;
A61K 2035/122 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 37/06 20060101 A61P037/06; A61K 39/395 20060101
A61K039/395 |
Claims
1. A method of providing for stem cell engraftment in a mammalian
subject, the method comprising a conditioning regimen that
comprises: contacting said subject concomitantly with (i) an agent
that specifically binds to endogenous stem cells in a targeted
tissue and (ii) an agent that blocks interaction between CD47 and
SIRP.alpha.; in a dose effective to ablate targeted endogenous stem
cells from said subject; contacting said subject with (iii) an
agent that induces transient immunosuppression; introducing a
cellular composition comprising exogenous stem cells to said
subject following a wash-out period of time sufficient to reduce
the serum level of (i) and (ii) to non-toxic levels in the subject;
wherein the exogenous stem cells engraft in the absence of
myeloablative conditioning.
2. The method according to claim 1, wherein the targeted tissue is
bone marrow and the targeted stem cells are hematopoietic stem
cells.
3. The method of claim 1, wherein the exogenous stem cells are
autologous or allogeneic relative to the subject.
4. The method of claim 1, wherein the exogenous stem cells are
genetically engineered ex vivo.
5. The method of claim 1, wherein said agent that specifically
binds to endogenous stem cells in a targeted tissue is a monoclonal
antibody specific for c-kit.
6. The method of claim 1, wherein the agent that blocks interaction
between CD47 and SIRP.alpha. is selected from: a soluble
SIRP.alpha. polypeptide; an antibody specific for CD47, an antibody
specific for SIRP.alpha., and a soluble CD47 polypeptide.
7. The method of claim 1, wherein the agent that induces transient
immunosuppression is selected from an agent that inhibits
CD40/CD40L activity; mycophenolic acid, cyclosporine A, rapamycin,
FK506, and corticosteroids.
8. The method of claim 7, wherein the agent that induces transient
immunosuppression is an antibody specific to CD40L.
9. The method of claim 8, wherein the agent that induces transient
immunosuppression is administered concomitantly with the exogenous
stem cells.
10. The method of claim 1, wherein the subject is an
immunocompetent human.
11. The method of claim 1, wherein the subject is haploidentical
relative to the exogenous stem cells.
12. The method of claim 1, wherein the exogenous stem cells are
administered as a composition of whole bone marrow mononuclear
cells.
13. The method of claim 1, wherein the stem cells are MHC matched
to the recipient.
14. The method of claim 1, further comprising contacting the
subject with (iv) an agent the depletes one or both of T cells and
NK cells, wherein the agent (iv) is administered prior to
introduction of the exogenous stem cells, and optionally concurrent
with the introduction of the exogenous stem cells.
15. The method of claim 14, wherein the subject is HLA-mismatched
relative to the exogenous stem cells.
16. The method of claim 14, wherein the cellular composition
comprises hematopoietic stem cells selected for CD34.sup.+
expression from bone marrow, cord blood, or peripheral blood.
17. The method of claim 16, wherein the cellular composition
comprises at least 50% CD34+ cells.
18. The method of claim 14, wherein the cellular composition
comprises hematopoietic stem cells derived from pluripotent cells
in vitro.
19. The method of claim 14, wherein the cellular composition
comprises at least 10.sup.5 CD34.sup.+ cells/kg of recipient body
weight.
20. The method of claim 14, wherein the agent (iv) depletes T cells
and NK cells.
21. The method of claim 20, wherein the agent (iv) is an antibody
selected from an antibody specific for CD2, CD52, CD45; or
anti-thymocyte globulin (ATG).
22. The method of claim 14, wherein an agent (iv) that selectively
depletes T cells is selected from an antibody specific for one or
more of CD3, CD4, and CD8.
23. The method of claim 14, wherein an agent (iv) that selectively
depletes NK cells is selected from an antibody specific for one or
more of CD122 and CD56.
24. A method of providing for stem cell engraftment in a mammalian
subject, the method comprising: HLA typing a donor and recipient to
determine an HLA-matched or HLA-mismatched pair; obtaining
hematopoietic cells from the donor comprising CD34.sup.+
hematopoietic stem and progenitor cells (HSPC) and optionally
isolating HSPC of the desired phenotype; formulating an effective
dose of the HSPC cellular composition; selecting a set of agents
for non-genotoxic conditioning regimen on the recipient prior to
infusion of the hematopoietic cells, based on the number of donor
cells administered to the recipient; the purity of the donor cells;
the degree of major histocompatibility mismatch between donor and
recipient; and the immune status of the recipient; administering
the set of agents for non-genotoxic conditioning; infusing the
hematopoietic cells; and monitoring the recipient for hematopoietic
stem cell engraftment.
Description
CROSS REFERENCE
[0001] This application claims benefit of U.S. Provisional Patent
Application No. 62/452,218, filed Jan. 30, 2017, which applications
are incorporated herein by reference in their entirety.
[0002] Stem cells provide the means for organisms to maintain and
repair certain tissues, through the ability of these cells to
self-renew and to generate differentiated cells. Clinically, bone
marrow and hematopoietic stem cell transplantation are widely used
as a means of providing patients with the capacity to generate
blood cells, usually where the patient has been depleted of
endogenous stem cells by high dose chemotherapy or radiation.
[0003] Hematopoietic cell transplantation (HCT) generally involves
the intravenous infusion of autologous or allogeneic blood forming
cells, the active subset of which are hematopoietic stem cells
(HSC); these are collected from bone marrow, peripheral blood, or
umbilical cord blood and transplanted to reestablish hematopoietic
function in patients whose bone marrow or immune system is damaged
or defective. This procedure is often performed as part of therapy
to eliminate a bone marrow infiltrative process, such as leukemia,
or to correct congenital immunodeficiency disorders. In addition,
HCT is used to allow patients with cancer to receive higher doses
of chemotherapy than bone marrow can usually tolerate; bone marrow
function is then salvaged by replacing the marrow with previously
harvested stem cells. Enriched or purified populations of HSC can
also be transplanted, and are not contaminated with other cells,
many of which are deleterious to the host.
[0004] The preparative or conditioning regimen is a critical
element in hematopoietic cell transplantation (HCT). In a
successful transplantation, clearance of bone-marrow niches must be
achieved for donor hematopoietic stem cell (HSC) to engraft. The
preparative regimen may also provide immunosuppression sufficient
to prevent rejection of the transplanted graft, and to eradicate
the disease for which the transplantation is being performed.
Current methods to clear niche space rely on radiation and/or
chemotherapy, which can impart toxic adverse effects that greatly
limit the potential clinical utility of BMT. Traditionally,
myeloablative conditioning is performed.
[0005] Myeloablative regimens can be classified as
radiation-containing or non-radiation-containing regimens,
therapies that were developed by escalating the dose of radiation
or of a particular drug to the maximally tolerated dose. Total-body
irradiation and cyclophosphamide or busulfan and cyclophosphamide
are the commonly used myeloablative therapies. These regimens are
especially used in aggressive malignancies, such as leukemias.
However, such treatment carries a number of disadvantages in terms
of toxicity to the patient.
[0006] Improved methods for engraftment of stem cells, including
hematopoietic stem cells, are of great clinical interest. The
present invention addresses this need.
SUMMARY OF THE INVENTION
[0007] Methods and compositions are provided for the long term
multilineage engraftment of stem cells, including without
limitation hematopoietic stem cells, in a recipient, by: treating
the recipient with a pre-transplantation non-myeloablative,
non-genotoxic conditioning regimen; and administering an effective
dose of a cell population comprising exogenous stem cells. The
conditioning regimen comprises administration of agents that act on
endogenous cell populations for various purposes. The methods allow
engraftment to treat hematologic disorders, and can also be used to
tolerize a recipient to a donor-type HLA for future organ
transplantation.
[0008] Endogenous stem cells are depleted by the conditioning
regimen. Agents that deplete endogenous stem cells include, without
limitation, an antibody specific for c-kit and an agent that
blockades CD47 activity. These agents are also capable of depleting
the exogenous stem cells after administration, and so require a
"wash-out" period from the time the agents are administered to the
time the exogenous stem cells are administered. The wash-out period
is sufficient to reduce the serum levels of the agents that deplete
endogenous stem cells to a non-toxic level, that does not result in
depletion of stem cells.
[0009] In some embodiments at least one agent is included in the
conditioning regimen that provides transient immune suppression of
cytotoxic lymphocytes. A variety of biological and
non-myeloablative pharmaceutical agents are available for this
purpose, including without limitation an agent that inhibits
CD40/CD40L activity; mycophenolic acid, cyclosporine A, rapamycin,
FK506, corticosteroids, etc. In some embodiments an agent inhibits
CD40L, and is an antibody specific to CD40L. The transient
immunosuppressive agent can be administered prior to or
concomitantly with the exogenous stem cells, so long as the agent
is active when the exogenous stem cells are administered.
[0010] In some embodiments at least one agent is included in the
conditioning regimen that depletes one or both of T lymphocytes and
natural killer (NK) cells. Agents that deplete T cells specifically
include without limitation, agents, including antibodies, specific
for CD3, CD4, CD8, etc. Agents that deplete T cells and NK cells
include without limitation, agents, including antibodies, specific
for CD2, CD52, CD45, anti-thymocyte globulin (ATG), etc. Agents
that deplete NK cells specifically include without limitation,
agents, including antibodies, specific for CD122, CD56, etc. The
depleting agent(s) can be administered prior to infusion the
exogenous stem cells, and are optionally active after infusion, so
long as the targeted cells have been depleted when the exogenous
stem cells are administered.
[0011] In one embodiment, methods are provided for the selection
and administration of an appropriate set of agents for
non-genotoxic conditioning prior to transplantation. It is shown
herein that the requirements of a pre-conditioning regimen for
successful engraftment of stem cells varies according to certain
parameters, including the number of donor cells administered to the
recipient; the purity of the donor cells; the degree of major
histocompatibility mismatch between donor and recipient; and the
immune status of the recipient. Selecting the appropriate set of
agents for the individual, and the timing for administration of the
agents, can optimize the therapeutic results of the
transplantation.
[0012] In some embodiments, the methods described herein may
comprise the steps of: HLA typing a donor and recipient to
determine an HLA-matched or HLA-mismatched pair; obtaining
hematopoietic cells from the donor comprising CD34.sup.+
hematopoietic stem and progenitor cells, which may be referred to
as HSPC; optionally isolating HSPC of the desired phenotype, e.g.
CD34+ cells, and formulating an effective dose of the HSPC;
selecting a set of agents for non-genotoxic conditioning regimen on
the recipient prior to infusion of the hematopoietic cells based on
the number of donor cells administered to the recipient; the purity
of the donor cells; the degree of major histocompatibility mismatch
between donor and recipient; and the immune status of the
recipient; administering the set of agents for non-genotoxic
conditioning; infusing the hematopoietic cells; and monitoring the
recipient for hematopoietic stem cell engraftment. The methods
described herein apply to both HLA-matched and HLA-mismatched
transplantation conditions, for example HLA-mismatched and not
haploidentical transplantations, haploidentical transplantations;
etc.
[0013] In some embodiments the HSPC are obtained from a donor
hematopoietic cell sample. In some embodiments the hematopoietic
cell sample is bone marrow. In some embodiments the HSPC are
obtained from umbilical cord blood. In some embodiments, the
hematopoietic cell sample is obtained by apheresis from donor
mobilized peripheral blood. In some embodiments the HSPC are
generated in vitro. The HSPC donor may be allogeneic or autologous,
for example where the HSPC are genetically engineered by
introduction or deletion of genetic material prior to re-infusion,
for example during ex vivo culture. Allogeneic donors may be MHC
matched to the recipient. The donor may be haploidentical or not
haplo-identical to the recipient. The donor may be mismatched at
one or more MHC loci, e.g. mismatched at 1, 2, 3, 4, 5 or 6 of the
major loci for MHC matching.
[0014] The HSPC are optionally isolated from the hematopoietic cell
sample for expression of CD34. The isolation may further comprise
selection for expression of CD90. HSPC that are purified may be at
least about 45% pure, as defined by the percentage of cells that
are CD34+ in the population, may be at least about 50% pure, at
least about 60% pure, at least about 70% pure, at least about 80%
pure, at least about 90% pure. The effective dose of CD34+ cells
may be from about 10.sup.5 to about 10.sup.7 CD34.sup.+ cells/kg of
recipient body weight, and may be at least about 5.times.10.sup.5
CD34.sup.+ cells/kg of recipient body weight, at least about
10.sup.6 CD34.sup.+ cells/kg of recipient body weight, at least
about 3.times.10.sup.6 CD34+ cells/kg of recipient body weight, at
least about 5.times.10.sup.6 CD34.sup.+ cells/kg of recipient body
weight, and may be 10.sup.7 CD34.sup.+ cells/kg of recipient body
weight or more. The dose of CD34+ cells; the purity of the cells,
and the total number of cells delivered, i.e. the total dose of
both CD34.sup.+ and CD34.sup.- cells in the infusate, are important
parameters for selection of the non-genotoxic conditioning
agents.
[0015] The maximum number of CD3+ cells delivered with the HSPC
composition may be not more than about 10.sup.6 CD3.sup.+ cells/kg
of recipient body weight, not more than about 5.times.10.sup.5
CD3.sup.+ cells/kg of recipient body weight, not more than about
3.times.10.sup.5 CD3.sup.+ cells/kg of recipient body weight, not
more than about 10.sup.4 CD3.sup.+ cells/kg of recipient body
weight. The number of CD3+ cells in the infusate may be a parameter
for the selection of agents that inhibit cytotoxic lymphocytes,
where increased numbers of CD3+ cells may require administration
immediately prior to, or at the time of infusion, one or more
agents that ablate T cells, including without limitation antibodies
specific for CD3, for CD4, for CD8, etc.
[0016] In some embodiments, the transplantation is performed in the
absence of myeloablative conditioning. In some embodiments the
recipient is immunocompetent. The administration of the
pre-transplantation conditioning regimen is repeated as necessary
to achieve the desired level of ablation.
[0017] In some embodiments the CD47 blockade is accomplished by
administering a soluble SIRP.alpha. polypeptide, which may be a
high affinity SIRP.alpha. variant polypeptide. In other
embodiments, antibodies specific for one or both of SIRP.alpha. and
CD47 are administered.
[0018] Following transplantation with donor stem cells, the
recipient may be a chimera or mixed chimera for the donor cells.
