U.S. patent application number 16/754400 was filed with the patent office on 2020-10-01 for methods for cancer treatment using a radiolabeled anti-cd45 immunoglobulin and adoptive cell therapies.
This patent application is currently assigned to Actinium Pharmaceuticals, Inc.. The applicant listed for this patent is Actinium Pharmaceuticals, Inc.. Invention is credited to Mark Berger, Dale Lincoln Ludwig, Sandesh Seth, Keisha Thomas.
Application Number | 20200308280 16/754400 |
Document ID | / |
Family ID | 1000004952942 |
Filed Date | 2020-10-01 |
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United States Patent
Application |
20200308280 |
Kind Code |
A1 |
Berger; Mark ; et
al. |
October 1, 2020 |
METHODS FOR CANCER TREATMENT USING A RADIOLABELED ANTI-CD45
IMMUNOGLOBULIN AND ADOPTIVE CELL THERAPIES
Abstract
Methods for the treatment of a proliferative disorder include
administration of a radiolabeled anti-CD45 antibody in concert with
an adoptive cell therapy. The adoptive cell therapy may include
administration of cells expressing a chimeric antigen receptor or a
T-cell receptor (CAR/TCR). The radiolabeled anti-CD45 antibody may
be administered before administration of the population of cells
expressing the CAR/TCR, either alone or in conjunction with
standard lymphodepletion agents. The radiolabeled anti-CD45
antibody may be administered after administration of the population
of cells expressing the CAR/TCR in preparation for transplantation
of autologous stem cells and/or administration of a second
effective amount of the populations of cells expressing the
CAR/TCR. These methods may improve treatment outcomes for
hematological malignancies including solid tumors, and/or may
lessen side effects associated with adoptive cell therapies.
Inventors: |
Berger; Mark; (New York,
NY) ; Thomas; Keisha; (Brooklyn, NY) ; Seth;
Sandesh; (New York, NY) ; Ludwig; Dale Lincoln;
(Rockaway, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Actinium Pharmaceuticals, Inc. |
New York |
NY |
US |
|
|
Assignee: |
Actinium Pharmaceuticals,
Inc.
New York
NY
|
Family ID: |
1000004952942 |
Appl. No.: |
16/754400 |
Filed: |
October 25, 2018 |
PCT Filed: |
October 25, 2018 |
PCT NO: |
PCT/US2018/057468 |
371 Date: |
April 8, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62576879 |
Oct 25, 2017 |
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62675417 |
May 23, 2018 |
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62693517 |
Jul 3, 2018 |
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62700978 |
Jul 20, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 51/1027 20130101;
C07K 16/289 20130101; A61P 35/00 20180101; A61K 2039/505 20130101;
A61K 51/1096 20130101; A61K 9/0019 20130101; A61K 35/17
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00; A61K 35/17 20060101
A61K035/17; A61K 51/10 20060101 A61K051/10 |
Claims
1. A method for treating a subject having a hematological
malignancy or a solid cancer, the method comprising: administering
to the subject an effective amount of a radiolabeled anti-CD45
antibody, wherein the effective amount is an amount sufficient to
lymphodeplete the subject; and administering to the subject an
effective amount of a population of cells expressing a chimeric
antigen receptor or a T-cell receptor (CAR/TCR).
2. The method of claim 1, wherein the radiolabeled anti-CD45
antibody is radiolabeled BC8.
3. The method of claim 1, wherein the radiolabeled anti-CD45
antibody comprises .sup.131I, .sup.125I, .sup.123I, .sup.90Y,
.sup.177Lu, .sup.186Re, .sup.188Re, .sup.89Sr, .sup.153Sm,
.sup.32P, .sup.225Ac, .sup.213Bi, .sup.213Po, .sup.211At,
.sup.212Bi, .sup.213Bi, .sup.223Ra, .sup.227Th, .sup.149Tb,
.sup.137Cs, .sup.212Pb and .sup.103Pd.
4. The method of claim 1, wherein the effective amount of the
radiolabeled anti-CD45 antibody is administered as a single
dose.
5. The method of claim 2, wherein the radiolabeled BC8 is
.sup.131I-BC8, and the effective amount of .sup.131I-BC8 is from 10
mCi to 200 mCi.
6. The method of claim 2, wherein the radiolabeled BC8 is
.sup.131I-BC8, and the effective amount of .sup.131I-BC8 is from 25
mCi to 100 mCi.
7. The method of claim 2, wherein the radiolabeled BC8 is
.sup.225Ac-BC8, and the effective amount of .sup.225Ac-BC8 is 0.1
.mu.Ci/kg of subject weight to 5.0 .mu.Ci/kg of subject weight.
8. The method of claim 2, wherein the radiolabeled BC8 is
administered 6, 7, or 8 days before administration of the
population of cells expressing the CAR/TCR.
9. The method of claim 2, wherein the effective amount of the
radiolabeled BC8 depletes at least 50% of lymphocytes of the
subject.
10. The method of claim 2, wherein the effective amount of the
radiolabeled BC8 does not induce myeloablation in the subject.
11. The method of claim 2, wherein the effective amount of the
radiolabeled BC8 provides a radiation dose of 2 Gy or less to the
bone marrow.
12. The method of claim 2, wherein the effective amount of the
radiolabeled BC8 depletes any of regulatory T cells, myeloid
derived suppressor cells, tumor associated macrophages, activated
macrophages secreting IL-1, activated macrophages secreting IL-6,
and combinations thereof.
13. The method of claim 1, wherein the population of cells
expressing the CAR/TCR are autologous cells.
14. The method of claim 1, wherein the population of cells
expressing the CAR/TCR are allogeneic cells.
15. The method of claim 1, wherein the population of cells
expressing the CAR/TCR target CD19, CD20, CD22, CD30, CD33, CD38,
CD123, CD138, CS-1, B-cell maturation antigen (BCMA), MAGEA3,
MAGEA3/A6, KRAS, CLL1, MUC-1, HER2, EpCam, GD2, GPA7, PSCA, EGFR,
EGFRvIII, ROR1, mesothelin, CD33/IL3Ra, c-Met, CD37, PSMA,
Glycolipid F77, GD-2, gp100, NY-ESO-1 TCR, FRalpha, CD24, CD44,
CD133, CD166, CA-125, HE4, Oval, estrogen receptor, progesterone
receptor, uPA, PAI-1, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5
or ULBP6, or a combination thereof.
16. The method of claim 1, wherein the population of cells
expressing the CAR/TCR target CD19, CD20, CD22, or a combination
thereof.
17. The method of claim 1, wherein administration of the population
of cells expressing the CAR/TCR target comprises administration of
gene-edited CAR T-cells, and wherein the gene-edited CAR T-cells
fail to properly express at least one checkpoint receptor, at least
one T-cell receptor, or both of the at least one checkpoint
receptor and the at least one T-cell receptor.
18. The method of claim 1, wherein the radiolabeled anti-CD45
antibody is administered after administration of the population of
cells expressing the CAR/TCR.
19. The method of claim 18, wherein the radiolabeled anti-CD45
antibody is administered 1 to 3 months after administration of the
population of cells expressing the CAR/TCR, and the effective
amount of the anti-CD45 antibody is an amount sufficient to induce
lymphodepletion in the subject.
20. The method of claim 18, wherein the subject has not shown a
complete response (CR) after administration of the population of
cells expressing the CAR/TCR, or the subject has relapsed or is
identified as having relapsed after administration of the
population of cells expressing the CAR/TCR.
21. The method of claim 17, further comprising, after
administration of the radiolabeled anti-CD45 antibody:
transplantation of autologous or allogeneic stem cells; or
administration of a second effective amount of the population of
cells expressing the CAR/TCR.
22. The method of claim 18, wherein the effective amount of the
radiolabeled anti-CD45 antibody is an amount sufficient to induce
myeloablation in the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a 371 National Stage filing of
PCT/US2018/057468 filed Oct. 25, 2018 which claims the benefit
under 35 U.S.C. .sctn. 119(e) of prior U.S. Provisional Application
Ser. No. 62/576,879, titled "Methods for Cancer Treatment Using A
radiolabeled anti-CD45 Immunoglobin and Adoptive Cell Therapies,"
filed Oct. 25, 2017; U.S. Provisional Application Ser. No.
62/675,417, titled "A radiolabeled anti-CD45-Based Lymphodepletion
Methods and Uses Thereof in Conjunction with Act-Based Cancer
Therapies," filed May 23, 2018; U.S. Provisional Application Ser.
No. 62/693,517, titled "A radiolabeled anti-CD45-Based Conditioning
Methods and Uses Thereof in Conjunction with Gene-Edited Cell-Based
Therapies," filed Jul. 3, 2018; and U.S. Provisional Application
Ser. No. 62/700,978, titled "A radiolabeled anti-CD45-Based
Lymphodepletion Methods and Uses Thereof in Conjunction with
Act-Based Cancer Therapies," filed Jul. 20, 2018, the contents of
which are all incorporated by reference here into this
application.
FIELD OF THE INVENTION
[0002] The present invention relates to methods for treating a
hematological malignancy including solid tumors comprising
administration of a radiolabeled anti-CD45 antibody and an adoptive
cell therapy such as a chimeric antigen receptor or T-cell receptor
modified T-cell, NK-cell, or dendritic-cell. The present invention
also relates to the administration of a radiolabeled anti-CD45
antibody with an autologous or allogeneic cell edited by
CRISPR/cas9, TALEN, or ZFN technology.
BACKGROUND OF THE INVENTION
[0003] Most patients with hematological malignancies or with
late-stage solid tumors are incurable with traditional therapies
such as surgery, radiation, and chemotherapy. Recent developments
which redirect a patient's own cells to recognize and control tumor
cell growth and proliferation show great promise. Such therapies,
referred to as adoptive cell therapies (ACTs), generally include
the transfusion of autologous cells that have been engineered to
recognize specific cell surface molecules on cancer cells.
[0004] Current ACTs include genetically modifying T-cells that
target antigens expressed on tumor cells through the expression of
chimeric antigen receptors (CARs). CAR T-cell therapy involves
genetically modifying autologous or allogenic T-cells to express
chimeric antigen receptors (CARs) that target tumor cell antigens.
CARs are antigen receptors that typically employ the single chain
fraction variable region of a monoclonal antibody designed to
recognize a cell surface antigen in a human leukocyte
antigen-independent manner. CARs directed against CD19 found on
normal B cells and over-expressed on certain forms of lymphoma have
recently been found to dramatically improve patient response rates
and, in some patients, provide a durable response. CARs are also
being developed for other blood cancers, targeting tumor-expressed
antigens including BCMA, CD33, CD22 and CD20. More recently, CAR-Ts
have been engineered to target antigens found on solid tumors,
including EGFR, EGFRvIII, Erb-B2, CEA, PSMA, MUC1, IL13-R.alpha.2
and GD2 (D'Aloia, et al., 2018, Cell Death and Disease.
9:282-293).
[0005] ACTs can also include recombinant T-cell receptor (TCR)
therapy. TCRs on lymphocytes can recognize tumor-specific proteins
typically found on the inside of cells. They do so by specifically
recognizing processed peptides (derived from those proteins) that
are complexed to major histocompatibility (MHC) antigens. In TCR
CAR-T therapy, a TCR is selected for specific recognition of a
tumor-expressed neoantigen and engineered for expression on a
patient's T-cells. In some cases, the TCR or the CAR may be
directed to the endogenous TCR locus. For example, the TRAC locus
(T-cell receptor gene) may be targeted via gene editing (e.g.,
CRISPR/cas9 technology, TALEN, or ZFN), effectively replacing the
endogenous TCR with the recombinant TCR gene.
[0006] In addition to autologous cells, allogeneic donor
lymphocytes may also be used for generating CAR-Ts using engineered
CARs or TCRs. In this case, the endogenous TCR on the donor cells
must be deleted to reduce the potential for graft-versus-host
disease. Gene editing technologies are an effective way to
introduce mutations to silence or ablate the endogenous TCR.
Finally, ACT methods further include administering
tumor-infiltrating lymphocytes (TILs).
[0007] Besides the ability to genetically modify T-cells to express
a CAR or a second TCR that recognizes and destroys respective
target cells in vitro/ex vivo, successful patient therapies with
engineered T-cells requires the T-cells to be capable of strong
activation, expansion, persistence over time, and, in the case of
relapsing disease, to enable a `memory` response. In fact, patient
outcomes are often linked to persistence or memory of the CAR
modified T-cells in the patient. Thus, methods which might improve
the persistence or memory of these cells may provide improved
cancer therapies.
[0008] Moreover, these engineered T-cells often show on-target, but
off-tissue toxicities which lead to some very serious side effects
that can be lethal. One such side effect is cytokine-release
syndrome (CRS), which results from the T-cell activation. CRS may
cause high fevers, low blood pressure or poor lung oxygenation.
Neurological toxicities have also been observed, such as delirium,
confusion, and seizure. Moreover, CAR T-cell therapy targeting
antigens found on the surface of B-cells not only destroy cancerous
B-cells but also normal B-cells. Therefore, B-cell aplasia (i.e.,
low numbers of B-cells or absent B-cells), while an expected side
effect, does result in a lowered ability to make the antibodies
that protect against infection. The lack of B-cells can also lead
to hypogammaglobulinemia, which may require treatment with long
term IVIG support. Tumor lysis syndrome (TLS), another known side
effect of CAR T-cell therapy, represents a group of metabolic
complications that can occur due to the breakdown of dying
cells.
[0009] Possible lessening of certain of these side effects may be
achieved through administration of lowered, but potentially less
effective, doses of the engineered cells in the ACT. Another
possible solution is hematopoietic stem cell transplantation
(HSCT), which may address certain of the long-term toxicities of
CAR T-cell therapies, such as the hypogammaglobulinemia. In
preparation for HSCT, agents may be administered to condition,
lymphodeplete, or ablate the stem cells and/or malignant cells.
Current non-targeted conditioning methods, which include, for
example, irradiation (e.g., total body irradiation) and DNA
alkylating/modifying agents, are highly toxic to multiple organ
systems, hematopoietic and non-hematopoietic cells, and the
hematopoietic microenvironment. These harsh conditioning regimens
effectively kill the patient's immune and niche cells and adversely
affect multiple organ systems, frequently leading to
life-threatening complications.
[0010] Lymphodepletion Generally
[0011] Before administering a dose of engineered immune cells to a
patient, it is common to lymphodeplete the patient. The
lymphodepletion process is considered important, indeed essential,
to the success of ACT methods. The process creates sufficient space
in the immune microenvironment (e.g., bone marrow) to allow the
transferred cells to engraft. It also creates a favorable immune
homeostatic environment for the successful engraftment,
proliferation, and persistence of the transferred cells by
eliciting a favorable cytokine profile. It elicits this cytokine
profile particularly in the peripheral immune niches (e.g., bone
marrow, spleen and lymph nodes) for the establishment and
proliferation of the engineered cells. (see, e.g., Maine, et al.,
2002, J. Clin. Invest, 110:157-159; Muranski, et al., 2006, Nat.
