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US 2015O132290A1

(19) United States


(12) Patent Application Publication (10) Pub. No.: US 2015/0132290 A1
Fuchs et al. (43) Pub. Date: May 14, 2015
(54) METHODS AND COMPOSITIONS FOR A6II 45/06 (2006.01)
INFUSION OF TRANSIENTLY ENGRAFTING, CI2N5/0783 (2006.01)
SELECTED POPULATIONS OF ALLOGENEC (52) U.S. Cl.
LYMPHOCYTES TO TREAT CANCER
CPC .............. A61K35/17 (2013.01); C12N5/0638
(71) Applicant: The Johns Hopkins University, (2013.01); A61 K3I/664 (2013.01); A61 K
Baltimore, MD (US) 45/06 (2013.01)
(72) Inventors: Ephraim Joseph Fuchs, Owings Mills,
MD (US); Heather Jills Symons, (57) ABSTRACT
Annapolis, MD (US); Lode Swinnen,
Lutherville, MD (US) The invention provides methods and compositions for admin
(21) Appl. No.: 14/398,724 istration of allogeneic lymphocytes as an an exogenous
source of CD4+ T cell help for endogenous, tumor-reactive
(22) PCT Filed: Mar. 15, 2013 CD8+ T cells. Depletion of CD8+ T cells from the donor
(86). PCT No.: PCT/US 13/32129 lymphocyte infusion reduces the risk of Sustained engraft
ment and graft-Versus-host disease. Removal of regulatory T
S371 (c)(1), cells from the infused population may augment the ability of
(2) Date: Nov. 3, 2014 non-regulatory T cells to provide help for endogenous effec
Related U.S. Application Data tors of anti-tumor immunity. Allogeneic T cell therapy is
(60) Provisional application No. 61/644,126, filed on May typically given in the context of allogeneic stem cell trans
8, 2012. plantation, in which the patient receives highly immunosup
pressive conditioning followed by an infusion of a stem cell
Publication Classification graft containing unselected populations of mature T cells. In
(51) Int. Cl. the treatment described here, the graft is engineered to mini
A 6LX 35/7 (2006.01) mize the possibility of Sustained donor cell engraftment, and
A6 IK3I/664 (2006.01) the anti-tumor effector T cells derive from the host.
Patent Application Publication May 14, 2015 Sheet 1 of 3 US 201S/O132290 A1

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Patent Application Publication May 14, 2015 Sheet 2 of 3 US 201S/O132290 A1

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Patent Application Publication May 14, 2015 Sheet 3 of 3 US 201S/O132290 A1

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US 2015/O 132290 A1 May 14, 2015

METHODS AND COMPOSITIONS FOR metastases and improved survival. However, these CD8+ T
INFUSION OF TRANSIENTLY ENGRAFTING, cells may not eliminate the cancer because of functional
SELECTED POPULATIONS OF ALLOGENEC paralysis of tumor-specific CD4+ T cells.
LYMPHOCYTES TO TREAT CANCER
SUMMARY OF THE INVENTION
BACKGROUND OF THE INVENTION
0008. The present invention is based on the seminal dis
0001 1. Field of the Invention covery that infusion of allogeneic lymphocytes containing
0002 The invention relates generally to immunology and CD4+ T cells can break tolerance in host anti-tumor CD8+ T
more specifically, to methods and compositions containing cells, even though the donor cells do not engraft long term in
allogeneic lymphocytes to treat cancer. the recipient. Administration of chemotherapy prior to the
0003 2. Background Information allogeneic cell infusion can augment the anti-tumor effect of
0004. The immune system of a host provides the means for the transiently engrafting lymphocytes, by promoting
quickly and specifically mounting a protective response to homeostatic expansion of the transferred lymphocytes, and/
pathogenic microorganisms and also for contributing to rejec or by depleting host regulatory T cells and myeloid-derived
tion of malignant tumors. Immune responses have been gen Suppressor cells. The immune response to cancerishampered
erally described as including humoral responses, in which by functional defects of the patient's CD4+ T cells. Infusions
antibodies specific for antigens are produced by differenti of allogeneic lymphocytes can provide an exogenous source
ated B lymphocytes, and cell mediated responses, in which of CD4+ T cell help for endogenous, tumor-reactive CD8+ T
various types of T lymphocytes eliminate antigens by a vari cells. Depletion of CD8+ T cells from the donor lymphocyte
ety of mechanisms. For example, CD4 (also called CD4+) infusion reduces the risk of Sustained engraftment and graft
helper T cells that are capable of recognizing specific antigens versus-host disease. Removal of regulatory T cells from the
may respond by releasing Soluble mediators such as cytok infused population may augment the ability of non-regulatory
ines to recruit additional cells of the immune system to par T cells to provide help for endogenous effectors of anti-tumor
ticipate in an immune response. CD8 (also called CD8+) immunity.
cytotoxic T cells are also capable of recognizing specific 0009 Allogeneic T cell therapy is typically given in the
antigens and may bind to and destroy or damage an antigen context of allogeneic stem cell transplantation, in which the
bearing cell or particle. In particular, cell mediated immune patient receives highly immunosuppressive conditioning fol
responses that include a cytotoxic T lymphocyte (CTL) lowed by an infusion of a stem cell graft containing unse
response can be important for elimination of tumor cells and lected populations of mature T cells. The goal of alloSCT is to
cells infected by a microorganism, such as virus, bacteria, or obtain Sustained engraftment of the donor cells and entails the
parasite. risk of mortality from graft-Versus-host disease. In the treat
0005 Cancer includes a broad range of diseases and ment described here, the graft is engineered to minimize the
affects approximately one in four individuals worldwide. A possibility of Sustained donor cell engraftment, and the anti
CTL response is a key feature of effective cancer vaccines: tumor effector T cells derive from the host. Thus the therapy
effective CD4 T cell help is also likely to play a critical role in entails a unique cooperation of host and donor lymphocytes
productive CD8 T cell activation and thus provide clinical during the period of transient donor cell engraftment.
benefit. 0010. This is a treatment that can be applied to any human
0006 With respect to microbial infections, malaria, tuber or animal cancer. Variations of the present invention include:
culosis, HIV-AIDS and other viral infections such as Herpes 1) variations of the chemotherapy regimen that is given prior
Simplex Virus (HSV) infections (the leading cause of genital to infusion of allogeneic lymphocytes (may include cyclo
ulcers worldwide) continue to contribute to global health phosphamide, 5-fluorouracil, gemcitabine, dasatinib, combi
concerns. HSV-2 prevalence is increasing at an alarming rate nations thereof; 2) variations in the source of donor lympho
across the globe. In the United States, the overall HSV-2 sero cytes (may be from related or unrelated donors, may include
prevalence rate exceeds 20%, and in developing nations defined mismatches at HLA Class I or Class II genetic loci; 3)
HSV-2 prevalence is estimated between 30% and 50%. In variations in the types of cells selected for infusion, such as
addition to the profound burden of HSV-2 infection in adults, depletion of CD4+CD25+ regulatory T cells, depletion of
the incidence of neonatal HSV-2 infection is increasing. Even CD8+ T cells. Donors may be immunized to defined antigens
when treated, neonatalencephalitis from HSV-2 infection has prior to lymphocyte infusions or may be polarized with cytok
a mortality >15%, and the neurological morbidity among ines ex vivo to enrich for Type I (IFN-gamma producing) or
HSV-2 infected infants is an additional 30 to 50% of surviving Type 17 (IL-17-producing) T cells.
cases. Concomitant with the HSV-2 epidemic is a stark real 0011. In a first embodiment, the invention provides A
ization that HSV-2 infection substantially increases the risk method of making an allogenic lymphocyte composition
for HIV-1 acquisition and transmission. Data from Africa comprising: providing a peripheral blood cell composition
show that HSV-2 infection can increase the risk for HIV from a human donor allogenic to the recipient, the peripheral
transmission by as much as 7-fold and that as many as half of blood cell composition comprising CD4+ T-cells, CD8+
newly acquired HIV cases are directly attributed to HSV-2 T-cells, and natural killer cells, wherein (i) the donor com
infection. Overall, the relative risk of HIV acquisition prises at least one human leukocyte antigen (HLA) Class II
increases more than 2-fold in HSV-2-infected individuals. allele mismatch relative to the recipient in the donor versus
0007 Emerging evidence Suggests that cancers induce a the recipient direction and the HLA Class II allele mismatch
state of unresponsiveness in lymphocytes that are specific for is at a gene selected from the group consisting of HLA
antigens uniquely expressed by the cancer. However, this DRB1, HLA-DQB1, and HLA-DPB1, and (ii) the recipient
unresponsiveness should be able to be reversed. Several does not have detectable antibodies reactive against human
human tumors are infiltrated by CD8+ T cells, and the degree leukocyte antigens of the donor, and making the allogenic
of CD8+ T cell infiltration often correlates with absence of lymphocyte composition from the peripheral blood cell com
US 2015/O 132290 A1 May 14, 2015

position by reducing the number of CD8+ T-cells in the proviso that the CD4+ T-cells of the first allogenic lympho
peripheral blood cell composition by at least one order of cyte composition are not activated ex vivo.
magnitude, wherein (a) the number of CD4+ T-cells in the 0014. In one aspect, subsequent to administering the first
allogenic lymphocyte composition differs from the number allogenic lymphocyte composition to the Subject, the method
of CD4+ T-cells in the peripheral blood cell composition by further comprises administering a Successive lymphoreduc
less than about 50%, and (b) the number of natural killer cells tive non-lymphoablative treatment to the subject to induce
in the allogenic lymphocyte composition is less than or equal transient lymphopenia in the Subject; and Subsequently
to the number of natural killer cells in the peripheral blood administering to the Subject a successive allogenic lympho
cell composition, with the proviso that the CD4+ T-cells of the cyte composition derived from an additional peripheral blood
allogenic lymphocyte composition are not activated ex vivo. cell composition of an additional human, allogenic donor, the
0012. In another embodiment, the invention provides a Successive allogenic lymphocyte composition comprising a
composition made by the invention method. In yet another number of CD4+ T-cells and a number of natural killer cells
embodiment, the invention provides An allogenic human from the additional peripheral blood cell composition of the
lymphocyte composition comprising: CD4+ T-cells and natu additional donor, wherein (i) the additional donor comprises
ral killer cells from the peripheral blood cell composition of a at least one human leukocyte antigen (HLA) Class II allele
donor, wherein (i) the donor comprises at least one human mismatch relative to the subject in the additional donor versus
leukocyte antigen (HLA) Class II allele mismatch relative to the subject direction and the HLA Class II allele mismatch is
the recipient in the donor versus the recipient direction and at a gene selected from the group consisting of HLA-DRB1,
the HLA Class II allele mismatch is at a gene selected from HLA-DQB1, and HLA-DPB1, (ii) the subject does not have
the group consisting of HLA-DRB1, HLA-DQB1, and HLA detectable antibodies reactive against human leukocyte anti
DPB1, (ii) the recipient does not have detectable antibodies gens of the additional donor, (iii) the number of CD4+ T-cells
reactive against human leukocyte antigens of the donor, (iii) in the Successive allogenic lymphocyte composition differs
the number of CD4+ T-cells in the allogenic lymphocyte from the number of CD4+ T-cells in the additional peripheral
composition differs from the number of CD4+ T-cells in the blood cell composition by less than about 50%, (iv) the num
peripheral blood cell composition by less than about 50%, ber of additional donor CD4+ T-cells based on an ideal body
(iv) the number of donor CD4+ T-cells based on an ideal body weight of the Subject in kilograms (kg) is between about
weight of the recipient in kilograms (kg) is between about 1x10CD4+ T-cells/kg and about 1x10CD4+ T-cells/kg, (v)
1x10CD4+ T-cells/kg and about 1x10CD4+ T-cells/kg, (v) the number of natural killer cells in the successive allogenic
the number of natural killer cells in the allogenic lymphocyte lymphocyte composition is less than or equal to the number of
composition is less than or equal to the number of natural natural killer cells in the additional peripheral blood cell
killer cells in the peripheral blood cell composition, and (vi) composition, and (vi) the Successive allogenic lymphocyte
the allogenic lymphocyte composition has at least one order composition has at least one order of magnitude fewer CD8+
of magnitude fewer CD8+ T-cells relative to the peripheral T-cells relative to the additional peripheral blood cell compo
blood cell composition; with the proviso that the CD4+ sition.
T-cells of the allogenic lymphocyte composition are not acti 0015 The invention also provides a method of making an
vated ex vivo. allogenic lymphocyte composition for administration to a
0013 The invention also provides a method of treating a human recipient, comprising: providing a peripheral blood
disease or condition in a human Subject, comprising: admin cell composition from a human donor allogenic to the recipi
istering a lymphoreductive non-lymphoablative treatment to ent, the peripheral blood cell composition comprising a num
the Subject to induce transient lymphopenia in the Subject; ber of CD4+ T-cells, a number of CD8+ T-cells, and a number
and Subsequently administering to the Subject a first allogenic of natural killer cells, wherein (i) the donor has CD4+ T-cell
lymphocyte composition derived from a peripheral blood cell immunity against an antigen present in the recipient, (ii) the
composition of a human, allogenic donor, the first allogenic donor comprises at least one human leukocyte antigen (HLA)
lymphocyte composition comprising a number of CD4+ Class II allele match relative to the recipient and the HLA
T-cells and a number of natural killer cells from the peripheral Class II allele match is at a gene selected from the group
blood cell composition of the donor, wherein (i) the donor consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1,
comprises at least one human leukocyte antigen (HLA) Class and (iii) the recipient does not have detectable antibodies
II allele mismatch relative to the subject in the donor versus reactive against human leukocyte antigens of the donor, and
the subject direction and the HLA Class II allele mismatch is making the allogenic lymphocyte composition from the
at a gene selected from the group consisting of HLA-DRB1, peripheral blood cell composition by reducing the number of
HLA-DQB1, and HLA-DPB1, (ii) the subject does not have CD8+ T-cells in the peripheral blood cell composition by at
detectable antibodies reactive against human leukocyte anti least one order of magnitude, wherein (a) the number of
gens of the donor, (iii) the number of CD4+ T-cells in the first CD4+ T-cells in the allogenic lymphocyte composition dif
allogenic lymphocyte composition differs from the number fers from the number of CD4+ T-cells in the peripheral blood
of CD4+ T-cells in the peripheral blood cell composition by cell composition by less than about 50%, and (b) the number
less than about 50%, (iv) the number of donor CD4+ T-cells of natural killer cells in the allogenic lymphocyte composi
based on an ideal body weight of the Subject in kilograms (kg) tion is less than or equal to the number of natural killer cells
is between about 1x10 CD4+ T-cells/kg and about 1x10 in the peripheral blood cell composition, with the proviso that
CD4+ T-cells/kg, (v) the number of natural killer cells in the the CD4+ T-cells of the allogenic lymphocyte composition
first allogenic lymphocyte composition is less than or equal to are not activated ex vivo.
the number of natural killer cells in the peripheral blood cell 0016. In another embodiment, the invention provides an
composition, and (vi) the first allogenic lymphocyte compo allogenic lymphocyte composition derived from a peripheral
sition has at least one order of magnitude fewer CD8+ T-cells blood cell composition of a human, allogenic donor for
relative to the peripheral blood cell composition, with the administration to a human recipient, the allogenic lympho
US 2015/O 132290 A1 May 14, 2015