The methods of the invention allow effective stem cell engraftment
in the absence of non-selective ablation methods, e.g. radiation or
chemotherapy, which have the undesirable effect of ablating
differentiated cells involved in the function of the targeted
tissue as well as undesirable side effects upon other tissues (e.g.
on cells of the gastrointestinal system, hair growth), as well as
increasing risk of secondary malignancies.
[0019] In one embodiment of the invention, the stem cells are one
or more of autologous hematopoietic stem cells, genetically
modified hematopoietic stem cells, and allogeneic hematopoietic
stem cells, usually allogeneic stem cells. Such stem cells find use
in the treatment of a variety of blood disorders, e.g. genetic
disorders including aplastic anemia; sickle cell disease;
thalassemias; severe immunodeficiency; bone marrow failure states,
immune deficiencies, hemoglobinopathies, leukemias, lymphomas,
immune-tolerance induction, genetic disorders treatable by bone
marrow transplantation and other blood disorders, and the like.
[0020] The methods of the invention are also useful in the
induction of tolerance in a patient, for example tolerance to donor
tissue, e.g. in organ transplants; tolerance to autoantigens, e.g.
in the context of treatment of autoimmune disease; and the like. In
one embodiment of the invention, a method is provided for inducing
tolerance in a patient, comprising administering to a patient
administration of an agent that targets stem cells, including
without limitation an antibody specific for c-kit and an agent that
blockades CD47 activity; performed in combination with
administration of an effective dose of one or a set of agents that
reduce the number or activity of cytotoxic lymphocytes, which
cytotoxic lymphocytes may include without limitation T cells, and
natural killer (NK) cells. In some embodiments at least one agent
is included that provides transient immune suppression of cytotoxic
lymphocytes, including without limitation an agent that inhibits
CD40/CD40L activity. In some embodiments the agent is an antibody
specific to CD40L. In some embodiments the methods are performed in
the absence of genotoxic conditioning. Following the conditioning
regimen, the recipient is infused with an effective dose of
hematopoietic stem and progenitor cells, thereby providing immune
tolerance to the donor cells for future organ transplants.
BRIEF DESCRIPTION OF THE FIGURES
[0021] The invention is best understood from the following detailed
description when read in conjunction with the accompanying
drawings. The patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawing(s) will be provided by
the Office upon request and payment of the necessary fee. It is
emphasized that, according to common practice, the various features
of the drawings are not to-scale. On the contrary, the dimensions
of the various features are arbitrarily expanded or reduced for
clarity. Included in the drawings are the following figures.
[0022] FIG. 1A-1F. ACK2, Clone 3, MR-1 and CD122 enable efficient
engraftment of haploidentical whole bone marrow into immune
competent animals. FIG. 1A 30e6 AKR.times.Hz F1 whole bone marrow
was harvested and retro-orbitally transplanted into each
Balb/c.times.C57BL/6 recipient. Chimerism was determined by CD45
allelic differences. FIG. 1B Each antibody was given on the marked
day for conditioning. On day -8, 100 ug of Clone 3 is given; on all
subsequent days 500 ug of Clone 3 is given. On day -6, 500 ug of
ACK2 is given. On day -1, 250 ug of Tm-b1 is given. On day 0, 500
ug of MR1 is given. FIG. 1C-1F. Mice were conditioned using
different combinations of each antibody. Total donor chimerism was
measured over 13 weeks FIG. 1C, in addition to T-cell FIG. 1D,
B-cell FIG. 1E, and granulocytes FIG. 1F chimerism. Unchimeric
mouse in ACK2+Clone 3+MR1 cohort is censored.
[0023] FIG. 2. 16 Week donor chimerism of antibody conditioned
mice. Figure shows percentage of mice that are chimeric per cohort
and the average levels of total donor, T-cell, B-cell, and
granulocyte chimerism. Unchimeric mouse in ACK2+Clone 3+MR1 cohort
is censored.
[0024] FIG. 3A-3F. NK-Cell depletion is required for engraftment of
low cell dose bone marrow. FIG. 3A Various amounts of AKR.times.Hz
F1 whole bone marrow was harvested and retroorbitally transplanted
into each Balb/c.times.C57BL/6 recipient. Chimerism was determined
by CD45 allelic differences. FIG. 3B Each antibody was given on the
marked day for conditioning. On day -8, 100 ug of Clone 3 is given;
on all subsequent days 500 ug of Clone 3 is given. On day -6, 500
ug of ACK2 is given. On day -2, 250 ug of Tm-b1 is given. On day 0,
500 ug of MR1 is given. C-F. Each group was conditioned at the
minimum with Clone 3, ACK2, and MR1. CD122 was also added to the
two noted cohorts; thus receiving all four antibodies. Conditioned
mice received either 30.times.10.sup.6, 10.sup.6, 3.times.10.sup.6
or 10.sup.5 whole bone marrow. Total donor chimerism was measured
at 3 weeks FIG. 3C, in addition to T-cell FIG. 3D, B-cell FIG. 3E,
and granulocytes FIG. 3F chimerism.
[0025] FIG. 4A-4I. A monoclonal antibody cocktail can induce
long-term multi-lineage hematopoietic reconstitution. FIG. 4A
Haploidentical transplantation schema using AKRB6F1 donors and
CB6F1 recipients. FIG. 4B Flow cytometric analysis of MHC Class I
on donor and recipient strains. FIG. 4C Dosing schedule for
conditioning regimen. FIG. 4D Donor chimerism in the long-term HSC
compartment (Lin- c-KIT+ Sca1+ CD150+ Flk2- CD34-) following Ab
conditioning. FIG. 4E-4G Ab conditioning allows for long-term
multi-lineage chimerism after WBM transplantation. FIG. 4H CBC
following WBM Ab conditioning on Day 0. FIG. 4I Percentage of
animals which are chimeric at various WBM doses, with or without NK
cell depletion.
[0026] FIG. 5A-5F. A monoclonal antibody cocktail can induce
long-term multi-lineage hematopoietic reconstitution of low dose
purified HSCs. FIG. 5A Sorting scheme used to calculate and isolate
LSK and c-KIT+ cells for transplantation. FIG. 5B Granulocyte
chimerism following various hematopoietic cell grafts. FIG. 5C
Dosing schedule for LSK Ab conditioning. FIG. 5D Mature immune cell
population abundances in the bone marrow following LSK Ab
conditioning. FIG. 5E Total peripheral blood donor chimerism
following LSK transplantation. FIG. 5F Percentage of animals which
are chimeric following exclusion of individual components of the
LSK Ab cocktail.
[0027] FIG. 6A-6E. Low dose LSK transplantation via a non-genotoxic
conditioning regimen allows for tolerance to donor tissue. FIG.
6A-6B Abundance of donor-reactive host T-cells in peripheral blood
following WBM FIG. 6A and LSK FIG. 6B transplantation. FIG. 6C
Ear-heart graft schematic. FIG. 6D Donor heart survival. FIG. 6E
Gross examination, H&E, and IF of representative ear-heart
grafts 34 days following tissue transplant.
[0028] FIG. 7A-7D. Hematopoietic stem cells can be engrafted
despite a full MHC mismatch. FIG. 7A Transplantation schematic
where DBA1/J are the donor and CB6F1 are the host. FIG. 7B Percent
of donor engraftment following WBM and LSK transplantation after 8
weeks. FIG. 7C Overall survival of transplanted animals. FIG. 7D
Overview describing an all-antibody conditioning regimen which can
deplete endogenous HSCs, and provide transient immune suppression
by targeting host T and NK cells.
[0029] FIG. 8A-8D. FIG. 8A Sorting scheme to determine peripheral
blood chimerism by CD45 allelic differences between the host and
the donor. Multi-lineage peripheral blood chimerism 16 weeks
following WBM transplant is shown for FIG. 8B T-cells, FIG. 8C B
cells and FIG. 8D granulocytes.
[0030] FIG. 9. Peripheral blood donor chimerism following
monotherapeutic conditioning using monoclonal antibodies.
[0031] FIG. 10A-10C. Complete blood count FIG. 10A, peripheral
blood subpopulations FIG. 10B and splenic subpopulations FIG. 10C
from animals one day after conditioning is completed without
transplantation.
[0032] FIG. 11. 16-week peripheral blood chimerism following
variations of LSK Ab conditioning.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0033] Methods are provided for the engraftment of stem cells in a
subject by treatment with a non-genotoxic, non-myeloablative
condition prior to infusion of a cellular composition comprising
the stem and progenitor cells.
[0034] It is an objective of the present invention to provide a
clinically applicable method of stem cell transplantation which
facilitates engraftment and reconstitutes immunocompetence of the
recipient without requiring radiotherapy or chemotherapy, or
development of GVHD or graft rejection. Guidelines are also
provided for selecting an appropriate conditioning regimen based on
the nature and dose of the donor stem cell population, and the
degree of HLA matching between the donor and recipient.
[0035] Aspects of the present invention are based on the discovery
that a depletion of the endogenous stem cell niche that facilitates
efficient engraftment of hematopoietic stem cells (HSCs) is
accomplished by combining the use of an agent that targets the
endogenous stem cells, e.g. anti-c-kit antibody, with an agent that
enhances the killing of endogenous stem cells by blocking the
interaction of CD47 and SIRP.alpha., optionally combined with
transient immunosuppression; and optionally combined with agents
that deplete T and/or NK cells, allows safe engraftment of the
donor cells. In particular, the present invention combines this
improved selective ablation of endogenous stem cells, in
combination with the administration to the recipient of exogenous
stem cells, resulting in efficient, long-term engraftment and
tolerance.
[0036] It is to be understood that this invention is not limited to
the particular methodology, products, apparatus and factors
described, as such methods, apparatus and formulations may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by appended claims.
[0037] It must be noted that as used herein and in the appended
claims, the singular forms "a," "and," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a drug candidate" refers to one or mixtures
of such candidates, and reference to "the method" includes
reference to equivalent steps and methods known to those skilled in
the art, and so forth.
[0038] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
publications mentioned herein are incorporated herein by reference
for the purpose of describing and disclosing devices, formulations
and methodologies which are described in the publication and which
might be used in connection with the presently described
invention.
[0039] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges may independently be
included in the smaller ranges is also encompassed within the
invention, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either both of those included limits are also
included in the invention.
[0040] In the following description, numerous specific details are
set forth to provide a more thorough understanding of the present
invention. However, it will be apparent to one of skill in the art
that the present invention may be practiced without one or more of
these specific details. In other instances, well-known features and
procedures well known to those skilled in the art have not been
described in order to avoid obscuring the invention.
[0041] Generally, conventional methods of protein synthesis,
recombinant cell culture and protein isolation, and recombinant DNA
techniques within the skill of the art are employed in the present
invention. Such techniques are explained fully in the literature,
see, e.g., Maniatis, Fritsch & Sambrook, Molecular Cloning: A
Laboratory Manual (1982); Sambrook, Russell and Sambrook, Molecular
Cloning: A Laboratory Manual (2001); Harlow, Lane and Harlow, Using
Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold
Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory; (1988).
Definitions
[0042] Conditioning regimen. Patients undergoing an allogeneic
hemopoietic stem cell transplant (HSCT), are prepared with a so
called conditioning regimen that may suppress the recipient's
immune system and deplete endogenous stem cells, in order to allow
engraftment of the donor stem cells.
[0043] The intensity of conventional conditioning regimens can vary
significantly. Description of the regimens can refer to genotoxic
or non-genotoxic regimens, which may overlap with reference to
myeloablative or non-myeloablative regimens. See, for example,
Bacigalupo et al. (2009) Biol Blood Marrow Transplant.
15(12):1628-1633, herein specifically incorporated by
reference.
[0044] Genotoxic regimens comprise, at least in part, the
administration of agents with direct or indirect effects on the
DNA: the induction of mutations, mistimed event activation, and
direct DNA damage leading to mutations. Examples of genotoxic
agents include radiation and certain chemotherapeutic drugs, such
as alkylating agents, intercalating agents and inhibitors of
enzymes involved in DNA replication. The methods of the invention
are non-genotoxic, and thus exclude the use of such agents.
[0045] Myeloablative conditioning regimens are combination of
agents expected to produce profound pancytopenia and myeloablation
within 1-3 weeks from administration; pancytopenia is long lasting,
usually irreversible and in most instances fatal, unless
hematopoiesis is restored by hemopoietic stem cell infusion.
Examples include total body irradiation and/or administration of
alkylating agents; fludarabine, dimethylbusulfan, etoposide (VP16);
etc. There is significant overlap in genotoxic and myeloablative
agents.
[0046] Non-myeloablative conditioning regiments typically cause
minimal cytopenia, and little early toxicity, but are
immunosuppressive to the extent that, when followed by
administration of an effective dose of HSPC, will result in
engraftment of donor lympho-hemopoietic stem cells.
[0047] The conditioning regimens provided herein are non-genotoxic
and non-myeloablative, and primarily utilize targeted agents for
depletion of endogenous cells that prevent engraftment, without
causing log-lasting pancytopenia. The methods do not utilize
genotoxic chemotherapeutic agents or radiation, although in some
instances non-genotoxic, targeted immunosuppressive agents, such as
cyclosporine A, corticosteroids, etc. can find use for transient
immunosuppression.
[0048] "Concomitant administration" of active agents in the methods
of the invention means administration with the reagents at such
time that the agents will have a therapeutic effect at the same
time. Such concomitant administration may involve concurrent (i.e.
at the same time), prior, or subsequent administration of the
agents. A person of ordinary skill in the art would have no
difficulty determining the appropriate timing, sequence and dosages
of administration for particular drugs and compositions of the
present invention.
[0049] Stem cell markers. Exemplary markers for antibody mediated
ablation of human hematopoietic stem cells include CD34; CD90
(thy-1); CD59; CD110 (c-mpl); c-kit (CD-117); etc. Markers useful
for the ablation of mesodermal stem cells include Fc.gamma.RII,
Fc.gamma.RIII, Thy-1, CD44, VLA-4.alpha., LFA-1.beta., HSA, ICAM-1,
CD45, Aa4.1, Sca-1, etc. Neural crest stem cells may be positively
selected with antibodies specific for low-affinity nerve growth
factor receptor (LNGFR). Neural stem/progenitor cells have been
described in the art, and their use in a variety of therapeutic
protocols has been widely discussed. For example, inter alia,
Uchida et al. (2000) Proc Natl Acad Sci USA. 97(26):14720-5. U.S.
