U.S. patent application number 17/378629 was filed with the patent office on 2022-01-27 for methods of treating immunotherapy-related toxicity using a gm-csf antagonist.
This patent application is currently assigned to HUMANIGEN, INC.. The applicant listed for this patent is HUMANIGEN, INC.. Invention is credited to Dale CHAPPELL, Cameron DURRANT.
Application Number | 20220025034 17/378629 |
Document ID | / |
Family ID | 1000005898488 |
Filed Date | 2022-01-27 |
United States Patent
Application |
20220025034 |
Kind Code |
A1 |
DURRANT; Cameron ; et
al. |
January 27, 2022 |
METHODS OF TREATING IMMUNOTHERAPY-RELATED TOXICITY USING A GM-CSF
ANTAGONIST
Abstract
Methods for reducing blood-brain barrier disruption in a subject
treated with immunotherapy, the method comprising administering a
recombinant GM-CSF antagonist to the subject. Methods for
preserving blood-brain barrier integrity in a subject treated with
immunotherapy, the method comprising administering a recombinant
hGM-CSF antagonist to the subject. Methods for decreasing or
preventing CAR-T cell therapy-induced neuroinflammation in a
subject in need thereof, the method comprising administering a
recombinant GM-CSF antagonist to the subject. Methods for
preventing or reducing blood-brain barrier in a subject treated
with immunotherapy, the method comprising administering CAR-T cells
having a GM-CSF gene knockout (GM-CSF.sup.k/o CAR-T cells) to the
subject.
Inventors: |
DURRANT; Cameron; (Oxford,
FL) ; CHAPPELL; Dale; (Nidwalden, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HUMANIGEN, INC. |
Burlingame |
CA |
US |
|
|
Assignee: |
HUMANIGEN, INC.
Burlingame
CA
|
Family ID: |
1000005898488 |
Appl. No.: |
17/378629 |
Filed: |
July 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16283694 |
Feb 22, 2019 |
11130805 |
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17378629 |
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16248762 |
Jan 15, 2019 |
10927168 |
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16283694 |
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16204220 |
Nov 29, 2018 |
10899831 |
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16248762 |
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16149346 |
Oct 2, 2018 |
10870703 |
|
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16204220 |
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62567187 |
Oct 2, 2017 |
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62729043 |
Sep 10, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/243 20130101;
C07K 2317/54 20130101; A61K 2039/505 20130101; C07K 2317/41
20130101; C07K 2317/92 20130101; C07K 2317/56 20130101; C07K
2317/569 20130101; C07K 2317/24 20130101; C07K 2317/21 20130101;
C07K 2317/55 20130101; C07K 2317/34 20130101; C07K 2317/565
20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; A61K 39/395 20060101 A61K039/395 |
Claims
1. A method for reducing blood-brain-barrier disruption in a
subject treated with immunotherapy, the method comprising
administering a recombinant GM-CSF antagonist to the subject.
2. The method of claim 1, wherein the subject has an incidence of
immunotherapy-related toxicity.
3. The method of claim 1, wherein the immunotherapy comprises
adoptive cell transfer, administration of monoclonal antibodies,
administration of cytokines, administration of a cancer vaccine, T
cell engaging therapies, or any combination thereof.
4. The method of claim 3, wherein the adoptive cell transfer
comprises administering chimeric antigen receptor-expressing
T-cells (CAR T-cells), T-cell receptor (TCR) modified T-cells,
tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor
(CAR)-modified natural killer cells, or dendritic cells, or any
combination thereof.
5. The method of claim 4, wherein the CAR T-cells are CD19 CAR-T
cells.
6. The method of claim 1, wherein the recombinant GM-CSF antagonist
is an hGM-CSF antagonist.
7. The method of claim 1, wherein the recombinant GM-CSF antagonist
is an anti-GM-CSF antibody.
8. The method of claim 7, wherein the anti-GM-CSF antibody binds
mammalian GM-CSF or binds primate GM-CSF.
9. The method of claim 8, wherein the primate is a monkey, a
baboon, a macaque, a chimpanzee, a gorilla, a lemur, a lorise, a
tarsier, a galago, a potto, a sifaka, an indri, an aye-ayes an ape
or a human.
10. The method of claim 7, wherein the anti-GM-CSF antibody is an
anti-hGM-CSF antibody.
11. The method of claim 10, wherein the anti-hGM-CSF antibody is
administered prior to, concurrent with, following immunotherapy or
a combination thereof.
12. The method of claim 10, wherein the anti-hGM-CSF antibody binds
human GM-CSF.
13. The method of claim 10, wherein the anti-hGM-CSF antibody is a
monoclonal antibody.
14. The method of claim 10, wherein the anti-hGM-CSF antibody is an
antibody fragment that is a Fab, a Fab', a F(ab')2, a scFv, or a
dAB.
15. The method of claim 10, wherein the anti-hGM-CSF antibody is a
human GM-CSF neutralizing antibody.
16. The method of claim 10, wherein the anti-hGM-CSF antibody is a
recombinant or chimeric antibody.
17. The method of claim 10, wherein the anti-hGM-CSF antibody is a
human antibody.
18. The method of claim 10, wherein the anti-hGM-CSF antibody binds
to the same epitope as chimeric 19/2 antibody.
19. The method of claim 10, wherein the anti-hGM-CSF antibody
comprises the VH region CDR3 and VL region CDR3 of chimeric 19/2
antibody.
20. The method of claim 10, wherein the anti-hGM-CSF antibody
comprises the VH region and VL region CDR1, CDR2, and CDR3 of
chimeric 19/2 antibody.
21. The method of claim 10, wherein the anti-hGM-CSF antibody
comprises a VH region that comprises a CDR3 binding specificity
determinant RQRFPY (SEQ ID NO: 12) or RDRFPY (SEQ ID NO: 13), a J
segment, and a V-segment, wherein the J-segment comprises at least
95% identity to human JH4 (YFD YWGQGTL VTVSS) and the V-segment
comprises at least 90% identity to a human germ line VH1 1-02 or
VH1 1-03 sequence; or a VH region that comprises a CDR3 binding
specificity determinant RQRFPY (SEQ ID NO: 12).
22. The method of claim 21, wherein the J segment comprises
YFDYWGQGTLVTVSS (SEQ ID NO: 14).