cyte composition comprising: a number of CD4+ T-cells and antigen present in the Subject, (ii) the donor comprises at least
a number of natural killer cells from the peripheral blood cell one human leukocyte antigen (HLA) Class II allele match
composition of the donor, wherein (i) the donor has CD4+ relative to the subject and the HLA Class II allele match is at
T-cell immunity against an antigen present in the recipient, a gene selected from the group consisting of HLA-DRB1,
(ii) the donor comprises at least one human leukocyte antigen HLA-DQB1, and HLA-DPB1, (iii) the subject does not have
(HLA) Class II allele match relative to the recipient and the detectable antibodies reactive against human leukocyte anti
HLA Class II allele match is at a gene selected from the group gens of the donor, (iv) the number of CD4+ T-cells in the
consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, allogenic lymphocyte composition differs from the number
(iii) the recipient does not have detectable antibodies reactive of CD4+ T-cells in the peripheral blood cell composition by
against human leukocyte antigens of the donor, (iv) the num less than about 50%, (v) the number of donor CD4+ T-cells
ber of CD4+ T-cells in the allogenic lymphocyte composition based on an ideal body weight of the Subject in kilograms (kg)
differs from the number of CD4+ T-cells in the peripheral is between about 1x105 CD4+ T-cells/kg and about 1x109
blood cell composition by less than about 50%, (v) the num CD4+ T-cells/kg, (vi) the number of natural killer cells in the
ber of donor CD4+ T-cells based on an ideal body weight of allogenic lymphocyte composition is less than or equal to the
the recipient in kilograms (kg) is between about 1x105 CD4+ number of natural killer cells in the peripheral blood cell
T-cells/kg and about 1x109 CD4+ T-cells/kg, (vi) the number composition, and (vii) the allogenic lymphocyte composition
of natural killer cells in the allogenic lymphocyte composi has at least one order of magnitude fewer CD8+ T-cells rela
tion is less than or equal to the number of natural killer cells tive to the peripheral blood cell composition; with the proviso
in the peripheral blood cell composition, and (vii) the allo that the CD4+ T-cells of the allogenic lymphocyte composi
genic lymphocyte composition has at least one order of mag tion are not activated ex vivo.
nitude fewer CD8+ T-cells relative to the peripheral blood cell 0020. In one aspect, the invention provides a method of
composition; with the proviso that the CD4+ T-cells of the treating a disease or condition in a human Subject, comprising
allogenic lymphocyte composition are not activated ex vivo. injecting a nanoparticle composition into a tumor, wherein
0017. The invention also provides an allogenic lympho the nanoparticle composition comprises nanoparticles com
cyte composition derived from a peripheral blood cell com prising an antigen not present in the Subject, thus introducing
position of a human, allogenic donor for administration to a the antigen into the Subject; administering to the Subject an
human recipient, the allogenic lymphocyte composition com allogenic lymphocyte composition derived from a peripheral
prising: a number of CD4+ T-cells and a number of natural blood cell composition of a human, allogenic donor, the allo
killer cells from the peripheral blood cell composition of the genic lymphocyte composition comprising a number of
donor, wherein (i) the donor has CD4+ T-cell immunity CD4+ T-cells and a number of natural killer cells from the
against an antigen not present in the recipient, (ii) the donor peripheral blood cell composition of the donor, wherein (i)
comprises at least one human leukocyte antigen (HLA) Class the donor has CD4+ T-cell immunity against the antigen, (ii)
II allele match relative to the recipient and the HLA Class II the donor comprises at least one human leukocyte antigen
allele match is at a gene selected from the group consisting of (HLA) Class II allele match relative to the subject and the
HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the recipi HLA Class II allele match is at a gene selected from the group
ent does not have detectable antibodies reactive against consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1,
human leukocyte antigens of the donor, (iv) the number of (iii) the subject does not have detectable antibodies reactive
CD4+ T-cells in the allogenic lymphocyte composition dif against human leukocyte antigens of the donor, (iv) the num
fers from the number of CD4+ T-cells in the peripheral blood ber of CD4+ T-cells in the allogenic lymphocyte composition
cell composition by less than about 50%, (v) the number of differs from the number of CD4+ T-cells in the peripheral
donor CD4+ T-cells based on an ideal body weight of the blood cell composition by less than about 50%, (v) the num
recipient in kilograms (kg) is between about 1x105 CD4+ ber of donor CD4+ T-cells based on an ideal body weight of
T-cells/kg and about 1x109 CD4+ T-cells/kg, (vi) the number the subject in kilograms (kg) is between about 1x105 CD4+
of natural killer cells in the allogenic lymphocyte composi T-cells/kg and about 1x109 CD4+ T-cells/kg, (vi) the number
tion is less than or equal to the number of natural killer cells of natural killer cells in the allogenic lymphocyte composi
in the peripheral blood cell composition, and (vii) the allo tion is less than or equal to the number of natural killer cells
genic lymphocyte composition has at least one order of mag in the peripheral blood cell composition, and (vii) the allo
nitude fewer CD8+ T-cells relative to the peripheral blood cell genic lymphocyte composition has at least one order of mag
composition; with the proviso that the CD4+ T-cells of the nitude fewer CD8+ T-cells relative to the peripheral blood cell
allogenic lymphocyte composition are not activated ex vivo. composition; with the proviso that the CD4+ T-cells of the
0018. In one embodiment, the invention provides a kit for allogenic lymphocyte composition are not activated ex vivo.
use in treating a disease or condition in a Subject, the kit
comprising: a lymphocyte composition wherein the Subject is BRIEF DESCRIPTION OF THE DRAWINGS
the human recipient; and a nanoparticle composition com
prising nanoparticles comprising the antigen not present in 0021 FIG. 1 shows the hematocrits of the same five
the human recipient. patients after BMT, with the jagged portions reflecting the
0019. Further, there is a method of treating a disease or effect of transfusion (graph).
condition in a human Subject, comprising: administering to 0022 FIG. 2 shows non-engrafting DLI induces anti-tu
the Subject an allogenic lymphocyte composition derived mor immunity (graph).
from a peripheral blood cell composition of a human, allo
genic donor, the allogenic lymphocyte composition compris 0023 FIG.3a shows engraftment of donor cells as vaccine
ing a number of CD4+ T-cells and a number of natural killer plus alloCD4s prolong survival (graph). FIG. 3b shows a
cells from the peripheral blood cell composition of the donor, graph with donor CD4+ T cell chimerism and days post
wherein (i) the donor has CD4+ T-cell immunity against an transplantation.
US 2015/O 132290 A1 May 14, 2015

0024 FIG. 4 shows validation runs for CD8 depletion ing doses per kilogram of recipient ideal body weight: a
using leukapheresis product and phlebotomy specimens lymphocyte composition comprising 105 CD4+ cells typi
(flow cytometry results). cally has no greater than 3.2x10 CD8+ cells, 10°CD4+ cells
typically has no greater than 3.2x10 CD8+ cells, 107 CD4+
DETAILED DESCRIPTION OF THE INVENTION cells typically has no greater than 3.2x10 CD8+ cells, 10
0025. The present disclosure arises at least in part from the CD4+ cells typically has no greater than 3.2x10 CD8+ cells,
seminal discovery that the immune response to cancer is and 5x10 CD4+ cells typically has no greater than 1.6x107
CD8+ cells.
hampered by functional defects of the patient's CD4+ T cells.
Infusions of allogeneic lymphocytes can provide an exog 0028. The number of natural killer cells in the allogenic
enous source of CD4+ T cell help for endogenous, tumor lymphocyte composition is less than or equal to the number of
reactive CD8+ T cells. Depletion of CD8+ T cells from the natural killer cells in the peripheral blood cell composition,
donor lymphocyte infusion reduces the risk of Sustained with the proviso that the CD4+ T-cells of the allogenic lym
engraftment and graft-Versus-host disease. Removal of regu phocyte composition are not activated ex vivo. reduced by
latory T cells from the infused population may augment the one order of magnitude, preferably about two orders of mag
ability of non-regulatory T cells to provide help for endog nitude, more preferably to about five orders of magnitude. In
enous effectors of anti-tumor immunity. Allogeneic T cell one embodiment, the number of CD8+ cells is reduced by
therapy is typically given in the context of allogeneic stem about 2.5 orders of magnitude (e.g., using the magnetic bead
cell transplantation, in which the patient receives highly cell sorter method).
immunosuppressive conditioning followed by an infusion of 0029. In one aspect, wherein if the donor and recipient are
a stem cell graft containing unselected populations of mature ABO blood type incompatible and the peripheral blood cell
T cells. In the treatment described here, the graft is engineered composition comprises a number of red blood cells, then
to minimize the possibility of Sustained donor cell engraft making the allogenic lymphocyte composition further com
ment, and the anti-tumor effector T cells derive from the host. prises reducing the number red blood cells. “ABO blood type
Thus, the therapy entails a unique cooperation of host and incompatible.” as used herein, refers to when the recipient has
donor lymphocytes during the period of transient donor cell a major ABO red blood cell incompatibility against the donor,
engraftment. e.g., the recipient is blood type O and the donor is blood type
0026. In one embodiment, the invention provides a A, B, or AB, the recipient is type A and the donor is type B or
method of making an allogenic lymphocyte composition for AB, or the recipient is type B and the donor is type A or AB.
administration to a human recipient, preferably, the donorand 0030. In one aspect, the number of red blood cells com
recipient are not the same human. The method includes pro prises less than or equal to about 50 ml in packed Volume. e.g.,
viding a peripheral blood cell composition from a human less than or equal to about 50 ml in packed volume, preferably
donor allogenic to the recipient, the peripheral blood cell less than or equal to about 30 ml in packed volume, further
composition comprising a number of CD4+ T-cells, a number “packed volume' should be defined, for example, centrifuga
of CD8+ T-cells, and a number of natural killer cells, some tion of the lymphocyte composition would result is a packed
natural killer cells have CD8+ antigen and may be removed Volume of 50 ml or less of red blood cells; a measured volume
by the “reducing step; however, preferred lymphocyte com sample of the lymphocyte composition could also be
positions of the present invention comprise at least some screened to provide a proportionally representative Volume of
natural killer cells from the donor. In one aspect, (i) the donor packed blood cells.
comprises at least one human leukocyte antigen (HLA) Class 0031. In one aspect, the number of CD4+ T-cells in the
II allele mismatch relative to the recipient in the donor versus allogenic lymphocyte composition differs from the number
the recipient direction (an HLA Class II allele mismatch in the of CD4+ T-cells in the peripheral blood cell composition by
donor versus recipient direction, i.e., 'graft-versus-host less than about 20%. In some embodiments, the CD4+ T-cells
direction) and the HLA Class II allele mismatch is at a gene are less than about 50%, less than about 40%, preferably less
such as HLA-DRB1, HLA-DQB1, or HLA-DPB1. The than about 20%, more preferably less than about 10%. In
recipient does not have detectable antibodies reactive against Some embodiments, ex vivo expansion of CD4+ T-cells may
human leukocyte antigens of the donor (“detectable antibod be performed, in such embodiments the number of CD4+
ies' in this context are defined using standard methods of T-cells can greatly exceed the original number. Such expan
making this determination (for example, the recipient does sion is an alternative embodiment.
not have antibodies against donor HLA molecules that are 0032. In some embodiments, CD4+ T-cells obtained from
detectable by complement-dependent cytotoxicity, in flow the donor are not intentionally expanded or intentionally dif
cytometric cross-match assays a positive result is undesir ferentiated ex vivo. Intentionally expanded or intentionally
able, or mean fluorescence intensity (MFI) of 3000 or greater differentiated is distinguished from expansion or differentia
in a Solid phase immunoassay is unacceptable). tion of the CD4+ T-cells that is merely a side effect (not
0027. The allogenic lymphocyte composition is made intentional, inadvertent) of the method, for example, CD4+
from the peripheral blood cell composition by reducing the T-cells can sometimes undergo differentiation by coming into
number of CD8+ T-cells in the peripheral blood cell compo contact with plastic, other examples of Such inadvertent
sition by at least one order of magnitude, wherein (a) the events. In another embodiment, there is a further proviso that
number of CD4+ T-cells in the allogenic lymphocyte compo stem cells have not been mobilized in the peripheral blood
sition differs from the number of CD4+ T-cells in the periph cell composition donor who is allogenic to the recipient.
eral blood cell composition by less than about 50%. In a 0033. In some aspects, reducing the CD8+ T-cells in the
preferred embodiment the ratio of the number of CD4+/the peripheral blood cell composition comprises using an anti
number of CD8+ T cells in the lymphocyte composition is CD8+ antibody associated with magnetic particles oran anti
preferably greater than or equal to about 30. Examples of CD8+ antibody plus complement. The peripheral blood cell
some embodiments include, but are not limited to, the follow composition can be a whole blood product or an apheresis
US 2015/O 132290 A1 May 14, 2015