Pat. No. 6,638,501, Bjornson et al.; U.S. Pat. No. 6,541,255,
Snyder et al.; U.S. Pat. No. 6,498,018, Carpenter; U.S. Patent
Application 20020012903, Goldman et al.; Palmer et al. (2001)
Nature 411(6833):42-3; Palmer et al. (1997) Mol Cell Neurosci.
8(6):389-404; Svendsen et al. (1997) Exp. Neurol. 148(1):135-46 and
Shihabuddin (1999) Mol Med Today. 5(11):474-80; each herein
specifically incorporated by reference. Human mesenchymal stem
cells may be ablated using the markers such as SH2 (CD105), SH3 and
SH4 and Stro-1.
[0050] In one embodiment of the invention, the marker for depletion
is c-kit (CD117). CD117 is a receptor tyrosine kinase type III,
which binds to stem cell factor (a substance that causes certain
types of cells to grow), also known as "steel factor" or "c-kit
ligand". When this receptor binds to stem cell factor (SCF) it
forms a dimer that activates its intrinsic tyrosine kinase
activity, that in turn phosphorylates and activates signal
transduction molecules that propagate the signal in the cell. See,
for example, the human refseq entries Genbank NM_000222; NP_000213.
CD117 is an important cell surface marker used to identify certain
types of hematopoietic (blood) progenitors in the bone marrow.
Hematopoietic stem cells (HSC), multipotent progenitors (MPP), and
common myeloid progenitors (CMP) express high levels of CD117. A
number of antibodies that specifically bind human CD117 are known
in the art and commercially available, including without limitation
SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, etc. Of interest is the
humanized form of SR1, AMG 191, described in U.S. Pat. Nos.
8,436,150, and 7,915,391 which is an aglycosylated IgG1 humanized
antibody.
[0051] An effective dose of an anti-CD117 antibody may be
administered in one or more doses, including a single dose, which
may be at least about one week prior to transplantation, at least
about 5 days prior to transplantation, at least about 3 days prior
to transplantation. The period of time between dosing and
transplantation is sufficient to substantially eliminate the
anti-CD117 antibody from the circulation of the recipient. For
example the decrease in peak serum levels following administration
is usually the time sufficient for the level to decrease as least
about 10-fold from peak levels, usually at least about 100-fold,
1000-fold, 10,000-fold, or more. It is preferable to introduce the
donor stem cells within the empty niche "window" following the
wash-out period, usually within about 3 days, about 2 days, about 1
day, or at the time of clearance.
[0052] In some embodiments, an effective dose of an anti-CD117
antibody is up to about 10 mg/kg, up to about 5 mg/kg; up to about
1 mg/kg, up to about 0.5 mg/kg; up to about 0.1 mg/kg; up to about
0.05 mg/kg; where the dose may vary with the specific antibody and
recipient.
[0053] Anti-CD47 agent. As used herein, the term "anti-CD47 agent"
or "agent that provides for CD47 blockade" refers to any agent that
reduces the binding of CD47 (e.g., on a target cell) to SIRP.alpha.
(e.g., on a phagocytic cell). Non-limiting examples of suitable
anti-CD47 reagents include SIRP.alpha. reagents, including without
limitation high affinity SIRP.alpha. polypeptides, anti-SIRP.alpha.
antibodies, soluble CD47 polypeptides, and anti-CD47 antibodies or
antibody fragments. In some embodiments, a suitable anti-CD47 agent
(e.g. an anti-CD47 antibody, a SIRP.alpha. reagent, etc.)
specifically binds CD47 to reduce the binding of CD47 to
SIRP.alpha..
[0054] The effective dose of an anti-CD47 agent can vary with the
agent, but will generally range from up to about 50 mg/kg, up to
about 40 mg/kg, up to about 30 mg/kg, up to about 20 mg/kg, up to
about 10 mg/kg, up to about 5 mg/kg; up to about 1 mg/kg, up to
about 0.5 mg/kg; up to about 0.1 mg/kg; up to about 0.05 mg/kg;
where the dose may vary with the specific antibody and recipient.
Agents that bind to CD47, e.g. soluble SIRP.alpha. polypeptides and
anti-CD47 antibodies, may be administered at higher doses due to
the larger number of CD47 expressing cells in the body.
[0055] The anti-CD47 agent may be administered one or a plurality
of days prior to transplantation, and in some embodiments is
administered daily for a period of from about 1, about 2, about 3,
about 4, about 5, about 6, about 7 or more days, i.e. from about 1
to 7 days, from about 1 to 5 days, from about 1 to 3 days, etc. As
with the anti-c-kit agent, targeting CD47 can affect the donor stem
cells after infusion, and therefore a wash-out period is required
before infusion of hematopoietic cells. The washout period may be
shorter than with the c-kit antibody, but is typically at least
about 24 hours, at least 36 hours, at least 48 hours, and may be up
to about one week, up to about 5 days, up to about 3 days, etc.
[0056] In some embodiments, a suitable anti-CD47 agent (e.g., an
anti-SIRP.alpha. antibody, a soluble CD47 polypeptide, etc.)
specifically binds SIRP.alpha. to reduce the binding of CD47 to
SIRP.alpha.. A suitable anti-CD47 agent that binds SIRP.alpha. does
not activate SIRP.alpha. (e.g., in the SIRP.alpha.-expressing
phagocytic cell). The efficacy of a suitable anti-CD47 agent can be
assessed by assaying the agent. In an exemplary assay, target cells
are incubated in the presence or absence of the candidate agent. An
agent for use in the methods of the invention will up-regulate
phagocytosis by at least 5% (e.g., at least 10%, at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 100%, at least 120%, at least
140%, at least 160%, at least 180%, at least 200%, at least 500%,
at least 1000%) compared to phagocytosis in the absence of the
agent. Similarly, an in vitro assay for levels of tyrosine
phosphorylation of SIRP.alpha. will show a decrease in
phosphorylation by at least 5% (e.g., at least 10%, at least 15%,
at least 20%, at least 30%, at least 40%, at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, or 100%) compared to
phosphorylation observed in absence of the candidate agent.
[0057] In some embodiments, the anti-CD47 agent does not activate
CD47 upon binding. When CD47 is activated, a process akin to
apoptosis (i.e., programmed cell death) may occur (Manna and
Frazier, Cancer Research, 64, 1026-1036, Feb. 1, 2004). Thus, in
some embodiments, the anti-CD47 agent does not directly induce cell
death of a CD47-expressing cell.
[0058] SIRP.alpha. reagent. A SIRP.alpha. reagent comprises the
portion of SIRP.alpha. that is sufficient to bind CD47 at a
recognizable affinity, which normally lies between the signal
sequence and the transmembrane domain, or a fragment thereof that
retains the binding activity. A suitable SIRP.alpha. reagent
reduces (e.g., blocks, prevents, etc.) the interaction between the
native proteins SIRP.alpha. and CD47. The SIRP.alpha. reagent will
usually comprise at least the d1 domain of SIRP.alpha..
[0059] In some embodiments, a subject anti-CD47 agent is a "high
affinity SIRP.alpha. reagent", which includes SIRP.alpha.-derived
polypeptides and analogs thereof (e.g., CV1-hIgG4, and CV1
monomer). High affinity SIRP.alpha. reagents are described in
international application PCT/US13/21937, which is hereby
specifically incorporated by reference. High affinity SIRP.alpha.
reagents are variants of the native SIRP.alpha. protein. The amino
acid changes that provide for increased affinity are localized in
the d1 domain, and thus high affinity SIRP.alpha. reagents comprise
a d1 domain of human SIRP.alpha., with at least one amino acid
change relative to the wild-type sequence within the d1 domain.
Such a high affinity SIRP.alpha. reagent optionally comprises
additional amino acid sequences, for example antibody Fc sequences;
portions of the wild-type human SIRP.alpha. protein other than the
d1 domain, including without limitation residues 150 to 374 of the
native protein or fragments thereof, usually fragments contiguous
with the d1 domain; and the like. High affinity SIRP.alpha.
reagents may be monomeric or multimeric, i.e. dimer, trimer,
tetramer, etc. In some embodiments, a high affinity SIRP.alpha.
reagent is soluble, where the polypeptide lacks the SIRP.alpha.
transmembrane domain and comprises at least one amino acid change
relative to the wild-type SIRP.alpha. sequence, and wherein the
amino acid change increases the affinity of the SIRP.alpha.
polypeptide binding to CD47, for example by decreasing the off-rate
by at least 10-fold, at least 20-fold, at least 50-fold, at least
100-fold, at least 500-fold, or more.
[0060] Optionally the SIRP.alpha. reagent is a fusion protein,
e.g., fused in frame with a second polypeptide. In some
embodiments, the second polypeptide is capable of increasing the
size of the fusion protein, e.g., so that the fusion protein will
not be cleared from the circulation rapidly. In some embodiments,
the second polypeptide is part or whole of an immunoglobulin Fc
region. The Fc region aids in phagocytosis by providing an "eat me"
signal, which enhances the block of the "don't eat me" signal
provided by the high affinity SIRP.alpha. reagent. In other
embodiments, the second polypeptide is any suitable polypeptide
that is substantially similar to Fc, e.g., providing increased
size, multimerization domains, and/or additional binding or
interaction with Ig molecules.
[0061] Anti-CD47 antibodies. In some embodiments, a subject
anti-CD47 agent is an antibody that specifically binds CD47 (i.e.,
an anti-CD47 antibody) and reduces the interaction between CD47 on
one cell (e.g., an infected cell) and SIRP.alpha. on another cell
(e.g., a phagocytic cell). In some embodiments, a suitable
anti-CD47 antibody does not activate CD47 upon binding. Some
anti-CD47 antibodies do not reduce the binding of CD47 to
SIRP.alpha. (and are therefore not considered to be an "anti-CD47
agent" herein) and such an antibody can be referred to as a
"non-blocking anti-CD47 antibody." A suitable anti-CD47 antibody
that is an "anti-CD47 agent" can be referred to as a "CD47-blocking
antibody". Non-limiting examples of suitable antibodies include
clones B6H12, 5F9, 8B6, and C3 (for example as described in
International Patent Publication WO 2011/143624, herein
specifically incorporated by reference). Suitable anti-CD47
antibodies include fully human, humanized or chimeric versions of
such antibodies. Humanized antibodies (e.g., hu5F9-G4) are
especially useful for in vivo applications in humans due to their
low antigenicity. Similarly caninized, felinized, etc. antibodies
are especially useful for applications in dogs, cats, and other
species respectively. Antibodies of interest include humanized
antibodies, or caninized, felinized, equinized, bovinized,
porcinized, etc., antibodies, and variants thereof.
[0062] Anti-SIRP.alpha. antibodies. In some embodiments, a subject
anti-CD47 agent is an antibody that specifically binds SIRP.alpha.
(i.e., an anti-SIRP.alpha. antibody) and reduces the interaction
between CD47 on one cell (e.g., an infected cell) and SIRP.alpha.
on another cell (e.g., a phagocytic cell). Suitable
anti-SIRP.alpha. antibodies can bind SIRP.alpha. without activating
or stimulating signaling through SIRP.alpha. because activation of
SIRP.alpha. would inhibit phagocytosis. Instead, suitable
anti-SIRP.alpha. antibodies facilitate the preferential
phagocytosis of inflicted cells over normal cells. Those cells that
express higher levels of CD47 (e.g., infected cells) relative to
other cells (non-infected cells) will be preferentially
phagocytosed. Thus, a suitable anti-SIRP.alpha. antibody
specifically binds SIRP.alpha. (without activating/stimulating
enough of a signaling response to inhibit phagocytosis) and blocks
an interaction between SIRP.alpha. and CD47. Suitable
anti-SIRP.alpha. antibodies include fully human, humanized or
chimeric versions of such antibodies. Humanized antibodies are
especially useful for in vivo applications in humans due to their
low antigenicity. Similarly caninized, felinized, etc. antibodies
are especially useful for applications in dogs, cats, and other
species respectively. Antibodies of interest include humanized
antibodies, or caninized, felinized, equinized, bovinized,
porcinized, etc., antibodies, and variants thereof.
[0063] Soluble CD47 polypeptides. In some embodiments, a subject
anti-CD47 agent is a soluble CD47 polypeptide that specifically
binds SIRP.alpha. and reduces the interaction between CD47 on one
cell (e.g., an infected cell) and SIRP.alpha. on another cell
(e.g., a phagocytic cell). A suitable soluble CD47 polypeptide can
bind SIRP.alpha. without activating or stimulating signaling
through SIRP.alpha. because activation of SIRP.alpha. would inhibit
phagocytosis. Instead, suitable soluble CD47 polypeptides
facilitate the preferential phagocytosis of infected cells over
non-infected cells. Those cells that express higher levels of CD47
(e.g., infected cells) relative to normal, non-target cells (normal
cells) will be preferentially phagocytosed. Thus, a suitable
soluble CD47 polypeptide specifically binds SIRP.alpha. without
activating/stimulating enough of a signaling response to inhibit
phagocytosis.
[0064] In some cases, a suitable soluble CD47 polypeptide can be a
fusion protein (for example as structurally described in US Patent
Publication US20100239579, herein specifically incorporated by
reference). However, only fusion proteins that do not
activate/stimulate SIRP.alpha. are suitable for the methods
provided herein. Suitable soluble CD47 polypeptides also include
any peptide or peptide fragment comprising variant or naturally
existing CD47 sequences (e.g., extracellular domain sequences or
extracellular domain variants) that can specifically bind
SIRP.alpha. and inhibit the interaction between CD47 and
SIRP.alpha. without stimulating enough SIRP.alpha. activity to
inhibit phagocytosis.
[0065] In certain embodiments, soluble CD47 polypeptide comprises
the extracellular domain of CD47, including the signal peptide,
such that the extracellular portion of CD47 is typically 142 amino
acids in length. The soluble CD47 polypeptides described herein
also include CD47 extracellular domain variants that comprise an
amino acid sequence at least 65%-75%, 75%-80%, 80-85%, 85%-90%, or
95%-99% (or any percent identity not specifically enumerated
between 65% to 100%), which variants retain the capability to bind
to SIRP.alpha. without stimulating SIRP.alpha. signaling.