23. The method of claim 21, wherein the CDR3 comprises RQRFPYYFDY
(SEQ ID NO: 15) or RDRFPYYFDY (SEQ ID NO: 16).
24. The method of claim 21, wherein the VH region CDR1 is a human
germline VH1 CDR1; the VH region CDR2 is a human germline VH1 CDR2;
or both the CDR1 and CDR2 are from a human germline VH1
sequence.
25. The method of claim 21, wherein the anti-hGM-CSF antibody
comprises a VH CDR1, or a VH CDR2, or both a VH CDR1 and a VH CDR2
as shown in a VH region set forth in FIG. 1.
26. The method of claim 21, wherein the V-segment sequence has a VH
V segment sequence shown in FIG. 1.
27. The method of claim 21, wherein the VH has the sequence of VH
#1, VH #2, VH #3, VH #4, or VH #5 set forth in FIG. 1.
28. The method of claim 10, wherein the anti-hGM-CSF antibody
comprises a VL-region that comprises a CDR3 comprising the amino
acid sequence FNK or FNR.
29. The method of claim 28, wherein the anti-hGM-CSF antibody
comprises a human germline JK4 region.
30. The method of claim 28, wherein the VL region CDR3 comprises
QQFN(K/R)SPL.
31. The method of claim 30, wherein the anti-hGM-CSF antibody
comprises a VL region that comprises a CDR3 comprising QQFNKSPLT
(SEQ ID NO: 18).
32. The method of claim 28, where the VL region comprises a CDR1,
or a CDR2, or both a CDR1 and CDR2 of a VL region shown in FIG.
1.
33. The method of claim 28, wherein the VL region comprises a V
segment that has at least 95% identity to the VKIII A27 V-segment
sequence as shown in FIG. 1.
34. The method of claim 28, wherein the VL region has the sequence
of VK #1, VK #2, VK #3, or VK #4 set forth in FIG. 1.
35. The method of claim 10, wherein the anti-hGM-CSF antibody has a
VH region CDR3 binding specificity determinant RQRFPY (SEQ ID NO:
12) or RDRFPY (SEQ ID NO: 13) and a VL region that has a CDR3
comprising QQFNKSPLT (SEQ ID NO: 18).
36. The method of claim 35, wherein the anti-hGM-CSF antibody has a
VH region sequence set forth in FIG. 1 and a VL region sequence set
forth in FIG. 1.
37. The method of claim 35, wherein the VH region or the VL region,
or both the VH and VL region amino acid sequences comprise a
methionine at the N-terminus.
38. The method of claim 6, wherein the hGM-CSF antagonist is
selected from the group comprising of an anti-hGM-CSF receptor
antibody or a soluble hGM-CSF receptor or receptor sub-unit, a
cytochrome b562 antibody mimetic, a hGM-CSF peptide analog, an
adnectin, a lipocalin scaffold antibody mimetic, a calixarene
antibody mimetic, and an antibody like binding peptidomimetic.
39. The method of claim 2, wherein the immunotherapy-related
toxicity is CAR-T related toxicity.
40. The method of claim 2, wherein the CAR-T related toxicity is
cytokine release syndrome, neurotoxicity, neuro-inflammation or a
combination thereof.
41. A method for preserving blood-brain barrier integrity in a
subject treated with immunotherapy, the method comprising
administering a recombinant hGM-CSF antagonist to the subject.
42. The method of claim 41, wherein the recombinant hGM-CSF
antagonist is an anti-GM-CSF antibody.
43. The method of claim 42, wherein the anti-GM-CSF antibody binds
mammalian GM-CSF or binds primate GM-CSF.
44. The method of claim 43, wherein the primate is a monkey, a
baboon, a macaque, a chimpanzee, a gorilla, a lemur, a lorise, a
tarsier, a galago, a potto, a sifaka, an indri, an aye-ayes an ape
or a human.
45. The method of claim 42, wherein the anti-GM-CSF antibody is an
anti-hGM-CSF antibody.
46. The method of claim 45, wherein the anti-hGM-CSF antibody is
administered prior to, concurrent with, following immunotherapy or
a combination thereof.
47. The method of claim 45, wherein the anti-hGM-CSF antibody binds
human GM-CSF.
48. The method of claim 45, wherein the anti-hGM-CSF antibody is a
monoclonal antibody.
49. The method of claim 45, wherein the anti-hGM-CSF antibody is an
antibody fragment that is a Fab, a Fab', a F(ab')2, a scFv, or a
dAB.
50. The method of claim 45, wherein the anti-hGM-CSF antibody is a
human GM-CSF neutralizing antibody.
51. The method of claim 45, wherein the anti-hGM-CSF antibody is a
recombinant or chimeric antibody.
52. The method of claim 45, wherein the anti-hGM-CSF antibody is a
human antibody.
53. The method of claim 45, wherein the anti-hGM-CSF antibody binds
to the same epitope as chimeric 19/2 antibody.
54. The method of claim 45, wherein the anti-hGM-CSF antibody
comprises the VH region CDR3 and VL region CDR3 of chimeric 19/2
antibody.
55. The method of claim 45, wherein the anti-hGM-CSF antibody
comprises the VH region and VL region CDR1, CDR2, and CDR3 of
chimeric 19/2 antibody.
56. The method of claim 45, wherein the anti-hGM-CSF antibody
comprises a VH region that comprises a CDR3 binding specificity
determinant RQRFPY (SEQ ID NO: 12) or RDRFPY (SEQ ID NO: 13), a J
segment, and a V-segment, wherein the J-segment comprises at least
95% identity to human JH4 (YFD YWGQGTL VTVSS) and the V-segment
comprises at least 90% identity to a human germ line VH1 1-02 or
VH1 1-03 sequence; or a VH region that comprises a CDR3 binding
specificity determinant RQRFPY (SEQ ID NO: 12).
57. The method of claim 55, wherein the J segment comprises
YFDYWGQGTLVTVSS (SEQ ID NO: 14).
58. The method of claim 55, wherein the CDR3 comprises RQRFPYYFDY
(SEQ ID NO: 15) or RDRFPYYFDY (SEQ ID NO: 16).