product, for example. Further, the HLA Class II allele mis DPB1) and completely matched for Class I alleles (to maxi
match in the donor versus the recipient direction can be a mize Survival of donor cells in the recipient and minimize
mismatch at HLA-DRB1. This limitation with an HLA Class alloantibody formation against Class I molecules). Further,
II allele mismatch in the donor versus recipient direction is for the ideal donor is completely mismatched with unshared
example 'graft-versus-host direction', wherein the at least HLAs of first-degree relatives of the recipient who are poten
one HLA Class II allele(s) mismatch in the direction of the tial donors for allogenic stem cell transplantation.
allogenic donor versus the recipient further comprises the 0040. In one embodiment, there is an allogenic lympho
same HLA Class II allele(s) mismatch between the allogenic cyte composition derived from a peripheral blood cell com
donor versus one or more first degree relatives of the recipi position of a human, allogenic donor for administration to a
ent, which is desirable to preserve the opportunity for bone human recipient, the allogenic lymphocyte composition com
marrow transplantation from the first degree relatives to the prising a number of CD4+ T-cells and a number of natural
recipient; ideally, all of the mismatches between donor versus killer cells from the peripheral blood cell composition of the
recipient do not exist between a potential family bone marrow donor, wherein (i) the donor comprises at least one human
donor versus the recipient. leukocyte antigen (HLA) Class II allele mismatch relative to
0034. In some aspects, screening for one or more selection the recipient in the donor versus the recipient direction and
characteristic(s) is done and the screening is carried out on a the HLA Class II allele mismatch is at a gene selected from
Subject selected from the group consisting the recipient, the the group consisting of HLA-DRB1, HLA-DQB1, and HLA
donor, and one or more potential allogenic donor(s). For DPB1, (ii) the recipient does not have detectable antibodies
example, a selection characteristic is screening for serologi reactive against human leukocyte antigens of the donor, (iii)
cal reactivity to an infectious agent antigen. An infectious the number of CD4+ T-cells in the allogenic lymphocyte
agent antigen is selected from the group consisting of a composition differs from the number of CD4+ T-cells in the
Human Immunodeficiency Virus (HIV) antigen, a Hepatitis peripheral blood cell composition by less than about 50%,
Virus antigen, and a Cytomegalovirus antigen. Important (including but not limited to less than about 50%, less than
agents to be screened for include, for example, HIV-1 antigen about 40%, preferably less than about 20%, more preferably
(S), HIV-2 antigen?s), hepatitis A virus antigencs), hepatitis B less than about 10%). Further, “differs from the number of
virus antigen(s), hepatitis C virus antigenCs), CMV antigens, CD4+ T-cells in the peripheral blood cell composition by less
infectious diseases, etc. If the virus or infectious agent or an than about 50% means plus or minus less than 50% of the
antigen thereof is the target of the therapy, one would not rule number of CD4+ T-cells in the peripheral blood cell compo
out a donor having the desired CD4+ mediated immune sition. For example, if the number of CD4+ T-cells in the
response against that agent. peripheral blood cell composition is 1x10 CD4+ cells, then
0035. In one aspect, the infectious agent antigen is a “differs from the number of CD4+ T-cells in the peripheral
Cytomegalovirus antigen, the recipient and the donor are blood cell composition by less than about 50% means the
screened, and there is no serological reactivity to the Cytome number of CD4+ T-cells is between 1.5x10 and 0.5x10. In
galovirus antigen in the recipient or the donor. In one aspect, the composition, the number of donor CD4+ T-cells based on
the viral antigen is an influenza antigen and the influenza an ideal body weight (ideal body weight (IBW) is based on
antigen is a hemagglutinin antigen or a neuraminidase anti height. For men, IBW=50+2.3 kg/inch over 5 feet. For
gen. women, IBW-45.5+2.3 kg/inch over 5 feet) of the recipient in
0036. In another aspect, a selection characteristic is kilograms (kg) is between about 1x10CD4+ T-cells/kg and
screening for more than one HLA Class II alleles. In certain about 1x10 CD4+ T-cells/kg (in a preferred embodiment,
instances, a potential allogenic donor is selected based on between about 1x10 CD4+ T-cells/kg and about 5x10
maximizing mismatch between the potential allogenic donor CD4+ T-cells/kg); (v) the number of natural killer cells in the
Versus the recipient, in the potential allogenic donor versus allogenic lymphocyte composition is less than or equal to the
recipient direction, at the more than one HLA Class II alleles, number of natural killer cells in the peripheral blood cell
and the potential allogenic donor is chosen as the donor. In composition, and (vi) the allogenic lymphocyte composition
certain instances, a selection characteristic is screening for has at least one order of magnitude fewer CD8+ T-cells rela
one or more HLA Class I allele(s). tive to the peripheral blood cell composition; with the proviso
0037. A potential allogenic donor can be selected based on that the CD4+ T-cells of the allogenic lymphocyte composi
minimizing mismatch between the potential allogenic donor tion are not activated ex vivo.
and the recipient at the more than one HLA Class I allele(s), 0041. Also provided is a method of treating a disease or
and the potential allogenic donor is chosen as the donor. condition in a human Subject, comprising administering a
0038. In one embodiment, the invention provides an allo lymphoreductive (in some embodiments of this aspect of the
genic lymphocyte composition for administration to a human present invention, it is desirable to provide alymphoreductive
recipient obtained by the method of the invention as described non-lymphoablative treatment to promote the homeostatic
herein. expansion and differentiation of the administered lympho
0039. If the recipient is seropositive for the CMV antigen, cyte composition; in other embodiments it is desirable that the
then the status of the donor does not matter. In certain treatment also be myeloreductive (i.e., inhibiting or depleting
embodiments wherein the donor is not immunized to an anti Suppressive myeloid populations including myeloid-derived
gen that is present in, or will be delivered to, the recipient, the Suppressor cells, tumor associate macrophage, and or N2
delivery of CD4+ T-cell help is contingent upon donor CD4+ neutrophils) non-lymphoablative treatment to the subject to
T-cell recognition of allogenic HLA Class II molecules on the induce transient lymphopenia in the Subject; and Subse
recipient’s cells. An example of an “ideal donor” for the quently administering to the Subject a first allogenic lympho
purpose of exemplifying these embodiments of the present cyte composition derived from a peripheral blood cell com
invention is then completely mismatched at HLA Class II position of a human, allogenic donor, the first allogenic
alleles (in particular, HLA-DRB1, HLA-DQB1, and HLA lymphocyte composition comprising a number of CD4+
US 2015/O 132290 A1 May 14, 2015

T-cells and a number of natural killer cells from the peripheral anti-CD40 antibodies oraptamers). Further, the nanoparticles
blood cell composition of the donor, wherein (i) the donor may include an agent that targets the nanoparticles to tumor
comprises at least one human leukocyte antigen (HLA) Class cells or antigen-presenting cells.
II allele mismatch relative to the subject in the donor versus 0046. In one aspect, method further comprises administra
the subject direction and the HLA Class II allele mismatch is tion of the anti-tumor monoclonal antibody and the anti
at a gene selected from the group consisting of HLA-DRB1, tumor monoclonal antibody is selected from the group con
HLA-DQB1, and HLA-DPB1, (ii) the subject does not have sisting of rituximab, cetuximab, trastuzumab, and
detectable antibodies reactive against human leukocyte anti pertuzumab. In one aspect, the invention comprises adminis
gens of the donor, (iii) the number of CD4+ T-cells in the first tration of the anti-tumor monoclonal antibody/drug conjugate
allogenic lymphocyte composition differs from the number and the anti-tumor monoclonal antibody/drug conjugate is
of CD4+ T-cells in the peripheral blood cell composition by selected from the group consisting of brentuximab vedotin,
less than about 50%, (iv) the number of donor CD4+ T-cells gemtuzumab ozogamicin, trastuzumab emtansine, inotu
based on an ideal body weight of the Subject in kilograms (kg) Zumab ozogamicin, glembatumumab vedotin, lorVotuZumab
is between about 1x10 CD4+ T-cells/kg and about 1x10 mertansine, cantuzumab mertansine, and milatuZumab
CD4+ T-cells/kg, (v) the number of natural killer cells in the doxorubicin. In some aspects, admistration is first the allo
first allogenic lymphocyte composition is less than or equal to genic lymphocyte composition and then administration of a
the number of natural killer cells in the peripheral blood cell chemotherapeutic agent to the Subject. For example, the che
composition, and (vi) the first allogenic lymphocyte compo motherapeutic agent is selected from the group consisting of
sition has at least one order of magnitude fewer CD8+ T-cells dasatinib, nilotinib, ponatinib, imatinib, lapatinib, and Vismo
relative to the peripheral blood cell composition, with the degib.
proviso that the CD4+ T-cells of the first allogenic lympho 0047. In one aspect, subsequent to administering the first
cyte composition are not activated ex vivo. allogenic lymphocyte composition to the Subject, the method
0042. While not wishing to be bound by a particular further comprises administration of a monoclonal antibody/
theory, the infused CD4+ cells provide signals to other cell CD4+ T-cell epitope conjugate to the Subject. In one aspect,
types, predominately the Subject's CD8+, macrophage, and/ Subsequent to administering the first allogenic lymphocyte
orantigen presenting cells that augment cytotoxic function of composition to the Subject, the method further comprises
these cells in the subject (tolerized CD4+ of subject/ex administering a Successive lymphoreductive non-lymphoab
hausted CD8+ of subject); an example of treatment of a lative treatment to the Subject to induce transient lymphope
disease or condition by this method should be exemplified, at nia in the subject; and subsequently administering to the
least treatment of myelodysplastic syndrome. Subject a Successive allogenic lymphocyte composition
0043. In one aspect, the lymphoreductive non-lymphoab derived from an additional peripheral blood cell composition
lative treatment comprises treating the Subject with one or of an additional human, allogenic donor, the Successive allo
more cytoreductive agent selected from the group consisting genic lymphocyte composition comprising a number of
of alkylating agents, alkyl Sulphonates, nitrosoureas, triaz CD4+ T-cells and a number of natural killer cells from the
enes, antimetabolites, pyrimidine analogs, purine analogs, additional peripheral blood cell composition of the additional
Vinca alkaloids, epipodophyllotoxins, antibiotics, dibromo donor, wherein (i) the additional donor comprises at least one
mannitol, deoxyspergualine, dimethyl myleran and thiotepa. human leukocyte antigen (HLA) Class II allele mismatch
In one aspect, the lymphoreductive non-lymphoablative relative to the subject in the additional donor versus the sub
treatment comprises treating the Subject with an alkylating ject direction and the HLA Class II allele mismatch is at a
agent and the alkylating agent is cyclophosphamide. In one gene selected from the group consisting of HLA-DRB1,
aspect, Subsequent to administering the first allogenic lym HLA-DQB1, and HLA-DPB1, (ii) the subject does not have
phocyte composition to the Subject, the method further com detectable antibodies reactive against human leukocyte anti
prises administration of an anti-tumor monoclonal antibody gens of the additional donor, (iii) the number of CD4+ T-cells
or anti-tumor monoclonal antibody/drug conjugate to the in the Successive allogenic lymphocyte composition differs
Subject. from the number of CD4+ T-cells in the additional peripheral
0044. In one aspect, kits are provided for use in treating a blood cell composition by less than about 50%, (iv) the num
disease or condition in a subject, the kit comprising: a lym ber of additional donor CD4+ T-cells based on an ideal body
phocyte composition as described herein wherein the Subject weight of the Subject in kilograms (kg) is between about
is the human recipient; and a nanoparticle composition com 1x10CD4+ T-cells/kg and about 1x10CD4+ T-cells/kg, (v)
prising nanoparticles comprising the antigen not present in the number of natural killer cells in the successive allogenic
the human recipient. In one aspect, the nanoparticles further lymphocyte composition is less than or equal to the number of
comprise a cytokine, for example, an interleukin or an inter natural killer cells in the additional peripheral blood cell
feron. The cytokine can be an interleukin and is selected from composition, and (vi) the Successive allogenic lymphocyte
the group consisting of IL-2, IL-7, IL-12, and IL-15. The composition has at least one order of magnitude fewer CD8+
cytokine is may be an interferon e.g., interferon gamma, T-cells relative to the additional peripheral blood cell compo
interferon beta, interferon alpha, interferon, tau, interferon sition. With this method there is the proviso that the CD4+
omega, and consensus interferon. T-cells of the Successive allogenic lymphocyte composition
0045. The nanoparticles may further comprise a com are not activated ex vivo. Subsequent to administering the first
pound selected from the group consisting of a chemokine, an allogenic lymphocyte composition to the Subject, the method
imaging agent, a photo antenna molecule, a thermal antenna further comprises administration of an agent that blocks
molecule, and a Toll-like receptor ligand, ligands that pro negative signaling in T-cells. The agent that blocks negative
mote differentiation of CD4+ T-cells into Type I (e.g., IFN signaling in T-cell is selected from the group consisting of an
gamma producing) CD4+ memory T-cells, ligands for recep anti-PD-1 antibody, ipilimumab, an anti-PD-L2 antibody,
tors that induce activation of antigen presenting cells (e.g., and a PD-1 fusion protein. The disease or condition is selected
US 2015/O 132290 A1 May 14, 2015