[0066] In certain embodiments, the signal peptide amino acid
sequence may be substituted with a signal peptide amino acid
sequence that is derived from another polypeptide (e.g., for
example, an immunoglobulin or CTLA4). For example, unlike
full-length CD47, which is a cell surface polypeptide that
traverses the outer cell membrane, the soluble CD47 polypeptides
are secreted; accordingly, a polynucleotide encoding a soluble CD47
polypeptide may include a nucleotide sequence encoding a signal
peptide that is associated with a polypeptide that is normally
secreted from a cell.
[0067] In other embodiments, the soluble CD47 polypeptide comprises
an extracellular domain of CD47 that lacks the signal peptide. As
described herein, signal peptides are not exposed on the cell
surface of a secreted or transmembrane protein because either the
signal peptide is cleaved during translocation of the protein or
the signal peptide remains anchored in the outer cell membrane
(such a peptide is also called a signal anchor). The signal peptide
sequence of CD47 is believed to be cleaved from the precursor CD47
polypeptide in vivo.
[0068] In other embodiments, a soluble CD47 polypeptide comprises a
CD47 extracellular domain variant. Such a soluble CD47 polypeptide
retains the capability to bind to SIRP.alpha. without stimulating
SIRP.alpha. signaling. The CD47 extracellular domain variant may
have an amino acid sequence that is at least 65%-75%, 75%-80%,
80-85%, 85%-90%, or 95%-99% identical (which includes any percent
identity between any one of the described ranges) to the native
CD47 sequence.
[0069] Transient immunosuppressive agent. A transient
immunosuppressive agent blocks the activity of immune cells,
particularly T lymphocytes, for a short period of time, usually the
period of time at or shortly before the administration of the donor
cells. Transient immunosuppression, i.e. an effective serum level
of the immunosuppressive agent(s) may be maintained for at least
about 3 days, at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, at least about 6 weeks, and may be maintained for up to 1
month, up to 2 months, up to 3 months, up to 4 months, up to 5
months, up to 6 months, or more. In some embodiments a single dose
of the agent is administered immediately prior to, or
concomitantly, with the donor cells. Such agents are usually
suppressive, without ablation of the immune cell population. The
initial dose of the agent may be made within about 3 days, within
about 2 days, within about 1 day, or at the time of administration
of the donor cells.
[0070] Transient immunosuppression can be achieved by
administration of a pharmacologic immunosuppressive agent,
including without limitation calcineurin inhibitors, which combine
with binding proteins to inhibit calcineurin activity, and which
include, for example, tacrolimus, cyclosporine A, etc. Levels of
both cyclosporine and tacrolimus must be carefully monitored.
Initially, levels can be kept in the range of 10-20 ng/mL, but,
after 3 months, levels may be kept lower (5-10 ng/mL) to reduce the
risk of nephrotoxicity. Other pharmacologic agents for this purpose
include steroids, azathioprine, mycophenolate mofetil, and
sirolimus, etc.
[0071] In some embodiments a transient immunosuppressive agent
blocks the interaction of CD40 and CD40 ligand. CD40 is a
costimulatory protein found on antigen presenting cells (APCs) and
is required for their activation. These APCs include phagocytes
(macrophages and dendritic cells) and B cells. CD40 is part of the
TNF receptor family. The primary activating signaling molecules for
CD40 are IFN.gamma. and CD40 ligand (CD40L).
[0072] "CD40 ligand" ("CD40L", also called "CD154") is a type II
transmembrane protein. CD40L was originally considered restricted
to activated T lymphocytes, functioning as a mediator of T
cell-dependent B cell activation, proliferation, and
differentiation. Expression of CD40L plays a functional role as a
central mediator of immunity and inflammation of the tumor necrosis
factor (TNF) gene superfamily. CD40/CD40L interaction is essential
for the development of thymus-dependent humoral immune responses.
CD40L modulates physiologic processes, such as T cell-mediated
effector functions and general immune responses required for
appropriate host defense, but also triggers the expression of
pro-inflammatory mediators, such as cytokines, adhesion molecules,
and matrix degrading activities, all of which are associated with
the pathogenesis of chronic inflammatory diseases, e.g., autoimmune
disorders, arthritis, atherosclerosis, and cancer.
[0073] Given its critical role in mediating many aspects of immune
responses, the CD40/CD40L pathway provides a therapeutic target for
the prevention of transplantation rejection. Interrupting the
CD40/CD40L signal pathway with anti-CD40L antibody can be effective
in preventing acute allograft rejection and alloantibody responses
in animal models and in clinical use. Subsequent studies have
demonstrated the beneficial effect of anti-CD40L on the
prolongation of graft survival in a number of rodent models (islet,
limb, corneal and marrow).
[0074] As used herein, the term "anti-CD40L agent" or "agent that
provides for CD40L blockade" refers to any agent that reduces the
binding of CD40L (e.g., on a target cell) to CD40. Non-limiting
examples of suitable anti-CD40L reagents include anti-CD40
antibodies, and anti-CD40L antibodies or antibody fragments. Agents
of interest also include, without limitation, fragments of
antibodies and small molecules. For example, CDP7657 is a high
affinity PEGylated monovalent Fab' anti-CD40L antibody fragment. An
effective dose of an antibody may be up to about 50 mg/kg, up to
about 25 mg/kg; up to about 10 mg/kg, up to about 5 mg/kg; up to
about 1 mg/kg; up to about 0.5 mg/kg; or less, where the dose may
vary with the specific antibody and recipient. As an alternative to
antibodies, small molecule inhibitors are described, for example in
Chen et al. (2017) J. Med. Chem. 60, 8906-8922, herein specifically
incorporated by reference.
[0075] T cell ablation. For some transplant situations, as outlined
in Table 1, it is desirable to delete endogenous T cells. In some
embodiments the ablative agent is specific for T cells, in others
it also acts on NK cells. Antibodies that target T cells include,
for example, antibodies specific for CD2, CD3, CD4, CD8, CD52
(campath), CD45, and ATG.
[0076] With respect to timing, a T cell depleting agent is
desirably active in the period of time at or shortly before the
administration of the donor cells. Therapeutic levels of the
depletion agent may be maintained for at least about 3 days, at
least about 1 week, at least about 2 weeks, at least about 3 weeks,
at least about 4 weeks, at least about 5 weeks, at least about 6
weeks, and may be maintained for up to 1 month, up to 2 months, up
to 3 months, up to 4 months, up to 5 months, up to 6 months, or
more following administration of the donor cells. In some
embodiments a dose of the agent is administered within about 3
days, within about 2 days, within about 1 day, or at the time of
administration of the donor cells, and depending on the antibody,
may be administered daily for several days, e.g. 2, 3 4 etc., prior
to infusion. An effective dose of an antibody may be up to about 50
mg/kg, up to about 25 mg/kg; up to about 10 mg/kg, up to about 5
mg/kg; up to about 1 mg/kg; up to about 0.5 mg/kg; or less, for
example up to about 100 .mu.g/kg, up to about 50 .mu.g/kg, up to
about 10 .mu.g/kg, up to about 1 .mu.g/kg, where the dose may vary
with the specific antibody and recipient. Antibody-based therapy
may use monoclonal (e.g., muromonab-CD3) or polyclonal antibodies;
anti-CD25 antibodies (e.g., basiliximab, daclizumab), etc.
Antibodies include, for example, an ATG preparation, aKT3,
BTI-322.RTM. (U.S. Pat. No. 5,730,979 the disclosure of which is
hereby incorporated by reference).
[0077] Multiple anti-human CD3 mAb are in clinical development,
including Teplizumab, and MGA031, is a humanized IgG1 antibody that
was developed by grafting the complementarity determining region of
OKT3 into a human IgG1 backbone. Otelixizumab (ChAglyCD3, TRX4,
GSK2136525) is derived from the rat antibody YTH12.5, and is a
humanized IgG1 with a single mutation in the .gamma.1 Fc portion to
avoid glycosylation and thus inhibit FcR binding. Visilizumab
(Nuvion, HuM291) is a humanized IgG2 antibody rendered non
mitogenic by two point mutations in its Fc region. Foralumab
(28F11-AE; NI-0401) is an entirely human anti-CD3 mAb.
[0078] A useful agent for depletion of T cells and NK cells is an
anti-CD52 antibody, exemplified by the clinically approved antibody
Campath (alemtuzumab), which is a recombinant DNA-derived humanized
monoclonal antibody directed against the 21-28 kD cell surface
glycoprotein, CD52. Campath-1H is an IgG1 kappa antibody with human
variable framework and constant regions, and
complementarity-determining regions from a murine (rat) monoclonal
antibody (Campath-1G). Campath may be administered, for example, at
the currently accepted clinical dose, e.g. escalating to the
maximum single dose of 30 mg over a period of from about 3 to about
7 days.
[0079] NK cell ablation. For some transplant situations, as
outlined in Table 1, it is desirable to also delete endogenous NK
cells. As indicated above, some agents act on both T cells and NK
cells, e.g. antibodies to CD2, CD52, etc. Other agents are specific
for NK cells and may be administered in combination with T cell
targeted agents. Antibodies that selectively target NK cells
include, for example, antibodies specific for CD122 and CD56.
[0080] With respect to timing, an NK cell depleting agent is
desirably active in the period of time at or shortly before the
administration of the donor cells. Therapeutic levels of the
depletion agent may be maintained for at least about 3 days, at
least about 1 week, at least about 2 weeks, at least about 3 weeks,
at least about 4 weeks, at least about 5 weeks, at least about 6
weeks, and may be maintained for up to 1 month, up to 2 months, up
to 3 months, up to 4 months, up to 5 months, up to 6 months, or
more following administration of the donor cells. In some
embodiments a dose of the agent is administered within about 3
days, within about 2 days, within about 1 day, or at the time of
administration of the donor cells, and depending on the antibody,
may be administered daily for several days, e.g. 2, 3, 4 etc.,
prior to infusion. An effective dose of an antibody may be up to
about 50 mg/kg, up to about 25 mg/kg; up to about 10 mg/kg, up to
about 5 mg/kg; up to about 1 mg/kg; up to about 0.5 mg/kg; or less,
for example up to about 100 .mu.g/kg, up to about 50 .mu.g/kg, up
to about 10 .mu.g/kg, up to about 1 .mu.g/kg, where the dose may
vary with the specific antibody and recipient.
[0081] "CD122" (also called "interleukin-2 receptor subunit beta",
IL2RB) is a type I membrane protein. CD122 is a subunit of the
interleukin 2 receptor (IL2R), which is involved in T cell-mediated
immune responses, and is present in 3 forms with respect to ability
to bind interleukin 2. The low affinity form of IL2R is a monomer
of the alpha subunit and is not involved in signal transduction.
The intermediate affinity form consists of an alpha/beta subunit
heterodimer, while the high affinity form consists of an
alpha/beta/gamma subunit heterotrimer. Both the intermediate and
high affinity forms of the receptor are involved in
receptor-mediated endocytosis and transduction of mitogenic signals
from interleukin 2. The use of alternative promoters results in
multiple transcript variants encoding the same protein.
[0082] As used herein, the term "anti-CD122 agent" or "agent that
provides for CD122 blockade" refers to any agent that depletes
CD122 positive cells, including natural killer (NK) cells.
Non-limiting examples of suitable anti-CD122 reagents include
anti-IL-2 antibodies, and anti-CD122 antibodies or antibody
fragments.
[0083] Antibodies that target CD56 are in clinical development and
find use in NK cell depletion. For example, IMGN901 is a
CD56-targeting antibody-drug conjugate designed for selective
delivery of the cytotoxic maytansinoid DM1 with a maximum tolerated
dose (MTD) of about 75 mg/m.sup.2. and which may be administered at
doses of, for example, from about 1 to about 60 mg/m.sup.2.
[0084] "Major histocompatibility complex antigens" ("MHC", also
called "human leukocyte antigens", HLA) are protein molecules
expressed on the surface of cells that confer a unique antigenic
identity to these cells. MHC/HLA antigens are target molecules that
are recognized by T-cells and natural killer (NK) cells as being
derived from the same source of hematopoietic stem cells as the
immune effector cells ("self") or as being derived from another
source of hematopoietic reconstituting cells ("non-self"). Two main
classes of HLA antigens are recognized: HLA class I and HLA class
II. HLA class I antigens (A, B, and C in humans) render each cell
recognizable as "self," whereas HLA class II antigens (DR, DP, and
DQ in humans) are involved in reactions between lymphocytes and
antigen presenting cells. Both have been implicated in the
rejection of transplanted organs.
[0085] An important aspect of the HLA gene system is its
polymorphism. Each gene, MHC class I (A, B and C) and MHC class II
(DP, DQ and DR) exists in different alleles. HLA alleles are
designated by numbers and subscripts. For example, two unrelated
individuals may carry class I HLA-B, genes B5, and Bw41,
respectively. Allelic gene products differ in one or more amino
acids in the .alpha. and/or .beta. domain(s). Large panels of
specific antibodies or nucleic acid reagents are used to type HLA
haplotypes of individuals, using leukocytes that express class I
and class II molecules. The genes most important for HLA typing are
the six MHC Class I and Class II proteins, two alleles for each of
HLA-A; HLA-B and HLA-DR.
[0086] The HLA genes are clustered in a "super-locus" present on
chromosome position 6p21, which encodes the six classical
transplantation HLA genes and at least 132 protein coding genes
that have important roles in the regulation of the immune system as
well as some other fundamental molecular and cellular processes.
The complete locus measures roughly 3.6 Mb, with at least 224 gene
loci. One effect of this clustering is that "haplotypes", i.e. the
set of alleles present on a single chromosome, which is inherited
from one parent, tend to be inherited as a group. The set of
alleles inherited from each parent forms a haplotype, in which some
alleles tend to be associated together. Identifying a patient's
haplotypes can help predict the probability of finding matching
donors and assist in developing a search strategy, because some
alleles and haplotypes are more common than others and they are
distributed at different frequencies in different racial and ethnic
groups.
[0087] As used herein, the term "HLA matched" refers to a donor
recipient pair in which none of the HLA antigens are mismatched
between the donor and recipient. HLA matched (i.e., where all of
the 6 alleles are matched) donor/recipient pairs have a decreased
risk of graft v. host disease (GVHD) relative to mismatched pairs
(i.e. where at least one of the 6 alleles is mismatched). HLA
haploidentical refers to a match where one chromosome is matched at
least at HLA-A; HLA-B and HLA-DR, and may be matched at minor
histocompatibility loci on the chromosome; but is not necessarily
matched on the second chromosome. Such donors frequently occur in
families, e.g. a parent is haploidentical to a child; and siblings
may be haploidentical.