59. The method of claim 55, wherein the VH region CDR1 is a human
germline VH1 CDR1; the VH region CDR2 is a human germline VH1 CDR2;
or both the CDR1 and CDR2 are from a human germline VH1
sequence.
60. The method of claim 55, wherein the anti-hGM-CSF antibody
comprises a VH CDR1, or a VH CDR2, or both a VH CDR1 and a VH CDR2
as shown in a VH region set forth in FIG. 1.
61. The method of claim 55, wherein the V-segment sequence has a VH
V segment sequence shown in FIG. 1.
62. The method of claim 55, wherein the VH has the sequence of VH
#1, VH #2, VH #3, VH #4, or VH #5 set forth in FIG. 1.
63. The method of claim 45, wherein the anti-hGM-CSF antibody
comprises a VL-region that comprises a CDR3 comprising the amino
acid sequence FNK or FNR.
64. The method of claim 63, wherein the anti-hGM-CSF antibody
comprises a human germline JK4 region.
65. The method of claim 63, wherein the VL region CDR3 comprises
QQFN(K/R)SPL.
66. The method of claim 65, wherein the anti-hGM-CSF antibody
comprises a VL region that comprises a CDR3 comprising QQFNKSPLT
(SEQ ID NO: 18).
67. The method of claim 63, where the VL region comprises a CDR1,
or a CDR2, or both a CDR1 and CDR2 of a VL region shown in FIG.
1.
68. The method of claim 63, wherein the VL region comprises a V
segment that has at least 95% identity to the VKIII A27 V-segment
sequence as shown in FIG. 1.
69. The method of claim 63, wherein the VL region has the sequence
of VK #1, VK #2, VK #3, or VK #4 set forth in FIG. 1.
70. The method of claim 45, wherein the anti-hGM-CSF antibody has a
VH region CDR3 binding specificity determinant RQRFPY (SEQ ID NO:
12) or RDRFPY (SEQ ID NO: 13) (SEQ ID NO: 13) and a VL region that
has a CDR3 comprising QQFNKSPLT (SEQ ID NO: 18).
71. The method of claim 70, wherein the anti-hGM-CSF antibody has a
VH region sequence set forth in FIG. 1 and a VL region sequence set
forth in FIG. 1.
72. The method of claim 70, wherein the VH region or the VL region,
or both the VH and VL region amino acid sequences comprise a
methionine at the N-terminus.
73. The method of claim 41, wherein the hGM-CSF antagonist is
selected from the group comprising of an anti-hGM-CSF receptor
antibody or a soluble hGM-CSF receptor or receptor sub-unit, a
cytochrome b562 antibody mimetic, a hGM-CSF peptide analog, an
adnectin, a lipocalin scaffold antibody mimetic, a calixarene
antibody mimetic, and an antibody like binding peptidomimetic.
74. The method of claim 41, wherein the subject has an
immunotherapy-related toxicity.
75. The method of claim 74, wherein the immunotherapy-related
toxicity is CAR-T related toxicity.
76. The method of claim 75, wherein the CAR-T related toxicity is
cytokine release syndrome, neurotoxicity, neuro-inflammation or a
combination thereof.
77. A method for preventing or reducing blood-brain barrier
disruption in a subject treated with immunotherapy, the method
comprising administering CAR-T cells having a GM-CSF gene knockout
(GM-CSF.sup.k/o CAR-T cells) to the subject.
78. The method of claim 77, further comprising administering a
recombinant hGM-CSF antagonist to the subject.
79. The method of claim 78, wherein the recombinant GM-CSF
antagonist is an hGM-CSF antagonist.
80. The method of claim 79, wherein the recombinant GM-CSF
antagonist is an anti-GM-CSF antibody.
81. The method of claim 80, wherein the anti-hGM-CSF antibody is an
antibody fragment that is a Fab, a Fab', a F(ab')2, a scFv, or a
dAB.
82. The method of claim 80, wherein the anti-hGM-CSF antibody has a
VH region sequence set forth in FIG. 1 and a VL region sequence set
forth in FIG. 1.
83. The method of claim 80, wherein the VH region or the VL region,
or both the VH and VL region amino acid sequences comprise a
methionine at the N-terminus.
84. The method of claim 78, wherein the hGM-CSF antagonist is
selected from the group comprising of an anti-hGM-CSF receptor
antibody or a soluble hGM-CSF receptor or receptor sub-unit, a
cytochrome b562 antibody mimetic, a hGM-CSF peptide analog, an
adnectin, a lipocalin scaffold antibody mimetic, a calixarene
antibody mimetic, and an antibody-like binding peptidomimetic.
85. The method of claim 77, wherein the subject further has a CAR-T
related toxicity selected from cytokine release syndrome,
neurotoxicity, or a combination thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 16/283,694, filed on Feb. 22, 2019, which is a
continuation-in-part application of U.S. application Ser. No.
16/248,762, filed on Jan. 15, 2019, now U.S. Pat. No. 10,927,168,
which is a continuation-in-part application of U.S. application
Ser. No. 16/204,220, filed on Nov. 29, 2018, now U.S. Pat. No.
10,899,831, which is a continuation-in-part application of U.S.
application Ser. No. 16/149,346, filed on Oct. 2, 2018, now U.S.
Pat. No. 10,870,703, which claims priority to U.S. Provisional
Application Nos. 62/567,187, filed Oct. 2, 2017, and 62/729,043,
filed Sep. 10, 2018, which are hereby incorporated by
reference.
SEQUENCE LISTING INCORPORATION
[0002] The ".txt" Sequence Listing filed with this application by
EFS and which is entitled P-566450-US15D-SQL-14JUL21_ST25.txt, is
25.5 kilobytes in size and which was created on Jul. 14, 2021, is
hereby incorporated by reference.