Clin. Pract. Oncol., 3(12):668-681; Klebanoff, et al., 2005, Trends
Immunol., 26(2): 111-117)
[0012] Chemotherapy-Based Lymphodepletion
[0013] It is common to use a combination of highly cytotoxic
chemotherapy agents, especially cyclophosphamide and fludarabine,
to lymphodeplete patients prior to ACT methods like CAR T-cell
therapy. These agents reduce lymphoid cell number. However, they
are highly toxic. They not only deplete the immune system in a
non-targeted manner but may also damage other normal cells and
tissues. Not all patients can tolerate them. Further, particularly
in CAR T-cell therapy, durable response rates are typically less
than 50%. Many patients eventually relapse after receiving CAR
T-cell therapy and require further therapeutic intervention or a
stem cell transplant (e.g., a bone marrow transplant).
[0014] Antibody-Based Lymphodepletion
[0015] Antibodies have greater cell-targeting specificity than
chemotherapeutics. Antibodies to immune cell-specific antigens are
therefore of interest as potential substitutes for
chemotherapeutics as lymphodepletion agents. CD45 is an immune
cell-specific antigen. In general, all cells of hematopoietic
origin, with the exception of mature erythrocytes and platelets,
express CD45. High expression of CD45 is also seen on most acute
lymphoid and myeloid leukemias. For example, CD45 is expressed at a
density of approximately 200,000 to 300,000 sites per cell on
circulating leukocytes and malignant B cells.
[0016] Anti-CD45 antibody-based lymphodepletion is known (see,
e.g., Louis, et al., 2009, Blood, 113:2442-2450). However, this
approach too has shortcomings. For example, in the Louis, et al.
study, eight patients were lymphodepleted with anti-CD45 antibody
and showed an increase in peripheral blood frequency of desired
T-cells after infusion. However, only three patients had clinical
benefits, and only one had a complete response.
[0017] Accordingly, what is needed are alternative methods and
therapy protocols which may improve the efficacy of ACTs such as
CAR-T therapies, and/or may reduce certain of the side effects of
the current ACT protocols. Additionally, myeloconditioning regimens
which reduce or eliminate undesirable toxicity are needed. Thus,
objects of the presently disclosed invention include methods which
improve the efficacy of ACT, methods which reduce certain side
effects of the current ACT protocols, and methods which may address
limitations of current ACT protocols caused by these side
effects.
SUMMARY OF THE INVENTION
[0018] The present invention provides solutions to the
aforementioned problems by providing methods for the treatment of a
hematological malignancy including solid tumors which include a
combination therapy comprising administration of a radiolabeled
anti-CD45 antibody as a lymphodepleting or conditioning regimen and
administration of an adoptive cell therapy (ACT). The ACT may
include administration of cells expressing a chimeric antigen
receptor (CAR), or a T-cell receptor (TCR), or may include
tumor-infiltrating lymphocytes (TIL).
[0019] According to certain aspects of the present invention, the
radiolabeled anti-CD45 antibody may be administered before
administration of the population of cells expressing the CAR/TCR or
the TIL, either alone or in conjunction with standard
lymphodepletion agents.
[0020] According to certain aspects of the present invention, the
radiolabeled anti-CD45 antibody may be administered after
administration of the population of cells expressing the CAR/TCR or
the TIL in preparation for transplantation of autologous or
allogeneic stem cells and/or administration of a second effective
amount of the populations of cells expressing the CAR/TCR or the
TIL.
[0021] According to certain aspects of the present invention, the
radiolabeled anti-CD45 antibody may be administered both before
administration of the population of cells expressing the CAR/TCR or
the TIL, either alone or in conjunction with standard
lymphodepletion agents, and after administration of the population
of cells expressing the CAR/TCR or the TIL in preparation for
transplantation of autologous or allogeneic stem cells and/or
administration of a second effective amount of the populations of
cells expressing the CAR/TCR or the TIL.
[0022] According to certain aspects of the present invention, the
population of cells expressing the CAR/TCR or the TIL may be
autologous cells, allogeneic cells derived from another human
patient, or xenogeneic cells derived from an animal of a different
species.
[0023] According to certain aspects of the present invention, the
population of cells expressing the CAR/TCR or the TIL may be
isolated by leukapheresis, transduced and selected approximately 4
weeks immediately prior to administration, as in the case of
autologous stem cells, or may be isolated from a healthy donor and
prepared in advance then stored, such as a frozen preparation, for
one or more patients as in the case of so called "off-the-shelf"
allogeneic CAR-T stem cell therapies.
[0024] According to certain aspects of the present invention, the
population of cells expressing the CAR/TCR may comprise a
population of activated T-cells or natural killer (NK) cells or
dendritic cells expressing the CAR/TCR which recognize an antigen.
Dendritic cells are capable of antigen presentation, as well as
direct killing of tumors. Further, according to certain aspects of
the invention, the population of cells may be a pluripotent stem
cell population that can be differentiated into a variety of
different blood cell types.
[0025] These methods may improve treatment outcomes for
hematological disorders and solid tumors, and/or may lessen side
effects associated with ACT, such as neurotoxicity, cytokine
release syndrome (CRS), hypogammaglobulinemia, cytopenias,
capillary leak syndrome (CLS), macrophage activation syndrome
(MAS), tumor lysis syndrome (TLS), and combinations thereof.
Additionally, the presently disclosed methods of combination
therapy may prolong persistence of the population of cells
expressing the CAR/TCR, the TIL, or the genetically modified stem
cells when compared to a method absent administration of the
radiolabeled anti-CD45 antibody.
[0026] According to certain aspects of the present invention, the
radiolabeled anti-CD45 antibody may comprise a monoclonal antibody
composition, wherein the composition may comprise a labeled
fraction and an unlabeled fraction. The radiolabeled anti-CD45
antibody may be provided as a single dose, wherein the single dose
of the radiolabeled anti-CD45 antibody may comprise unlabeled
anti-CD45 antibody in an amount of from 0.1:10 to 1:1
labeled:unlabeled.
[0027] According to certain aspects of the present invention, the
antigen may be hematopoietic in origin, such as an antigen present
on a hematological cell or a hematological tumor cell or may be an
antigen on a solid tumor cell.
[0028] According to certain aspects of the present invention, the
antigen may be one that is expressed only on cancer cells or one
that is preferentially expressed on cancer cells, such as a
neo-antigen.
[0029] According to certain aspects of the present invention, the
radiolabeled anti-CD45 antibody may be a radiolabeled BC8. The
radiolabel may be any of .sup.131I, .sup.125I, .sup.123I, .sup.90Y,
.sup.177Lu, .sup.186Re, .sup.188Re, .sup.89Sr, .sup.153Sm,
.sup.32P, .sup.225Ac, .sup.213Bi, .sup.213Po, .sup.211At,
.sup.212Bi, .sup.213Bi, .sup.223Ra, .sup.227Th, .sup.149Th,
.sup.137Cs, .sup.212Pb and .sup.103Pd.
[0030] According to certain aspects of the invention, the
radiolabeled BC8 is .sup.131I-BC8, and the effective amount of
.sup.131I-BC8 is from 10 mCi to 200 mCi, or wherein the effective
amount of .sup.131I-BC8 is less than 200 mCi. According to certain
aspects of the invention, the radiolabeled BC8 is .sup.225Ac-BC8,
and the effective amount of .sup.225Ac-BC8 is 0.1 .mu.Ci/kg of
subject weight to 5.0 .mu.Ci/kg of subject weight.
[0031] According to certain aspects of the invention, the
radiolabeled BC8 is administered 6, 7, or 8 days before
administration of the population of cells expressing the CAR/TCR.
The radiolabeled BC8 may be provided in an amount effective to
deplete at least 50% of lymphocytes of the subject, or at least 70%
of lymphocytes of the subject, or at least 80% of lymphocytes of
the subject.
[0032] According to certain aspects of the present invention, the
antigen may be selected from the group comprising CD19, CD20, CD22,
CD30, CD33, CD38, CD123, CD138, CS-1, B-cell maturation antigen
(BCMA), MAGEA3, MAGEA3/A6, KRAS, CLL1, MUC-1, HER2, EpCam, GD2,
GPA7, PSCA, EGFR, EGFRvIII, ROR1, mesothelin, CD33/IL3Ra, c-Met,
CD37, PSMA, Glycolipid F77, GD-2, gp100, NY-ESO-1 TCR, FRalpha,
CD24, CD44, CD133, CD166, CA-125, HE4, Oval, estrogen receptor,
progesterone receptor, uPA, PAI-1, MICA, MICB, ULBP1, ULBP2, ULBP3,
ULBP4, ULBP5 or ULBP6, or a combination thereof.
[0033] The various aspects of the present invention will be
realized and attained by means of the combinations specifically
outlined in the appended claims. The foregoing general description
and the following detailed description and examples of this
invention are provided to illustrate various aspects of the present
invention, and by no means are to be viewed as limiting any of the
described embodiments.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIG. 1A depicts a prior art adoptive cell therapy protocol
which includes leukapheresis, cytoreduction using standard
chemotherapeutic agents, CAR/TCR T-cell infusion, and possible
myeloconditioning in preparation for optional hematopoietic stem
cell transplantation (HSCT).
[0035] FIG. 1B depicts an autologous or allogeneic adoptive cell
therapy treatment of the present invention which includes
administration of an anti-CD45 antibody (e.g., Iomab-B) instead of,
or in addition to, the standard cytoreduction agents.
[0036] FIG. 1C depicts an autologous or allogeneic adoptive cell
therapy treatment of the present invention which includes
administration of an anti-CD45 antibody (e.g., Iomab-B) as a
myeloconditioning agent prior to HSCT.
[0037] FIG. 1D depicts an autologous or allogeneic adoptive cell
therapy treatment of the present invention which includes
administration of an anti-CD45 antibody (e.g., Iomab-B) instead of,
or in addition to, the standard cytoreduction agents, and
administration of an anti-CD45 antibody (e.g., Iomab-B) as a
myeloconditioning agent prior to HSCT.
[0038] FIG. 1E depicts pharmo-kinetic data demonstrating exemplary
clearance and dosing times for a lymphodepletion protocol according
to the presently disclosed invention.
[0039] FIG. 2 shows the median change in absolute neutrophil count
following dosing with .sup.131I-BC8.
[0040] FIGS. 3A-3E shows results of immune cell analysis following
.sup.131I-anti-CD45 antibody targeted lymphodepletion in a mouse
model using surrogate antibody 30F11.
[0041] FIGS. 4A-4D show results from immunophenotyping of
lymphocyte populations following .sup.131I-anti-CD45 antibody
targeted lymphodepletion in mice.
[0042] FIG. 5A shows depletion of splenic T-reg cells, FIG. 5B
shows depletion of myeloid derived suppressor cells (MDSC), and
FIG. 5C shows depletion of bone marrow HSC after targeted
lymphodepletion with .sup.131I-anti-CD45 antibody in mice.
[0043] FIG. 6 shows selected published trials of autologous
anti-CD19 CAR T-cell therapy for patients with B-cell non-Hodgkin's
lymphoma (NHL).
[0044] FIG. 7 shows a schematic of preclinical studies of the
effects in mice of low dose .sup.131I-anti-CD45 radioimmunotherapy
(surrogate 30F11) investigating the lymphodepletive response on
particular immune cell types. Controls include chemotherapeutic
lymphodepletive treatments, cyclophosphamide (Cy) or
cyclophosphamide/fludarabine (Flu/Cy), and no lymphodepletive
treatment.
[0045] FIG. 8 shows a preclinical model of adoptive T-cell transfer
following anti-CD45 radioimmunotherapy-mediated
conditioning/lymphodepletion in mice. In this model, E.G7 lymphoma
tumor-bearing mice will be conditioned by a single selected dose of
.sup.131I-anti-CD45 radioimmunotherapy prior to adoptive cell
transfer of OVA-specific CD8+ T-cells, and monitored for
engraftment of the transferred cells and resulting anti-tumor
response.
[0046] FIG. 9 shows clinical data from a low dose .sup.131I-BC8
study demonstrating lymphodepletion.
[0047] FIG. 10 shows pharmo-kinetic data demonstrating clearance
rate (<25 cGy) of .sup.131I-BC8.
[0048] FIG. 11 shows pharmo-kinetic data demonstrating cumulative
dose to spleen of .sup.131I-BC8 after administration of 100
mCi.
[0049] FIG. 12 shows blood clearance of .sup.131I-BC8.
DETAILED DESCRIPTION OF THE INVENTION
[0050] This invention provides radiolabeled anti-CD45
antibody-based methods for lymphodepleting a subject, and related
methods and articles of manufacture. When these methods precede
certain cell-based therapies, the methods are able to enhance the
outcome of the cell-based therapies while minimizing adverse
effects.
Definitions
[0051] In this application, certain terms are used which shall have
the meanings set forth as follows.
[0052] The singular forms "a," "an," "the" and the like include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "an" antibody includes both a
single antibody and a plurality of different antibodies.
[0053] The term "about" when used before a numerical designation,
e.g., temperature, time, amount, and concentration, including a
range, indicates approximations which may vary by .+-.10%, .+-.5%,
or .+-.1%.
[0054] As used herein, "administer", with respect to an antibody,
means to deliver the antibody to a subject's body via any known
method suitable for antibody delivery. Specific modes of
administration include, without limitation, intravenous,
transdermal, subcutaneous, intraperitoneal, intrathecal and
intra-tumoral administration. Exemplary administration methods for
antibodies may be as substantially described in International
Publication No. WO 2016/187514, incorporated by reference
herein.
[0055] In addition, in this invention, antibodies can be formulated
using one or more routinely used pharmaceutically acceptable
carriers. Such carriers are well known to those skilled in the art.
For example, injectable drug delivery systems include solutions,
suspensions, gels, microspheres and polymeric injectables, and can
comprise excipients such as solubility-altering agents (e.g.,
ethanol, propylene glycol and sucrose) and polymers (e.g.,
polycaprylactones and PLGA's).
[0056] As used herein, the term "antibody" includes, without
limitation, (a) an immunoglobulin molecule comprising two heavy
chains and two light chains and which recognizes an antigen; (b)
polyclonal and monoclonal immunoglobulin molecules; (c) monovalent
and divalent fragments thereof (e.g., di-Fab), and (d) bi-specific
forms thereof. Immunoglobulin molecules may derive from any of the
commonly known classes, including but not limited to IgA, secretory
IgA, IgG and IgM. IgG subclasses are also well known to those in
the art and include, but are not limited to, human IgG1, IgG2, IgG3
and IgG4. Antibodies can be both naturally occurring and
non-naturally occurring (e.g., IgG-Fc-silent). Furthermore,
antibodies include chimeric antibodies, wholly synthetic
antibodies, single chain antibodies, and fragments thereof.
Antibodies may be human, humanized or nonhuman.
[0057] As used herein, an "anti-CD45 antibody" is an antibody that
binds to any available epitope of CD45. According to certain
aspects, the anti-CD45 antibody binds to the epitope recognized by
the monoclonal antibody "BC8." BC8 is known, as are methods of
making it. Likewise, methods of labeling BC8 with .sup.131I or
.sup.225Ac are known. These methods are described, for example, in
International Publication No. WO 2017/155937. As used herein,
"cancer" includes, without limitation, a solid cancer (e.g., a
tumor) and a hematologic malignancy.