[0088] As used herein, the term "HLA mismatched" refers to a donor
recipient pair in which at least one HLA antigen, in particular
with respect to HLA-A, HLA-B and HLA-DR, is mismatched between the
donor and recipient. In some cases, one haplotype is matched and
the other is mismatched. This situation is frequently found with
organs from living or deceased donors. HLA mismatched
donor/recipient pairs have an increased risk of GVHD relative to
perfectly matched pairs (i.e. where all 6 alleles are matched).
[0089] HLA alleles are typically noted with a variety of levels of
detail. Most designations begin with HLA- and the locus name, then
* and some (even) number of digits specifying the allele. The first
two digits specify a group of alleles. Older typing methodologies
often could not completely distinguish alleles and so stopped at
this level. The third through fourth digits specify a synonymous
allele. Digits five through six denote any synonymous mutations
within the coding frame of the gene. The seventh and eighth digits
distinguish mutations outside the coding region. Letters such as L,
N, Q, or S may follow an allele's designation to specify an
expression level or other non-genomic data known about it. Thus, a
completely described allele may be up to 9 digits long, not
including the HLA-prefix and locus notation.
[0090] As used herein, a "recipient" is an individual to whom an
organ, tissue or cells from another individual (donor), commonly of
the same species, has been transferred. For the purposes of the
present disclosure, a recipient and a donor are either HLA-matched
or HLA-mismatched.
[0091] As used herein, "antibody" includes reference to an
immunoglobulin molecule immunologically reactive with a particular
antigen, and includes both polyclonal and monoclonal antibodies.
The term also includes genetically engineered forms such as
chimeric antibodies (e.g., humanized murine antibodies) and
heteroconjugate antibodies. The term "antibody" also includes
antigen binding forms of antibodies, including fragments with
antigen-binding capability (e.g., Fab', F(ab').sub.2, Fab, Fv and
rIgG. The term also refers to recombinant single chain Fv fragments
(scFv). The term antibody also includes bivalent or bispecific
molecules, diabodies, triabodies, and tetrabodies.
[0092] Selection of antibodies for endogenous stem cell ablation
and transient immunosuppression may be based on a variety of
criteria, including selectivity, affinity, cytotoxicity, etc. The
phrase "specifically (or selectively) binds" to an antibody or
"specifically (or selectively) immunoreactive with," when referring
to a protein or peptide, refers to a binding reaction that is
determinative of the presence of the protein, in a heterogeneous
population of proteins and other biologics. Thus, under designated
immunoassay conditions, the specified antibodies bind to a
particular protein sequences at least two times the background and
more typically more than 10 to 100 times background. In general,
antibodies of the present invention bind antigens on the surface of
target cells in the presence of effector cells (such as natural
killer cells or macrophages). Fc receptors on effector cells
recognize bound antibodies. The cross-linking of Fc receptors
signals the effector cells to kill the target cells by cytolysis or
apoptosis. In one embodiment, the induction is achieved via
antibody-dependent cellular cytotoxicity (ADCC). In alternative
embodiments, the antibodies are active in growth inhibition of the
targeted cells, an ablation is achieved by interfering with growth
factor signaling, e.g. antibodies specific for growth factor
receptors such as c-kit.
[0093] An antibody immunologically reactive with a particular
antigen can be generated by recombinant methods such as selection
of libraries of recombinant antibodies in phage or similar vectors,
or by immunizing an animal with the antigen or with DNA encoding
the antigen. Methods of preparing polyclonal antibodies are known
to the skilled artisan. The antibodies may, alternatively, be
monoclonal antibodies. Monoclonal antibodies may be prepared using
hybridoma methods. In a hybridoma method, an appropriate host
animal is typically immunized with an immunizing agent to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the immunizing agent. Alternatively,
the lymphocytes may be immunized in vitro. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell.
[0094] Human antibodies can be produced using various techniques
known in the art, including phage display libraries. Similarly,
human antibodies can be made by introducing of human immunoglobulin
loci into transgenic animals, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which
closely resembles that seen in humans in all respects, including
gene rearrangement, assembly, and antibody repertoire.
[0095] Antibodies also exist as a number of well-characterized
fragments produced by digestion with various peptidases. Thus
pepsin digests an antibody below the disulfide linkages in the
hinge region to produce F(ab)'.sub.2, a dimer of Fab which itself
is a light chain joined to V.sub.H-C.sub.H1 by a disulfide bond.
The F(ab)'.sub.2 may be reduced under mild conditions to break the
disulfide linkage in the hinge region, thereby converting the
F(ab)'.sub.2 dimer into an Fab' monomer. The Fab' monomer is
essentially Fab with part of the hinge region. While various
antibody fragments are defined in terms of the digestion of an
intact antibody, one of skill will appreciate that such fragments
may be synthesized de novo either chemically or by using
recombinant DNA methodology. Thus, the term antibody, as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries.
[0096] A "humanized antibody" is an immunoglobulin molecule which
contains minimal sequence derived from non-human immunoglobulin.
Humanized antibodies include human immunoglobulins (recipient
antibody) in which residues from a complementary determining region
(CDR) of the recipient are replaced by residues from a CDR of a
non-human species (donor antibody) such as mouse, rat or rabbit
having the desired specificity, affinity and capacity. In some
instances, Fv framework residues of the human immunoglobulin are
replaced by corresponding non-human residues. Humanized antibodies
may also comprise residues which are found neither in the recipient
antibody nor in the imported CDR or framework sequences. In
general, a humanized antibody will comprise substantially all of at
least one, and typically two, variable domains, in which all or
substantially all of the CDR regions correspond to those of a
non-human immunoglobulin and all or substantially all of the
framework (FR) regions are those of a human immunoglobulin
consensus sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin.
[0097] Antibodies of interest for ablation may be tested for their
ability to induce ADCC (antibody-dependent cellular cytotoxicity).
Antibody-associated ADCC activity can be monitored and quantified
through detection of either the release of label or lactate
dehydrogenase from the lysed cells, or detection of reduced target
cell viability (e.g. annexin assay). Assays for apoptosis may be
performed by terminal deoxynucleotidyl transferase-mediated
digoxigenin-11-dUTP nick end labeling (TUNEL) assay (Lazebnik et
al., Nature: 371, 346 (1994). Cytotoxicity may also be detected
directly by detection kits known in the art, such as Cytotoxicity
Detection Kit from Roche Applied Science (Indianapolis, Ind.).
Preferably, the antibodies of the present invention induce at least
10%, 20%, 30%, 40%, 50%, 60%, or 80% cytotoxicity of the target
cells.
[0098] In some embodiments, the antibody is conjugated to an
effector moiety. The effector moiety can be any number of
molecules, including labeling moieties such as radioactive labels
or fluorescent labels, or can be a cytotoxic moiety. Cytotoxic
agents are numerous and varied and include, but are not limited to,
cytotoxic drugs or toxins or active fragments of such toxins.
Suitable toxins and their corresponding fragments include
diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain,
curcin, crotin, phenomycin, enomycin, saporin, auristatin-E and the
like. Cytotoxic agents also include radiochemicals made by
conjugating radioisotopes to antibodies. Targeting the cytotoxic
moiety to transmembrane proteins serves to increase the local
concentration of the cytotoxic moiety in the targeted area.
[0099] The term stem cell is used herein to refer to a mammalian
cell that has the ability both to self-renew, and to generate
differentiated progeny (see Morrison et al. (1997) Cell
88:287-298). Generally, stem cells also have one or more of the
following properties: an ability to undergo asynchronous, or
symmetric replication, that is where the two daughter cells after
division can have different phenotypes; extensive self-renewal
capacity; capacity for existence in a mitotically quiescent form;
and clonal regeneration of all the tissue in which they exist, for
example the ability of hematopoietic stem cells to reconstitute all
hematopoietic lineages.
[0100] For engraftment purposes, a composition comprising
hematopoietic stem cells, is administered to a patient. Such
methods are well known in the art. The stem cells are optionally,
although not necessarily, purified. Abundant reports explore
various methods for purification of stem cells and subsequent
engraftment, including flow cytometry; an isolex system (Klein et
al. (2001) Bone Marrow Transplant. 28(11):1023-9; Prince et al.
(2002) Cytotherapy 4(2):137-45); immunomagnetic separation (Prince
et al. (2002) Cytotherapy 4(2):147-55; Handgretinger et al. (2002)
Bone Marrow Transplant. 29(9):731-6; Chou et al. (2005) Breast
Cancer. 12(3):178-88); and the like. Each of these references is
herein specifically incorporated by reference, particularly with
respect to procedures, cell compositions and doses for
hematopoietic stem cell transplantation.
[0101] Hematopoietic stem cells can be obtained by harvesting from
bone marrow or from peripheral blood. Bone marrow is generally
aspirated from the posterior iliac crests while the donor is under
either regional or general anesthesia. Additional bone marrow can
be obtained from the anterior iliac crest. A dose of
1.times.10.sup.8 and 2.times.10.sup.8 marrow mononuclear cells per
kilogram is generally considered desirable to establish engraftment
in autologous and allogeneic marrow transplants, respectively. Bone
marrow can be primed with granulocyte colony-stimulating factor
(G-CSF; filgrastim [Neupogen]) to increase the stem cell count.
Reference to "whole bone marrow" for the purposes described herein
generally refers to a composition of mononuclear cells derived from
bone marrow that have not been selected for specific immune cell
subsets. "Fractionated bone marrow" may be, for example, depleted
of T cells, e.g. CD8.sup.+ cells, CD52.sup.+ cells, CD3.sup.+
cells, etc.; enriched for CD34+ cells, etc.
[0102] Hematopoietic stem cells are also obtained from cord blood.
Cord blood is an almost unlimited source of hematopoietic stem
cells for allogeneic hematopoietic stem cell transplant. Cord blood
banks (CBB) have been established for related or unrelated UCBT
with more than 400,000 units available and more than 20,000
umbilical cord blood transplants performed in children and in
adults. UCB hematopoietic progenitors are enriched in primitive
stem/progenitor cells able to produce in vivo long-term
repopulating stem cells. However, the number of cells available
from any single donor can be relatively low in comparison with
other sources.
[0103] Mobilization of stem cells from the bone marrow into
peripheral blood by cytokines such as G-CSF or GM-CSF has led to
the widespread adoption of peripheral blood progenitor cell
collection by apheresis for hematopoietic stem cell
transplantation. The dose of G-CSF used for mobilization is 10
.mu.g/kg/day. In autologous donors who are heavily pretreated,
however, doses of up to 40 .mu.g/kg/day can be given. Mozobil may
be used In conjunction with G-CSF to mobilize hematopoietic stem
cells to peripheral blood for collection.
[0104] The dose of stem cells administered may depend on the
desired purity of the infused cell composition, and the source of
the cells. Current guidelines indicate that the minimum dose
required for engraftment is 1-2.times.10.sup.6 CD34.sup.+ cells/kg
body weight for autologous and allogeneic transplants. Higher doses
can include, for example, 3.times.10.sup.6, 4.times.10.sup.6,
5.times.10.sup.6, 6.times.10.sup.6, 7.times.10.sup.6,
8.times.10.sup.6, 9.times.10.sup.6, 10.sup.7 or more. Frequently
the dose is limited by the number of available cells. Typically,
regardless of the source, the dose is calculated by the number of
CD34+ cells present. The percent number of CD34.sup.+ cells can be
low for unfractionated bone marrow or mobilized peripheral blood;
in which case the total number of cells administered is much
higher.
[0105] The CD34+ cells may be selected by affinity methods,
including without limitation magnetic bead selection, flow
cytometry, and the like from the donor hematopoietic cell sample.
The HSPC composition may be at least about 50% pure, as defined by
the percentage of cells that are CD34+ in the population, may be at
least about 75% pure, at least about 85% pure, at least about 95%
pure, or more. Preferable a maximum number of CD3+ cells delivered
with the HSPC composition is not more than about 10.sup.6 CD3.sup.+
cells/kg of recipient body weight, not more than about 10.sup.5
CD3.sup.+ cells/kg of recipient body weight, not more than about
10.sup.4 CD3.sup.+ cells/kg of recipient body weight. Alternatively
cell populations may be tandemly selected for expression of CD34
and CD90, which cell populations may be highly purified, e.g. at
least about 85% CD34.sup.+ CD90.sup.+ cells, at least about 90%
CD34.sup.+ CD90.sup.+ cells, at least about 95% CD34.sup.+
CD90.sup.+ cells and may be up to about 99% CD34.sup.+ CD90.sup.+
cells or more. Alternatively unmanipulated bone marrow or mobilized
peripheral blood populations are used.
[0106] Hematopoietic stem cells can also be generated in vitro, for
example from pluripotent embryonic stem cells, induced pluripotent
cells, and the like. For example, see Sugimura et al. (2017) Nature
545:432-438, herein specifically incorporated by reference, which
details a protocol for generation of hematopoietic progenitors.
[0107] The cells which are employed may be fresh, frozen, or have
been subject to prior culture. They may be fetal, neonate, adult,
etc. Hematopoietic stem cells may be obtained from fetal liver,
bone marrow, blood, particularly G-CSF or GM-CSF mobilized
peripheral blood, or any other conventional source. Cells for
engraftment are optionally isolated from other cells, where the
manner in which the stem cells are separated from other cells of
the hematopoietic or other lineage is not critical to this
invention. If desired, a substantially homogeneous population of
stem or progenitor cells may be obtained by selective isolation of
cells free of markers associated with differentiated cells, while
displaying epitopic characteristics associated with the stem
cells.
[0108] Cells may be genetically altered in order to introduce genes
useful in the differentiated cell, e.g. repair of a genetic defect
in an individual, selectable marker, etc., or genes useful in
selection against undifferentiated ES cells. Cells may also be
genetically modified to enhance survival, control proliferation,
and the like. Cells may be genetically altering by transfection or
transduction with a suitable vector, homologous recombination, or
other appropriate technique, so that they express a gene of
interest. In one embodiment, cells are transfected with genes
encoding a telomerase catalytic component (TERT), typically under a
heterologous promoter that increases telomerase expression beyond
what occurs under the endogenous promoter, (see International
Patent Application WO 98/14592). In other embodiments, a selectable
marker is introduced, to provide for greater purity of the desired
differentiating cell. Cells may be genetically altered using vector
containing supernatants over an 8-16 h period, and then exchanged
into growth medium for 1-2 days. Genetically altered cells are
selected using a drug selection agent such as puromycin, G418, or
blasticidin, and then recultured.