FIELD OF THE DISCLOSURE
[0003] The invention relates to methods for reducing blood-brain
barrier disruption in a subject treated with immunotherapy, the
methods comprising administering a recombinant GM-CSF antagonist to
the subject. The invention also relates to methods for preserving
blood-brain barrier integrity in a subject treated with
immunotherapy, the methods comprising administering a recombinant
hGM-CSF antagonist to the subject. The invention further relates to
methods for decreasing or preventing CAR-T cell therapy-induced
neuroinflammation in a subject in need thereof, the method
comprising administering a recombinant GM-CSF antagonist to the
subject. The invention relates to relates to methods for reducing
relapse rate or preventing occurrence of tumor relapse in a subject
treated with immunotherapy in an absence of an incidence of
immunotherapy-related toxicity. The invention also relates to
methods for reducing relapse rate or preventing occurrence of tumor
relapse in a subject treated with immunotherapy in a presence of an
incidence of immunotherapy-related toxicity. The invention further
relates to methods for reducing a level of a cytokine or chemokine
other than GM-CSF in a subject having an incidence of
immunotherapy-related toxicity, the methods comprising
administering a recombinant GM-CSF antagonist to the subject. The
invention also relates to methods for treating or preventing
immunotherapy-related toxicity in a subject, the method comprising
administering to the subject chimeric antigen receptor-expressing
T-cells (CAR-T cells), the CAR-T cells having a GM-CSF gene
knockout (GM-CSF.sup.k/o CAR-T cells), and a recombinant hGM-CSF
antagonist. The disclosure herein also provides methods of
inhibiting or reducing the incidence and/or the severity of
immunotherapy-related toxicity in a subject, the method comprising
administering a recombinant GM-CSF antagonist to the subject.
BACKGROUND
[0004] Granulocyte-macrophage colony-stimulating factor (GM-CSF) is
a cytokine secreted by various cell types including macrophages, T
cells, mast cells, natural killer cells, endothelial cells and
fibroblasts. GM-CSF stimulates the differentiation of granulocytes
and of monocytes. Monocytes, in turn, migrate into tissue and
mature into macrophages and dendritic cells. Thus, secretion of
GM-CSF leads to a rapid increase in macrophage numbers. GM-CSF is
also involved in the inflammatory response in the Central Nervous
System (CNS) causing influx of blood-derived monocytes and
macrophages, and the activation of astrocytes and microglia.
Immuno-related toxicities comprise potentially life-threatening
immune responses that occur as a result of the high levels of
immune activation occurring from different immunotherapies.
Immuno-related toxicity is currently a major complication for the
application of immunotherapies in cancer patients. Chimeric antigen
receptor T (CAR-T) cell therapy has emerged as a novel and
potentially revolutionary therapy to treat cancer. Based on
unprecedented responses in B cell malignancies, two CD19 targeted
CAR-T (CART19) cell products were approved by the FDA in 2017.
However, the wider application of CAR-T cell therapy is limited by
the emergence of unique and potentially fatal toxicities. These
include the development of cytokine release syndrome (CRS) and
neurotoxicity (NT). Up to 50% of patients treated with CART19 cells
develop grade 3 or higher CRS or NT and several deaths have been
reported. These toxicities are associated with prolonged
hospitalization, intensive care unit (ICU) stays, and the long-term
effects of NT are unknown. Thus, controlling these CART19 cell
related toxicities is imperative to lessen morbidity, mortality,
duration of hospitalization, ICU admissions, supportive care
required and the significant indirect costs associated with CAR-T
cell therapy. It is clear that there remains a critical need for
methods of preventing and treating immuno-related toxicity. An
ideal method will minimize the risk of these life-threatening
complications without affecting the efficacy of the immunotherapy
and could potentially even improve the efficacy by allowing, for
example, safe increased dosing of immunotherapeutic compounds
and/or an expansion of T cells.
BRIEF SUMMARY OF THE INVENTION
[0005] In one aspect, this invention provides methods for reducing
blood-brain barrier disruption in a subject treated with
immunotherapy, the methods comprising administering a recombinant
GM-CSF antagonist to the subject.
[0006] In another aspect, this invention provides methods for
preserving blood-brain barrier integrity in a subject treated with
immunotherapy, the methods comprising administering a recombinant
hGM-CSF antagonist to the subject.
[0007] In still another aspect, this invention provides methods for
decreasing or preventing CAR-T cell therapy-induced
neuroinflammation in a subject in need thereof, the method
comprising administering a recombinant GM-CSF antagonist to the
subject.
[0008] In another aspect, this invention provides methods for
preventing or reducing blood-brain barrier in a subject treated
with immunotherapy, the method comprising administering CAR-T cells
having a GM-CSF gene knockout (GM-CSF.sup.k/o CAR-T cells) to the
subject.
[0009] In an aspect, this invention provides methods for reducing
relapse rate or preventing or delaying occurrence of tumor relapse
in a subject treated with immunotherapy in an absence of an
incidence of immunotherapy-related toxicity, the method comprising
administering to the subject a recombinant hGM-CSF antagonist. In a
related aspect, this invention provides methods for reducing
relapse rate or preventing occurrence of tumor relapse in a subject
treated with immunotherapy in a presence of an incidence of
immunotherapy-related toxicity, the method comprising administering
to the subject a recombinant hGM-CSF antagonist.
[0010] In another aspect, this invention provides a method reducing
a level of a cytokine or chemokine other than GM-CSF in a subject
having an incidence of immunotherapy-related toxicity, the method
comprising administering to the subject a recombinant hGM-CSF
antagonist, wherein the level of the cytokine or chemokine is
reduced compared to the level thereof in a subject during the
incidence of immunotherapy-related toxicity.
[0011] In a further aspect, this invention provides a method for
treating or preventing immunotherapy-related toxicity in a subject,
the method comprising administering to the subject chimeric antigen
receptor-expressing T-cells (CAR-T cells), the CAR-T cells having
their GM-CSF genes `knocked-out` (GM-CSF.sup.k/o CAR-T cells), and
a recombinant hGM-CSF antagonist.
[0012] In one aspect, disclosed herein is a method of inhibiting or
reducing the incidence or the severity of immunotherapy-related
toxicity in a subject, the method comprising a step of
administering a recombinant hGM-CSF antagonist to the subject.
[0013] In a related aspect, said immunotherapy comprises adoptive
cell transfer, administration of monoclonal antibodies,
administration of cytokines or chemokines, administration of a
cancer vaccine, T cell engaging therapies, or any combination
thereof.