from the group consisting of a cancer, an autoimmune disor sition differs from the number of CD4+ T-cells in the periph
der, an organ transplantation, an allograft rejection, and a eral blood cell composition by less than about 20%.
viral infection. For example, the disease or condition is a 0052. In one embodiment is provided an allogenic lym
cancer and the cancer is myelodysplastic syndrome. phocyte composition derived from a peripheral blood cell
0048. In one embodiment, the invention provides a composition of a human, allogenic donor for administration
method of making an allogenic lymphocyte composition for to a human recipient, the allogenic lymphocyte composition
administration to a human recipient, comprising providing a comprising a number of CD4+ T-cells and a number of natural
peripheral blood cell composition from a human donor allo killer cells from the peripheral blood cell composition of the
genic to the recipient, the peripheral blood cell composition donor, wherein (i) the donor has CD4+ T-cell immunity
comprising a number of CD4+ T-cells, a number of CD8+ against an antigen present in the recipient, (ii) the donor
T-cells, and a number of natural killer cells, wherein (i) the comprises at least one human leukocyte antigen (HLA) Class
donor has CD4+ T-cell immunity againstan antigen present in II allele match relative to the recipient and the HLA Class II
the recipient, (ii) the donor comprises at least one human allele match is at a gene selected from the group consisting of
leukocyte antigen (HLA) Class II allele match relative to the HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the recipi
recipient and the HLA Class II allele match is at a gene ent does not have detectable antibodies reactive against
selected from the group consisting of HLA-DRB1, HLA human leukocyte antigens of the donor, (iv) the number of
DQB1, and HLA-DPB1, and (iii) the recipient does not have CD4+ T-cells in the allogenic lymphocyte composition dif
detectable antibodies reactive against human leukocyte anti fers from the number of CD4+ T-cells in the peripheral blood
gens of the donor, and making the allogenic lymphocyte cell composition by less than about 50%, (v) the number of
composition from the peripheral blood cell composition by donor CD4+ T-cells based on an ideal body weight of the
reducing the number of CD8+ T-cells in the peripheral blood recipient in kilograms (kg) is between about 1x10 CD4+
cell composition by at least one order of magnitude, wherein T-cells/kg and about 1x10CD4+ T-cells/kg, (vi) the number
(a) the number of CD4+ T-cells in the allogenic lymphocyte of natural killer cells in the allogenic lymphocyte composi
composition differs from the number of CD4+ T-cells in the tion is less than or equal to the number of natural killer cells
peripheral blood cell composition by less than about 50%, in the peripheral blood cell composition, and (vii) the allo
and (b) the number of natural killer cells in the allogenic genic lymphocyte composition has at least one order of mag
lymphocyte composition is less than or equal to the number of nitude fewer CD8+ T-cells relative to the peripheral blood cell
natural killer cells in the peripheral blood cell composition, composition; with the proviso that the CD4+ T-cells of the
with the proviso that the CD4+ T-cells of the allogenic lym allogenic lymphocyte composition are not activated ex vivo.
phocyte composition are not activated ex vivo. 0053. The invention provides an allogenic lymphocyte
0049. In one aspect, the antigen present in the recipient is composition derived from a peripheral blood cell composi
selected from the group consisting of a neoplastic antigen tion of a human, allogenic donor for administration to a
(neoplastic antigen is an antigen associated with a neoplasm human recipient, the allogenic lymphocyte composition com
where neoplasm is defined as any new and abnormal cellular prising a number of CD4+ T-cells and a number of natural
growth, specifically one in which cellular replication is killer cells from the peripheral blood cell composition of the
uncontrolled and progressive. Neoplasms may be benign, donor, wherein (i) the donor has CD4+ T-cell immunity
pre-malignant or malignant, and Cancers are malignant neo against an antigen not present in the recipient, (ii) the donor
plasms. Thus all cancer antigens are neoplastic antigens but comprises at least one human leukocyte antigen (HLA) Class
not all neoplastic antigens are cancer antigens. Neoplastic II allele match relative to the recipient and the HLA Class II
idiotype (Id) is a tumor-specific target, for example, in those allele match is at a gene selected from the group consisting of
B cell malignancies that express this molecule on their cell HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the recipi
Surface, for example, lymphoma or multiple myeloma, and ent does not have detectable antibodies reactive against
include a viral antigen, a bacterial antigen, a fungal antigen, a human leukocyte antigens of the donor, (iv) the number of
parasitic antigen, and a non-human animal antigen. CD4+ T-cells in the allogenic lymphocyte composition dif
0050. In one aspect, a potential allogenic donor is selected fers from the number of CD4+ T-cells in the peripheral blood
from the one or more potential allogenic donor(s) based on cell composition by less than about 50%, (v) the number of
minimizing mismatch between the potential allogenic donor donor CD4+ T-cells based on an ideal body weight of the
and the recipient at the more than one HLA Class II alleles, recipient in kilograms (kg) is between about 1x105 CD4+
and the potential allogenic donor is chosen as the donor. A T-cells/kg and about 1x109 CD4+ T-cells/kg, (vi) the number
potential allogenic donor is selected from the one or more of natural killer cells in the allogenic lymphocyte composi
potential allogenic donor(s) based on minimizing mismatch tion is less than or equal to the number of natural killer cells
between the potential allogenic donor and the recipient at the in the peripheral blood cell composition, and (vii) the allo
one or more HLA Class I allele(s), and the potential allogenic genic lymphocyte composition has at least one order of mag
donor is chosen as the donor. nitude fewer CD8+ T-cells relative to the peripheral blood cell
0051. In one aspect, the antigen present in the recipient composition; with the proviso that the CD4+ T-cells of the
against which the donor has immunity is a viral antigen and allogenic lymphocyte composition are not activated ex vivo.
the viral antigen is selected from the group consisting of a 0054 The invention provides a method of treating a dis
human papillomavirus antigen, an Epstein Barr Virus antigen, ease or condition in a human Subject, comprising administer
a Kaposi's sarcoma-associated herpesvirus (KSHV) antigen, ing to the Subject an allogenic lymphocyte composition
a Hepatitis A virus antigen, a Hepatitis B virus antigen, and a derived from a peripheral blood cell composition of a human,
Hepatitis C virus antigen. For example, the viral antigen is a allogenic donor, the allogenic lymphocyte composition com
human papillomavirus antigen and the human papillomavirus prising a number of CD4+ T-cells and a number of natural
antigen is an E6 or an E7 antigenic peptide. In one aspect, the killer cells from the peripheral blood cell composition of the
number of CD4+ T-cells in the allogenic lymphocyte compo donor, wherein (i) the donor has CD4+ T-cell immunity
US 2015/O 132290 A1 May 14, 2015

against an antigen present in the Subject, (ii) the donor com nant giant cell tumor chordoma, osteochrondroma
prises at least one human leukocyte antigen (HLA) Class II (osteocartilaginous exostoses), benign chondroma, chondro
allele match relative to the subject and the HLA Class II allele blastoma, chondromyxofibroma, osteoid osteoma and giant
match is at a gene selected from the group consisting of cell tumors; nervous system cancers, including, for example,
HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the subject cancers of the skull, e.g., osteoma, hemangioma, granuloma,
does not have detectable antibodies reactive against human Xanthoma, and oSteitis defoinians; cancers of the meninges,
leukocyte antigens of the donor, (iv) the number of CD4+ e.g., meningioma, meningiosarcoma, and gliomatosis, can
T-cells in the allogenic lymphocyte composition differs from cers of the brain, e.g., astrocytoma, medulloblastoma, glioma,
the number of CD4+ T-cells in the peripheral blood cell ependymoma, germinoma (pinealoma), glioblastoma multi
composition by less than about 50%, (v) the number of donor form, oligodendroglioma, Schwannoma, retinoblastoma, and
CD4+ T-cells based on an ideal body weight of the subject in congenital tumors; and cancers of the spinal cord, e.g., neu
kilograms (kg) is between about 1x10CD4+ T-cells/kg and rofibroma, meningioma, glioma, and sarcoma; gynecological
about 1x10 CD4+ T-cells/kg, (vi) the number of natural cancers, including, for example, cancers of the uterus, e.g.,
killer cells in the allogenic lymphocyte composition is less endometrial carcinoma; cancers of the cervix, e.g., cervical
than or equal to the number of natural killer cells in the carcinoma, and pre tumor cervical dysplasia; cancers of the
peripheral blood cell composition, and (vii) the allogenic ovaries, e.g., ovarian carcinoma, including serous cystadeno
lymphocyte composition has at least one order of magnitude carcinoma, mucinous cystadenocarcinoma, unclassified car
fewer CD8+ T-cells relative to the peripheral blood cell com cinoma, granulosa thecal cell tumors, Sertoli Leydig cell
position; with the proviso that the CD4+ T-cells of the allo tumors, dysgerminoma, and malignant teratoma; cancers of
genic lymphocyte composition are not activated ex vivo. the Vulva, e.g., squamous cell carcinoma, intraepithelial car
0055. The compositions and methods of the invention can cinoma, adenocarcinoma, fibrosarcoma, and melanoma; can
be used against a broad range of cancers and tumor types, cers of the vagina, e.g., clear cell carcinoma, squamous cell
including but not limited to bladder cancer, brain cancer, carcinoma, botryoid sarcoma, and embryonal rhabdomyosa
breast cancer, colorectal cancer, cervical cancer, gastrointes rcoma; and cancers of the fallopian tubes, e.g., carcinoma;
tinal cancer, genitourinary cancer, head and neck cancer, lung hematologic cancers, including, for example, cancers of the
cancer, ovarian cancer, prostate cancer, renal cancer, skin blood, e.g., acute myeloid leukemia, chronic myeloid leuke
cancer, and testicular cancer. More particularly, cancers that mia, acute lymphoblastic leukemia, chronic lymphocytic leu
may be treated by the compositions and methods described kemia, myeloproliferative diseases, multiple myeloma, and
myelodysplastic syndrome, Hodgkin’s lymphoma, non
herein include, but are not limited to, the following: cardiac Hodgkin’s lymphoma (malignant lymphoma) and Walden
cancers, including, for example sarcoma, e.g., angiosarcoma, strom's macroglobulinemia; skin cancers, including, for
fibrosarcoma, rhabdomyosarcoma, and liposarcoma; example, malignant melanoma, basal cell carcinoma, squa
myxoma, rhabdomyoma; fibroma; lipoma and teratoma; lung mous cell carcinoma, Kaposi's sarcoma, moles dysplastic
cancers, including, for example, bronchogenic carcinoma, nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis;
e.g., squamous cell, undifferentiated Small cell, undifferenti and adrenal gland cancers, including, for example, neuroblas
ated large cell, and adenocarcinoma; alveolar and bronchiolar toma. In certain embodiments, when the disease is cancer, it
carcinoma; bronchial adenoma; sarcoma; lymphoma; chon may include a lung cancer tumor, a breast cancer tumor, a
dromatous hamartoma; and mesothelioma; gastrointestinal prostate cancer tumor, a brain cancer tumor, or a skin cancer
cancer, including, for example, cancers of the esophagus, e.g., tumor for example.
squamous cell carcinoma, adenocarcinoma, leiomyosar
coma, and lymphoma; cancers of the stomach, e.g., carci 0056. The compositions of the invention can also be
noma, lymphoma, and leiomyosarcoma; cancers of the pan administered in combination with existing methods of treat
creas, e.g., ductal adenocarcinoma, insulinoma, ing cancers, for example by chemotherapy, irradiation, or
glucagonoma, gastrinoma, carcinoid tumors, and Vipoma; surgery. Thus, there is further provided a method of treating
cancers of the Small bowel, e.g., adenocarcinoma, lymphoma, cancer comprising administering an effective amount of an
carcinoid tumors, Kaposi's sarcoma, leiomyoma, heman invention composition to an individual in need of Such treat
gioma, lipoma, neurofibroma, and fibroma; cancers of the ment, wherein an effective amount of at least one further
large bowel, e.g., adenocarcinoma, tubular adenoma, Villous cancer chemotherapeutic agent is administered to the indi
adenoma, hamartoma, and leiomyoma; genitourinary tract vidual. Examples of Suitable chemotherapeutic agents
cancers, including, for example, cancers of the kidney, e.g., include any of abarelix, aldesleukin, alemtuzumab, alitretin
adenocarcinoma, Wilm's tumor (nephroblastoma), lym oin, allopurinol, altretamine, anastrozole, arsenic trioxide,
phoma, and leukemia; cancers of the bladder and urethra, e.g., asparaginase, azacitidine, bevacizumab, bexarotene, bleomy
squamous cell carcinoma, transitional cell carcinoma, and cin, bortezombi, bortezomib, busulfan intravenous, busulfan
adenocarcinoma; cancers of the prostate, e.g., adenocarci oral, calusterone, capecitabine, carboplatin, carmustine,
noma, and sarcoma; cancer of the testis, e.g., seminoma, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine,
teratoma, embryonal carcinoma, teratocarcinoma, choriocar cyclophosphamide, cytarabine, dacarbazine, dactinomycin,
cinoma, sarcoma, interstitial cell carcinoma, fibroma, dalteparin Sodium, dasatinib, daunorubicin, decitabine,
fibroadenoma, adenomatoid tumors, and limphoma; liver denileukin, denileukin diftitox, dexraZOxane, docetaxel,
cancers, including, for example, hepatoma, e.g., hepatocellu doxorubicin, dromostanolone propionate, eculizumab, epiru
lar carcinoma; cholangiocarcinoma; hepatoblastoma; bicin, erlotinib, estramustine, etoposide phosphate, etopo
angiosarcoma; hepatocellular adenoma, and hemangioma; side, exemestane, fentanyl citrate, filgrastim, floXuridine, flu
bone cancers, including, for example, osteogenic sarcoma darabine, fluorouracil, fulvestrant, gefitinib, gemcitabine,
(osteosarcoma), fibrosarcoma, malignant fibrous histiocy gemtuzumab ozogamicin, goserelin acetate, histrelin acetate,
toma, chondrosarcoma, Ewing's sarcoma, malignant lym ibritumomabtiuxetan, idarubicin, ifosfamide, imatinib mesy
phoma (reticulum cell sarcoma), multiple myeloma, malig late, interferon alfa 2a, irinotecan, lapatinib ditosylate, lena
US 2015/O 132290 A1 May 14, 2015