[0109] The cells of this invention can also be genetically altered
in order to enhance their ability to be involved in tissue
regeneration, or to deliver a therapeutic gene to a site of
administration. A vector is designed using the known encoding
sequence for the desired gene, operatively linked to a promoter
that is constitutive, pan-specific, specifically active in a
differentiated cell type, etc. Suitable inducible promoters are
activated in a desired target cell type, either the transfected
cell, or progeny thereof. By transcriptional activation, it is
intended that transcription will be increased above basal levels in
the target cell by at least about 100 fold, more usually by at
least about 1000 fold. Various promoters are known that are induced
in different cell types.
[0110] Many vectors useful for transferring exogenous genes into
target mammalian cells are available. The vectors may be episomal,
e.g. plasmids, virus derived vectors such cytomegalovirus,
adenovirus, etc., or may be integrated into the target cell genome,
through homologous recombination or random integration, e.g.
retrovirus derived vectors such MMLV, HIV-1, ALV, etc. For
modification of stem cells, lentiviral vectors are preferred.
Lentiviral vectors such as those based on HIV or FIV gag sequences
can be used to transfect non-dividing cells, such as the resting
phase of human stem cells. Combinations of retroviruses and an
appropriate packaging line may also find use, where the capsid
proteins will be functional for infecting the target cells.
Usually, the cells and virus will be incubated for at least about
24 hours in the culture medium. The cells are then allowed to grow
in the culture medium for short intervals in some applications,
e.g. 24-73 hours, or for at least two weeks, and may be allowed to
grow for five weeks or more, before analysis. Commonly used
retroviral vectors are "defective", i.e. unable to produce viral
proteins required for productive infection. Replication of the
vector requires growth in the packaging cell line. The vectors may
include genes that must later be removed, e.g. using a recombinase
system such as Cre/Lox, or the cells that express them destroyed,
e.g. by including genes that allow selective toxicity such as
herpesvirus TK, bcl-xs, etc.
[0111] Chimerism, as used herein, generally refers to chimerism of
the hematopoietic system, unless otherwise noted. A determination
of whether an individual is a full chimera, mixed chimera, or
non-chimeric made be made by an analysis of a hematopoietic cell
sample from the graft recipient, e.g. peripheral blood, bone
marrow, etc. as known in the art. Analysis may be done by any
convenient method of typing. In some embodiments the degree of
chimerism amongst all mononuclear cells, T cells, B cells, CD56+NK
cells, and CD15+ neutrophils is regularly monitored, using PCR with
probes for microsatellite analysis. For example, commercial kits
that distinguish polymorphisms in short terminal repeat lengths of
donor and host origin are available. Automated readers provide the
percentage of donor type cells based on standard curves from
artificial donor and host cell mixtures.
[0112] Individuals who exhibited more than a 95% donor cells in a
given blood cell lineage by such analysis at any time
post-transplantation are referred to as having full donor chimerism
in this transplant patient group. Mixed chimerism is defined as
greater than 1% donor but less than 95% donor DNA in such analysis.
Individuals who exhibit mixed chimerism may be further classified
according to the evolution of chimerism, where improving mixed
chimerism is defined as a continuous increase in the proportion of
donor cells over at least a 6-month period. Stable mixed chimerism
is defined as fluctuations in the percentage of recipient cells
over time, without complete loss of donor cells.
[0113] A "patient" for the purposes of the present invention
includes both humans and other animals, particularly mammals,
including pet and laboratory animals, e.g. mice, rats, rabbits,
etc. Thus the methods are applicable to both human therapy and
veterinary applications. In one embodiment the patient is a mammal,
preferably a primate. In other embodiments the patient is
human.
[0114] Additional terms. The terms "treatment", "treating", "treat"
and the like are used herein to generally refer to obtaining a
desired pharmacologic and/or physiologic effect. The effect can be
prophylactic in terms of completely or partially preventing a
disease or symptom(s) thereof and/or may be therapeutic in terms of
a partial or complete stabilization or cure for a disease and/or
adverse effect attributable to the disease. The term "treatment"
encompasses any treatment of a disease in a mammal, particularly a
human, and includes: (a) preventing the disease and/or symptom(s)
from occurring in a subject who may be predisposed to the disease
or symptom but has not yet been diagnosed as having it; (b)
inhibiting the disease and/or symptom(s), i.e., arresting their
development; or (c) relieving the disease symptom(s), i.e., causing
regression of the disease and/or symptom(s). Those in need of
treatment include those already inflicted (e.g., those with cancer,
those with an infection, etc.) as well as those in which prevention
is desired (e.g., those with increased susceptibility to cancer,
those with an increased likelihood of infection, those suspected of
having cancer, those suspected of harboring an infection,
etc.).
Methods for Engraftment
[0115] The methods of the invention provide for improved
engraftment of stem cells after transplantation into a recipient.
The recipient may be immunocompetent, and the transplantation may
be performed in the absence of myeloablative conditioning, i.e. in
the absence of radiation and/or chemotherapeutic drugs. The
recipient is conditioned with the combined administration a set of
agents selected according to the cells and HLA match. The selection
of agents is indicated in Table 1, which provides guidelines for
optimized conditioning protocols. A "+" indicates that for the
indicated agent, HLA match and cell source, the agent should be
included; and a "-" indicates it is not required, although
optionally can be included. As disclosed above, certain agents can
deplete both T cells and NK cells, and therefore only the single
agent is required for both. The timing and dose for the different
agents is as indicated above. The conditioning regimens of the
invention selectively ablate endogenous stem cells and provide for
suitable, selected suppression of endogenous immune responses,
which allow for engraftment even in non-matched recipients.
TABLE-US-00001 TABLE 1 Autologous Haploindentical Unmatched/Xeno
SC.sup.@ TI.sup.# NK* T.sup.& SC.sup.@ TI.sup.# NK* T.sup.&
SC.sup.@ TI.sup.# NK* T.sup.& Bone Marrow + - - - + + - - + + +
+ high dose Bone marrow + - - - + + - - + + + + low dose Cord Blood
+ - - - + + + + + + + + Unfractionated + - - - + + + + + + + + PBMC
PBMC + - - - + + + + + + + + enriched HSPC engineered + + - - + + +
+ + + + + cell populations SC.sup.@ administer a combination of
agents to deplete endogenous stem cells, and allow a washout period
prior to infusion of the indicated exogenous cell population.
TI.sup.# administer an agent active at the time of infusion to
provide for transient immunosuppression NK* administer an agent
prior to and optionally at the time of infusion to deplete NK cells
T.sup.& administer an agent prior to and optionally at the time
of infusion to deplete T cells Bone marrow high dose is equivalent
to .gtoreq.150 .times. 10.sup.7 CD34.sup.+ cells/kg Bone marrow low
dose is equivalent to >150 .times. 10.sup.6 CD34.sup.+ cells/kg
PBMC enriched HSPC have a purity of greater than 50% CD34.sup.+
cells Engineered cell populations are genetically altered ex vivo,
or derived from pluripotent progenitors in vitro
[0116] Following the conditioning regimen, an effective dose of a
cellular composition comprising exogenous stem cells is
administered to the recipient during a period of transient
immunosuppression. The stem cells may be autologous, allogeneic or
xenogeneic, including without limitation allogeneic haploidentical
stem cells, mismatched allogeneic stem cells, genetically
engineered autologous cells, etc.
[0117] The infusion of HSPC is a relatively simple process that is
performed at the bedside. A bone marrow product is generally used
fresh and is infused through a central vein over a period of
several hours. Autologous products are frequently cryopreserved; if
so they are thawed at the bedside and infused rapidly over a period
of several minutes. PBMC may be stored briefly overnight or
frozen.
[0118] Where the donor is allogeneic to the recipient, the HLA type
of the donor and recipient may be tested for a match, or
haploidentical cells are used. HLA-haploidentical donors can be
manipulated by CD34 or CD34CD90 selection. Moreover,
HLA-haplo-identical donors are now widely used (and may surpass
HLA-identical) for other indications. For HLA matching,
traditionally, the loci critical for matching are HLA-A, HLA-B, and
HLA-DR. HLA-C and HLA-DQ are also now considered when determining
the appropriateness of a donor. A completely matched sibling donor
is generally considered the ideal donor. For unrelated donors, a
complete match or a single mismatch is considered acceptable for
most transplantation, although in certain circumstances, a greater
mismatch is tolerated. Preferably matching is both serologic and
molecular. Where the donor is umbilical cord blood the degree of
tolerable HLA disparity is much greater, and a match of 3-4 out of
the 6 HLA-A, HLA-B and HLA-DRB1 antigens is sufficient for
transplantation. Immunocompetent donor T cells may be removed using
a variety of methods to reduce or eliminate the possibility that
graft versus host disease (GVHD) will develop.
[0119] In some embodiments, success of the procedure is monitored
by determining the presence of host-derived myeloid cells, e.g.
CD15.sup.+ cells, in circulation of the recipient. Blood myeloid
chimerism is indicator of true HSC engraftment due to the
short-lived nature of myeloid cells. After about 8 weeks post-HCT,
methods described herein have provided for measurable and sustained
levels of blood myeloid chimerism, e.g. of at least about 1% donor
type CD15.sup.+ cells, at least about 2% donor type CD15.sup.+
cells, at least about 4% donor type CD15.sup.+ cells, at least
about 8% donor type CD15.sup.+ cells, or more.
[0120] The conditioning agents, which may be provided in the
absence of myeloablative radiation or chemotherapy, are
administered according to the specific requirements discussed
above. Some agents are administered to be active following
administration of the HSPC, while other agents require a washout
period.
[0121] The transient immunosuppressive agent is provided in a dose
that decreases activated T cell activity by at least 10-fold, at
least 100-fold, at least 1000-fold, at least 100,000-fold or more.
The effective dose will depend on the individual and the specific
agent, but will where the agent is an antibody, the dose may be at
least about 50 .mu.g/kg body weight, at least about 250 .mu.g/kg,
at least about 500 .mu.g/kg, at least about 750 .mu.g/kg, at least
about 1 mg/kg, and up to about 2.5 mg/kg, up to about 5 mg/kg, up
to about 7.5 mg/kg, up to about 10 mg/kg, up to about 15 mg/kg, up
to about 25 mg/kg, up to about 50 mg/kg, up to about 100 mg/kg.
[0122] The conditioning agents are formulated in pharmaceutical
compositions. The exact dose will depend on the purpose of the
treatment, and will be ascertainable by one skilled in the art
using known techniques (e.g., Ansel et al., Pharmaceutical Dosage
Forms and Drug Delivery; Lieberman, Pharmaceutical Dosage Forms
(vols. 1-3, 1992), Dekker, ISBN 0824770846, 082476918X, 0824712692,
0824716981; Lloyd, The Art, Science and Technology of
Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations
(1999)). As is known in the art, adjustments for patient condition,
systemic versus localized delivery, as well as the age, body
weight, general health, sex, diet, time of administration, drug
interaction and the severity of the condition may be necessary, and
will be ascertainable with routine experimentation by those skilled
in the art.
[0123] The administration of the agents can be done in a variety of
ways as discussed above, including, but not limited to, orally,
subcutaneously, intravenously, intranasally, transdermally,
intraperitoneally, intramuscularly, or intraocularly. Antibodies
may be delivered by intravenous injection.
[0124] In one embodiment, the pharmaceutical compositions are in a
water soluble form, such as being present as pharmaceutically
acceptable salts, which is meant to include both acid and base
addition salts. "Pharmaceutically acceptable acid addition salt"
refers to those salts that retain the biological effectiveness of
the free bases and that are not biologically or otherwise
undesirable, formed with inorganic acids such as hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and
the like, and organic acids such as acetic acid, propionic acid,
glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic
acid, succinic acid, fumaric acid, tartaric acid, citric acid,
benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,
ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the
like. "Pharmaceutically acceptable base addition salts" include
those derived from inorganic bases such as sodium, potassium,
lithium, ammonium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum salts and the like. Particularly useful are the
ammonium, potassium, sodium, calcium, and magnesium salts. Salts
derived from pharmaceutically acceptable organic non-toxic bases
include salts of primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted
amines, cyclic amines and basic ion exchange resins, such as
isopropylamine, trimethylamine, diethylamine, triethylamine,
tripropylamine, and ethanolamine.
[0125] The pharmaceutical compositions may also include one or more
of the following: carrier proteins such as serum albumin; buffers;
fillers such as microcrystalline cellulose, lactose, corn and other
starches; binding agents; sweeteners and other flavoring agents;
coloring agents; and polyethylene glycol.
[0126] The pharmaceutical compositions can be administered in a
variety of unit dosage forms depending upon the method of
administration. For example, unit dosage forms suitable for oral
administration include, but are not limited to, powder, tablets,
pills, capsules and lozenges. It is recognized that compositions of
the invention when administered orally, should be protected from
digestion. This is typically accomplished either by complexing the
molecules with a composition to render them resistant to acidic and
enzymatic hydrolysis, or by packaging the molecules in an
appropriately resistant carrier, such as a liposome or a protection
barrier. Means of protecting agents from digestion are well known
in the art.
[0127] The compositions for administration will commonly comprise
an antibody or other agent dissolved in a pharmaceutically
acceptable carrier, preferably an aqueous carrier. A variety of
aqueous carriers can be used, e.g., buffered saline and the like.
These solutions are sterile and generally free of undesirable
matter. These compositions may be sterilized by conventional, well
known sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, e.g.,
sodium acetate, sodium chloride, potassium chloride, calcium
chloride, sodium lactate and the like. The concentration of active
agent in these formulations can vary widely, and will be selected
primarily based on fluid volumes, viscosities, body weight and the
like in accordance with the particular mode of administration
selected and the patient's needs (e.g., Remington's Pharmaceutical
Science (15th ed., 1980) and Goodman & Gillman, The
Pharmacological Basis of Therapeutics (Hardman et al., eds.,
1996)).