[0014] In another aspect, adoptive cell transfer comprises
administering chimeric antigen receptor-expressing T-cells (CAR
T-cells), T-cell receptor (TCR) modified T-cells,
tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor
(CAR)-modified natural killer cells, or dendritic cells, or any
combination thereof. In a related aspect, the monoclonal antibody
is selected from a group comprising: anti-CD3, anti-CD52, anti-PD1,
anti-PD-L1, anti-CTLA4, anti-CD20, anti-BCMA antibodies,
bi-specific antibodies, or bispecific T-cell engager (BiTE)
antibodies, or any combination thereof. In a related aspect, the
cytokines are selected from a group comprising: IFN.alpha.,
IFN.beta., IFN.gamma., IFN.lamda., IL-1, IL-2, IL-6, IL-7, IL-15,
IL-21, IL-11, IL-12, IL-18, GM-CSF, TNF.alpha., or any combination
thereof.
[0015] In another aspect, inhibiting or reducing the incidence or
the severity of immunotherapy-related toxicity comprises reducing
the concentration of at least one inflammation-associated factor in
the serum, tissue fluid, or in the CSF of the subject. In a related
aspect, the inflammation-associated factor is selected from a group
comprising: C-reactive protein, GM-CSF, IL-1, IL-2, sIL2R.alpha.,
IL-5, IL-6, IL-8, IL-10, IP10, IL-15, MCP-1 (AKA CCL2), MIG,
MIP1.beta., IFN.gamma., CX3CR1, or TNF.alpha., or any combination
thereof. In another aspect, the administration of recombinant
GM-CSF antagonist does not reduce the efficacy of said
immunotherapy. In another aspect, the administration of recombinant
GM-CSF antagonist increases the efficacy of said immunotherapy. In
another aspect, administration of recombinant GM-CSF antagonist
occurs prior to, concurrent with, or following immunotherapy. In a
related aspect, the recombinant GM-CSF antagonist is
co-administered with corticosteroids, anti-IL-6 antibodies,
tocilizumab, anti-IL-1 antibodies, cyclosporine, antiepileptics,
benzodiazepines, acetazolamide, hyperventilation therapy, or
hyperosmolar therapy, or any combination thereof.
[0016] In another aspect, the immunotherapy-related toxicity
comprises a brain disease, damage or malfunction. In a related
aspect, the brain disease, damage or malfunction comprises CAR-T
cell related NT or CAR-T cell related encephalopathy syndrome
(CRES). In a related aspect, inhibiting or reducing incidence of a
brain disease, damage or malfunction comprises reducing headaches,
delirium, anxiety, tremor, seizure activity, confusion, alterations
in wakefulness, hallucinations, dysphasia, ataxia, apraxia, facial
nerve palsy, motor weakness, seizures, nonconvulsive EEG seizures,
altered levels of consciousness, coma, endothelial activation,
vascular leak, intravascular coagulation, or any combination
thereof in the subject. In another aspect, the
immunotherapy-related toxicity comprises CAR-T induced Cytokine
Release Syndrome (CRS). In a related aspect, inhibiting or reducing
incidence of CRS comprises reducing or inhibiting, without
limitation, high fever, myalgia, nausea, hypotension, hypoxia, or
shock, or a combination thereof. In a related aspect, the
immunotherapy-related toxicity is life-threatening.
[0017] In another aspect, the serum concentration of ANG2 or VWF,
or the serum ANG2:ANG1 ratio of the subject is reduced. In a
related aspect, the subject has a body temperature above 38.degree.
C., an IL-6 serum concentration >16 pg/ml, or an MCP-1 serum
concentration above 1,300 pg/ml during the first 36 hours after
infusion of said CAR-T cells. In a related aspect, the subject is
predisposed to have said brain disease, damage or malfunction. In a
related aspect, the subject has an ANG2:ANG1 ratio in serum above 1
prior to the infusion of said CAR-T cells.
[0018] In another aspect, the immunotherapy-related toxicity
comprises hemophagocytic lymphohistiocytosis (HLH) or
macrophage-activation syndrome (MAS). In a related aspect,
inhibiting or reducing incidence of HLH or MAS comprises increasing
survival time and/or time to relapse, reducing macrophage
activation, reducing T cell activation, reducing the concentration
of IFN.gamma. in the peripheral circulation, or reducing the
concentration of GM-CSF in the peripheral circulation, or any
combination thereof.
[0019] In another aspect, the subject presents with fever,
splenomegaly, cytopenias involving two or more lines,
hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis, low or
absent NK-cell activity, ferritin serum concentration above 500
U/ml, or soluble CD25 serum concentration above 2400 U/ml, or any
combination thereof. In a related aspect, the subject is
predisposed to acquiring HLH or MAS. In a related aspect, the
subject carries a mutation in a gene selected from: PRF1, UNC13D,
STX11, STXBP2, or RAB27A, or has reduced expression of perforin, or
any combination thereof.
[0020] In one embodiment, the GM-CSF antagonist is an anti-hGM-CSF
antibody. In another embodiment, the anti-hGM-CSF antibody blocks
binding of hGM-CSF to the alpha subunit of the hGM-CSF receptor. In
another embodiment, the anti-hGM-CSF antibody is a polyclonal
antibody. In another embodiment, the anti-hGM-CSF antibody is a
monoclonal antibody. In another embodiment, the anti-hGM-CSF
antibody is an antibody fragment that is a Fab, a Fab', a F(ab')2,
a scFv, or a dAB. In some embodiments, the monoclonal anti-hGM-CSF
antibody, the single-chain Fv, and the Fab may be generated in the
chicken; chicken IgY are avian equivalents of mammalian IgG
antibodies. (Park et al., Biotechnology Letters (2005) 27:289-295;
Finley et al., Appl. Environ. Microbiol., May 2006, p. 3343-3349).