[0058] As used herein, "depleting", with respect to a subject's
lymphocytes, shall mean to lower the population of at least one
type of the subject's lymphocytes (e.g., at least one type of the
subject's peripheral blood lymphocytes or at least one type of the
subject's bone marrow lymphocytes). According to certain preferred
aspects of this invention, a subject's lymphocyte decrease is
determined by measuring the subject's peripheral blood lymphocyte
level. As such, and by way of example, a subject's lymphocyte
population is depleted if the population of at least one type of
the subject's peripheral blood lymphocytes is lowered by no more
than 99%. For example, a subject's lymphocytes are depleted if the
subject's peripheral blood T-cell level is lowered by 50%, the
subject's peripheral blood NK cell level is lowered by 40%, and/or
the subject's peripheral blood B cell level is lowered by 30%. In
this example, the subject's lymphocytes are depleted even if the
level of another immune cell type, such as neutrophils, is not
lowered. According to certain aspects, depleting a subject's
lymphocytes is reflected by a peripheral blood lymphocyte
population reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90% or 99%.
[0059] Methods for measuring peripheral blood lymphocyte
populations are routine. They include, for example, flow cytometry
on whole blood samples to determine lymphocyte counts based on
labeling with a fluorescent antibody directed against a specific a
cell surface marker such as CD45, CD4 or CD8. Methods for measuring
peripheral blood neutrophil populations are also routine. They
include, for example, flow cytometry on whole blood samples to
determine neutrophil counts based on labeling with a fluorescent
antibody directed against a specific a cell surface marker such as
Ly6G.
[0060] As used herein, an amount of a radiolabeled anti-CD45
antibody, when administered, is "effective" if the subject's
peripheral blood lymphocytes are depleted, such as by at least 50%,
or 60%, 70%, 80%, 90%, or 99%. An amount of radiolabeled anti-CD45
antibody, when administered, is "effective" if the subject's
peripheral blood lymphocytes are depleted without depletion of the
subject's neutrophils, or with less than 10% or 20% reduction in
the subject's neutrophils. An "effective" amount of radiolabeled
anti-CD45 antibody is an amount that will deplete the subject's
regulatory T cells, myeloid derived suppressor cells, tumor
associated macrophages, activated macrophages secreting IL-1 and/or
IL-6, and combinations thereof, such as by at least 20%, Or 30R,
40%, 50%, 60%, 70%, 80%, 90%, or 99%.
[0061] According to certain aspects, when the radiolabeled
anti-CD45 antibody is .sup.131I-BC8, the effective amount is below,
for example, 300 mCi (i.e., where the amount of .sup.131I-BC8
administered to the subject delivers a total body radiation dose of
below 300 mCi). According to certain aspects, when the antibody is
.sup.131I-BC8, the effective amount is below 250 mCi, below 200
mCi, below 150 mCi, below 100 mCi, below 50 mCi, below 40 mCi,
below 30 mCi, below 20 mCi or below 10 mCi. According to certain
aspects, when the antibody is .sup.131I-BC8, the effective amount
is from 1 mCi to 10 mCi, from 1 mCi to 200 mCi, from 10 mCi to 20
mCi, from 10 mCi to 30 mCi, from 10 mCi to 40 mCi, from 10 mCi to
50 mCi, from 10 mCi to 100 mCi, from 10 mCi to 150 mCi, from 10 mCi
to 200 mCi, from 20 mCi to 30 mCi, from 30 mCi to 40 mCi, from 40
mCi to 50 mCi, from 50 mCi to 100 mCi, from 50 mCi to 150 mCi, from
50 mCi to 200 mCi, from 60 mCi to 140 mCi, from 70 mCi to 130 mCi,
from 80 mCi to 120 mCi, from 90 mCi to 110 mCi, from 100 mCi to 150
mCi, from 150 mCi to 200 mCi, or from 200 mCi to 250 mCi. According
to certain aspects, when the antibody is .sup.131I-BC8, the
effective amount is from 10 mCi to 120 mCi, from 20 mCi to 110 mCi,
from 25 mCi to 100 mCi, from 30 mCi to 100 mCi, from 40 mCi to 100
mCi, or from 75 mCi to 100 mCi. According to certain aspects, when
the antibody is .sup.131I-BC8, the effective amount is 1 mCi, 10
mCi, 20 mCi, 30 mCi, 40 mCi, 50 mCi, 60 mCi, 70 mCi, 80 mCi, 90
mCi, 100 mCi, 110 mCi, 120 mCi, 130 mCi, 140 mCi, 150 mCi, or 200
mCi.
[0062] According to certain aspects, when the radiolabeled
anti-CD45 antibody is .sup.225Ac-BC8, the effective amount is
below, for example, 5.0 .mu.Ci/kg (i.e., where the amount of
.sup.225Ac-BC8 administered to the subject delivers a radiation
dose of below 5.0 .mu.Ci per kilogram of subject's body weight).
According to certain aspects, when the antibody is .sup.225Ac-BC8,
the effective amount is below 4.5 .mu.Ci/kg, 4.0 .mu.Ci/kg, 3.5
.mu.Ci/kg, 3.0 .mu.Ci/kg, 2.5 .mu.Ci/kg, 2.0 .mu.Ci/kg, 1.5
.mu.Ci/kg, 1.0 .mu.Ci/kg, 0.9 .mu.Ci/kg, 0.8 .mu.Ci/kg, 0.7
.mu.Ci/kg, 0.6 .mu.Ci/kg, 0.5 .mu.Ci/kg, 0.4 .mu.Ci/kg, 0.3
.mu.Ci/kg, 0.2 .mu.Ci/kg, 0.1 .mu.Ci/kg or 0.05 .mu.Ci/kg.
According to certain aspects, when the antibody is .sup.225Ac-BC8,
the effective amount is from 0.05 .mu.Ci/kg to 0.1 .mu.Ci/kg, from
0.1 .mu.Ci/kg to 0.2 .mu.Ci/kg, from 0.2 .mu.Ci/kg to 0.3
.mu.Ci/kg, from 0.3 .mu.Ci/kg to 0.4 .mu.Ci/kg, from 0.4 .mu.Ci/kg
to 0.5 .mu.Ci/kg, from 0.5 .mu.Ci/kg to 0.6 .mu.Ci/kg, from 0.6
.mu.Ci/kg to 0.7 .mu.Ci/kg, from 0.7 .mu.Ci/kg to 0.8 .mu.Ci/kg,
from 0.8 .mu.Ci/kg to 0.9 .mu.Ci/kg, from 0.9 .mu.Ci/kg to 1.0
.mu.Ci/kg, from 1.0 .mu.Ci/kg to 1.5 .mu.Ci/kg, from 1.5 .mu.Ci/kg
to 2.0 .mu.Ci/kg, from 2.0 .mu.Ci/kg to 2.5 .mu.Ci/kg, from 2.5
.mu.Ci/kg to 3.0 .mu.Ci/kg, from 3.0 .mu.Ci/kg to 3.5 .mu.Ci/kg,
from 3.5 .mu.Ci/kg to 4.0 .mu.Ci/kg, from 4.0 .mu.Ci/kg to 4.5
.mu.Ci/kg, or from 4.5 .mu.Ci/kg to 5.0 .mu.Ci/kg. According to
certain aspects, when the antibody is .sup.225Ac-BC8, the effective
amount is 0.05 .mu.Ci/kg, 0.1 .mu.Ci/kg, 0.2 .mu.Ci/kg, 0.3
.mu.Ci/kg, 0.4 .mu.Ci/kg, 0.5 .mu.Ci/kg, 0.6 .mu.Ci/kg, 0.7
.mu.Ci/kg, 0.8 .mu.Ci/kg, 0.9 .mu.Ci/kg, 1.0 .mu.Ci/kg, 1.5
.mu.Ci/kg, 2.0 .mu.Ci/kg, 2.5 .mu.Ci/kg, 3.0 .mu.Ci/kg, 3.5
.mu.Ci/kg, 4.0 .mu.Ci/kg or 4.5 .mu.Ci/kg.
[0063] For an antibody labeled with a radioisotope, the majority of
the drug administered to a subject typically consists of
non-labeled antibody, with the minority being the labeled antibody.
The ratio of labeled to non-labeled antibody can be adjusted using
known methods. Thus, accordingly to certain aspects of the present
invention, the anti-CD45 antibody may be provided in a total
protein amount of up to 60 mg, such as 5 mg to 45 mg, or a total
protein amount of between 0.2 mg/kg patient weight to 0.6 mg/kg
patient weight.
[0064] According to certain aspects of the present invention, the
radiolabeled anti-CD45 antibody may comprise a monoclonal antibody
composition, wherein the composition may comprise a labeled
fraction and an unlabeled fraction. The radiolabeled anti-CD45
antibody may be provided as a single dose, wherein the single dose
of the radiolabeled anti-CD45 antibody may comprise unlabeled
anti-CD45 antibody in an amount of from 0.1:10 to 1:1
labeled:unlabeled.
[0065] The adoptive cell therapy may include administration of
cells expressing a chimeric antigen receptor (CAR), or a T-cell
receptor (TCR), or may include tumor-infiltrating lymphocytes
(TIL). The population of cells expressing the CAR/TCR may comprise
a population of activated T-cells or natural killer (NK) cells or
dendritic cells expressing the CAR/TCR which recognize an antigen.
Dendritic cells are capable of antigen presentation, as well as
direct killing of tumors. The population of cells expressing the
CAR/TCR may comprise a population of gene-edited cells.
[0066] As used herein, the term "gene-edited" CAR T-cell is
synonymous with the terms "genetically engineered" CAR T-cell and
"engineered" CAR T-cell. A gene-edited CAR T-cell that "fails to
properly express" a checkpoint receptor (e.g., PD1, Lag3 or TIM3)
does not express the full-length, functional checkpoint receptor.
For example, a gene-edited CAR T-cell that fails to properly
express PD1 may fail to do so because, without limitation, (i) the
cell's PD1 gene has been ablated, or (ii) the cell's PD1 gene has
been otherwise altered so as not to yield a fully or even partially
functional PD1 product. In other words, according to certain
aspects, a gene-edited CAR T-cell that fails to properly express
PD1 may fail to do so because the cell's PD1 gene has been altered
to diminish PD1 expression. Similarly, a gene-edited CAR T-cell
that "fails to properly express" a T-cell receptor does not express
the full-length, functional T-cell receptor.
[0067] According to certain aspects, the functional endogenous
T-cell receptor is replaced through editing by a "knock-in" to the
native TCR locus of an exogenously transduced CAR or recombinant
TCR. The gene-edited CAR T-cells may include, without limitation,
the following: (i) allogenic gene-edited CAR T-cells that fail to
properly express PD1 but do properly express all other checkpoint
receptors and T-cell receptors; (ii) allogenic gene-edited CAR
T-cells that fail to properly express a particular T-cell receptor
but do properly express all checkpoint receptors and all other
T-cell receptors; and (iii) allogenic gene-edited CAR T-cells that
fail to properly express PD1 and fail to properly express a
particular T-cell receptor, but do properly express all other
checkpoint receptors and all other T-cell receptors.
[0068] Examples of T-cell gene editing to generate allogeneic,
universal CAR T-cells include the work of Eyquem and colleagues
(Eyquem, et. al., 2017, Nature. 543:113-117). In that study, the
endogenous T-cell receptor alpha constant locus (TRAC) was
effectively replaced by a recombinant CAR gene construct. By this
method, the recombinant CAR was placed effectively under the
control of the cell's native TCR regulatory signals. By this same
strategy, CARs or recombinant TCRs may be effectively inserted by
knock-in into the T-cell receptor beta constant gene locus (TRBC)
or into the beta-2 microglobulin (B2M) MHC-I-related gene locus,
known to be expressed in all T-cells. Another example includes the
work of Ren and colleagues (Ren, et. al., 2017, Clin. Cancer Res
23:2255-2266). Recognizing that checkpoint receptors are
immune-suppressive and may blunt the stimulation of exogenous
autologous or allogeneic CAR T-cells, this group exploited
CRISPR/cas9 technology to ablate the endogenous TCR .alpha. and
.beta. loci (TRAC and TRBC) and the B2M gene, while also silencing
the endogenous PD1 gene. With this approach, the engineered cells
did not elicit graft-versus-host disease, but did resist immune
checkpoint receptor suppression.
[0069] A "hematologic malignancy", also known as a blood cancer, is
a cancer that originates in blood-forming tissue, such as the bone
marrow or other cells of the immune system. Hematologic
malignancies include, without limitation, leukemias (such as acute
myeloid leukemia (AML), acute promyelocytic leukemia, acute
lymphoblastic leukemia (ALL), acute mixed lineage leukemia, chronic
myeloid leukemia, chronic lymphocytic leukemia (CLL), hairy cell
leukemia and large granular lymphocytic leukemia), myelodysplastic
syndrome (MDS), myeloproliferative disorders (polycythemia vera,
essential thrombocytosis, primary myelofibrosis and chronic myeloid
leukemia), lymphomas, multiple myeloma, MGUS and similar disorders,
Hodgkin's lymphoma, non-Hodgkin lymphoma (NHL), primary mediastinal
large B-cell lymphoma, diffuse large B-cell lymphoma, follicular
lymphoma, transformed follicular lymphoma, splenic marginal zone
lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell
malignancies.
[0070] As used herein, a subject's "peripheral blood lymphocytes"
shall mean the mature lymphocytes circulating in the subject's
blood. Examples of peripheral blood lymphocytes include, without
limitation, peripheral blood T-cells, peripheral blood NK cells and
peripheral blood B cells. A subject's peripheral blood lymphocyte
population is readily measurable. Thus, by measuring a decrease in
the level of at least one type of peripheral blood lymphocyte
following a depleting event (e.g., the administration of a low
.sup.131I-BC8 dose), one can easily determine that lymphodepletion
has occurred in a subject.
[0071] "Solid cancers" include, without limitation, bone cancer,
pancreatic cancer, skin cancer, cancer of the head or neck,
cutaneous or intraocular malignant melanoma, uterine cancer,
ovarian cancer, prostate cancer, rectal cancer, cancer of the anal
region, stomach cancer, testicular cancer, uterine cancer,
carcinoma of the fallopian tubes, carcinoma of the endometrium,
carcinoma of the cervix, carcinoma of the vagina, carcinoma of the
vulva, cancer of the esophagus, cancer of the small intestine,
cancer of the endocrine system, cancer of the thyroid gland, cancer
of the parathyroid gland, cancer of the adrenal gland, sarcoma of
soft tissue, cancer of the urethra, cancer of the penis, pediatric
tumors, cancer of the bladder, cancer of the kidney or ureter,
carcinoma of the renal pelvis, neoplasm of the central nervous
system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis
tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,
epidermoid cancer, squamous cell cancer, environmentally-induced
cancers including those induced by asbestos.