lidomide, letrozole, leucovorin, leuprolide acetate, levami one order of magnitude fewer CD8+ T-cells relative to the
sole, lomustine, meclorethamine, megestrol acetate, peripheral blood cell composition; with the proviso that the
melphalan, mercaptopurine, methotrexate, methoXSalen, CD4+ T-cells of the allogenic lymphocyte composition are
mitomycin C, mitotane, mitoxantrone, nandrolone phenpro not activated ex vivo. In one aspect, the antigen is a non
pionate, nelarabine, nofetumomab, oxaliplatin, paclitaxel, human animal antigen and the non-human animal antigen is a
pamidronate, panitumumab, pegaspargase, pegfilgrastim, keyhole limpet hemocyanin antigen. In another aspect, the
pemetrexed disodium, pentostatin, pipobroman, plicamycin, antigen is a viral antigen and the viral antigen is selected from
procarbazine, quinacrine, rasburicase, rituximab, Sorafenib, the group consisting of a human papillomavirus antigen, an
streptozocin, Sunitinib, Sunitinib maleate, tamoxifen, temo Epstein Barr Virus antigen, a Kaposi's sarcoma-associated
Zolomide, teniposide, testolactone, thalidomide, thioguanine, herpesvirus (KSHV) antigen, a Hepatitis. A virus antigen, a
thiotepa, topotecan, toremifene, to situmomab, trastuzumab, Hepatitis B virus antigen, and a Hepatitis C virus antigen.
tretinoin, uracil mustard, valrubicin, vinblastine, Vincristine, 0059 Compositions of the invention may be administered
Vinorelbine, Vorinostat, and Zoledronate. to the individual by a variety of routes, for example, orally,
0057. In methods of the invention described herein, topically, parenterally, intravaginally, systemically, intramus
optionally before administering to the Subject the allogenic cularly, rectally or intravenously. In certain embodiments, the
lymphocyte composition the method further comprises composition is formulated with a pharmaceutical carrier.
administering a treatment to deplete or inhibit myeloid-de Preferably, the composition is administered intravenously.
rived suppressor cells. The treatment to deplete or inhibit
myeloid-derived suppressor cells may comprise administra 0060. In some embodiments, the composition is combined
tion of a drug selected from the group consisting of dasatinib, with other anti-viral or anti-cancer therapies, such as admin
5-fluorouracil, taxotere, clodronate, and gemcitabine for istration of an anti-viral or anti-cancer agent, radiation
example. Optionally, before administering to the subject the therapy, phototherapy or immunotherapy. The anti-viral or
allogenic lymphocyte composition the method further com anti-cancer agent can be administered with an invention com
prises administering a treatment to deplete or inhibit tumor position either in the same formulation or in separate formu
associated macrophage cells. Optionally, before administer lations, to enhance treatment. In these embodiments, the com
ing to the Subject the allogenic lymphocyte composition the position and the other therapies can be administered at the
method further comprises administering a treatment to same time (simultaneously) or at separate times (sequen
deplete regulatory T cells. The treatment to deplete regulatory tially), provided that they are administered in Such a manner
T cells may include administration of a drug selected from the and sufficiently close in time to have the desired effect.
group consisting of cyclophosphamide, denileukin diftitox, 0061 The following examples are intended to illustrate
and daclizumab. but not limit the invention.
0058. In another embodiment, the invention provides a
method of treating a disease or condition in a human Subject EXAMPLES
comprising injecting a nanoparticle composition into a tumor
(nanoparticles can be injected into or infused into the Subject, 0062. The myelodysplastic syndromes (MDS) are a
wherein the nanoparticles further comprise a targeting agent, diverse group of malignant stem cell disorders characterized
and the targeting agent binds to a target cell Such as a dis by dysplastic and ineffective bone marrow production of
persed/non-localized neoplasm (e.g., a lymphoma or leuke blood cells and a variable risk of transformation to acute
mia) where direct injection to all possible sites is not practical leukemia. These disorders may develop denovo or arise years
or feasible), wherein the nanoparticle composition comprises after exposure to potentially mutagenic chemotherapy.
nanoparticles comprising an antigen not present in the Sub 0063. Approximately 12,000-20,000 new cases of MDS
ject, thus introducing the antigen into the Subject; adminis will be diagnosed in the United States this year with a median
tering to the Subject an allogenic lymphocyte composition
derived from a peripheral blood cell composition of a human, age of onset of between 60 and 72. Current treatment out
allogenic donor, the allogenic lymphocyte composition com comes for the myelodysplastic syndromes have been disap
prising a number of CD4+ T-cells and a number of natural pointing. Age, performance status, and disease risk category,
killer cells from the peripheral blood cell composition of the as determined by the International Prognostic Scoring Sys
donor, wherein (i) the donor has CD4+ T-cell immunity tem (IPSS), usually determine the choice of treatment modal
against the antigen, (ii) the donor comprises at least one ity. Patients <60 years of age, who have good or excellent
human leukocyte antigen (HLA) Class II allele match relative performance status and who are in the IPSS intermediate-2 or
to the subject and the HLA Class II allele match is at a gene high risk categories, would predominantly be considered for
selected from the group consisting of HLA-DRB1, HLA high intensity therapies, since these IPSS categories confer a
DQB1, and HLA-DPB1, (iii) the subject does not have detect median survival of 1.2 and 0.4 years, respectively. High
able antibodies reactive against human leukocyte antigens of intensity therapies are defined as treatments requiring hospi
the donor, (iv) the number of CD4+ T-cells in the allogenic talization, including intensive combination chemotherapy
lymphocyte composition differs from the number of CD4+ and hematopoetic cell transplantation.
T-cells in the peripheral blood cell composition by less than 0064 Patients in the low or intermediate-1 category would
about 50%, (v) the number of donor CD4+ T-cells based on an generally be considered for low intensity therapies. These
ideal body weight of the Subject in kilograms (kg) is between include treatments that can be administered in the outpatient
about 1x10 CD4+ T-cells/kg and about 1x10CD4+ T-cells/ clinic, such as hematopoetic growth factors, differentiation
kg, (vi) the number of natural killer cells in the allogenic inducing agents, biologic response modifiers, and low inten
lymphocyte composition is less than or equal to the number of sity chemotherapy. Patients with poor performance status
natural killer cells in the peripheral blood cell composition, would be considered for supportive care or low intensity
and (vii) the allogenic lymphocyte composition has at least therapies.
US 2015/O 132290 A1 May 14, 2015

Example 1 TABLE 1
Donor
IDE Device Patientii chimerism Blast 9% Blast 96 Post
(age in years) Diagnosis (Day 30) Pre-BMT BMT (day)
0065. The investigational agent to be used in this trial is the
CliniMACS system with CliniMACS(R) CD8 reagent, a medi 1 (39) RAEB-t O 22 O (181+)
cal device that is used to enrich or deplete CD8+ T cells from 2 (62) AA-> RAEB O 15 () (378)
human blood products. The CliniMACS(R) System intended 3 (62) PCV-e RAEB O 8 O (342)
4 (56) RAEB O 5 2
for selection of CD8+ cells comprises four primary compo 5 (59) RAEB-t O 2O 4 (73)
nents: 1) CliniMACS(R) CD8 Reagent—colloidal super para 50 (78)
magnetic iron-dextran beads linked to a murine antibody
against human CD8; 2) CliniMACSplus Instrument—a soft
ware controlled instrument that processes the blood sample 0067 FIG. 1 shows the hematocrits of the same five
(cell product); 3) CliniMACSR) Tubing Set, (Standard or patients after BMT, with the jagged portions reflecting the
LS)—a single-use, sterile, disposable tubing set with two
proprietary cell selection columns; and 4) CliniMACS(R) effect of transfusion. Three of five patients became transfu
PBS/EDTA Buffer a sterile, isotonic phosphate buffered, 1 sion independent, with patient #1 remaining in morphologic
mM EDTA, saline solution, used as external wash and trans and hematologic remission for at least three years. More
port fluid for the in vitro preparation of blood cells. The interestingly, patient #2 demonstrated a delayed hematologic
system utilizes magnetic cell sorting (MACS(R), a powerful response, becoming transfusion independent four months
tool for the isolation of many cell types, to selectively enrich after BMT and three months after documentation of graft
or deplete the cell population of interest. In this case, CD8+ T rejection. In light of the sensitivity of MDS to immuno
cells are labeled with a monoclonal antibody linked to super therapy, we raise the possibility that an endogenous (i.e. host
paramagnetic particles and then are depleted from the blood derived) anti-tumor immune response is reawakened by the
product by passage through the CliniMACS system, which immunological perturbation provided by the transiently
incorporates a strong permanent magnet and a separation engrafting donor lymphocytes. This postulated mechanism,
column with a ferromagnetic matrix to remove the labeled the awakening of an endogenous anti-response following
cells. It is worth nothing that the therapeutic agent in this trial, graft rejection, may also account for the induction of leuke
CD8+ T cell-depleted blood cells, comes out of the device and mia remission in patients receiving white blood cell transfu
is not intended to contain any component of the device. sions after either no conditioning or only 100 cGy total body
irradiation. Interestingly, in the latter study, three major clini
Example 2 cal responses occurred in the absence of measurable donor
chimerism. Clinical responses of both non-Hodgkin’s and
FIG. 2 shows a graph with percent survival and days after
Transiently Engrafting Donor Lymphocytes Induce tumor inoculation to show non-engrafting DLI induces anti
Clinical Tumor Responses tumor immunity. BALB/cxC57BL/6 F1 mice (H-2b/d:=107
group) were conditioned with Cy 200 mg/kg IP on day-1. On
0066. To date, only two therapies are capable of prolong day 0, they received 106 A20 lymphoma cells IV+/-5x107
ing the survival of patients with MDS. The first, allogeneic spleen cells from partially MHC-mismatched B6xC3H F1
BMT has achieved some long term cures, as well as delay in donors (H-2b/k)+/-autologous tumor cell vaccine sc (106
disease progression. This therapy is only applicable to a small irradiated A20+2x105 irr. B78H1-GM-CSF). Hodgkin’s
fraction of affected patients due to age, donor availability, and lymphoma despite the loss of donor chimerism following
comorbidities. The second is the methyltransferase inhibitor, allogeneic BMT have also been described; in some of these
5-azacitidine. This therapy has been shown to prolong median cases, tumor regression following transiently engrafting
survival by 7 months compared to supportive care alone10. donor lymphocyte infusions was observed.24 While it is cer
Some patients with MDS respond to immunosuppressive tainly possible that donor T cells induced an anti-leukemic
regimens, such as cyclosporine, antithymocyte globulin effect before they were rejected, it is also possible that a
(ATG) or steroids, with a sustained increase in blood counts. transient GVH reaction broke functional tolerance to leuke
This finding is similar to aplastic anemia, where immunosup mia in host T cells. We have recently tested the hypothesis that
pression treats the autoimmune component leading to the transiently engrafting donor lymphocyte infusions (DLI) can
cytopenias. The favorable results obtained with agents that induce anti-tumor immune responses from host T cells in a
specifically target the immune system Suggest that MDS is a mouse model. BALB? cxC57BL/6 F1 mice were treated with
disease that is susceptible to immune modulation. One poten Cy on day-1, and on day 0 they received 106A20 lymphoma
tial explanation for the benefit of ATG, cyclosporine, and cells (of BALB/c origin) IV together with nothing, haploiden
steroids is that these drugs unmask the activity of an endog tical DLI alone, autologous tumor cell vaccine alone, or DLI+
enous anti-tumor immune response by selectively inhibiting vaccine. Compared to animals receiving either no treatment
or killing lymphocytes that Suppress anti-tumor immunity. or vaccine alone after Cy conditioning, those that were con
Further potential evidence for the existence of a cryptic, ditioned with Cy and then treated with DLI alone or DLI+
endogenous immune response against MDS was seen in our vaccine Survived significantly longer, with apparent cure
trial of nonmyeloablative, partially HLA-mismatched (hap achieved in five and four animals, respectively (FIG.2). None
loidentical) allogeneic bone marrow transplantation, in of the nine cured animals had any detectable donor chimerism
which five patients experienced disease responses despite when tested >100 days after DLI, suggesting that the donor T
graft rejection. All five patients had at least transient reduc cells were rejected. These results demonstrated that the com
tions in the percentage of bone marrow blasts, and three of bination of Cy followed by partially MHC-mismatched DLI
five patients, each of whom was dependent upon red blood induced significant anti-tumor effects. In order to character
cell--/-platelet transfusions prior to transplantation, became ize the role of donor CD4+ versus CD8+ T cells in the anti
transfusion independent. Table 1 demonstrates that, despite tumor effect, the experiment was repeated in recipients of
absence of donor cell engraftment on day 30 after BMT, at Cy+vaccine--50 million mismatched spleen cells that were
least three of five patients had a reduction of marrow blasts either untreated or depleted of CD4+ T cells, CD8+ T cells, or
lasting at least six months after BMT. both. In this experiment, recipients of whole spleen DLI all
US 2015/O 132290 A1 May 14, 2015