Chicken IgY antibodies have the following advantages: higher
avidity, i.e., overall strength of binding between an antibody and
an antigen, higher specificity (less cross reactivity with
mammalian proteins other than the immunogen); high yield in the egg
yolk, and lower background (the structural difference in the Fc
region of IgY and IgG results in less false positive staining). In
another embodiment, the anti-hGM-CSF antibody may be a camelid,
e.g., a llama-derived single variable domain on a heavy chain
antibodies lacking light chains (also called sdAbs, VHHs and
Nanobodies.RTM.); the VHH domain (about 15 kDa) is the smallest
known antigen recognition site that occurs in mammals having full
binding capacity and affinities (equivalent to conventional
antibodies). (Garaicoechea et al. (2015) PLoS ONE 10(8): e0133665;
Arbabi-Ghahroudi M (2017) Front. Immunol. 8:1589; Wu et al.,
Translational Oncology (2018) 11, 366-373). In another embodiment,
the antibody fragment is conjugated to polyethylene glycol. In
another embodiment, the anti-hGM-CSF antibody has an affinity
ranging from about 5 pM to about 50 pM. In another embodiment,
anti-hGM-CSF antibody is a neutralizing antibody. In another
embodiment, the anti-hGM-CSF antibody is a recombinant or chimeric
antibody. In another embodiment, the anti-hGM-CSF antibody is a
human antibody. In another embodiment, the anti-hGM-CSF antibody
comprises a human variable region. In another embodiment, the
anti-hGM-CSF antibody comprises an engineered human variable
region. In another embodiment the anti-hGM-CSF antibody comprises a
humanized variable region. In another embodiment, the anti-hGM-CSF
antibody comprises an engineered human variable region. In another
embodiment the anti-hGM-CSF antibody comprises a humanized variable
region.
[0021] In one embodiment, the anti-hGM-CSF antibody comprises a
human light chain constant region. In another embodiment, the
anti-hGM-CSF antibody comprises a human heavy chain constant
region. In another embodiment, the human heavy chain constant
region is a gamma chain. In another embodiment, the anti-hGM-CSF
antibody binds to the same epitope as chimeric 19/2 antibody. In
another embodiment, the anti-hGM-CSF antibody comprises the VH
region CDR3 and VL region CDR3 of chimeric 19/2 antibody. In
another embodiment, the anti-GM-CSF antibody comprises the VH
region and VL region CDR1, CDR2, and CDR3 of chimeric 19/2
antibody.
[0022] In one embodiment, the anti-hGM-CSF antibody comprises a
heavy chain variable region that comprises a CDR3 binding
specificity determinant RQRFPY (SEQ ID NO: 12) or RDRFPY (SEQ ID
NO: 13), a J segment, and a V-segment, wherein the J-segment
comprises at least 95% identity to human JH4 (YFDYWGQGTLVTVSS) [SEQ
ID NO: 14] and the V-segment comprises at least 90% identity to a
human germ line VH1 1-02 (SEQ ID NO: 19) or VH1 1-03 (SEQ ID NO:
20) sequence; or a heavy chain variable region that comprises a
CDR3 binding specificity determinant comprising RQRFPY (SEQ ID NO:
12). In another embodiment, the J segment comprises YFDYWGQGTLVTVSS
(SEQ ID NO: 14). In another embodiment, the CDR3 comprises
RQRFPYYFDY (SEQ ID NO: 15) or RDRFPYYFDY (SEQ ID NO: 16). In
another embodiment, the heavy chain variable region CDR1 or CDR2
can be a human germline VH1 sequence; or both the CDR1 and CDR2 can
be human germline VH1. In another embodiment, the antibody
comprises a heavy chain variable region CDR1 or CDR2, or both CDR1
and CDR2, as shown in a V.sub.H region set forth in FIG. 1. In
another embodiment, the anti-hGM-CSF antibody has a V-segment that
has a V.sub.H V-segment sequence shown in FIG. 1. In another
embodiment, the V.sub.H that has the sequence of VH #1, VH #2, VH
#3, VH #4, or VH #5 set forth in FIG. 1.
[0023] In another embodiment, the anti-hGM-CSF antibody, e.g., that
has a heavy chain variable region as described in the paragraph
above, comprises a light chain variable region that comprises a
CDR3 binding specificity determinant comprising the amino acid
sequence FNK or FNR.
[0024] In another embodiment, the anti-hGM-CSF antibody comprises a
VL region that comprises a CDR3 comprising the amino acid sequence
FNK or FNR. In one embodiment, the anti-GM-CSF antibody comprises a
human germline JK4 region. In another embodiment, the antibody
V.sub.L region CDR3 comprises QQFN(K/R)SPLT (SEQ ID NO: 17). In
another embodiment, the anti-GM-CSF antibody comprises a VL region
that comprises a CDR3 comprising QQFNKSPLT (SEQ ID NO: 18). In
another embodiment, the VL region comprises a CDR1, or a CDR2, or
both a CDR1 and CDR2, of a V.sub.L region shown in FIG. 1. In
another embodiment, the V.sub.L region comprises a V segment that
has at least 95% identity to the VKIIIA27 V-segment sequence as
shown in FIG. 1. In another embodiment, the V.sub.L region has the
sequence of VK #1, VK #2, VK #3, or VK #4 set forth in FIG. 1.
[0025] In one embodiment, the anti-hGM-CSF antibody has a VH region
CDR3 binding specificity determinant RQRFPY (SEQ ID NO: 12) or
RDRFPY (SEQ ID NO: 13) and a VL region that has a CDR3 comprising
QQFNKSPLT (SEQ ID NO: 18). In another embodiment, the anti-hGM-CSF
antibody has a VH region sequence set forth in FIG. 1 and a VL
region sequence set forth in FIG. 1. In another embodiment, the VH
region or the VL region, or both the VH and VL region amino acid
sequences comprise a methionine at the N-terminus. In another
embodiment, the GM-CSF antagonist is selected from the group
comprising of an anti-hGM-CSF receptor antibody or receptor
sub-unit or a soluble GM-CSF receptor, a cytochrome b562 antibody
mimetic, a hGM-CSF peptide analog, an adnectin, a lipocalin
scaffold antibody mimetic, a calixarene antibody mimetic, and an
antibody like binding peptidomimetic.
[0026] In one embodiment, disclosed herein is a method of
increasing the efficacy of CAR-T immunotherapy in a subject, the
method comprising a step of administering a recombinant hGM-CSF
antagonist to the subject, wherein said administering increases the
efficacy of CAR-T immunotherapy in said subject. In another
embodiment, said administering a recombinant hGM-CSF antagonist
occurs prior to, concurrent with, or following said CAR-T
immunotherapy. In another embodiment, said increased efficacy
comprises increased CAR-T cell expansion, reduced myeloid-derived
suppressor cell (MDSC) number that inhibit T-cell function, synergy
with a checkpoint inhibitor, or any combination thereof. In another
embodiment, said increased CAR-T cell expansion comprises at least
a 50% increase compared to a control. In another embodiment, said
increased CAR-T cell expansion comprises at least a one quarter log
expansion compared to a control. In another embodiment, said
increased cell expansion comprises at least a one-half log
expansion compared to a control. In another embodiment, said
increased cell expansion comprises at least a one log expansion
compared to a control. In another embodiment, said increased cell
expansion comprises a greater than one log expansion compared to a
control.