[0072] As used herein, the term "subject" includes, without
limitation, a mammal such as a human, a non-human primate, a dog, a
cat, a horse, a sheep, a goat, a cow, a rabbit, a pig, a rat and a
mouse. Where the subject is human, the subject can be of any age.
For example, the subject can be 60 years or older, 65 or older, 70
or older, 75 or older, 80 or older, 85 or older, or 90 or older.
Alternatively, the subject can be 50 years or younger, 45 or
younger, 40 or younger, 35 or younger, 30 or younger, 25 or
younger, or 20 or younger. For a human subject afflicted with
cancer, the subject can be newly diagnosed, or relapsed and/or
refractory, or in remission.
[0073] As used herein, a "suitable time period" after administering
a radiolabeled anti-CD45 antibody to a subject and before
performing adoptive cell therapy on the subject is a time period
sufficient to permit the administered antibody to deplete the
subject's lymphocytes and/or for the subject's lymphocytes to
remain depleted. According to certain aspects, the suitable time
period is fewer than 10 days, fewer than 9 days, fewer than 8 days,
fewer than 7 days, fewer than 6 days, fewer than 5 days, fewer than
4 days, or fewer than 3 days. According to certain aspects, the
suitable time period is 1 day, 2 days, 3 days, 4 days, 5 days, 6
days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days,
14 days, 15 days, or greater than 15 days.
[0074] As used herein, a "radioisotope" can be an alpha-emitting
isotope, a beta-emitting isotope, and/or a gamma-emitting isotope.
Examples of radioisotopes include the following: .sup.131I,
.sup.125I, .sup.123I, .sup.90Y, .sup.177Lu, .sup.186Re, .sup.188Re,
.sup.89Sr .sup.153Sm, .sup.32P, .sup.225Ac, .sup.213Bi .sup.213Po,
.sup.211At, .sup.212Bi, .sup.213Bi, .sup.223Ra, .sup.227Th,
.sup.149Tb, .sup.137Cs, .sup.212Pb an .sup.103Pd. Methods for
affixing a radioisotope to an antibody (i.e., "labeling" an
antibody with a radioisotope) are well known.
[0075] As used herein, "treating" a subject afflicted with a cancer
shall include, without limitation, (i) slowing, stopping or
reversing the cancer's progression, (ii) slowing, stopping or
reversing the progression of the cancer's symptoms, (iii) reducing
the likelihood of the cancer's recurrence, and/or (iv) reducing the
likelihood that the cancer's symptoms will recur. According to
certain preferred aspects, treating a subject afflicted with a
cancer means (i) reversing the cancer's progression, ideally to the
point of eliminating the cancer, and/or (ii) reversing the
progression of the cancer's symptoms, ideally to the point of
eliminating the symptoms, and/or (iii) reducing or eliminating the
likelihood of relapse (i.e., consolidation, which ideally results
in the destruction of any remaining cancer cells).
[0076] Throughout this application, various publications are cited.
The disclosure of these publications is hereby incorporated by
reference into this application to describe more fully the state of
the art to which this invention pertains. Unless otherwise defined,
all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art
to which the present invention belongs. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing described herein, suitable methods
and materials are described below.
ASPECTS OF THE INVENTION
[0077] Lymphodepletion in Combination with Adoptive Cell
Therapy
[0078] The present invention solves an unmet need in the art by
providing an unexpectedly superior way to lymphodeplete a subject
prior to a cell-based therapy like CAR T-cell therapy or TCR cell
therapy. This invention employs a radiolabeled anti-CD45 antibody
such as .sup.131I-BC8 to lymphodeplete the subject. The antibody
can lymphodeplete the subject at surprisingly low doses. This
approach avoids certain adverse effects caused by less specific
agents like chemotherapeutics. Also, using this approach, at least
some types of CD45+ immune cells, such as neutrophils, surprisingly
avoid significant depletion.
[0079] This lymphodepletion method is useful, for example, for
improving the outcome of a subsequent therapy wherein the depletion
of lymphocytes is desirable. According to certain preferred aspects
of this method, the subject is afflicted with cancer and is about
to undergo adoptive cell therapy to treat the cancer (e.g.,
hematological malignancy or solid cancer). Adoptive cell therapies
are known, and include, for example, CAR T-cell therapy (e.g.,
autologous cell therapy and allogeneic cell therapy). Preferred are
CAR T-cell therapies for treating hematologic malignancies such as
ALL, AML and CLL. Examples of approved CAR T-cell therapies
include, without limitation, KYMRIAH.RTM. (tisagenlecleucel) for
treating NHL and DLBCL, and YESCARTA.RTM. (axicabtagene ciloleucel)
for treating NHL.
[0080] These presently disclosed methods may improve treatment
outcomes for hematological malignancies including solid tumors,
and/or may lessen side effects associated with the adoptive cell
therapies, such as the CAR T-cell therapies KYMRIAH.RTM. and/or
YESCARTA.RTM.. For example, side effects of adoptive cell therapies
include neurotoxicity, cytokine release syndrome (CRS),
hypogammaglobulinemia, cytopenias, capillary leak syndrome (CLS),
macrophage activation syndrome (MAS), tumor lysis syndrome (TLS),
and combinations thereof. Moreover, the presently disclosed methods
may prolong persistence of the population of cells expressing the
CAR/TCR or the TIL when compared to a method absent administration
of the radiolabeled anti-CD45 antibody.
[0081] According to certain aspects of the method, the subject is
afflicted with cancer (e.g., hematological malignancy or solid
cancer) and is about to undergo adoptive cell therapy to treat the
cancer. Adoptive cell therapy is known, and includes, for example,
CAR T-cell therapy (e.g., autologous cell therapy and allogeneic
cell therapy). Adoptive cell therapies provide a method of
promoting regression of a cancer in a subject, and generally
comprise (i) collecting autologous T-cells (leukapheresis); (ii)
expanding the T-cells (culturing); (iii) administering to the
subject non-myeloablative lymphodepleting chemotherapy; and (iv)
after administering non-myeloablative lymphodepleting chemotherapy,
administering to the subject the expanded T-cells (see FIG. 1A).
The methods of the presently disclosed invention include using a
radiolabeled anti-CD45 antibody in lieu of the lymphodepleting
chemotherapy (FIG. 1B), after administration of the expanded cells
(e.g., T-cell, NK-cells, dendritic cells, etc.) (FIG. 1C), or both
before administration of the expanded cells to lymphodeplete and
after administration of the expanded cells (FIG. 1D). The later
administration of the anti-CD45 antibody (i.e., after
administration of the expanded cells) may be used in preparation
for transplantation of autologous stem cells (HSCT), or
administration of a second effective amount of expanded cells.
[0082] Accordingly, the present invention provides methods for the
treatment of a proliferative disease, such as a hematological
malignancy or a solid cancer, which include administration of a
radiolabeled anti-CD45 antibody and an adoptive cell therapy. The
adoptive cell therapy may generally include apheresis of autologous
cells which may be gene edited prior to reinfusion (adoptive cell
therapy such as CAR T-cell therapy) after lymphodepletion by the
radiolabeled anti-CD45 antibody. Alternatively, allogeneic cells
may be reinfused after lymphodepletion to provide the adoptive cell
therapy. According to methods of the present invention, the
radiolabeled anti-CD45 antibody may be provided as a single dose 3
to 9 days, such as 6 to 8 days, prior to the adoptive cell therapy,
as shown in FIG. 1E.
[0083] Thus, the present invention provides a method for treating a
subject afflicted with cancer comprising (i) administering to the
subject an amount of a radiolabeled anti-CD45 antibody effective to
deplete the subject's lymphocytes, and (ii) after a suitable time
period, performing adoptive cell therapy on the subject to treat
the subject's cancer. Preferably, the subject is human.
[0084] Targeted Lymphodepletion to Deplete Suppressor Cells
[0085] The present invention further provides methods for targeted
lymphodepletion of immune suppressor cells such as regulatory T
(T-reg) cells and myeloid-derived suppressor cells (MDSC). Both
cells types (i.e., T-regs and MDSC) can dampen the activation and
efficacy of CAR T-cell therapies. Moreover, the present invention
also provides methods for targeted lymphodepletion of immune
suppressor cells such as monocytes and tumor-associated macrophages
(TAMs) that have been implicated in cytokine release contributing
to toxicities such as cytokine release syndrome (CRS) and
neurotoxicity associated with CAR T-cells.
[0086] Tumors, both solid and liquid have evolved methods to hijack
and/or evade the immune system as a means to perpetuate and thrive.
This has been called the hostile tumor immune microenvironment
(TME). A classical and relevant example is the up-regulated
expression of the ligand PD-L1 on the tumor cell surface to bind
PD1 on the surface of T cells, leading to down-modulation of immune
cell activation. Interestingly, although blockade of this mechanism
has led to remarkable response rates and durable survival in
several different types of cancer, most patients do not respond to
this form of therapy (i.e., anti-PD1/PD-L1), implying that immune
evasion in the tumor microenvironment is multi-faceted and complex.
To this end, the tumor, in part through oncogenic expression,
signaling, and cytokine production, can confer challenges on the
immune system, hindering the mounting of an effective anti-tumor
response. This can lead to an environment characterized by
oxidative stress, nutrient depletion, an acidic pH, and hypoxia.
Further, the presence of these suppressive immune cells (T-regs and
MDSC), and tumor-associated macrophages (TAM) can effectively blunt
immune cell activation through direct contact or release of
suppressive soluble factors and cytokines.
[0087] While a patient's endogenous immune system may encounter
such an environment and lead to a compromised anti-tumor immune
response, adoptive cell therapies such as CAR T-cell therapy may
also be susceptible to these immune suppressive mechanisms,
restricting the ability of these novel cell therapies to mount an
effective response to the tumor.
[0088] CAR T therapies, and adoptive cell therapy (ACT) in general,
represents one of the most promising anti-cancer strategies
emerging from clinical research. Response rates have been
extraordinary, on the range of 80% across these tumors, although
durable responses have only ranged around 40-50% (see, for example,
studies listed in FIG. 6). Nevertheless, these results represent a
significant improvement in outcomes for these patients. It is
unclear why some patients respond, and others do not, though the
tumor immune microenvironment is a likely contributor to modulate
the response to cell therapy.
[0089] To this end, preclinical and clinical studies have shown
that regulatory T cells (T-regs) have an impact on the response to
ACT in mice and in patients suffering from melanoma (Gattinoni, et
al., 2005, JEM, 202:907; Yao, et al., 2012, Blood, 119:5688). In
these studies, depletion of T-regs, whether by intentional
depletion or via conditioning with external beam radiation, had a
favorable impact on the anti-tumor response to ACT. Interestingly,
these and other studies suggest that T-reg depletion is more
sustained following treatment with radiation as opposed to
chemotherapy-induced conditioning, where a rapid rebound of T-regs
was seen with the latter chemotherapy conditioning and poorer
outcomes.
[0090] MDSCs and TAMs are other cell types implicated in creating a
poor tumor immune microenvironment. Through upregulation of
metabolic gene expression, such as Indoleamine 2,3-dioxygenase
(IDO), adenosine A2A receptor, and CD73, tumors can effectively
create a nutrient deprivation in the tumor environment which can
blunt T-cell activation. For example, tryptophan metabolism by IDO
from tumors and MDSCs leads to T-cell anergy and death, as well as
T-reg accumulation at the tumor site. Further, these immune
suppressive cells may secrete immune modulatory cytokines such as
TGF-.beta. which can also exert a negative effect on T-cell
activation.
[0091] The negative impact of the hostile tumor immune
microenvironment may exist for both liquid and solid tumors, though
may be even more pronounced in solid tumors. To this end, early
clinical results suggest that the robust response to CAR-T therapy
in liquid tumors such as lymphoma, has not been observed in solid
tumors, suggesting that factors or conditions exist in solid tumors
that may present physical or metabolic barriers to mounting an
effective CAR-T-mediated immune response (Newick, et al., 2016,
Mol. Ther. --Oncolytics, 3:16006; D'Aloia, et al., 2018, Cell Death
and Disease. 9:282-293).
[0092] The tumor immune microenvironment has also been implicated
in the two primary adverse events associated with CAR-T
administration, namely cytokine release syndrome (CRS) and
neurotoxicity. Recent preclinical studies have shown that cytokine
release leading to CRS or neurotoxicity is due to activated
macrophages following recruitment to the site of CAR-T and tumor
cells. Mouse study result. (Giavadris, et al., 2018, Nat. Med.,
24:731) documented that macrophages secrete IL-1 or IL-6 following
recruitment and activation by CAR-T cells at the tumor site.
[0093] Conditioning has been shown to improve the immune
homeostatic environment to enable successful ACT or CAR-T
engraftment and expansion in vivo following infusion. However, the
use of cytotoxic non-specific chemotherapy can elicit off-target
toxicities and has been identified as a risk factor in CRS and
neurotoxicity following CAR-T administration (Hay, et al., 2016).
Interestingly, most CAR-T programs exploit the use of the
combination of fludarabine and cyclophosphamide (flu/cy) as a
conditioning regimen prior to CAR-T. These drugs are often
administered 2-7 days prior to ACT infusion, using 2-5 day course
of therapy.
[0094] The targeted therapy for conditioning of the present
invention offers a much improved strategy for enhancing outcomes
with CAR-T. In the invention described herein, not only may
lymphocytes be targeted for depletion, but also those immune cell
types implicated in mediating a hostile tumor immune
microenvironment, and those implicated in CAR-T adverse events such
as CRS and neurotoxicity. The present invention targets normal
immune cells including T-regs, MDSCs, TAMs, and activated
macrophages secreting IL-1 and/or IL-6. In doing so, the invention
may have a dramatic improvement in CAR-T outcomes and safety.
[0095] Furthermore, the invention will target, primarily in
hematopoietic tumors, patient cancer cells to reduce tumor burden
and increase the probability of CAR-T anti-tumor response. More
specifically, the invention provides a therapeutic strategy
targeting the CD45 antigen, which is found on all normal nucleated
immune cells with the exception of red blood cells and platelets.
CD45 is also expressed on most lymphoid and leukemic tumor cells.
While naked antibodies have shown some impact on reducing immune
cell populations, the radiolabeled anti-CD45 antibody of the
present invention will effect a more pronounced and sustained
suppression of immune cells implicated in modulating CAR-T
responses, consistent with, but in a targeted manner, to external
beam radiation. In this way, the radiation is targeted and
impactful on the CD45 cell populations while sparing normal
tissues. More specifically, the radiolabeled anti-CD45 antibody may
be provided as a single dose at a level sufficiently effective to
deplete circulating immune cells within the spleen, lymph nodes,
and peripheral blood, but limited in impact on hematopoietic stem
cells in the bone marrow. Importantly, in addition to lymphocyte
depletion, macrophages, MDSCs and T-regs will be depleted to
improve the activation and response to CAR-T therapy and mitigate
adverse events CRS and neurotoxicity.
[0096] The Radiolabeled Anti-CD45 Antibody
[0097] The CD45 antigen is a 180-240 kD trans-membrane glycoprotein
which is a member of the protein tyrosine phosphatase family. CD45
plays a key role in T-cell and B-cell receptor signal transduction.