died of GVHD before day 20 (FIG. 3). In contrast, mice that cells/kg of recipient body weight but in none of fifteen
received vaccine plus CD8+ T cell depleted spleen cells lived patients receiving >3.1x10 CD8+ T cells/kg. Thus, even
significantly longer than mice receiving vaccine plus pan-T after myeloablative conditioning, CD8+ T cell depletion sig
cell depleted spleen cells (p=0.04), indicating that depletion nificantly increases the risk of graft rejection, which nullifies
of CD8+ T cells abrogated lethal GVHD without abrogating the risk of graft-induced aplasia and GVHD. In the second
anti-tumor immunity. In order to understand why depletion of study patients received T cell-depleted, haploidentical
CD8+ T cells from the allogeneic DLI abrogated GVHD, we peripheral blood stem cell (PBSC; n=15) or PBSC plus mar
studied the survival of donor cells in mice conditioned with row grafts (n=28) containing a mean of 2.7x10" or 3.5x10'
Cy and then infused with mismatched spleen cells, either CD3+ T cells/kg, respectively, which translates to CD8+ T
untreated or depleted of either or both T cell subsets. Inter cell doses of approximately 1-1.5x10/kg. To facilitate
estingly, CD8+ T cell-depleted spleen cells engrafted only engraftment in the face of T cell depletion, patients were
transiently, with donor CD4+ T cell chimerism peaking at 7 conditioned intensively and received “mega-dose” stem cell
days after DLI and declining to undetectable levels by day 21 grafts containing a mean of 14.0x10° or 10.6x10 CD34+
(below). In contrast, Sustained engraftment of donor cells was cells/kg for recipients of PBSC only versus PBSC plus mar
seen in all mice receiving DLI containing CD8+ T cells, and row, respectively. In light of the low T cell content, no GVHD
most of these animals eventually died of GVHD. Taken prophylaxis was administered. Engraftment occurred in all 43
together, the animal studies demonstrate that Cy followed by patients, and no patient experienced acute or chronic GVHD
CD8+ T cell depleted DLI induces transient engraftment of as a result of the transplantation procedure. These data dem
donor cells and significant anti-tumor effects without induc onstrate that even when Sustained engraftment occurs in
ing acute GVHD. More recently, we have found that deple patients receiving myeloablative conditioning, haploidentical
tion of host CD8+ T cells prior to “Cy+DLI” significantly grafts containing <10 CD8+ T cells/kg are unlikely to cause
diminishes the therapeutic effect, strongly implicating host GVHD. Since the device usually achieves >2 log depletion of
CD8+ T cells as critical mediators of the anti-tumor effect. CD8+ T cells, the starting dose of CD8+ T cell depleted PBCs
on the trial will likely contain fewer than 10 CD8+ T cells/kg.
Example 3 It is likely, then, that no patients receiving this dose will
engraft or experience GVHD, even if sustained engraftment
Clinical Experience with CD8+ T Cell Depleted OCCU.S.

Allogeneic StemCell or Lymphocyte Infusions Example 4


0068. There are no reports of patients treated with Cy CD8 Depletion Using the CliniMACS System with
followed by an infusion of CD8+ T cell depleted PBCs from
haploidentical donors, so it is not possible to provide prelimi CliniMACS-CD8 Reagent
nary safety data. However, there have been reports of patients (0070 For this trial, CD8+ T cells will be depleted using the
undergoing alloBMT who have received CD8+ T cell-de CliniMACS system with the CliniMACS CD8 Reagent
pleted grafts or of patients in relapse after alloBMT who have (Miltenyi Biotec, Woburn, Mass.), under an investigator
received CD8+ T cell depleted PBMC infusions. The goal of sponsored IDE. We have performed three validation runs, the
CD8+ T cell depletion was to reduce the incidence of GVHD first using a leukapheresis product, and the last two using
while preserving the anti-leukemia effect of the infusion. phlebotomy specimens. Flow cytometry results from the last
With regard to GVHD, the studies did not yield a conclusive depletion are shown below, demonstrating excellent deple
answer, with some showing a possible benefit and others tion of CD8+ T cells, from 8.40% to 0.03% of total cells, and
showing none. Interestingly, infusion of CD8+ T cell-de a corresponding enrichment of CD4+ T cells and CD16+ or
pleted DLI induced the activation of endogenous CD8+ T CD56+ NK cells (FIG. 4). Table 2 demonstrates all three
cells, a finding that is consistent with the hypothesis that products meet the protocol criterion of having a CD8+ T cell
CD8+ T cell-depleted DLI can effectively awaken a host number that is <3.2% of the CD4+ T cell number. Had prod
CD8+ T cell response against cancer. ucts #2 and #3 been used to deliver a dose of 106 CD4+ T
0069. The results of two other studies are germane to cells/kg to a recipient with an ideal body weight of 70kg, they
considerations of the safety of the proposed clinical trial. In would have contained 2.6x103 and 7.7x102 CD8+ T cells/kg
the first study, patients with various hematologic malignan IBW, respectively, doses that are well below the threshold for
cies received marrow from unrelated donors that were mis engraftment or GVHD induction. In comparison, the five
matched for either one HLA-DR allele or one HLA Class I patients who responded despite graft rejection received mar
(HLA-A or HLA-B) antigen. Patients received CD4+ T cell rows containing a median of 1.43x107 CD4+ T cells/kg
depleted grafts containing titrated doses of CD8+ T cells. The (range 0.84-3.14x10/kg) and 1.86x107 CD8+ T cells/kg
major finding relevant to the proposed study was that graft (range 0.43-2.16x10/kg). Therefore, MDS patients are
rejection occurred despite myeloablative conditioning in six capable of rejecting haploidentical cell infusions containing
often patients receiving grafts containing <3.1x10°CD8+ T this many T cells.
TABLE 2

CE8 Depleted Cell Product Validation Results


Total Total CD8+
% CD4- CD4+ % CD8+ CD8+ Cells Log Percent Sterility
Cells Cells Cells Cells Depletion Viable Result

Depletion #1 39.6% 7.6 x 108 0.5% 9.5 x 10 1.9S 98% Negative


#2 11.6% 2.5 x 108 0.03% 6.5 x 10 2.43 90% Negative
#3 39.9% 3.0 x 108 0.03% 2.3 x 10 2.67 97% Negative
US 2015/O 132290 A1 May 14, 2015

Example 5 (0074 Experience with haploidentical DLI, without CD8+


T-cell depletion, has been recently published. In a phase I/II
Correlative Laboratory Studies to Predict Response trial, 41 patients with relapsed/refractory malignancies
to Therapy received nonablative conditioning with 100 cGy total body
0071. In light of the potential toxicities of immunosup
irradiation, followed by infusion of 1x10° to 2x108 hap
loidentical CD3+ cells/kg, with 29 patients receiving the
pressive therapies, such as antithymocyte globulin, in patients highest dose. Objective responses were achieved at the higher
with MDS35, numerous investigators have endeavored to dose levels. Notably, 1x10 CD3+ cells/dose was the mini
identify patient characteristics that correlate with response to mum dose associated with response (25% response rate, or 2
therapy. Such characteristics that predict disease response of 8 patients), with 2x10 CD3+ cells/dose (the highestevalu
include hypocellular marrow, abnormal T cell receptor rep ated) associated with the greatest response rate (nearly 50%,
ertoire by T cell receptor beta chain variable region CDR3 in 10 of 21 patients). As proof of principle, all responses
size by spectratype analysis, presence of cells with a pheno occurred in the absence of Sustained donor chimerism. In the
type characteristic of paroxysmal nocturnal hemoglobinuria highest dose cohort, transient donor chimerism was seen but
(PNH), expression of HLADR15, trisomy 8, younger age, disappeared by 2 weeks in most patients, with one of two
and shorter transfusion history. A major goal of this trial is to patients who converted to full donor chimerism developing
determine whether characteristics that predict response to severe acute GVHD (steroid responsive, with subsequent
immunosuppressive therapy can also predict response to development of fatal sepsis). An acute clinical syndrome
Cy+CD8+ T cell-depleted haploidentical DLI. Patients termed “haplo immunostorm’ likely secondary to cytokine
entered onto this trial will have examination of T cell receptor flux (characterized by 1 or more of the following: fever,
spectratype both before and after therapy. Cytogenetics and malaise, LFT abnormalities, rash and diarrhea) was seen
HLA typing will be performed routinely on all patients. commonly at the higher dose levels and was exquisitely
0072 Recent studies have uncovered a role for donor natu responsive to steroids. This study demonstrated the biological
ral killer cell alloreactivity in preventing relapse of acute activity and manageable safety profile of this approach. The
leukemia after haploidentical stem cell transplantation 36-38. minimum CD3+ T-cell dose (not CD8+ depleted) required for
More recently, alloreactive natural killer cells of donor origin response in that study was 1x10 cells/kg.
have been found to prevent relapse of AML and MDS after
HLA-identical stem cell transplantation39. These results Example 7
underscore the need to characterize the expressed repertoire
of killer immunoglobulin-like receptors, or KIRs, on donor Patent Selection
NK cells using both molecular and flow cytometric methods 0075 Patients must have pathologically confirmed:
So as to identify donors expressing KIRs whose HLA ligands Myelodysplastic syndrome (MDS), IPSS score of Int-2 or
are missing on recipient cells. These studies will be per high (see Appendix A for IPSS scoring system). Patients must
formed retrospectively in the Immunogenetics Laboratory have failed or be ineligible or intolerant of treatment with
and the results correlated with response to therapy. 5-azacitidine.
Example 6 Example 8
Rationale for the Proposed Trial Design Treatment Plan
0073. This is a standard phase I/II trial design that seeks to 0076 All patients will require documentation of a detailed
determine, in the phase I portion of the trial, the maximally history and physical examination and standard evaluation of
tolerated dose (MTD) of CD8+ T cell-depleted haploidentical cardiac, liver and renal function, as designated in section 10.
peripheral blood cells (CD8- PBCs) when infused after All patients will undergo a bone marrow aspirate and biopsy
cyclophosphamide (Cy), and then to estimate, in the phase II for morphological, cytogenetic (if applicable) and flow cyto
portion of the trial, the efficacy of treatment with Cy plus the metric (if applicable) evaluation no more than one month
MTD of CD8- PBCs. High dose Cy (>100 mg/kg) has been prior to registration on protocol, along with other standard
used extensively as part of transplantation conditioning for disease evaluations (e.g., CT of chest, abdomen, pelvis)
patients with hematologic malignancies, and its safety is where applicable.
well-documented in this population, including elderly
patients (ages 55-66) with myelodysplastic syndrome. The Pre-Treatment Evaluation
most serious risks of treatment, because they have the poten 0077 Cyclophosphamide will be administered as an iv
tial to cause death, are prolonged aplasia and graft-Versus host infusion over 1-2 hr., (depending on Volume) on days -2 and
disease, both of which require Sustained engraftment of the -1. The dose of cyclophosphamide is 50 mg/kg/day. Dose is
donor cells. The choice of the initial cell dose, 10 CD4+ T calculated based on the adjusted ideal body weight or actual
cells/kg and <3.2x10 CD8+ T cells/kg, was based solely body weight whichever is less. Body weight and height are
upon safety considerations. Specifically, grafts containing measured directly. An approximate weight for height would
<104 CD8+ T cells/kg do not cause severe GVHD, even be calculated from a standard table or equations that reflect
among patients receiving lethal conditioning and no pharma ideal “values.
cologic immunosuppression after transplantation. Moreover,
partial depletion of CD8+ T cells from standard marrow Cyclophosphamide and Pre-DLI Regimen
grafts significantly increases the risk of graft rejection33.
which is the desired outcome of treatment in this trial. For 0078 Patients will be instructed to increase fluids over
these reasons, it is felt that a DLI product containing <104 night before cyclophosphamide administration. Hydration
CD8+ T cells/kg is unlikely to cause serious adverse events. with normal saline at 3 cc/kg/hriv will be started 2 hr prior to
US 2015/O 132290 A1 May 14, 2015