[0027] In an embodiment, the hGM-CSF antagonist comprises a
neutralizing antibody. In another embodiment, the neutralizing
antibody is a monoclonal antibody.
[0028] In an embodiment, disclosed herein is a method of inhibiting
or reducing the incidence or the severity of CAR-T related toxicity
in a subject, the method comprising a step of administering a
recombinant hGM-CSF antagonist to the subject, wherein said
administering inhibits or reduces the incidence or the severity of
CAR-T related toxicity in said subject. In an embodiment, said
CAR-T related toxicity comprises NT, CRS, or a combination thereof.
In some embodiments, the CAR-T cell related NT is reduced by about
50% compared to a reduction in NT in a subject treated with CAR-T
cells and a control antibody. In various embodiments, the
recombinant hGM-CSF antagonist is a hGM-CSF neutralizing antibody
in accordance with embodiments described herein.
[0029] In another embodiment, said inhibiting or reducing incidence
of CRS comprises increasing survival time and/or time to relapse,
reducing macrophage activation, reducing T cell activation, or
reducing the concentration of circulating hGM-CSF, or any
combination thereof. In another embodiment, said subject presents
with fever (with or without rigors, malaise, fatigue, anorexia,
myalgia, arthralgia, nausea, vomiting, headache, skin rash,
diarrhea, tachypnea, hypoxemia, hypoxia, shock, cardiovascular
tachycardia, widened pulse pressure, hypotension, capillary leak,
increased early cardiac output, diminished late cardiac output,
elevated D-dimer, hypofibrinogenemia with or without bleeding,
azotemia, transaminitis, hyperbilirubinemia, mental status changes,
confusion, delirium, frank aphasia, hallucinations, tremor,
dysmetria, altered gait, seizures, organ failure, or any
combination thereof.
[0030] In another embodiment, the inhibiting or reducing the
incidence or the severity of CAR-T related toxicity comprises
preventing the onset of CAR-T related toxicity.
[0031] In another embodiment, disclosed herein is a method of
blocking or reducing GM-CSF expression in a cell, comprising
knocking out or silencing GM-CSF gene expression in a cell. In an
embodiment, the blocking or reducing of GM-CSF expression comprises
short interfering RNS (siRNA), CRISPR, RNAi, DNA-directed RNA
interference (ddRNAi), which is a gene-silencing technique that
uses DNA constructs to activate an animal cell's endogenous RNA
interference (RNAi) pathways, or targeted genome editing with
engineered transcription activator-like effector nucleases
(TALENs), i.e., artificial proteins composed of a customizable
sequence-specific DNA-binding domain fused to a nuclease that
cleaves DNA in a nonsequence-specific manner. (Joung and Sander,
Nat Rev Mol Cell Biol. 2013 January; 14(1): 49-55), which is
incorporated herein in its entirety by reference. In an embodiment,
the cell is a CAR-T cell.
[0032] In one embodiment, the subject is a human.
[0033] In one embodiment, disclosed herein is a hGM-CSF antagonist
for use in a method of inhibiting or reducing the incidence or
severity of immunotherapy-related toxicity in a subject, the method
comprising a step of administering a recombinant hGM-CSF antagonist
to the subject. In one embodiment, disclosed herein is a
pharmaceutical composition comprising an anti-hGM-CSF
antagonist.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 provides exemplary V.sub.H and V.sub.L sequences of
anti-GM-CSF antibodies.
[0035] FIGS. 2A-2B illustrates binding of GM-CSF to Ab1 (FIG. 2A)
or Ab2 (FIG. 2B) determined by surface plasmon resonance analysis
at 37.degree. C. (Biacore 3000). Ab1 and Ab2 were captured on anti
Fab polyclonal antibodies immobilized on the Biacore chip.
Different concentrations of GM-CSF were injected over the surface
as indicated. Global fit analysis was carried out assuming a 1:1
interaction using Scrubber2 software.
[0036] FIGS. 3A-3B illustrates binding of Ab and Ab2 to
glycosylated (FIG. 3A) and non-glycosylated GM-CSF (FIG. 3B).
Binding to glycosylated GM-CSF expressed from human 293 cells or
non-glycosylated GM-CSF expressed in E. coli was determined by
ELISA. Representative results from a single experiment are shown
(exp 1). Two-fold dilutions of Ab1 and Ab2 starting from 1500 ng/ml
were applied to GM-CSF coated wells. Each point represents
mean.+-.standard error for triplicate determinations. Sigmoidal
curve fit was performed using Prism 5.0 Software (Graphpad).
[0037] FIGS. 4A-4B illustrates competition ELISA demonstrating
binding of Ab1 and Ab2 to a shared epitope. ELISA plates coated
with 50 ng/well of recombinant GM-CSF were incubated with various
concentrations of antibody (Ab2, Ab1 or isotype control antibody)
together with 50 nM biotinylated Ab2. Biotinylated antibody binding
was assayed using neutravidin-HRP conjugate. Competition for
binding to GM-CSF was for 1 hr (FIG. 4A) or for 18 hrs (FIG. 4B).
Each point represents mean.+-.standard error for triplicate
determinations. Sigmoidal curve fit was performed using Prism 5.0
Software (Graphpad).
[0038] FIG. 5 illustrates inhibition of GM-CSF-induced IL-8
expression. Various amounts of each antibody were incubated with
0.5 ng/ml GM-CSF and incubated with U937 cells for 16 hrs. IL-8
secreted into the culture supernatant was determined by ELISA.
[0039] FIG. 6 illustrates dose-dependent inhibition of
GM-CSF-stimulated CD11b on human granulocytes by anti-GM-CSF
antibody.
[0040] FIG. 7 illustrates dose-dependent inhibition of
GM-CSF-induced HLA-DR on CD14+ human, primary monocytes/macrophages
by anti-GM-CSF antibody.