Different isoforms of CD45 exist due to variable splicing of 3 of
its 34 exons, and these isoforms are very specific to the
activation and maturation state of the cell as well as the cell
type. These various isoforms have the same trans-membrane and
cytoplasmic segments, but different extra-cellular domains, and are
differentially expressed on subpopulations of B- and T-cell
lymphocytes. The primary ligands described for CD45 include
galectin-1, CD1, CD2, CD3, CD4, TCR, CD22 and Thy-1.
[0098] Depending on which of the alternatively spliced exons (A, B
or C) is recognized, antibodies restricted to recognizing one or
the other isoform have been identified (termed CD45RA, CD45RB,
CD45RC, CD45RAB, etc.). In addition, monoclonal antibodies (mAbs)
which bind an epitope common to all the different isoforms have
also been identified (CD45RABC), as well as mAbs which selectively
bind to the 180 kD isoform without any of the variable exons A, B
or C (CD45RO). This latter mAb is restricted to a subset of
cortical thymocytes, activated T cells and memory cells, and is
absent on B cells.
[0099] In general, all cells of hematopoietic origin, with the
exception of mature erythrocytes and platelets, express CD45. High
expression of CD45 is seen with most acute lymphoid and myeloid
leukemias. Since CD45 is not found on tissues of non-hematopoietic
origin, its specific expression in leukemia has made it a good
target for developing therapeutics, including
radio-immunotherapeutics. For example, CD45 is expressed at a
density of approximately 200,000 to 300,000 sites per cell on
circulating leukocytes and malignant B cells.
[0100] Among several clones of the anti-CD45 murine antibody, BC8
recognizes all the human isoforms of the CD45 antigen, and thus
provides an excellent target for the development of therapeutics
for human malignancies of hematopoietic origin, including leukemias
and lymphomas. CAR T-cell therapies have also found success in the
treatment of cancers of hematopoietic origin, such as leukemias and
lymphomas.
[0101] The anti-CD45 antibody of the presently disclosed methods
may be any known in the art. According to certain aspects of the
present invention, the anti-CD45 antibody may comprise a BC8
monoclonal antibody, such as substantially detailed in U.S. patent
application Ser. No. 15/603,817, incorporated by reference herein.
An exemplary composition comprising the BC8 monoclonal antibody
includes those compositions as detailed in WO 2017/155937.
[0102] The anti-CD45 antibody may be administered intravenously,
intramuscularly, or subcutaneously to a patient. Exemplary
administration amounts and rates for the compositions may be as
substantially described in WO 2016/187514, incorporated by
reference herein.
[0103] Doses considered effective in safe depletion of circulating
immune cells would be doses that deliver 2 Gy or less to the bone
marrow, thereby reducing the negative impact of targeting CD45 on
hematopoietic stem cells. Such doses should deplete lymphocytes,
immune cells implicated in the hostile immune tumor
microenvironment, and tumor cells all leading to enhanced response
to ACT or CAR-T therapy. As shown in Table 1, calculations from
dosimetry performed in patients receiving .sup.131I-BC8 indicate
that doses below 100 mCi will result in the delivery of a targeted
radiation dose to bone marrow, the dose limiting organ, in the
range of 200 cGy (2 Gy). Such doses are also found to deliver a
higher amount of radiation to the spleen, the site of lymphocytes
and myeloid cells for targeted lymphodepletion (see Table 2 and
FIGS. 3A-3F and 4A-4D).
[0104] According to certain aspects of this method, the
radiolabeled anti-CD45 antibody is radiolabeled BC8. Radiolabeled
antibodies envisioned in this invention include, without
limitation, .sup.131I-BC8, .sup.125I-BC8, .sup.123I-BC8,
.sup.90Y-BC8, .sup.177Lu-BC8, .sup.186Re-BC8, .sup.188Re-BC8,
.sup.89Sr-BC8, .sup.153Sm-BC8, .sup.32P-BC8, .sup.225Ac-BC8,
.sup.213Bi-BC8, .sup.213Po-BC8, .sup.211At-BC8, .sup.212Bi-BC8,
.sup.213Bi-BC8, .sup.223Ra-BC8, .sup.227Th-BC8, .sup.149Tb-BC8,
.sup.131I-BC8, .sup.137Cs-BC8, .sup.212Pb-BC8 and .sup.103Pd-BC8.
Preferably, the radiolabeled BC8 is .sup.131I-BC8 or
.sup.225Ac-BC8.
[0105] According to certain aspects of this method, the effective
amount of .sup.131I-BC8 is from 10 mCi to 200 mCi. Examples of
effective amounts include, without limitation, from 50 mCi to 100
mCi, from 50 mCi to 150 mCi, from 50 mCi to 200 mCi, from 60 mCi to
140 mCi, from 70 mCi to 130 mCi, from 80 mCi to 120 mCi, from 90
mCi to 110 mCi, from 100 mCi to 150 mCi, 50 mCi, 60 mCi, 70 mCi, 80
mCi, 90 mCi, 100 mCi, 110 mCi, 120 mCi, 130 mCi, 140 mCi, 150 mCi,
or 200 mCi. According to certain aspects, when the antibody is
.sup.131I-BC8, the effective amount is from 10 mCi to 120 mCi, from
20 mCi to 110 mCi, from 25 mCi to 100 mCi, from 30 mCi to 100 mCi,
from 40 mCi to 100 mCi, from 50 mCi to 100 mCi, or from 75 mCi to
100 mCi. These low lymphodepletive doses of .sup.n1I-BC8 are
surprising over the known myeloablative doses of .sup.131I-BC8,
such as 300 mCi to 1,200 mCi. For example, it was unexpected that
these lower doses would yield a drop in lymphocyte levels.
Moreover, these lower doses permit the patient to go home
immediately after the .sup.131I-BC8 is administered. This would not
be possible for a patient receiving, say, a 1,200 mCi dose due to
the radiation risk posed to others in close physical proximity to
the patient.
[0106] According to certain aspects of this method, the effective
amount of .sup.225Ac-BC8, is from 0.05 .mu.Ci/kg to 5.0 .mu.Ci/kg
of subject's body weight. Examples of effective amounts include,
without limitation, from 0.05 .mu.Ci/kg to 5.0 .mu.Ci/kg, such as
from 0.1 .mu.Ci/kg to 0.2 .mu.Ci/kg, from 0.2 .mu.Ci/kg to 0.3
.mu.Ci/kg, from 0.3 .mu.Ci/kg to 0.4 .mu.Ci/kg, from 0.4 .mu.Ci/kg
to 0.5 .mu.Ci/kg, from 0.5 .mu.Ci/kg to 0.6 .mu.Ci/kg, from 0.6
.mu.Ci/kg to 0.7 .mu.Ci/kg, from 0.7 .mu.Ci/kg to 0.8 .mu.Ci/kg,
from 0.8 .mu.Ci/kg to 0.9 .mu.Ci/kg, from 0.9 .mu.Ci/kg to 1.0
.mu.Ci/kg, from 1.0 .mu.Ci/kg to 1.5 .mu.Ci/kg, from 1.5 .mu.Ci/kg
to 2.0 .mu.Ci/kg, from 2.0 .mu.Ci/kg to 2.5 .mu.Ci/kg, from 2.5
.mu.Ci/kg to 3.0 .mu.Ci/kg, from 3.0 .mu.Ci/kg to 3.5 .mu.Ci/kg,
from 3.5 .mu.Ci/kg to 4.0 .mu.Ci/kg, from 4.0 .mu.Ci/kg to 4.5
.mu.Ci/kg, or from 4.5 .mu.Ci/kg to 5.0 .mu.Ci/kg.
[0107] The effective amount of the radiolabeled anti-CD45 antibody
may be provided as a single dose. A majority of the anti-CD45
antibody administered to a subject typically consists of
non-labeled antibody, with the minority being the labeled antibody.
The ratio of labeled to non-labeled antibody can be adjusted using
known methods. Thus, accordingly to certain aspects of the present
invention, the anti-CD45 antibody may be provided in a total
protein amount of up to 100 mg, such as less than 60 mg, or from 5
mg to 45 mg, or a total protein amount of between 0.1 mg/kg patient
weight to 1.0 mg/kg patient weight, such as from 0.2 mg/kg patient
weight to 0.6 mg/kg patient weight.
[0108] According to certain aspects of the present invention, the
radiolabeled anti-CD45 antibody may comprise a labeled fraction and
an unlabeled fraction, wherein the ratio of labeled:unlabeled may
be from about 0.01:10 to 1:1, such as 0.1:10 to 1:1
labeled:unlabeled. Moreover, the radiolabeled anti-CD45 antibody
may be provided as a single dose composition tailored to a specific
patient, wherein the amount of labeled and unlabeled anti-CD45
antibody in the composition may depend on at least a patient
weight, age, and/or disease state or health status.
[0109] According to certain aspects, the suitable time period after
administering the radiolabeled anti-CD45 antibody that the ACT may
be performed is 3 days, 4 days, 5 days, 6 days, 7 days, 8 days or 9
days, such as preferably 6, 7 or 8 days.
[0110] According to certain aspects, the method for treating a
subject afflicted with cancer consists of (i) administering to the
subject a single dose of .sup.131I-BC8 effective to deplete the
subject's lymphocytes, and (ii) after a suitable time period (e.g.,
6, 7 or 8 days), performing adoptive cell therapy on the subject to
treat the subject's cancer. According to certain aspects, the
method for treating a subject afflicted with cancer consists of (i)
administering to the subject a single dose of .sup.22SAc-BC8
effective to deplete the subject's lymphocytes, and (ii) after a
suitable time period (e.g., 6, 7 or 8 days), performing adoptive
cell therapy on the subject to treat the subject's cancer.
[0111] Adoptive Cell Therapy
[0112] Adoptive cell therapies (ACT) are a potent approach for
treating cancer but also for treating other diseases such as
infections and graft versus host disease. ACT is the passive
transfer of ex vivo grown cells, most commonly immune-derived
cells, into a host with the goal of transferring the immunologic
functionality and characteristics of the transplant.
[0113] ACT can be autologous (e.g., isolated by leukapheresis,
transduced and selected approximately 4 weeks immediately prior to
administration), as is common in adoptive T-cell therapies, or
allogeneic as typical for treatment of infections or
graft-versus-host disease. Moreover, the ACT may be xenogeneic.
[0114] ACT may also comprise transfer of autologous tumor
infiltrating lymphocytes (TILs) which may be used to treat patients
with advanced solid tumors such as melanoma and hematologic
malignancies.
[0115] ACT may also comprise transfer of allogeneic lymphocytes
isolated, prepared, and stored (e.g., frozen) "off-the-shelf" from
a healthy donor which may be used to treat patients with advanced
solid tumors such as melanoma and hematologic malignancies.
[0116] The ACT may use cell types such as T-cells, natural killer
(NK) cells, delta-gamma T-cells, regulatory T-cells, dendritic
cells, and peripheral blood mononuclear cells. The ACT may use
monocytes with the purpose of inducing differentiation to dendritic
cells subsequent to contact with tumor antigens. Given that
monocytes have a fixed mitotic index, permanent manipulation of the
host may be diminished.
[0117] According to certain aspects, the adoptive cell therapy may
be a CAR T-cell therapy. The CAR T-cell can be engineered to target
a tumor antigen of interest by way of engineering a desired antigen
binding domain that specifically binds to an antigen on a tumor
cell. In the context of the present invention, "tumor antigen" or
"proliferative disorder antigen" or "antigen associated with a
proliferative disorder," refers to antigens that are common to
specific proliferative disorders such as cancer. The antigens
discussed herein are merely included by way of example and are not
intended to be exclusive, and further examples will be readily
apparent to those of skill in the art.
[0118] According to certain aspects, the CAR T-cell therapy employs
CAR T-cells that target CD19, CD20, CD22, CD30, CD33, CD38, CD123,
CD138, CS-1, B-cell maturation antigen (BCMA), MAGEA3, MAGEA3/A6,
KRAS, CLL1, MUC-1, HER2, EpCam, GD2, GPA7, PSCA, EGFR, EGFRvIII,
ROR1, mesothelin, CD33/IL3Ra, c-Met, CD37, PSMA, Glycolipid F77,
GD-2, gp100, NY-ESO-1 TCR, FRalpha, CD24, CD44, CD133, CD166,
CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA,
PAI-1, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 or ULBP6, or a
combination thereof (e.g., both CD33 and CD123). It is envisioned
in this invention that, according to certain aspects, the subject
afflicted with cancer is a patient with a higher burden of disease
(.gtoreq.5% bone marrow blasts) with a greater incidence of adverse
events such as cytokine release syndrome and shorter long-term
survival after CAR T.
[0119] The CAR T-cell may comprise an antigen binding domain
capable of targeting two or more different antigens (i.e.,
bispecific or bivalent, trispecific or trivalent, tetraspecific,
etc.). As such, the CAR T-cell may comprise a first antigen binding
domain that binds to a first antigen and a second antigen binding
domain that binds to a second antigen (e.g., tandem CAR). For
example, the CAR T-cell may comprise a CD19 binding domain and a
CD22 binding domain and may thus recognize and bind to both CD19
and CD22. Or further, the CAR T-cell may comprise a CD19 binding
domain and a CD20 binding domain and may thus recognize and bind to
both CD19 and CD20.
[0120] Alternatively, each cell in the population of cells, or the
overall population of cells, may comprise more than one distinct
CAR T-cell (e.g., construct), wherein each CAR T-cell construct may
recognize a different antigen. For example, the population of CAR
T-cells may target three antigens such as, for example, HER2,
IL13R.alpha.2, and EphA2.
[0121] According to certain aspects of the present invention, the
population of cells, whether autologous or allogeneic, may be
engineered using gene editing technology such as by CRISPR/cas9
(clustered regularly interspaced short palindromic repeats/CRISPR
associated protein 9), Zinc Finger Nucleases (ZFN), or
transcription activator-like effector nuclease (TALEN). These
technologies, recognized and practiced in the art of genetic
engineering, enable selective editing, disruption, or insertion of
targeted sequences to modify the genome of the cell of interest.
Accordingly, isolated autologous or allogeneic cells for adoptive
transfer practiced in the current invention may be edited to delete
or replace a known gene or sequence. For example, the T cell
receptor (TCR) in an allogeneic T cell population may be deleted or
replaced prior to or after CAR-T transduction as a means to
eliminate graft-versus-host disease in recipient patients.
[0122] According to certain aspects of the present invention, the
population of cells may comprise a population of T-cells, NK-cells,
or dendritic cells expressing a CAR, wherein the CAR comprises an
extracellular antibody or antibody fragment that includes a
humanized anti-CD19 binding domain or a humanized anti-BCMA binding
domain, a transmembrane domain, and one or more cytoplasmic
co-stimulatory signaling domains. The population of cells may
comprise a population of cells expressing a CAR, wherein the CAR
comprises an extracellular antibody or antibody fragment that
includes two or more binding domains, such as a humanized anti-CD19
binding domain, a humanized anti-CD22 binding domain, and/or a
humanized anti-BCMA binding domain, and a transmembrane domain and
one or more cytoplasmic co-stimulatory signaling domains.