cyclophosphamide, then the rate will be reduced to 2 cc/kg/hr infused lymphocytes is expected to induce anamnestic immu
for 1 hr precyclophosphamide and continued for 8 hr post nity to cells of the donor or even to other close relatives.
cyclophosphamide. Mesna will be given in divided doses iv Patient’s peripheral blood will be obtained on day 60 and
30 min pre- and at 3, 6, and 8 hr post cyclophosphamide. tested for the presence of human anti-mouse antibody
0079 Mesna dose will be based on the cyclophosphamide (HAMA) and for cytotoxic antibodies against donor cells.
dose being given. The total daily dose of mesna is equal to
80% of the total daily dose of cyclophosphamide. Duration of Follow Up
0080 Prophylactic anti-microbial therapy will be started I0086 Patients will be followed for a minimum of 60 days
on Day 0 and will follow institutional practice. after DLI, and then until death or disease progression, which
0081 Antifungal prophylaxis will be administered as fol ever occurs first. Patients removed from study for unaccept
lows: Fluconazole 400 mg poor IV qd, beginning Day 0 and able adverse events or who develop treatment-related adverse
continuing until the ANC is >500 for 3 consecutive days (or events will be followed until resolution or stabilization of the
for 2 consecutive measurements overa 3 day period). Another adverse event.
appropriate prophylactic antifungal agent may be substituted.
Pneumocystis carinii pneumonia (PCP) prophylaxis will start Post DLI Monitoring:
on Day 0 and should continue until Day 60. Patients intolerant
of trimethoprim/sulfamethoxazole (Bactrim) will receive I0087 Patients remaining on study will have blood drawn
either dapsone or pentamidine as PCP prophylaxis. Viral pro on days 14, 28, and 60, and six months after DLI. A CBC with
phylaxis will consist of vallacyclovir oracyclovir from Day 0 manual differential will be obtained with these blood draws.
to Day 60. An oral quinolone (e.g...moxifloxacin or norfloxa Lymphocyte Subsets, including the percentage of cells
cin) will be administered according to institutional preference expressing CD4 or CD8, will be analyzed by flow cytometry.
until the ANC is >500 for 3 consecutive days (or for 2 con After day 60, the patient will have monthly complete blood
secutive measurements over a 3 day period) following DLI. counts with white blood cell differential as long as there is no
0082 All patients will receive infusion of haploidentical documented disease progression, until 6 months after DLI.
PBCs depleted of CD8+ T cells using the CliniMACS(R) sys 0088 Disease Assessment.
tem with CliniMACS(R) CD8 reagent. The numbering of the I0089. In addition to disease assessments specified above,
dose levels is from the lowest to the highest cell dose. The first results of additional disease assessments performed as stan
cohort of patients (dose level 1) will receive Cy plus CD8+ T dard of care will be collected for study purposes until death or
cell-depleted haploidentical PBCs (CD8- PBCs) containing disease progression, whichever occurs first.
an intended dose of 1x105 CD4+ T cells/kg of recipient IBW.
If criteria for dose escalation are met, patients on dose level 2, EXAMPLE 10
2b, 3, or 4 will receive CD8- PBCs containing an intended
dose of 1x10, 3x106, 1x107, or 5x107 CD4+ T cells/kg, Dosing Delays/Dose Modification
respectively.
0090 Cyclophosphamide dose will not be modified. DLI
DLI Dose Calculation dose will be modified in the event of excessive content of
CD8+ T cells.
0083. The formula for calculating the volume of final
(CD8-depleted) product that will deliver the intended dose of Adverse Events: List and Reporting Requirements
CD4+ T cells is as follows: Intended volume (m1)=Intended
CD4+ T cell dose (cells/kg)xRecipient IBW*(kg)/CD4+ T 0091. The following information shall be collected on all
cell concentration (cells/ml) *Note Ifactual weight<ideal, patients with acute GVHD: Date of onset (defined as the date
use actual weight. of first biopsy confirming GVHD) GVHD evaluation form at
However, the total number of CD8+ T cells that are infused the time of onset, weekly until GVHD resolves, and Day 60
may not exceed 3.2% of the intended number of CD4+ T cells Initial overall clinical grade Maximum overall clinical grade
to be infused (the numerator of the equation above). If the Date of onset of grade III-IV acute GVHD, if any The occur
ratio of CD4+/CD8+ cells in the depleted product is less than rence and severity of acute and chronic GVHD after Day 60
31.25 (=1/.032), then the volume of the product to be infused will be captured at the patient’s six month evaluation.
will be determined by the following formula: If CD4/CD8 0092 All instances of grade II-IV acute GVHD will be
ratio of final product31.25, then: Infused volume (ml)=In captured as adverse events. Grade III-IV GVHD will be
tended volumex(CD4/CD8 ratio)/31.25 If the ratio of CD4+/ reported as a serious adverse event.
CD8+ cells in the depleted product is equal to or greater than 0093 DLI-induced aplasia is defined as neutropenia (ab
31.25, then the volume of the product to be infused is the solute neutrophil count<500/ml) with any evidence of donor
intended volume (formula 1): If CD4/CD8 ratio of final prod chimerism on day 60 or later. All cases of DLIinduced aplasia
uct>31.25, then: Infused volume (ml)=Intended volume will be reported as serious adverse events.
Transfusion Support Example 11
0084 Platelet and packed red cell transfusions will be Pharmaceutical Information
given per current institutional recommendations.
Example 9 Cyclophosphamide (Cytoxan R.)
0094. Cyclophosphamide is commercially available.
Duration of Therapy Cyclophosphamide is an alkylating agent which prevents cell
0085 Patients are eligible for only one lymphocyte infu division primarily by cross-linking DNA strands. Cyclophos
Sion. This restriction is in place because rejection of the phamide is cell cycle non-specific. Cyclophosphamide for
US 2015/O 132290 A1 May 14, 2015

injection is available in 2000 mg vials which are reconstituted tec, Auburn, Calif.). Prior to CD8 depletion, whole blood
with 100 ml sterile water for injection. The concentration of products will initially be processed to prepare a buffy coat
the reconstituted product is 20 mg/ml. The calculated dose concentrate and for major ABO incompatible donor/recipient
will be diluted further in 250-500 ml of Dextrose 5% in water. pairs the buffy coat concentrate will be further processed
Each dose will be infused over 1-2 hr (depending on the total using lymphocyte separation medium to remove contaminat
Volume). ing red blood cells. Processed whole blood products or aph
0095 Clinical toxicities of cyclophosphamide include eresis products are then concentrated and resuspended in
alopecia, nausea and Vomiting, headache and dizziness, hem PBS/EDTA supplemented with 0.5% human serum albumin.
orrhagic cystitis, cardiotoxicity, immunosuppression, myclo 0103 Murine monoclonal CD8 antibody, conjugated to
Suppression, pulmonary fibrosis, increased hepatic enzymes iron-dextran Super-paramagnetic particles is added and incu
and syndrome of inappropriate anti-diuretic hormone bated at room temperature for 30 minutes. One vial of anti
(SIADH). Cyclophosphamide will be dispensed by the body will be used to treat up to 40x10 total white blood cells
Oncology Pharmacy and is produced by Mead Johnson Phar andup to 4x10 CD8+ cells. Excess antibody will be removed
maceuticals. by washing 1 time and the product volume will be adjusted to
0096 Mesna (sodium-2-mercapto ethane sulphonate) 100 ml with PBS/EDTA with albumin. It is then connected to
0097 Mesna is a prophylactic agent used to preventhem the CliniMACS Selection System using a sterile disposable
orrhagic cystitis induced by the tubing set. The run is initiated by a pre-set computer program
0098 oxasophosphorines (cyclophosphamide and ifos which controls (i) the flow of antibody-treated cells, (ii) wash
phamide). It has no intrinsic cytotoxicity and no antagonistic ing that removes residual unbound cells, (iii) removal of the
effects on chemotherapy. Mesna binds with acrolein, the uro magnetic field around the column to release selected cells,
toxic metabolite produced by the oXasophosphorines, to pro and (iv) the final collection of CD8 depleted cells into a bag.
duce a non-toxic thioether and slows the rate of acrolein The entire process takes approximately 2-6 hours from
formation by combining with 4-hydroxy metabolites of oxa completion of initial product concentration. The Subsequent
Sophosphorines. product will be analyzed for cell count, viability, CD3, CD4.
0099 Mesna is available in 200 mg. 400 mg and 1000 mg CD8, CD16, and CD56 content. The CD4 concentration will
vials containing a 100 mg/ml Solution. Each dose of mesna be used to calculate the patient dose. The calculated volume
will be diluted further in 50 ml of normal saline to be infused will be removed and prepared for infusion according to insti
over 15 min. Mesna dose will be based on the cyclophospha tutional standard operating procedures.
mide dose being given. The total daily dose of mesna is equal
to 80% of the total daily dose of cyclophosphamide. At the Correlative/Special Studies
doses used for uroprotection mesna is virtually non-toxic.
However, adverse effects which may be attributable to mesna Phenotypic Immune Reconstitution
include nausea and Vomiting, diarrhea, abdominal pain, 0104 Peripheral blood concentrations of lymphocyte sub
altered taste, rash, urticaria, headache, joint or limb pain, sets including CD4+ and CD8+ T cells will be determined
hypotension and fatigue. using the absolute lymphocyte count and flow cytometry on
CBERIDE Device days 14, 28, 60, and 6 months after DLI.
0105 Analysis of Host CD8+ T Cell Repertoire Diversity
0100 Donors will have their blood collected via periph by Spectratype Analysis. Recent studies indicate that the
eral whole blood collection (450 ml into CPDA-1) or a leu diversity of the T cell repertoire can be assessed by T cell
kapheresis procedure to collect peripheral white blood cells receptor V region spectratyping, which evaluates the CDR3/
under steady state conditions (without mobilization). Each diversity/joining regions (VB-D-J-CB) of cells expressing a
leukapheresis collection will be performed on a continuous given V gene. This region confers specificity of the T cell
flow cell separator (COBE Spectra, Gambro) using institu receptor. Immunoscoping or V spectratyping is remarkably
tional standard operating procedures for lymphocyte collec useful for evaluating anti-tumor immune responses following
tion. The method of blood donation, phlebotomy versus leu therapy and immune reconstitution following bone marrow
kapheresis, will be determined by obtaining a peripheral transplantation49. Moreover, spectratype analysis of MDS
blood absolute CD4+ T cell count within 30 days prior to patients before and after immunosuppressive therapy has
donation and by estimating the Volume of blood required to revealed skewing of the T cell repertoire that normalizes with
obtain the targeted CD4+ T cell dose. Since the normal range a response to treatment. We therefore hypothesize that
of peripheral blood CD4+ T cell counts is 0.5-1.5x10/ml, it patients with MDS and possibly CMML will have skewed T
is likely that simple phlebotomy will be sufficient for dose cell repertoires prior to treatment, that the DLI will initially
levels 1-2, pheresis may be required for levels 2b but leuka induce a population of alloreactive T cells, and that respond
pheresis will be required for dose level 3 and 4. ers will eventually acquire a normal T cell repertoire as
0101 Based upon extensive prior experience, a 4 hour revealed by spectratype analysis. Pre-treatment CD8+ T cells
leukapheresis procedure should be sufficient to obtain 5x107 will be obtained from patient peripheral blood mononuclear
CD4+ T cells/kg of recipient IBW. Target collections will be cells (PBMCs). To identify patientanti-donor reactive T cells,
at least 30% more than the desired dose to accommodate for pre-treatment PBMCs from the patient will be cultured for
cell loss during the depletion process. seven days with irradiated donor PBMCs prior to cell sorting.
0102 The product will undergo CD8 depletion in the The culture period allow for the clonal expansion of patient
Graft Engineering Laboratory. All standard operating proce anti-donor T cells. PBMCs will also be collected and CD8+ T
dures will be followed. The product will be analyzed for cells will be purified on days 14, 28, 60, and at six months.
nucleated cell count, CD3, CD4, CD8, CD16, and CD56. The 0106 Those of skill in the art should, in light of the present
product will be stored overnight and CD8 depletion will take disclosure, appreciate that many changes can be made in the
place on the CliniMACS(R) Selection System (Miltenyi Bio specific embodiments which are disclosed and still obtain a
US 2015/O 132290 A1 May 14, 2015