[0128] The compositions containing ablative agents, e.g.
antibodies, soluble SIRP.alpha., etc. can be administered for
therapeutic treatment. Compositions are administered to a patient
in an amount sufficient to substantially ablate targeted endogenous
stem cells, as described above. An amount adequate to accomplish
this is defined as a "therapeutically effective dose." Single or
multiple administrations of the compositions may be administered
depending on the dosage and frequency as required and tolerated by
the patient. The particular dose required for a treatment will
depend upon the medical condition and history of the mammal, as
well as other factors such as age, weight, gender, administration
route, efficiency, etc.
[0129] In the methods of the invention, the agents are administered
as a short course of therapy prior to transplantation. Usually the
treatment is completed at least about one week prior to
transplantation, at least about 5 days prior to transplantation, at
least about 3 days prior to transplantation. The process may be
repeated if necessary, e.g. may be repeated twice, three times,
four times, five times, or more, as necessary to clear the
niche.
Conditions for Treatment
[0130] The indications for stem cell transplantation vary according
to disease categories and are influenced by factors such as
cytogenetic abnormalities, response to prior therapy, patient age
and performance status, disease status (remission vs relapse),
disease-specific prognostic factors, availability of a suitable
graft source, time of referral, and time to transplant.
[0131] Autologous HSCT is currently used to treat the following
conditions: Multiple myeloma, Non-Hodgkin lymphoma, Hodgkin
disease, Acute myeloid leukemia, Neuroblastoma, Germ cell tumors,
Autoimmune disorders--Systemic lupus erythematosus (SLE), systemic
sclerosis, Amyloidosis.
[0132] Allogenic HSCT is currently used to treat the following
disorders: Acute myeloid leukemia, Acute lymphoblastic leukemia,
Chronic myeloid leukemia; Chronic lymphocytic leukemia,
Myeloproliferative disorders, Myelodysplastic syndromes, Multiple
myeloma, Non-Hodgkin lymphoma, Hodgkin disease, Aplastic anemia,
Pure red cell aplasia, Paroxysmal nocturnal hemoglobinuria, Fanconi
anemia, Thalassemia major, Sickle cell anemia, Severe combined
immunodeficiency (SCID), Wiskott-Aldrich syndrome, Hemophagocytic
lymphohistiocytosis (HLH), Inborn errors of metabolism--Eg,
mucopolysaccharidosis, Gaucher disease, metachromatic
leukodystrophies, and adrenoleukodystrophies, Epidermolysis
bullosa, Severe congenital neutropenia, Shwachman-Diamond syndrome,
Diamond-Blackfan anemia, Leukocyte adhesion deficiency, and the
like.
[0133] Embodiments of the invention include transplantation into a
patient suffering from a genetic blood disorder, where exogenous
stem cells of a normal phenotype are transplanted into the patient.
Such diseases include, without limitation, the treatment of anemias
caused by defective hemoglobin synthesis (hemoglobinopathies). The
stem cells may be allogeneic stem cells of a normal phenotype, or
may be autologous cells that have been genetically engineered to
delete undesirable genetic sequences, and/or to introduce genetic
sequences that correct the genetic defect.
[0134] Sickle cell diseases include HbS Disease; drepanocytic
anemia; meniscocytosis. Chronic hemolytic anemia occurring almost
exclusively in blacks and characterized by sickle-shaped RBCs
caused by homozygous inheritance of Hb S. Homozygotes have sickle
cell anemia; heterozygotes are not anemic, but the sickling trait
(sicklemia) can be demonstrated in vitro. In Hb S, valine is
substituted for glutamic acid in the sixth amino acid of the beta
chain. Deoxy-Hb S is much less soluble than deoxy-Hb A; it forms a
semisolid gel of rodlike tactoids that cause RBCs to sickle at
sites of low PO.sub.2. Distorted, inflexible RBCs adhere to
vascular endothelium and plug small arterioles and capillaries,
which leads to occlusion and infarction. Because sickled RBCs are
too fragile to withstand the mechanical trauma of circulation,
hemolysis occurs after they enter the circulation. In homozygotes,
clinical manifestations are caused by anemia and vaso-occlusive
events resulting in tissue ischemia and infarction. Growth and
development are impaired, and susceptibility to infection
increases. Anemia is usually severe but varies highly among
patients. Anemia may be exacerbated in children by acute
sequestration of sickled cells in the spleen.
[0135] Thalassemias are a group of chronic, inherited, microcytic
anemias characterized by defective Hb synthesis and ineffective
erythropoiesis, particularly common in persons of Mediterranean,
African, and Southeast Asian ancestry. Thalassemia is among the
most common inherited hemolytic disorders. It results from
unbalanced Hb synthesis caused by decreased production of at least
one globin polypeptide chain (.beta., .alpha., .gamma.,
.delta.).
[0136] Aplastic anemia results from a loss of RBC precursors,
either from a defect in stem cell pool or an injury to the
microenvironment that supports the marrow, and often with
borderline high MCV values. The term aplastic anemia commonly
implies a panhypoplasia of the marrow with associated leukopenia
and thrombocytopenia.
[0137] Combined immunodeficiency is a group of disorders
characterized by congenital and usually hereditary deficiency of
both B- and T-cell systems, lymphoid aplasia, and thymic dysplasia.
The combined immunodeficiencies include severe combined
immunodeficiency, Swiss agammaglobulinemia, combined
immunodeficiency with adenosine deaminase or nucleoside
phosphorylase deficiency, and combined immunodeficiency with
immunoglobulins (Nezelof syndrome). Most patients have an early
onset of infection with thrush, pneumonia, and diarrhea. If left
untreated, most die before age 2. Most patients have profound
deficiency of B cells and immunoglobulin. The following are
characteristic: lymphopenia, low or absent T-cell levels, poor
proliferative response to mitogens, cutaneous anergy, an absent
thymic shadow, and diminished lymphoid tissue. Pneumocystis
pneumonia and other opportunistic infections are common.
EXPERIMENTAL
Example 1
A Non-Genotoxic Conditioning Regimen for Haploidentical
Hematopoietic Stem Cell Transplantation Materials and Methods
[0138] Mice. All donor and recipient mice were between 8 and 12
weeks of age. Donor mice were AKR.times.Hz F1 mice bred by the
Shizuru lab. AKR.times.Hz F1 mice are double positive for 45.1 and
45.2, and H2Kb and H2Kk. Recipient mice were CB6F1 from JAX. CB6F1
mice are single positive for 45.2 and double positive for H2Kb and
H2Kd. All procedures were approved by the International Animal Care
and Use Committee. Mouse strains were maintained at Stanford
University's Research Animal Facility.
[0139] Antibodies. All antibodies for in vivo conditioning were
purchased from Bio X Cell, including anti-CD47 (clone 3/clone
mIAP410), anti-CD117 (clone ACK2), anti-CD40L (clone MR-1), and
anti-CD122 (clone TM-b1).
[0140] BM Transplant. Recipient CB6F1 mice were given a priming
dose of 100 ug of anti-CD47 intraperitoneally on Day -8. On Day -6,
mice were given a 500 .mu.g retro-orbital injection of anti-CD117.
Prior to anti-CD117 treatment, mice were given an intraperitoneal
injection of Benadryl. On Days -6 through -2, mice were also given
500 .mu.g daily intraperitoneal injections of anti-CD47. On Day -2,
mice were given up to 250 .mu.g of anti-CD122. On Day 0, 500 .mu.g
of anti-CD40L is given a few hours prior to transplantation.
[0141] For transplantation, whole bone marrow is harvested from
8-12 week old AKR.times.Hz mice. The whole bone marrow is taken
from tibia, femurs, hips, and spine. The red blood cells are lysed
and the remaining cells are counted and appropriately resuspended
prior to injection. The cells are delivered with a retro-orbital
injection.
[0142] Chimerism checks. Recipient mice are periodically bled with
a retro-orbital puncture to measure donor chimerism. The blood is
stained with fluorescent antibodies against CD45.1, CD45.2, CD3,
CD19, CD11 b, and Gr-1.
Results
[0143] As shown in FIGS. 1A-1F, a combination of antibodies
specific for c-kit, CD47, CD40L and CD122, with the protocol
described above, enabled efficient engraftment of haploidentical
whole bone marrow into immune competent animals. Shown in FIG. 2
are the percentage of mice that were chimeric per cohort and the
average levels of total donor, T-cell, B-cell, and granulocyte
chimerism. At low doses of cells, as shown in FIG. 3, NK cel
depletion with anti-CD122 is required.
Example 2
Antibody Conditioning Enables MHC-Mismatched Hematopoietic Stem
Cell Transplants and Organ Graft Tolerance
[0144] Replacing a patient's diseased blood system by hematopoietic
cell transplantation (HCT) can treat or cure genetic disorders of
the blood and immune system, including leukemia, autoimmune
diseases and immunodeficiencies. In HCT, a patient's blood and
immune systems are typically ablated using toxic "conditioning
regimens" (chemotherapy and/or radiation) and then replaced with
donor cells containing hematopoietic stem cells (HSCs) to
regenerate a healthy blood system. While HCT is a foundational
treatment, its use and safety are hindered by graft vs. host
disease (which can be overcome by transplanting purified HSCs
devoid of contaminating donor T cells) and lethal toxicities caused
by the conditioning regimens. Therefore, a decisive goal is to
achieve HCT conditioning with more specific, safer agents (e.g.,
monoclonal antibodies), obviating the need for toxic chemotherapy
or radiation.
[0145] Here we show that a combination of six monoclonal antibodies
can safely and specifically deplete host HSCs, T cells and NK cells
of immune-competent mice and permit foreign (allogeneic) HSC
engraftment. The engrafted donor HSCs were either mismatched at
half (haploidentical) or all the MHC genes, and in both cases
generated donor blood and immune systems that stably co-exist with
host blood cells. These chimeric immune systems were functional, as
exhibited by tolerance to HSC donor strain heart tissue and
rejection of 3.sup.rd party hearts. These studies demonstrate
antibody conditioning, which can be applied to purified human HSC
transplantation as a platform for regenerative medicine,
facilitating applications including foreign organ transplants and
treatment of diverse blood and immune system disorders.
[0146] A multitude of genetic blood and immune system disorders can
be treated by hematopoietic cell transplantation (HCT): examples
include thalassemia, sickle cell anemia, Fanconi's anemia,
inherited immunodeficiencies, autoimmune diseases (e.g., multiple
sclerosis), and metabolic storage disorders. These diseases can be
corrected when an individual's blood system is replaced by healthy,
transplanted blood cells, which stably derive from the transplanted
rare hematopoietic stem cells (HSCs) in HCT grafts. After
regeneration of a donor-derived blood and immune system, HCT
recipients are immunologically tolerant to organ transplants from
the HSC donor. While any single-gene or multi-gene genetic disorder
of the blood system could be cured by allogeneic HCT, treatment of
non-malignant hematological or immunological disorders only
accounted for 6% of total HCT cases reported in Europe in 2015.
[0147] To overcome the disproportionately infrequent use of HCT to
treat non-malignant blood disorders and extend its reach, two key
challenges must be addressed: safety concerns and donor
availability. At present, allogeneic HCT leads to clinical or
subclinical graft vs. host disease (GvHD) caused by contaminating
donor-derived T cells; but GvHD can be overcome by transplanting
purified HSCs devoid of T cells. Moreover, HCT conditioning
requires chemotherapy and radiation, which can induce
life-threatening side effects.
[0148] Another challenge confronting HCT for genetic blood
disorders is the current need for fully matched donors at the human
leukocyte antigen (HLA, otherwise known as major histocompatibility
complex [MHC]) loci; while 75% of Caucasian Americans currently
have matched donors, it is markedly harder to find fully-matched
donors for Black Americans (16-19% currently have a match) or other
under-represented ethnic groups. If it were possible to safely
perform HCT using haploidentical donors (which are matched at half
of HLA loci), this would significantly expand the availability of
donors to theoretically enable any individual to receive HCT from
their parent, child, or 75% of siblings. Finally, if it were
possible to safely transplant fully HLA-mismatched HSCs, this would
massively open the pool of available donors; with the added benefit
that recipients would be immunologically tolerant to foreign organs
or tissues obtained from the same donor. This would enable
HLA-mismatched organ transplants without the lifelong
immunosuppression commonly needed to prevent rejection for vital
organ transplants.
[0149] The safety of HCT would be considerably improved if toxic
conditioning regimens (chemotherapy and/or radiation) were replaced
by more specific agents, such as monoclonal antibodies depleting
components of the immune system. While prior antibody conditioning
regimens enable the transplantation of minor histocompatibility
antigen-mismatched HSCs (see, for example, patent publication WO
2016/033201), transplantation of MHC-mismatched HSCs using
antibody-based conditioning has not previously been shown.
[0150] Here we demonstrate that conditioning using six monoclonal
antibodies enables wild-type mice to receive partially-
(haploidentical) or fully-MHC mismatched HSCs, therefore enabling
blood system replacement and induction of tolerance to mismatched
donor organs without recourse to chemotherapy or radiation.
[0151] For haploidentical transplantation experiments,
AKR.times.C57BL/6 F.sub.1 (hereafter referred to as AB6F.sub.1)
mice were used as bone marrow or HSC donors and
BALB/C.times.C57BL/6 F.sub.1 (CB6F.sub.1) (FIG. 4a) mice served as
recipients; these mouse strains are only matched at the H2.sup.b
haplotype but mismatched for H2.sup.k and H2.sup.d (i.e., at half
of the Major Histocompatibility Complex [MHC] haplotypes) (FIG.
4b). We sought to determine if conventional conditioning could be
replaced with monoclonal antibodies (mAb). We previously
demonstrated that immune-deficient mice could be conditioned using
an anti-Kit antibody to enable syngeneic HSC engraftment, whereas
comparable conditioning of immune-competent mice required dual
administration of anti-Kit and anti-CD47 blocking agents. CD47
blockade enables macrophages to phagocytose antibody-bound
(opsonized) cells, such as KIT.sup.+ HSCs opsonized by anti-c-KIT
antibodies.