[0123] CAR cell therapy has shown unprecedented initial response
rates in advanced B-cell malignancies; however, relapse after CAR
cell infusion, and limited therapeutic success in solid tumors is a
major hurdle in successful CAR regimens. This latter limitation is
mainly attributable to the hostile microenvironment of a solid
tumor. Anatomical barriers such as the tumor stroma, and
immunosuppressive cytokines and immune cells which are harmful to
the infiltration of infused CAR modified cells into tumor sites,
both limit the success of CAR cell therapy. Armored CAR may be used
to circumvent certain of these limitations. These CAR cells are
further modified to express immune-modulatory proteins, such as
cytokines (e.g., IL-2, IL-12 or IL-15), which may stimulate T-cell
activation and recruitment, and may thus aid in combating the tumor
microenvironment. Thus, according to certain aspects of the present
invention, the population of cells may comprise a population of
cells expressing a CAR and further expressing an immune modulatory
protein such as, for example, IL-2, IL-12, or IL-15.
[0124] An ACT of the present invention includes a population of
cells expressing T-cell receptors (TCRs). TCRs are antigen-specific
molecules that are responsible for recognizing antigenic peptides
presented in the context of a product of the major
histocompatibility complex (MHC) on the surface of antigen
presenting cells or any nucleated cell (e.g., all human cells in
the body, except red blood cells). In contrast, antibodies
typically recognize soluble or cell-surface antigens, and do not
require presentation of the antigen by an MHC. This system endows
T-cells, via their TCRs, with the potential ability to recognize
the entire array of intracellular antigens expressed by a cell
(including virus proteins) that are processed intracellularly into
short peptides, bound to an intracellular MHC molecule, and
delivered to the surface as a peptide-MHC complex. This system
allows virtually any foreign protein (e.g., mutated cancer antigen
or virus protein) or aberrantly expressed protein to serve as a
target for T-cells.
[0125] As indicated above, on-target, off-tissue adverse events,
typical of a cytokine release syndrome (CRS); have been observed
for ACT. To maintain the benefit of these revolutionary treatments
while minimizing the risk, a tunable safety switch may be
incorporated which may control the activity level of CAR-expressing
or TCR expressing cells. That is, an inducible costimulatory
chimeric polypeptide may be included which may allow for a
sustained, modulated control of the CAR or TCR. The ligand inducer
may activate the CAR-expressing or TCR-expressing cell by
multimerizing an inducible chimeric signaling molecule, for
example, which may induce intracellular signaling pathways, leading
to the activation of the target cells (T-cell, NK-cell, TIL,
dendritic cell). In the absence of the ligand inducer, the target
cell is quiescent, or has a basal level of activity.
[0126] Thus, according to certain aspects of the present invention,
a switch may be added which activates the CAR or TCR (i.e., safety
switch CAR or TCR, goCAR or goTCR). The CAR or TCR expressing cell
may be designed to only be fully activated when exposed to both a
cancer cell (e.g., target antigen) and a chemical agent (e.g.,
rimiducid, rapamycin), thus providing a means to control the degree
of activation of the CAR or TCR cells by adjusting the
administration schedule of the chemical agent. The CAR or TCR would
still work in a target manner, but on a controllable schedule which
may reduce certain of the side effects of ACT. Thus, according to
certain aspects of the present invention, the ACT may include goCAR
or goTCR cell therapies.
[0127] According to certain aspects of the present invention, the
engineered CAR cell may be allogeneic from a healthy donor and be
further engineered to ablate or replace the endogenous TCR by gene
editing technology such as CRISPR/cas9, ZFN, or TALEN, wherein the
deletion of the endogenous TCR serves to eliminate CAR driven
graft-versus-host disease.
[0128] According to certain aspects of the present invention,
autologous cells (e.g., T-cell or NK-cells or dendritic cells) may
be collected from the subject. These cells may be obtained from a
number of sources, including peripheral blood mononuclear cells,
bone marrow, lymph node tissue, cord blood, thymus tissue, tissue
from a site of infection, ascites, pleural effusion, spleen tissue,
and tumors. According to certain aspects of the present invention,
allogeneic or xenogeneic cells may be used, typically isolated from
healthy donors. When the T-cells, NK cells, dendritic cells, or
pluripotent stem cells are allogeneic or xenogeneic cells, any
number of cell lines available in the art may be used.
[0129] The cells can be obtained from a unit of blood collected
from a subject using any number of techniques known to the skilled
artisan, such as Ficoll.TM. separation. According to certain
aspects of the present invention, cells from the circulating blood
of an individual may be obtained by apheresis. The apheresis
product typically contains lymphocytes, including T-cells,
monocytes, granulocytes, B-cells, other nucleated white blood
cells, red blood cells, and platelets.
[0130] Enrichment of a cell population by negative selection can be
accomplished with a combination of antibodies directed to surface
markers unique to the negatively selected cells. One method is cell
sorting and/or selection via negative magnetic immunoadherence or
flow cytometry that uses a cocktail of monoclonal antibodies
directed to cell surface markers present on the cells negatively
selected. For example, to enrich for CD4+ cells by negative
selection, a monoclonal antibody cocktail typically includes
antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. According
to certain aspects of the present invention, it may be desirable to
enrich for or positively select for a cell population. For example,
positive enrichment for a regulatory T-cell may use positive
selection for CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+.
[0131] The collected cells may be engineered to express the CAR or
TCR by any of a number of methods known in the art. Moreover, the
engineered cells may be expanded by any of a number of methods
known in the art. As detailed above, the CAR or TCR may be
bispecific, trispecific, or quadraspecific; the CAR or TCR may
include a switch such as a goCAR or goTCR, or a safety switch CAR
or TCR; the CAR or TCR may express immune-modulatory proteins such
as an armored CAR or TCR.
[0132] Additional agents which are known to suppress bone marrow,
or may act to lymphodeplete, or alter the microenvironment of a
tumor, may be used in combination with the CD45 antibody. For
example, gemcitabine may deplete myeloid derived suppressor cells
and/or bone marrow, while PD1, PD-L1, TIM3 or LAG-3 antagonist
antibodies, GITR or OX40 co-stimulatory antibodies, IDO inhibitors,
A2aR antagonists, or CD73 antagonists, may alter or shift the tumor
microenvironment.
[0133] According to certain aspects of the present invention, the
collection of blood samples or apheresis product from a subject may
be at any time period prior to when the expanded cells as described
herein might be needed. As such, the source of the cells to be
engineered and expanded (or simply expanded in the case of TILs)
can be collected at any time point necessary, and desired cells,
such as T-cells, NK-cells, dendritic cells, or TILs, can be
isolated and frozen for later use in ACT, such as those ACT
described herein.
[0134] Thus, according to certain aspects of the present invention,
the blood sample or apheresis may be from a generally healthy
subject, or a generally healthy subject who is at risk of
developing a disease, but who has not yet developed a disease, and
the cells of interest may be isolated and frozen for later use. The
cells may be expanded, frozen, and used at a later time. In certain
cases, the cell samples may be collected from a subject shortly
after diagnosis of a particular disease as described herein but
prior to any treatments.
[0135] According to certain aspects of the present invention, the
cells may be isolated from a subject and used fresh, or frozen for
later use, in conjunction with (e.g., before, simultaneously or
following) lymphodepletion using the radiolabeled anti-CD45
antibody of the present invention.
[0136] According to certain aspects of the present invention, the
population of cells expressing the CAR/TCR may be administered to
the subject by dose fractionation, wherein a first percentage of a
total dose is administered on a first day of treatment, a second
percentage of the total dose is administered on a subsequent day of
treatment, and optionally, a third percentage of the total dose is
administered on a yet subsequent day of treatment.
[0137] An exemplary total dose comprises 10.sup.3 to 10.sup.11
cells/kg body weight of the subject, such as 10.sup.3 to 10.sup.10
cells/kg body weight, or 10.sup.3 to 10.sup.9 cells/kg body weight
of the subject, or 10.sup.3 to 10.sup.8 cells/kg body weight of the
subject, or 10.sup.3 to 10.sup.7 cells/kg body weight of the
subject, or 10.sup.3 to 10.sup.6 cells/kg body weight of the
subject, or 10.sup.3 to 10.sup.5 cells/kg body weight of the
subject. Moreover, an exemplary total dose comprises 10.sup.4 to
10.sup.11 cells/kg body weight of the subject, such as 10.sup.5 to
10.sup.11 cells/kg body weight, or 10.sup.6 to 10.sup.11 cells/kg
body weight of the subject, or 10.sup.7 to 10.sup.11 cells/kg body
weight of the subject.
[0138] An exemplary total dose may be administered based on a
patient body surface area rather than the body weight. As such, the
total dose may include 10.sup.3 to 10.sup.13 cells per m.sup.2.
[0139] An exemplary dose may be based on a flat or fixed dosing
schedule rather than on body weight or body surface area.
Flat-fixed dosing may avoid potential dose calculation mistakes.
Additionally, genotyping and phenotyping strategies, and
therapeutic drug monitoring, may be used to calculate the proper
dose. That is, dosing may be based on a patient's immune repertoire
of immunosuppressive cells (e.g., T-regs, MDSC), and/or disease
burden. As such, the total dose may include 10.sup.3 to 10.sup.13
total cells.
[0140] According to certain aspects of the present invention, cells
may be obtained from a subject directly following a treatment. In
this regard, it has been observed that following certain cancer
treatments, in particular treatments with drugs that damage the
immune system, shortly after treatment during the period when
subjects would normally be recovering from the treatment, the
quality of certain cells (e.g., T-cells) obtained may be optimal or
improved for their ability to expand ex vivo. Likewise, following
ex vivo manipulation using the methods described herein, these
cells may be in a preferred state for enhanced engraftment and in
vivo expansion. Thus, it is contemplated within the context of the
present invention to collect blood cells, including T-cells,
NK-cells, dendritic cells, or other cells of the hematopoietic
lineage, during this recovery phase.
[0141] According to certain aspects of the present invention, the
radiolabeled anti-CD45 antibody may be administered after
administration of the effective dose of the population of cells
expressing the CAR/TCR, or the TIL. The effective amount of the
radiolabeled anti-CD45 antibody may be an amount sufficient to
induce lymphodepletion in the subject. According to certain
aspects, the effective amount of the radiolabeled anti-CD45
antibody may be an amount sufficient to induce myeloablation in the
subject.
[0142] According to certain aspects of the present invention, the
radiolabeled anti-CD45 antibody may be administered 1 to 3 months
after administration of the population of cells expressing the
CAR/TCR, or the TIL, and the effective amount of the radiolabeled
anti-CD45 antibody is an amount sufficient to induce
lymphodepletion in the subject. Such may be done in preparation for
transplantation of autologous stem cells; or administration of a
second effective amount of the population of cells expressing the
CAR/TCR.
[0143] According to certain aspects of the present invention, the
radiolabeled anti-CD45 antibody may be administered to the subject
both before and after ACT. That is, the method may comprise an
apheresis step to collect a population of cells that may engineered
to express a CAR/TCR, and expanded, followed by administration of a
first dose of an effective amount of a radiolabeled anti-CD45
antibody. The population of cells expressing the CAR/TCR may then
be administered to the subject, followed by administration of a
second dose of an effective amount of a radiolabeled anti-CD45
antibody. A second dose of the population of cells expressing the
CAR/TCR may then be administered. This second dose of the
population of cells expressing the CAR/TCR may be the same as the
first dose, or may be different. That is, the first and second dose
may be doses of the same population of cells expressing the same
CAR/TCR. Alternatively, the second dose may be a different
effective amount of the same population of cells expressing the
same CAR/TCR.
[0144] According to certain aspects of the present invention, the
second dose may be the same or a different effective amount of a
different population of cells expressing the same or a different
CAR/TCR. Differences in the CAR/TCR may be in any aspect of the
CAR/TCR such as, for example, different binding or antigen
recognition domains or co-stimulatory domains. The second dose may
additionally or alternatively include secreting cells with IL-12 or
may even include adjuvant immunotherapies with small molecule
inhibitors such as BTK, P13K, IDO inhibitors either concurrent or
sequential to the cell therapy infusion.
[0145] Thus, according to certain aspects of the present invention,
a second dose of the population of cells expressing the CAR/TCR may
include a second population of cells expressing a second CAR/TCR,
wherein the second CAR/TCR differs from the CAR/TCR in the first
dose. For example, a second dose may comprise a second population
of activated T-cells or NK-cells or dendritic cells expressing the
second CAR/TCR, wherein the second CAR/TCR may comprise a second
CAR having one or more of the extracellular, transmembrane, or
cytoplasmic co-stimulatory signaling domains which differ from the
CAR in the first dose. As example, the second CAR may recognize a
different antigen, or may be expressed on a different cell type, or
may include different cytoplasmic co-stimulatory signaling domains,
etc.
[0146] Administration of the second dose of the radiolabeled
anti-CD45 antibody after the population of cells expressing the
CAR/TCR have been administered may be used in subjects who have not
shown a complete response (CR) after administration of the
population of cells expressing the CAR/TCR. Moreover,
administration of the second dose of the radiolabeled anti-CD45
antibody after administration of the population of cells expressing
the CAR/TCR may be used when the subject has relapsed or is
identified as having relapsed, such as with antigen positive
disease (e.g., CD-19+) or antigen negative disease (CD-19-), after
administration of the population of cells expressing the
CAR/TCR.
[0147] According to certain aspects of the present invention, the
methods may comprise administration of one or more additional
therapeutic agents. Exemplary therapeutic agents include a
chemotherapeutic agent, an anti-inflammatory agent, an
immunosuppressive, an immunomodulatory agent, or a combination
thereof.
[0148] Therapeutic agents may be administered according to any
standard dose regime known in the field. Exemplary chemotherapeutic
agents include anti-mitotic agent, such as taxanes, for instance
docetaxel, and paclitaxel, and vinca alkaloids, for instance
vindesine, vincristine, vinblastine, and vinorelbine. Exemplary
chemotherapeutic agents include a topoisomerase inhibitor, such as
topotecan.
[0149] Exemplary chemotherapeutic agents include a growth factor
inhibitor, a tyrosine kinase inhibitor, a histone deacetylase
inhibitor, a P38a MAP kinase inhibitor, inhibitors of angiogenesis,
neovascularization, and/or other vascularization, a colony
stimulating factor, an erythropoietic agent, an anti-anergic
agents, an immunosuppressive and/or immunomodulatory agent, a
virus, viral proteins, immune checkpoint inhibitors, BCR inhibitors
(e.g., BTK, P13K, etc.), immune-metabolic agents (e.g., IDO,
arginase, glutaminase inhibitors, etc.), and the like. According to
certain aspects of the present invention, the one or more
therapeutic agents may comprise an antimyeloma agent. Exemplary
antimyeloma agents include dexamethasone, melphalan, doxorubicin,
bortezomib, lenalidomide, prednisone, carmustine, etoposide,
cisplatin, vincristine, cyclophosphamide, and thalidomide, several
of which are indicated above as chemotherapeutic agents,
anti-inflammatory agents, or immunosuppressive agents.