like or similar result without departing from the spirit and 13. The method of claim 9, wherein a selection character
scope of the invention. The present invention is not to be istic is screening for more than one HLA Class II alleles.
limited in scope by the specific embodiments described 14. The method of claim 13, wherein a potential allogenic
herein, which are intended as single illustrations of individual donoris selected based on maximizing mismatch between the
aspects of the invention, and functionally equivalent methods potential allogenic donor versus the recipient, in the potential
and components are within the scope of the invention. Indeed, allogenic donor versus recipient direction, at the more than
various modifications of the invention, in addition to those one HLA Class II alleles, and the potential allogenic donor is
shown and described herein, will become apparent to those chosen as the donor.
skilled in the art from the foregoing description. Accordingly, 15. The method of claim 9, wherein a selection character
the invention is limited only by the following claims. istic is screening for one or more HLA Class I allele(s).
1. A method of making an allogenic lymphocyte composi 16. The method of claim 15, wherein a potential allogenic
tion comprising: donor is selected based on minimizing mismatch between the
providing a peripheral blood cell composition from a potential allogenic donor and the recipient at the more than
human donor allogenic to the recipient, the peripheral one HLA Class I allele(s), and the potential allogenic donor is
blood cell composition comprising CD4+ T-cells, CD8+ chosen as the donor.
T-cells, and natural killer cells, wherein (i) the donor 17. An allogenic lymphocyte composition for administra
comprises at least one human leukocyte antigen (HLA) tion to a human recipient obtained by the method of claim 1.
Class II allele mismatch relative to the recipient in the 18. Analogenic human lymphocyte composition compris
donor versus the recipient direction and the HLA Class 1ng:
II allele mismatch is at a gene selected from the group CD4+ T-cells and natural killer cells from the peripheral
consisting of HLA-DRB1, HLA-DQB1, and HLA blood cell composition of a donor, wherein
DPB1, and (ii) the recipient does not have detectable (i) the donor comprises at least one human leukocyte
antibodies reactive against human leukocyte antigens of antigen (HLA) Class II allele mismatch relative to the
the donor. recipient in the donor versus the recipient direction
2. The method of claim 1, wherein if the donor and recipi and the HLA Class II allele mismatch is at a gene
ent are ABO blood type incompatible and the peripheral selected from the group consisting of HLA-DRB1,
blood cell composition comprises a number of red blood HLA-DQB1, and HLA-DPB1, and
cells, then making the allogenic lymphocyte composition (ii) the recipient does not have detectable antibodies
further comprises reducing the number red blood cells. reactive against human leukocyte antigens of the
3. The method of claim 2, wherein the number of red blood donor,
cells comprises less than or equal to about 50 ml in packed with the proviso that the CD4+ T-cells of the allogenic
Volume. lymphocyte composition are not activated ex vivo.
4. The method of claim 1, wherein the number of CD4+ 19. The lymphocyte composition of claim 18, wherein the
T-cells in the allogenic lymphocyte composition differs from peripheral blood cell composition comprises a number of red
the number of CD4+ T-cells in the peripheral blood cell blood cells and the number of red blood cells comprises less
composition by less than about 20%. than or equal to about 50 ml in packed volume.
5. The method of claim 1, with the further proviso that 20. The lymphocyte composition of claim 18, with the
CD4+ T-cells obtained from the donor are not intentionally further proviso that CD4+ T-cells obtained from the donor are
expanded or intentionally differentiated ex vivo. not intentionally expanded or intentionally differentiated ex
6. The method of claim 1, wherein reducing the CD8+ V1VO.
T-cells in the peripheral blood cell composition comprises 21. The lymphocyte composition of claim 18, wherein the
using an anti-CD8+ antibody associated with magnetic par peripheral blood cell composition is a whole blood productor
ticles or an anti-CD8+ antibody plus complement. an apheresis product.
7. The method of claim 1, wherein the peripheral blood cell 22. The lymphocyte composition of claim 18, wherein the
composition is a whole blood productoran apheresis product. HLA Class II allele mismatch is a mismatch of two or more
8. The method of claim 1, wherein the HLA Class II allele HLA Class II alleles selected from the group consisting of
mismatch in the donor versus the recipient direction is a HLA-DRB1, HLA-DPB1, and HLA-DQB1.
mismatch at HLA-DRB1. 23. The lymphocyte composition of claim 18, wherein the
9. The method of claim 1, wherein the providing further HLA Class II allele mismatch in the donor versus the recipi
comprises screening for one or more selection characteristic ent direction is a mismatch at HLA-DRB1.
(s) and the screening is carried out on a Subject selected from 24. A method of treating a disease or condition in a human
the group consisting the recipient, the donor, and one or more Subject, comprising:
potential allogenic donor(s). administering a lymphoreductive non-lymphoablative
10. The method of claim 9, wherein a selection character treatment to the Subject to induce transientlymphopenia
istic is screening for serological reactivity to an infectious in the Subject; and
agent antigen. Subsequently administering to the Subject a first allogenic
11. The method of claim 10, wherein the infectious agent lymphocyte composition derived from a peripheral
antigen is selected from the group consisting of a Human blood cell composition of a human, allogenic donor, the
Immunodeficiency Virus (HIV) antigen, a Hepatitis Virus first allogenic lymphocyte composition comprising a
antigen, and a Cytomegalovirus antigen. number of CD4+ T-cells and a number of natural killer
12. The method of claim 11, wherein the infectious agent cells from the peripheral blood cell composition of the
antigen is a Cytomegalovirus antigen, the recipient and the donor, wherein
donor are screened, and there is no serological reactivity to (i) the donor comprises at least one human leukocyte
the Cytomegalovirus antigen in the recipient or the donor. antigen (HLA) Class II allele mismatch relative to the
US 2015/O 132290 A1 May 14, 2015
16

subject in the donor versus the subject direction and (i) the additional donor comprises at least one human
the HLA Class II allele mismatch is at a gene selected leukocyte antigen (HLA) Class II allele mismatch
from the group consisting of HLA-DRB1, HLA relative to the subject in the additional donor versus
DQB1, and HLA-DPB1, the subject direction and the HLA Class II allele mis
(ii) the subject does not have detectable antibodies reac match is at a gene selected from the group consisting
tive against human leukocyte antigens of the donor, of HLA-DRB1, HLA-DQB1, and HLA-DPB1,
with the proviso that the CD4+ T-cells of the first allogenic (ii) the subject does not have detectable antibodies reac
lymphocyte composition are not activated ex vivo. tive against human leukocyte antigens of the addi
25. The method of claim 24, wherein the lymphoreductive tional donor,
non-lymphoablative treatment comprises treating the Subject (iii) the number of CD4+ T-cells in the successive allo
with one or more cytoreductive agent selected from the group genic lymphocyte composition differs from the num
consisting of alkylating agents, alkyl Sulphonates, ber of CD4+ T-cells in the additional peripheral blood
nitrosoureas, triaZenes, antimetabolites, pyrimidine analogs, cell composition by less than about 50%,
purine analogs, Vinca alkaloids, epipodophyllotoxins, antibi (iv) the number of additional donor CD4+ T-cells based
otics, dibromomannitol, deoxyspergualine, dimethyl myle on an ideal body weight of the Subject in kilograms
ran and thiotepa. (kg) is between about 1x10 CD4+ T-cells/kg and
26. The method of claim 25, wherein the lymphoreductive about 1x10 CD4+ T-cells/kg,
non-lymphoablative treatment comprises treating the Subject (v) the number of natural killer cells in the successive
with an alkylating agent and the alkylating agent is cyclo allogenic lymphocyte composition is less than or
phosphamide. equal to the number of natural killer cells in the addi
27. The method of claim 24, wherein subsequent to admin tional peripheral blood cell composition, and
istering the first allogenic lymphocyte composition to the (vi) the Successive allogenic lymphocyte composition
Subject, the method further comprises administration of an has at least one order of magnitude fewer CD8+
anti-tumor monoclonal antibody or anti-tumor monoclonal T-cells relative to the additional peripheral blood cell
antibody/drug conjugate to the Subject. composition.
28. The method of claim 27, wherein the method further 34. The method of claim33, with the proviso that the CD4+
comprises administration of the anti-tumor monoclonal anti T-cells of the Successive allogenic lymphocyte composition
body and the anti-tumor monoclonal antibody is selected are not activated ex vivo.
from the group consisting of rituximab, cetuximab, trastu 35. The method of claim 24, wherein subsequent to admin
Zumab, and pertuzumab. istering the first allogenic lymphocyte composition to the
29. The method of claim 27, wherein the method further Subject, the method further comprises administration of an
comprises administration of the anti-tumor monoclonal anti agent that blocks negative signaling in T-cells.
body/drug conjugate and the anti-tumor monoclonal anti 36. The method of claim 35, wherein the agent that blocks
body/drug conjugate is selected from the group consisting of negative signaling in T-cell is selected from the group con
brentuximab vedotin, gemtuzumab ozogamicin, trastuzumab sisting of an anti-PD-1 antibody, ipilimumab, an anti-PD-L2
emtansine, inotuZumab ozogamicin, glembatumumab antibody, and a PD-1 fusion protein.
Vedotin, lorVotuZumab mertansine, cantuzumab mertansine,
and milatuZumab-doxorubicin. 37. The method of claim 24, wherein the disease or condi
30. The method of claim 24, wherein subsequent to admin tion is selected from the group consisting of a cancer, an
istering the first allogenic lymphocyte composition to the autoimmune disorder, an organ transplantation, an allograft
Subject, the method further comprises administration of a rejection, and a viral infection.
chemotherapeutic agent to the Subject. 38. The method of claim 37, wherein the disease or condi
31. The method of claim30, wherein the chemotherapeutic tion is a cancer and the cancer is myelodysplastic syndrome.
agent is selected from the group consisting of dasatinib, nilo 39-129. (canceled)
tinib, ponatinib, imatinib, lapatinib, and Vismodegib. 130. The method of claim 1, further comprising making the
32. The method of claim 24, wherein subsequent to admin allogenic lymphocyte composition from the peripheral blood
istering the first allogenic lymphocyte composition to the cell composition by reducing the number of CD8+ T-cells in
Subject, the method further comprises administration of a the peripheral blood cell composition by at least one order of
monoclonal antibody/CD4+ T-cell epitope conjugate to the magnitude, wherein
Subject. (a) the number of CD4+ T-cells in the allogenic lympho
33. The method of claim 24, wherein subsequent to admin cyte composition differs from the number of CD4+
istering the first allogenic lymphocyte composition to the T-cells in the peripheral blood cell composition by less
subject, the method further comprises than about 50%, and
administering a successive lymphoreductive non-lym (b) the number of natural killer cells in the allogenic lym
phoablative treatment to the subject to induce transient phocyte composition is less than or equal to the number
lymphopenia in the Subject; and of natural killer cells in the peripheral blood cell com
Subsequently administering to the Subject a Successive position, with the proviso that the CD4+ T-cells of the
allogenic lymphocyte composition derived from an allogenic lymphocyte composition are not activated ex
additional peripheral blood cell composition of an addi vivo.
tional human, allogenic donor, the Successive allogenic 131. The composition of claim 18, wherein
lymphocyte composition comprising a number of CD4+ (iii) the number of CD4+ T-cells in the allogenic lympho
T-cells and a number of natural killer cells from the cyte composition differs from the number of CD4+
additional peripheral blood cell composition of the addi T-cells in the peripheral blood cell composition by less
tional donor, wherein than about 50%,
US 2015/O 132290 A1 May 14, 2015
17

(iv) the number of donor CD4+ T-cells based on an ideal


body weight of the recipient in kilograms (kg) is
between about 1x10CD4+ T-cells/kg and about 1x10
CD4+ T-cells/kg,
(v) the number of natural killer cells in the allogenic lym
phocyte composition is less than or equal to the number
of natural killer cells in the peripheral blood cell com
position, and
(vi) the allogenic lymphocyte composition has at least one
order of magnitude fewer CD8+ T-cells relative to the
peripheral blood cell composition.
132. The method of claim 24, wherein
(iii) the number of CD4+ T-cells in the first allogenic lym
phocyte composition differs from the number of CD4+
T-cells in the peripheral blood cell composition by less
than about 50%,
(iv) the number of donor CD4+ T-cells based on an ideal
body weight of the Subject in kilograms (kg) is between
about 1x10 CD4+ T-cells/kg and about 1x10 CD4+
T-cells/kg,
(v) the number of natural killer cells in the first allogenic
lymphocyte composition is less than or equal to the
number of natural killer cells in the peripheral blood cell
composition, and
(vi) the first allogenic lymphocyte composition has at least
one order of magnitude fewer CD8+ T-cells relative to
the peripheral blood cell composition.
k k k k k