[0152] In order to engraft allogeneic HSCs mismatched at the MHC
loci, it may require suppressing or eliminating both T cells and NK
cells, which reject cells expressing foreign major and minor
histocompatibility antigens or that lack "self" MHC. To eliminate
host NK cells we targeted CD122/II2R.beta. (which is expressed
throughout human and mouse NK cell development) using the
anti-CD122 mAb Tm-.beta.1 to deplete these cells. To prevent T-cell
mediated rejection we targeted CD40L (also known as CD154), which
is a co-stimulatory cell surface molecule expressed by activated
T-cells and is required for their signaling with CD40.sup.+ antigen
presenting cells. Interruption of the CD40-CD40L axis can help
induce tolerance to hematopoietic cells and skin grafts and
importantly, does not deplete all T cells since CD40L is
upregulated on activated T cells; we inhibited CD40L using the
anti-CD40L antibody MR1.
[0153] Mice were treated over the course of eight days (FIG. 4c)
with the four monoclonal antibodies (anti-CD122, anti-CD40L,
anti-Kit and anti-CD47; herein referred to as 4Ab conditioning) and
then transplanted with 30 million whole bone marrow (WBM) cells.
Chimerism was periodically measured by CD45 allelic differences
(FIG. 8a) and multi-lineage mixed chimerism was observed in all
animals receiving 4Ab conditioning (FIG. 8b-d). Importantly, mixed
chimerism was also observed in the long-term HSC (LT-HSC)
compartment (FIG. 4d), indicating that the donor chimerism did not
result from engraftment of long-lived mature immune cells, but was
being actively maintained by donor stem cells.
[0154] To identify the minimally necessary components of this
cocktail, we tested each antibody in isolation (FIG. 9) and then as
various combinations of the four antibodies. The minimally
necessary cocktail for 30 million WBM cells to engraft was
anti-CD47, anti-c-KIT, and anti-CD40L (FIG. 4e-g). However, only
75% of the mice in the group lacking anti-CD122 were chimeric. In
the group receiving the complete 4Ab conditioning, 100% of the mice
were chimeric. Interestingly, engrafted animals from both groups
showed similar levels of multi-lineage chimerism over twenty weeks.
Additionally, the 4Ab conditioning did not induce granulocytopenia
prior to transplantation (FIG. 4h).
[0155] We tested the lowest dose of WBM that could engraft by
titrating the dose of WBM while modulating the usage of anti-CD122.
The number of chimeric mice decreased as the amount of bone marrow
transplanted decreased (FIG. 4i). At 3 million WBM cells, 20% of
mice were chimeric without anti-CD122, while 80% of mice were
chimeric in this cell dose group with NK depletion.
[0156] To eliminate the possibility of GvHD, next we transplanted
enriched HSC populations (as opposed to WBM). In these experiments
Lineage.sup.- Sca1.sup.+ Kit.sup.+ (LSK) cells (FIG. 5a) were
transplanted, which are highly enriched for HSC and multipotent
progenitor (MPP) cells. Both Kit enriched and LSK cells were given
in quantities that corresponded to their abundance in 30 million
WBM cells (FIG. 5b). All three types of grafts showed complete,
long-term multi-lineage chimerism in irradiated controls.
Strikingly, while 4Ab-conditioned mice were successfully engrafted
long term by WBM, they were not reconstituted by Kit-enriched or
LSK transplants (FIG. 5b). This therefore indicates that additional
conditioning antibodies may be required for enriched HSC
populations to successfully engraft.
[0157] In order to facilitate LSK engraftment we attempted to
provide additional immune suppression by eliminating T cells using
anti-CD4 and anti-CD8 depleting antibodies (FIG. 5c). The addition
of anti-CD4 and anti-CD8 antibodies to the 4Ab regime robustly
depleted T-cells from peripheral blood, spleen and bone marrow
(FIG. 5d and FIG. 10). The usage of this six antibody cocktail,
which will be referred to as 6Ab conditioning (anti-CD122,
anti-CD40L, anti-Kit, anti-CD47, anti-CD4 and anti-CD8 mAbs),
induced long term chimerism in recipients transplanted with 9000
LSK cells (FIG. 5e). This cell dosage corresponds to approximately
360,000 LSK/kg, which is well below HSC doses seen in preclinical
testing for allografts in mice and clinical usage in autografts in
humans. In summary, 6Ab conditioning enables low doses of cells,
e.g. purified HSC, to engraft mice without recourse to chemotherapy
or radiation.
[0158] To determine if all six components of this cocktail were
necessary, we used a reductive process to identify the dispensable
antibodies. Removal of anti-CD40L, anti-CD4, and anti-CD8 resulted
in fewer chimeric animals and lower chimerism within each cohort,
as compared to the complete 6Ab conditioning cohort (FIG. 5f and
FIG. 11). However, removal of the anti-CD122 antibody did not
significantly change the percentage of chimeric animals as compared
to the control cohort. Unlike in the 4Ab conditioning regimen,
CD122 may be less necessary in the 6Ab conditioning due to NK
dependence on T-cell activation, which is lost in the 6Ab
conditioning regimen, as there is near complete depletion of
T-cells.
[0159] Importantly, 6Ab conditioning followed by HSC
transplantation induced centrally-mediated immunological tolerance
to the donor genetic strain. Central tolerance implies thymic
re-education of the host immune system to permit donor cell
engraftment. To gauge central tolerance in these animals, we
measured the presence of the V beta 6 (Vb6) TCR chain in peripheral
blood. The Vb6 is reactive to the Mtv-7 provirus-encoding
super-antigen, which is present in the AKR strain. Therefore, for
AB6F.sub.1 HSCs to coexist in CB6F.sub.1, the CB6F.sub.1 endogenous
Vb6+ T-cells must be clonally deleted. In both WBM- and
LSK-transplanted animals, chimeric animals showed deletion of host
Vb6+ T-cells (FIG. 6a-b). Interestingly, in the WBM cohort
conditioned with anti-Kit, anti-CD47, and anti-CD40L, the only
animal with a normal Vb6+ T-cell frequency also never achieved
chimerism (FIG. 6b).
[0160] Strikingly, we found that 6Ab-conditioned mice engrafted
with MHC-mismatched donor HSC were immunologically tolerant to
organs from the same donor strain. To this end, we transplanted
heart grafts from HSC donor (AB6F.sub.1) or third-party (DBA/1J
strain, which are homozygous for H2.sup.q) newborn pups into the
ear pinna of naive and LSK-Ab conditioned chimeric animals (FIG.
6c). In naive, unconditioned, untransplanted mice, both AB6F.sub.1
and DBA1/J hearts were rejected rapidly (FIG. 6d). In 6Ab
conditioned chimeric mice, DBA1/J hearts were rejected within 14
days while active, beating AB6F1 hearts persisted for at least 115
days. Representative ear-heart grafts were harvested at 34 days and
analyzed by immunohistochemistry. Upon gross examination, AB6F1
hearts are visible in the pinna while DBA/1J hearts are no longer
apparent (FIG. 6e). H&E analysis showed troponin+ cardiac
tissue lacking immune cell infiltrates in the AB6F1-engrafted
pinna; however, by this time there was no cardiac or troponin+
tissue within the pinna containing DBA/1J hearts (FIG. 6e). This
therefore indicates that MHC-mismatched donor HSC can induce
immunological tolerance of 6Ab-conditioned mice to heart grafts
from the same genetic donor.
[0161] Finally, we demonstrated that the 6Ab conditioning regimen
enabled successful engraftment of fully MHC-mismatched HSCs. We
used DBA1/J (H2.sup.q) mice as donors and CB6F.sub.1 (H2.sup.b/d)
hosts (FIG. 7a). After transplanting 9000 DBA1/J LSK cells, we
observed high donor-host chimerism by 8 weeks in all
6Ab-conditioned CB6F.sub.1 mice (FIG. 7b). Mice transplanted with 3
million WBM cells alone failed to establish donor chimerism
(confirming the necessity for conditioning), while 40% of
4Ab-conditioned mice receiving WBM achieved low levels of
chimerism. 80% of the irradiated CB6F1 mice transplanted with WBM
were dead by 9 weeks following transplantation (FIG. 7c), likely by
GvHD, which was not observed in LSK transplants.
[0162] In sum, here we have developed a method to transplant half-
(haploidentical) and fully-MHC mismatched hematopoietic cell
compositions, including purified HSC, into immune-competent
animals; importantly this is accomplished without the use of
chemotherapy and/or radiation, and without the GvHD that occurs in
most, if not all, other types of HCT transplants.
[0163] These findings are relevant to clinical use of hematopoietic
cells transplantation, for example for the treatment of blood and
immune system disorders. First, this antibody conditioning
regimen--combined with purified HSC transplants--improves the
safety of blood and immune system replacement by obviating the use
of chemotherapy/radiation and by eliminating GvHD. Second, by
facilitating transplantation of haploidentical, HLA-mismatched
HSCs, this increases significantly the donor pool to enable most
recipients to find a match, even if their age or clinical status
had prevented HCT under previous protocols. For instance, patients
with Fanconi's anemia are highly susceptible to DNA damage, and
therefore, conventional transplant conditioning regimens pose a
serious risk to this cohort.
[0164] Lastly, the ability to induce immunological tolerance to
foreign organs opens opportunities for all patients requiring
lifesaving organ transplants: specifically, it obviates the need
for lifelong immune suppression for patients to receive foreign
organ transplants. In particular, the immune systems in
antibody-conditioned, donor HSC-transplanted animals are tolerant
to donor (but not third-party) hearts. The coexistence of donor and
host T cells in these partially-chimeric animals can provide
MHC-restricted T cells for both donor and host tissues.
[0165] Today, a donor of an organ, tissue or HSC transplant is a
living or recently deceased person. A goal of regenerative medicine
is to differentiate a pluripotent (embryonic or induced
pluripotent) stem cell line into HSCs and other needed tissue stem
cells (such as those of the neural, bone and cartilage, or liver),
either in vitro or in vivo within a large-animal host (such as a
pig). This relieves the need for human beings to give up their HSCs
and organs for others. Antibody conditioning, followed by
co-transplantation of pluripotent stem cell-derived HSC and tissue
stem cells, could deliver lifesaving organs for patients without
recourse to long-term immunosuppression.
Methods
[0166] Animals. All experiments were performed according to
guidelines established by the Stanford University Administrative
Panel on Laboratory Animal Care. AKR.times.C57BL/6 F1 donors were
crossed and bred in house. CB6F1 and DBA1/J recipients were
purchased from the Jackson Laboratory. DBA1/J pregnant females were
purchased from Taconic Biosciences for ear heart grafts.
[0167] Antibodies. Anti-CD47 (mIAP410), anti-c-KIT (ACK2),
anti-CD122 (Tm-.beta.1), anti-CD40L (MR1), anti-CD4 (GK1.5), and
anti-CD8 (YTS169.4) were purchased from BioXCell. Anti-CD47 was
given intraperitoneally as a 100 .mu.g dose on Day -8 and then as a
500 .mu.g dose for subsequent injections throughout the
conditioning process. Retro-orbital anti-c-KIT and intraperitoneal
anti-CD40L were both given as one time 500 .mu.g boluses.
Anti-CD122 was given intraperitoneally as a 250 .mu.g dose while
anti-CD4 and anti-CD8 were given as 100 .mu.g intraperitoneal
doses. Mice receiving anti-c-KIT antibody were given 400 .mu.g of
diphenhydramine intraperitoneally 15 minutes prior to injection.
Anti-CD25 (PC-61.5.3) was purchased from BioXCell and given as a
one-time 100 .mu.g intraperitoneal injection.
[0168] Graft Preparation and Transplantation. Whole bone marrow was
extracted from donor mice tibia, femurs, hips, and spine. Bones
were crushed, filtered, and subsequently underwent red blood cell
(RBC) lysis. For c-Kit enriched transplants, RBC lysed whole bone
marrow were bound to Miltenyi CD117 MicroBeads as per the
manufacturer's instructions and collected after magnetic
separation. For LSK cell transplants, RBC lysed whole bone marrow
were bound to the Miltenyi Lineage Cell Depletion Kit cocktail as
per the manufacturer's instructions. Flow through from the magnetic
separation columns was collected and stained in PBS with 2% FBS
with optimal concentrations of the following antibodies: CD3 PE
(17A2), CD4 PE (GK1.5), CD5 PE (53-7.3), CD8a PE (53-6.7), B220 PE
(RA3-6B2), Gr-1 PE (RB6-8C5), Mac-1 PE (M1/70), Ter119 PE (TER119),
SCA1 Pe-Cy7 (D7), and CD117 APC (2B8). Propidium iodide was added
as a viability stain just prior to sorting on a BD Aria. All cells
for transplant were resuspended at the desired concentration in PBS
with 2% FBS. Irradiation control mice were lethally irradiated with
two doses of 6.5Gy prior to transplantation. All mice were
anesthetized using isoflurane and then transplanted with 100 uL of
cell suspension via retroorbital injection.
[0169] Peripheral Blood Chimerism. Mice were periodically bled via
retroorbital bleeding into EDTA coated tubes. Blood was then
incubated in 1% dextran with 5 mM EDTA at 37 C for 1 hour. The
supernatant from each tube was extracted, lysed and then stained
with optimal concentrations of the following antibodies: CD3 APC
(17A2), CD19 PE-Cy7 (ebio103), Gr-1 BV421 (RB6-8C5), Mac-1 APC-Cy7
(M1/70), CD45.1 FITC (A20), and CD45.2 PE (104). Samples were
analyzed on a BD Fortessa and donor versus host chimerism was
distinguished based on CD45 allelic differences.
[0170] Ear-Heart Graft. Neonatal mice were euthanized 1-2 days
after birth and their hearts were harvested. Recipient mice were
prepared by making a small incision on the dorsal side of their ear
near the skull. Afterward, using a trocar, a pouch was created by
tunneling from the incision site to the tip of the pinna. Neonatal
hearts were delivered at the distal end of the pouch with the
trocar. The tunnel was closed by gently pushing the lifted skin
back to the dermis. Heart viability was monitored for beating by
visualizing the graft through a dissecting microscope.
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[0187] Each publication cited in this specification is hereby
incorporated by reference in its entirety for all purposes.
[0188] It is to be understood that this invention is not limited to
the particular methodology, protocols, cell lines, animal species
or genera, and reagents described, as such may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention, which will be limited
only by the appended claims
[0189] As used herein the singular forms "a", "and", and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "a cell" includes a
plurality of such cells and reference to "the culture" includes
reference to one or more cultures and equivalents thereof known to
those skilled in the art, and so forth. All technical and
scientific terms used herein have the same meaning as commonly
understood to one of ordinary skill in the art to which this
invention belongs unless clearly indicated otherwise.
* * * * *