EXAMPLES
Example 1--.sup.131I-BC8 (Iomab-B)
[0150] The Iomab-B drug product is a radio-iodinated anti-CD45
murine monoclonal antibody (mAb) (.sup.131I-BC8). It is specific
for the hematopoietic CD45 antigen. The Iomab-B drug product is
supplied as a sterile formulation contained in a container closure
system consisting of depyrogenated Type 1 50 mL glass vial,
sterilized grey chlorobutyl rubber stopper, and open top style
aluminum seal. Each dose vial also contains a drug product fill
volume of 45 mL in a 50 mL vial. Similarly, it is provided as a
single use dose for complete infusion during intravenous
administration, and contains patient-specific radioactivity from 1
mCi to 200 mCi (e.g., 100 mCi or 150 mCi) of .sup.131I and 6.66-45
mg of BC8. BC8 antibody dose is determined according to the ideal
body weight at a level of 0.5 mg/kg. The drug product is
co-administered in-line with 0.9% Sodium Chloride Injection USP
(normal saline solution) to the patients at a ratio of 1:9 of drug
product to saline solution. The total drug product and saline
infusion volume of approximately 430-450 mL is administered over
varied durations, since the infusion rate depends on the amount of
BC8 antibody in the 45 mL drug product fill volume.
[0151] International Publication No. WO 2017/155937 teaches the
full structure of BC8, and methods for making .sup.131I-BC8.
Example 2--.sup.225Ac-BC8
[0152] Conjugation of Anti-CD45 BC8 with DOTA and Subsequent
Labeling with .sup.225Ac:
[0153] The antibody BC8 (2 mg) was equilibrated with conjugation
buffer (Na carbonate buffer with 1 mM EDTA, pH=8.5-9.0) by four
ultrafiltration spins using a Centricon filter with a MW cutoff of
50,000, or a Vivaspin ultrafiltration tube with a MW cutoff of
50,000. 1.5 ml of conjugation buffer per spin was used. For each
spin, the antibody was spun for 5-20 minutes, at 53,000 RPM and at
4.degree. C. to a residual retentate volume of 100-200 .mu.l. The
antibody was incubated at 4.degree. C. for 30 minutes following the
2.sup.nd and 3.sup.rd spins to allow for equilibration. For DOTA
conjugation, a solution of S-2-(4-Isothiocyanatobenzyl)-1,4,7,10
tetraazacyclododecanetetraacetic acid (p-SCN-Bz-DOTA; MW=687) at 3
mg/ml in 0.15M NH.sub.4OAc was prepared by dissolution and
vortexing. DOTA-Bz-pSCN and BC8 antibody (at >5 mg/ml) was mixed
together at a 7.5 molar ratio (DOTA:antibody) in an Eppendorf tube
and incubated for 15 hours at room temperature. For purification of
the DOTA-antibody conjugate, unreacted DOTA-Bz-pSCN was removed by
seven rounds of ultrafiltration with 1.5 ml of 0.15M NH.sub.4OAc
buffer, pH=6.5 to a volume of approximately 100 .mu.l. After the
final wash, 0.15 MNH.sub.4OAc buffer was added to bring the
material to a final concentration of approximately 1 mg/ml. The
final concentration of the DOTA-BC8 conjugate was measured and the
number of DOTA molecules conjugated to the antibody was determined
to be 1.2-1.5 DOTA to antibody.
[0154] Radiolabeling of DOTA-Antibody Conjugates with
.sup.225Ac:
[0155] To label the DOTA-BC8 conjugate with .sup.225AC, 15 .mu.L
0.15M NH.sub.4OAc buffer, pH=6.5, was mixed with 2 .mu.L (10 .mu.g)
DOTA-BC8 (5 mg/ml) in an Eppendorf reaction tube. Four .mu.L of
.sup.225AC (10 .mu.Ci) in 0.05 M HCl were subsequently added, the
contents of the tube were mixed with a pipette tip, and the
reaction mixture was incubated at 37.degree. C. for 90 minutes with
shaking at 100 rpm. At the end of the incubation period, 3 .mu.L of
1 mM DTPA solution was added to the reaction mixture and incubated
at room temperature for 20 minutes to bind un-complexed .sup.225Ac.
Instant thin layer chromatography (ITLC) was performed with a 10 cm
silica gel strip and a 10 mM EDTA/normal saline mobile phase to
determine the radiochemical purity of .sup.225Ac-BC8, separating
.sup.225Ac-labeled BC8 from .sup.225Ac-DTPA and counting sections
in a gamma counter equipped with a multichannel analyzer. The
radiolabeling efficiency over several runs was determined to be
greater than 80%.
Example 3--Change in Absolute Neutrophil Count
[0156] CD45 is a cell surface protein expressed on most immune cell
types, including both lymphocytes and neutrophils. Iomab-B
(.sup.131I-BC8) radioimmunotherapy targets and delivers its
beta-emitting payload to CD45-positive cells. It would have been
expected that all CD45-positive cell types would be equally
susceptible to depletion following dosing with .sup.131I-BC8. FIG.
2 depicts the relative absolute neutrophil counts at various time
points following a 10 mCi dose of .sup.131I-BC8, presented as
fold-increase or decrease. While absolute lymphocyte counts were
significantly reduced and exhibited sustained depletion over time,
median absolute neutrophil counts exhibited a minimal decrease
following .sup.131I-BC8 dosing, with rapid recovery. This is a
surprising finding. The limited impact of .sup.131I-BC8 on
neutrophils and their rapid rebound would be expected to benefit
patients by preventing infections that might otherwise occur.
Example 4--Clinical Data Demonstrating Lymphodepletion and
Clearance
[0157] Clinical data obtained from patients given low dose levels
of .sup.131I-BC8 show consistent peripheral lymphocyte reduction.
Pharmacokinetic data show fast clearance of .sup.131I-BC8. This
clearance limits interaction with CAR T products. Increased disease
control and prolonged lymphodepletion allow for a flexible window
of time between .sup.131I-BC8 administration and CAR T infusion,
which in turn prevents toxicity. For example, as shown in FIGS.
3A-3F, immune cell analysis following .sup.131I-anti-CD45 antibody
targeted lymphodepletion shows a selective depletion of WBC (FIG.
3A) in peripheral blood and splenic immune cell populations (FIG.
3B), with minimal impact on bone marrow compartment (FIG. 3C) and
sparing of RBCs and platelets (FIGS. 3D and 3E, respectively). FIG.
3F provides these data as a bar graph. FIG. 12 shows the rapid
clearance of the .sup.131I-BC8 from the circulating blood in
patients. On average, 59 percent of the radiolabeled BC8 antibody
cleared from blood with a biological half-time of 0.65 hours (39
minutes), and 41 percent cleared with a biological half-time of 31
hours.
Example 5--Various CAR T-Cell Therapies in Development, and Their
Lymphodepletion Regimens
[0158] There are numerous CAR T-cell therapies in development, each
with its own lymphodepletion regime. The chart in FIG. 4 shows
selected published trials of anti-CD19 CAR T-cell therapy for
patients with B-cell NHL. Most of these trials employ a
chemotherapeutic lymphodepletion regime.
Example 6--.sup.131I-BC8-Based Lymphodepletion Prior to
KYMRIAH.RTM.
[0159] In this example, the subject invention is used to treat a
human subject afflicted with NHL or DLBCL. In the first case, this
method comprises (i) administering to an NHL patient from 25 mCi to
200 mCi (e.g., 100 mCi or 150 mCi) of .sup.131I-BC8, and (ii) after
6, 7 or 8 days, performing KYMRIAH.RTM. (tisagenlecleucel) therapy
on the patient according to its known protocol. In the second case,
this method comprises (i) administering to a DLBCL patient from 25
mCi to 200 mCi (e.g., 100 mCi or 150 mCi) of .sup.131I-BC8, and
(ii) after 6, 7 or 8 days, performing KYMRIAH.RTM. therapy on the
patient according to its known protocol.
Example 7--.sup.131I-BC8-Based Lymphodepletion Prior to
YESCARTA.RTM.
[0160] In this example, the subject invention is used to treat a
human subject afflicted with NHL. This method comprises (i)
administering to an NHL patient from 25 mCi to 200 mCi (e.g., 100
mCi or 150 mCi) of .sup.131I-BC8, and (ii) after 6, 7 or 8 days,
performing YESCARTA.RTM. (axicabtagene ciloleucel) therapy on the
patient according to its known protocol.
Example 8--Preclinical Modeling of Anti-CD45
Radioimmunotherapy-Mediated Conditioning/Lymphodepletion
[0161] Summary:
[0162] Prior to a patient receiving a dose of an adoptive cell
transfer such as engineered autologous or allogeneic CAR T-cells,
it is common to perform a lymphodepletion step often using high
dose chemotherapy. This process is considered important to create
sufficient space in the immune microenvironment, e.g., bone marrow,
to allow the transferred cells to engraft. Further, it appears to
elicit a favorable cytokine profile for establishment and
proliferation of the donor lymphocytes. Anti-CD45
radioimmunotherapy is being investigated in a Phase III clinical
trial as a myeloablative regimen prior to allogeneic hematopoietic
cell transplantation in AML patients. Results from this trial
suggest that lower doses of .sup.131I-anti-CD45 radioimmunotherapy
may be sufficiently lymphodepleting but not myeloablative.
[0163] As example, studies investigating the cumulative radiation
dose absorbed in the spleens of human patients receiving single
doses of 50 mCi to 200 mCi of .sup.131I-anti-CD45 antibody have
been performed. As shown in Table 1, the time at which the
remaining absorbed dose to the spleen will not exceed 25 cGy varies
with the total dose of .sup.131I-anti-CD45 antibody. Moreover, FIG.
11 show the cumulative dose rate to the spleen for patients
administered 100 mCi CD45 antibody through infusion, wherein the
dose rate to the spleen is plotted against time-post infusion.
TABLE-US-00001 TABLE 1 Days Total spleen Time to remaining
(approximate) absorbed .sup.131I Activity absorbed dose less
post-infusion dose Administered than 25 cGy (nearest half-day)
(cGy) 50 mCi 117 hours 4.9 174 75 mCi 141 hours 5.9 261 100 mCi 157
hours 6.6 348 150 mCi 182 hours 7.6 522 200 mCi 199 hours 8.3
696
[0164] Preclinical studies of the effects of low dose
.sup.131I-anti-CD45 radioimmunotherapy (surrogate 30F11)
investigating the lymphodepletive response on particular immune
cell types, and not the changes in cytokine expression in response
to this form of conditioning, have been performed. For example,
FIG. 9 shows transient lymphodepletion in human patients receiving
5 to 20 mCi of .sup.131I-anti-CD45 antibody (median dose 8.4 mCi
for 13 patients). The results of such studies will be supportive in
developing and planning human clinical studies utilizing anti-CD45
radioimmunotherapy as a non-myeloablative conditioning regimen
prior to the administration of autologous or allogeneic adoptive
cell transfer (ACT).
[0165] Current studies include immune competent mice (e.g., female
C57Bl/6 mice 8 to 12 weeks old) using a CD45 radiolabeled
(.sup.131I) anti-mouse receptor antibody to deplete bone marrow and
peripheral blood of CD45+ immune cells for modeling of
CD45-radioimmunotherapy agents as conditioning regimens for
receiving ACT. The comparator will be treatment with the
chemotherapy combination of cyclophosphamide (Cy) with fludarabine
(Flu). At up to three time points post-treatment, animals will be
sacrificed, and peripheral blood, spleen, and bone marrow samples
collected for immunophenotyping to evaluate lymphoid and myeloid
subsets for lymphodepletion, and blood (serum) collected for
cytokine profiling in response to the various treatment
regimens.
[0166] Materials:
[0167] Mice (3 per each time point group), e.g., female adolescent
C57Bl/6 mice (30 mice total). Mouse surrogate anti-CD45 antibody
30F11 (vendor: Millipore Sigma: MABF321, rat IgG2b). .sup.131I for
labeling. Fludarabine (Flu)+ Cyclophosphamide (Cy) (treatment: 250
mg/kg Cy+50 mg/kg Flu)
[0168] Part I:
[0169] Labeling and in vitro characterization of surrogate CD45
antibody 30F11--Perform iodination of 30F11 antibody; perform
immunoreactivity to mouse-CD45+ cells (e.g., B6-Ly5a splenocytes
for 1 hour at 5 ng/ml: target>70% immunoreactivity)
[0170] Part II:
[0171] (a) (i) Prepare dose for in vivo study (0.5 mCi in 100 ug
administered in 200 ul; per Matthews, et al., 2001, Blood,
2:737-745) (ii) Matthews dosimetry results: 0.5 mCi dose: 54 Gy to
spleen, 17 Gy to marrow, 8 Gy to lung, and 5 Gy to liver. In other
studies, 0.05, 0.1 and 0.2 mCi in 100 ug were administered for
determination of dose response.
[0172] (b) The radioimmunotherapy regimen will be administered IP.
Nine mice per group will receive CD45-radioimmunotherapy or
chemotherapy (Cy/Flu) combination. Twelve mice will serve as
pre-treatment and no treatment controls.
[0173] (c) At three time points (i.e., 24 hours, 48 hours and 96
hours), mice will be sacrificed and sampled (bone marrow,
peripheral blood, and spleen; and serum collected). Refer to FIG.
7.
[0174] (d) Immunophenotyping will be performed on bone marrow and
peripheral blood evaluating: (i) Tregs (CD4, CD25, FoxP3); (ii) CD4
& CD8 T-cells, B-cell NK cells: (CD3, CD4, CD8, CD19, CD335);
(iii) HSC (Lin-, c-KIT, SCA1); and (iv) MDSC, DC, MAC,
granulocytes: (CD11b, CD11c, CD244, syglec F, Ly6G, Ly6C).
[0175] (e) Cytokine profiling will be performed using Panel 31-Plex
(MD31) by Luminex, including IL6, IL7, IL10, IL15, MIP1a, VEGF and
IFNg.
[0176] See for example, references: Wrzesinski, et al., 2010, J.
Immunother., 33:1-7; Gattinoni, et al., 2005, JEM, 202(7):907-912;
Bracci, et al., 2007, Clin. Cancer Res. 13:644-653; Matthews, et
al., 2001, Blood, 2:737-745.
Example 9--Preclinical Model of Adoptive T-cell Transfer Following
Anti-CD45 Radioimmunotherapy-Mediated Conditioning/Lymphodepletion
in Mice
[0177] Summary:
[0178] Lymphodepletion is considered a critical step to condition a
patient for receiving an autologous or allogeneic cell therapy such
as CAR T. A study has been proposed to evaluate the impact of
.sup.131I-anti-CD45 radioimmunotherapy on selective depletion of
immune cells and on modulation of the cytokine response in a mouse
model in preparation for adoptive cell transfer. In the present
study, the use of CD45 radioimmunotherapy will be evaluated as a
non-myeloablative conditioning regimen prior to adoptive T-cell
transfer. E.G7 lymphoma tumor-bearing mice will be conditioned by a
single selected dose of .sup.131I-anti-CD45 radioimmunotherapy
prior to adoptive cell transfer of OVA-specific CD8+ T-cells and
monitored for engraftment of the transferred cells and resulting
anti-tumor response. The comparator will be conditioning with the
chemotherapy combination of Cy with Flu or no prior conditioning.
Refer to FIG. 8.
[0179] Materials:
[0180] (i) Mice (5 per each group), female adolescent C57Bl/6
CD45.1 mice (3 groups; 15 mice total). (ii) Donor CD45.2 OT-1 mice
(about 5 mice--sufficient for donor T-cell pool). (iii) E.G7 tumor
cell line. (iv) Mouse surrogate anti-CD45 antibody 30F11 (vendor:
Millipore Sigma: MABF321 200 ug, rat IgG2b.kappa.). (v) .sup.111In
for labeling. (vi) Fludarabine+Cyclophosphamide (treatment: 250
mg/kg Cy+50 mg/kg Flu).
[0181] Methods:
[0182] (1) CD45.1 C57BL/6 mice will each be injected subcutaneously
with 2.times.10.sup.6 E.G7 tumor cells until an approximate 100
mm.sup.3 tumor volume is reached (No Matrigel).
[0183] (2) Approximately 7 days post-tumor cell injection, mice
will receive lymphodepletion either by .sup.131I-anti-CD45
radioimmunotherapy (single dose I.P. of 0.5 mCi .sup.131I-anti-CD45
antibody; 100 ug antibody in 200 ul) or Flu/Cy, or no
conditioning.
[0184] (3) CD8+ T-cells will be isolated from CD45.2 OT-1
transgenic mice and cultured and activated in vitro.
[0185] (4) Four days post-lymphodepletion, 2.times.10.sup.6 CD8+
T-cells will be administered to each cohort via tail vein
injection.
[0186] (5) Tumor volume and body weight will be measured daily as
well as behavior and well-being assessments.
[0187] (6) Based on Hsu, et al., 2015, Oncotarget, 6:44134-44150,
tumor responses should be noted within 10 days, post-T-cell
administration. Following measurement on day 10, mice will be
sacrificed and blood and tumors harvested.
[0188] (7) Tumors will be sectioned and stained for H&E, CD8+
cells, and Treg.
[0189] (8) Blood will be assessed by flow for the presence of
engrafted CD8 cells (CD45.2+) and populations of Tregs and
MDSCs.
[0190] (9) Cytokine profiling will be performed to assess terminal
levels of IL-10, IL-12, IL-15 and IFNg.
[0191] See for example, references: Matthews, et al., 2001, Blood,
2:737-745; and Louis, et al., 2015, Oncotarget, 6:44134-44150.
Example 10--Gene-Edited T-Cells
[0192] This example relates to lymphodepletion in cancer patients
preceding administration of one or more doses of an adoptive cell
therapy containing gene-edited T-cells.
[0193] CAR T-cells have shown considerable promise clinically, with
response rates in refractory lymphoma patient populations exceeding
80%, and durable responses lasting six months or more in nearly 50%
of treated patients.
[0194] However, these engineered T-cells remain susceptible to
immune regulatory control, such as the up-regulation of immune
checkpoint receptors like PD1, Lag3 or TIM3. These receptors
mediate a state of exhaustion and limit the activation potential of
the engineered cells. In the case of allogenic CAR T-cells, these
exogenously administered cells carry the risk of eliciting graft
versus host disease (GVHD). This risk is caused by the recognition
of mis-matched major histocompatibility antigens by native T-cell
receptors present on the engineered allogeneic T-cells.
[0195] These adverse outcomes can be effectively mitigated through
gene editing technology such as CRISPR/cas9. For example, ablation
of the gene encoding PD1 would eliminate the potential for
checkpoint regulation of the CAR T anti-tumor response. Further,
gene editing to ablate the endogenous TCR locus in allogenic CAR
T-cell preparations would effectively prevent GVHD (Ren, et al.).
Lymphodepletion is an important step in enabling successful
engraftment, proliferation and persistence of administered CAR
T-cells. However, safer and more effective methods for
lymphodepletion (such as the subject methods) are needed to replace
the use of non-specific chemotherapy and radiation. Known,
non-specific regimens may contribute to the emergence of CAR
T-related toxicities such as cytokine release syndrome (CRS), and
gene-edited CAR T-cells are not exempt from this risk. The subject
CD45-based lymphodepletion method is a safer, targeted and more
effective method for depleting lymphocytes prior to gene-edited CAR
T, whether the CAR T-cells are ablated for checkpoint receptors or
endogenous TCRs.
Example 11--Dosimetry of Red Marrow and Time to Reduced Activity
Levels for Multicenter Pivotal Phase 3 Study of Iomab-B
[0196] Calculations were performed to evaluate times post-infusion
for .sup.131I-anti-CD45 antibody (Iomab-B) activity levels to fall
to reduced or postulated "safe" levels needed to minimize radiation
dose effects in red marrow to enable and facilitate follow-on
cellular marrow recovery therapy.
[0197] After administration of Iomab-B, the activity of the
radiolabeled antibody decreases exponentially from each of the
major organs of pronounced uptake. Without wishing to be bound by
theory, one possible explanation is that the dose rate to red
marrow decreases exponentially over time due to the combined
effects of biological clearance plus radioactive decay, such that a
time point may be reached after which the total remaining dose to
marrow through infinite time does not exceed a value of 25 cGy. The
value 25 cGy represents one estimate of a relatively "safe"
absorbed dose that should not adversely affect red marrow cellular
regeneration and recovery after prior therapy with high-dose
Iomab-B.
[0198] Data Review:
[0199] In a subset of 25 patients who have received
.sup.131I-anti-CD45 antibody (.sup.131I-BC8) in a clinical trial,
the mean initial uptake of .sup.131I-BC8 in red marrow was 17.4
percent, which cleared with an average effective half-time of 45.1
hours. FIG. 10 shows average dose rate (cGy/hour) to red marrow in
Iomab-B patients for a 100 mCi infusion. Dose rate to marrow is
plotted against time-post infusion. The disappearance curve
represents a single exponential function having a retention
half-time of 45.1 hours. At 154 hours (about 6.5 days)
post-infusion, the area-under-curve shows that during all remaining
time, a total absorbed dose not to exceed 25 cGy will be imparted
to red marrow (representing about 9 percent of the total dose of
271 cGy).
[0200] FIG. 10 also shows that the average initial dose rate to red
marrow was 4.16 cGy/hour. Times to reach the 25 cGy point will
increase with increasing activity infused. Table 2 shows the 25 cGy
time points for infusion of 50 mCi, 75 mCi, 100 mCi, 150 mCi, and
200 mCi .sup.131I-anti-CD45 antibody.
TABLE-US-00002 TABLE 2 Time points after which 25 cGy is delivered
to red marrow, based on observed average biokinetic parameters for
25 patients. Days Total marrow Time to remaining (approximate)
absorbed .sup.131I Activity absorbed dose less post-infusion dose
Administered than 25 cGy (nearest half-day) (cGy) 50 mCi 110 hours
4.5 135 75 mCi 136 hours 5.5 203 100 mCi 154 hours 6.5 271 150 mCi
180 hours 7.5 406 200 mCi 198 hours 8 542
[0201] Preliminary Conclusions:
[0202] From these data, it appears that a waiting period of 6 to 8
days is likely sufficient, depending on the dose administered,
after infusion of the .sup.131I-anti-CD45 antibody for start of
cell-recovery therapy (ACT). For added safety, and to account for
patients having bio-kinetic uptakes and clearance half-times
greater than the average values, the safety time period could be
increased by one or two days (to 9 to 10 days for 200 mCi) after
.sup.131I-anti-CD45 antibody infusion.
[0203] Individual patient variability: Patients differed in Iomab
bio-distribution and clearance kinetics. Although the average
initial uptake in red marrow was 17.4 percent of the total
infusion, the highest observed red marrow uptake post-infusion was
36 percent in one patient. Whereas the average clearance half-time
from marrow was 45.1 hours, the longest observed clearance
half-time was 71 hours. Whereas the average absorbed dose to red
marrow was 2.71 cGy per mCi .sup.131I, the highest value observed
in the current protocol was 4.24 cGy/mCi.
Example 12--Immunophenotyping of T-Reg Cells Following
.sup.131I-BC8 Targeted Lymphodepletion in Mice
[0204] Studies of targeted lymphodepletion in mice using
.sup.131I-anti-CD45 antibody directed radioimmunotherapy have shown
the ability to define a dose at which lymphocytes including T-regs
can be targeted for depletion, while effectively sparing an impact
on the bone marrow. FIGS. 4A-4D and 5A-5C demonstrates that doses
of 50-200 .mu.Ci of .sup.131I-BC8 depletes lymphocytes and T-regs,
and does not have an appreciable effect on bone marrow cells. Dose
levels of 1.5-4 times higher than evaluated for lymphodepletion
have been shown in a mouse tumor model of B-cell lymphoma to direct
an anti-tumor effect. The lower lymphodepleting doses of this
invention will also target CD45 positive tumor cells and contribute
to an anti-tumor effect, reducing tumor burden including PD1
positive tumors, and improving ACT or CAR-T outcomes. In summary,
when used in preparation for ACT, the invention will target the
immune suppressive tumor microenvironment, leading to an
improvement in ACT engraftment, response and anti-tumor
outcome.
[0205] The following aspects are disclosed in this application:
[0206] Aspect 1. A method for treating a subject having a
hematological malignancy or a solid cancer, the method comprising:
administering to the subject an effective amount of a radiolabeled
anti-CD45 antibody; and administering to the subject an effective
amount of a population of cells expressing a chimeric antigen
receptor or a T-cell receptor (CAR/TCR).
[0207] Aspect 2. The method of Aspect 1, wherein the radiolabeled
anti-CD45 antibody is radiolabeled BC8, and the effective amount is
an amount sufficient to lymphodeplete the subject.
[0208] Aspect 3. The method of aspects 1 or 2, wherein the
radiolabeled anti-CD45 antibody comprises .sup.131I, .sup.125I,
.sup.123I, .sup.90Y, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.89Sr,
.sup.153Sm, .sup.32P, .sup.225Ac, .sup.213Bi, .sup.213Po,
.sup.211At, .sup.212Bi, .sup.213Bi, .sup.223Ra, .sup.227Th,
.sup.149Tb, .sup.137Cs, .sup.212Pb and .sup.103Pd.
[0209] Aspect 4. The method according to any one of aspects 1 to 3,
wherein the effective amount of the radiolabeled anti-CD45 antibody
is administered as a single dose.
[0210] Aspect 5. The method according to any one of aspects 2 to 4,
wherein the radiolabeled BC8 is .sup.131I-BC8, and the effective
amount of .sup.131I-BC8 is from 10 mCi to 200 mCi, or wherein the
effective amount of .sup.131I-BC8 is less than 200 mCi.
[0211] Aspect 6. The method according to any one of aspects 2 to 4,
wherein the radiolabeled BC8 is .sup.131I-BC8, and the effective
amount of .sup.131I-BC8 is from 25 mCi to 100 mCi, or wherein the
effective amount of .sup.131I-BC8 is less than 100 mCi.
[0212] Aspect 7. The method according to any one of aspects 2 to 4,
wherein the radiolabeled BC8 is .sup.225Ac-BC8, and the effective
amount of .sup.225Ac-BC8 is 0.1 .mu.Ci/kg of subject weight to 5.0
.mu.Ci/kg of subject weight.
[0213] Aspect 8. The method according to any one of aspects 2 to 7,
wherein the radiolabeled BC8 is administered 6, 7, or 8 days before
administration of the population of cells expressing the
CAR/TCR.
[0214] Aspect 9. The method according to any one of aspects 2 to 8,
wherein the effective amount of the radiolabeled BC8 depletes at
least 50% of lymphocytes of the subject, or at least 70% of
lymphocytes of the subject, or at least 80% of lymphocytes of the
subject.
[0215] Aspect 10. The method according to any one of aspects 2 to
9, wherein the effective amount of the radiolabeled BC8 does not
induce myeloablation in the subject.
[0216] Aspect 11. The method according to any one of aspects 2 to
10, wherein the effective amount of the radiolabeled BC8 provides a
radiation dose of 2 Gy or less to the bone marrow.
[0217] Aspect 12. The method according to any one of aspects 2 to
11, wherein the effective amount of the radiolabeled BC8 depletes
regulatory T cells, myeloid derived suppressor cells, tumor
associated macrophages, activated macrophages secreting IL-1 and/or
IL-6, and combinations thereof.
[0218] Aspect 13. The method according to any one of aspects 1 to
12, wherein the population of cells expressing the CAR/TCR are
autologous cells.
[0219] Aspect 14. The method according to any one of aspects 1 to
12, wherein the population of cells expressing the CAR/TCR are
allogeneic cells.
[0220] Aspect 15. The method according to any one of aspects 1 to
14, wherein the population of cells expressing the CAR/TCR target
CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CS-1, B-cell
maturation antigen (BCMA), MAGEA3, MAGEA3/A6, KRAS, CLL1, MUC-1,
HER2, EpCam, GD2, GPA7, PSCA, EGFR, EGFRvIII, ROR1, mesothelin,
CD33/IL3Ra, c-Met, CD37, PSMA, Glycolipid F77, GD-2, gp100,
NY-ESO-1 TCR, FRalpha, CD24, CD44, CD133, CD166, CA-125, HE4, Oval,
estrogen receptor, progesterone receptor, uPA, PAI-1, MICA, MICB,
ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 or ULBP6, or a combination
thereof.
[0221] Aspect 16. The method according to any one of aspects 1 to
15, wherein the population of cells expressing the CAR/TCR target
CD19, CD20, CD22, or a combination thereof.
[0222] Aspect 17. The method according to any one of aspects 1 to
16, wherein administration of the population of cells expressing
the CAR/TCR target comprises administration of gene-edited CAR
T-cells, and wherein the gene-edited CAR T-cells fail to properly
express at least one checkpoint receptor and/or at least one T-cell
receptor.
[0223] Aspect 18. The method according to any one of aspects 1 to
17, wherein the radiolabeled anti-CD45 antibody is administered
after administration of the population of cells expressing the
CAR/TCR.
[0224] Aspect 19. The method according to aspect 18, wherein the
radiolabeled anti-CD45 antibody is administered 1 to 3 months after
administration of the population of cells expressing the CAR/TCR,
and the effective amount of the anti-CD45 antibody is an amount
sufficient to induce lymphodepletion in the subject.
[0225] Aspect 20. The method according to aspects 18 or 19, wherein
the subject has not shown a complete response (CR) after
administration of the population of cells expressing the CAR/TCR,
or the subject has relapsed or is identified as having relapsed
after administration of the population of cells expressing the
CAR/TCR.
[0226] Aspect 21. The method according to any one of aspects 18 to
20, further comprising, after administration of the radiolabeled
anti-CD45 antibody: transplantation of autologous or allogeneic
stem cells; or administration of a second effective amount of the
population of cells expressing the CAR/TCR.
[0227] Aspect 22. The method according to any one of aspects 18 to
21, wherein the effective amount of the radiolabeled anti-CD45
antibody is an amount sufficient to induce myeloablation in the
subject.
* * * * *