[0041] FIG. 8 illustrates the role of GM-CSF (Myeloid Inflammatory
Factor) as a key cytokine in CAR-T-related activity and in
stimulation of white blood cell proliferation, which is a
characteristic feature in certain leukemias, e.g., acute myeloid
leukemia (AML).
[0042] FIG. 9 illustrates inhibition of GM-CSF-dependent human TF-1
cell proliferation (human erythroleukemia) by neutralization of
human GM-CSF with anti-GM-CSF antibody. KB003 is a recombinant
monoclonal antibody designed to target and neutralize human GM-CSF.
KB002 is a mouse/human chimeric monoclonal antibody, that targets
and neutralizes hGM-CSF.
[0043] FIG. 10 is a depiction of a chimeric antigen receptor.
[0044] FIG. 11 illustrates CAR-T19 therapy results in high response
rates in relapsed refractory ALL. Data show historic outcomes in
R/R ALL and outcomes in R/R ALL after CAR-T19 therapy. (Maude, et
al NEJM 2014).
[0045] FIG. 12 illustrates evidence showing a significant GM-CSF
link to NT. GM-CSF levels correlate with serious adverse effects
after CAR-T cell therapy. GM-CSF levels precede and modulate other
cytokines other than IL-15. Elevated GM-CSF is clearly associated
with .gtoreq.grade 3 NT. IL-2 is only other cytokine with this
association.
[0046] FIG. 13 illustrates an estimated time course of CRS and NT
following CD19 CAR-T cell therapy. Timing of symptom onset and CRS
severity depends on the inducing agent, type of cancer, age of
patient, and the magnitude of immune cell activation. CAR-T related
CRS symptom onset typically occurs days to occasionally weeks after
the T-cell infusion, coinciding with maximal T-cell expansion.
Similar to CRS associated with mAb therapy, CRS associated with
adoptive T-cell therapies has been consistently associated with
elevated IFN.gamma., IL-6, TNF.alpha., IL-1, IL-2, IL-6, GM-CSF,
IL-10, IL-8, and IL-5. No clear CAR-T cell dose-response
relationship for CRS exists, but very high doses of T cells may
result in earlier onset of symptoms.
[0047] FIG. 14 illustrates that GM-CSF is a key initiator of CAR-T
adverse effects. The figure depicts the central role of GM-CSF in
CRS and NT. Perforin allows granzymes to penetrate the tumor cell
membrane. CAR-T produced GM-CSF recruits CCR2+ myeloid cells to the
tumor site, which produce CCL2 (MCP1). CCL2 positively reinforces
its own production by CCR2+ myeloid cell recruitment. IL-1 and IL-6
from myeloid cells form another positive feedback loop with CAR-T
by inducing production of GM-CSF. Phosphatidyl serine is exposed as
a result of perforin and granzyme cell membrane destruction.
Phosphatidyl-serine stimulates myeloid cell production of CCL2,
IL-1, IL-6, and other inflammatory effectors. The final outcome of
this self-reinforcing feedback loop results in endothelial
activation, vascular permeability, and ultimately, CRS and NT.
Moreover, animal model evidence shows GM-CSF knockout mice show no
sign of CRS, but IL-6 knockout mice can still develop CRS. GM-CSF
receptor k/o from CCR2+ myeloid cells abrogates cascade in
neuro-inflammation models. (Sentman, et al., J. Immunol.; Coxford,
et al. Immunity 2015 (43)510-514; Ishii et al., Blood 2016
128:3358; Teachey, et al. Cancer Discov. 2016 Jun. 6(6): 664-679;
Lee, et al., Blood 2016 124:2:188; Barrett, et al., Blood 2016:
128-654, each of which is incorporated in its entirety herein by
reference.).
[0048] FIGS. 15A-15G illustrate that GM-CSF CRISPR knockout T-cells
exhibit reduced expression of GM-CSF but similar levels of other
cytokines and degranulation. a. Generation of GM-CSF knockout
CAR-Ts. (See Example 6).
[0049] FIGS. 16A-16J illustrate that GM-CSF neutralizing antibody
in accordance with embodiments described herein does not inhibit
CAR-T mediated killing, proliferation, or cytokine production but
successfully neutralizes GM-CSF (See Example 7).
[0050] FIGS. 17A-17B illustrate the protocol and results from a
mouse model of human CRS. (Example 5).
[0051] FIGS. 18A-18C illustrate CAR-T efficacy in a xenograft model
in combination with a GM-CSF neutralizing antibody in accordance
with embodiments described herein. The GM-CSF neutralizing antibody
is shown to not inhibit CAR-T efficacy in vivo. (See Example
8).
[0052] FIG. 19 illustrates in vitro and in vivo preclinical data
showing that a GM-CSF neutralizing antibody in accordance with
embodiments described herein did not impair CAR-T impact on
survival. The GM-CSF neutralizing antibody does not impede CAR-T
cell function in vivo in the absence of PBMCs. Survival was similar
for CAR-T+ control and CAR-T+GM-CSF neutralizing antibody. (See
Example 9).
[0053] FIGS. 20A-20B illustrate in vitro and in vivo preclinical
data showing that a GM-CSF neutralizing antibody in accordance with
embodiments described herein does increase CAR-T expansion. The
GM-CSF neutralizing antibody increases in vitro CAR-T cancer cell
killing. Antibody neutralization of GM-CSF increases proliferation
of CAR-T cells in the presence of PBMCs. CAR-T proliferation
increased by the GM-CSF neutralizing antibody in presence of PBMCs.
(It was not affected without PBMCs). The anti-GM-CSF antibody did
not inhibit CAR-T degranulation, intracellular GM-CSF production,
or IL-2 production. (See Example 10).
[0054] FIG. 21 illustrates that CAR-T expansion is associated with
improved overall response rate. CAR AUC (area under the curve)
defined as cumulative levels of CAR+ cells/.mu.L of blood over the
first 28 days post CAR-T administration. P values calculated by
Wilcoxon rank sum test. (Neelapu, et al ICML 2017 Abstract 8). (See
Example 11).
[0055] FIG. 22 illustrates a study protocol for GM-CSF neutralizing
antibody in accordance with embodiments described herein. (See
Example 12). CRS and NT to be assessed daily while hospitalized and
at clinic visit for first 30 days. Eligible subjects to receive
GM-CSF neutralizing antibody on days -1, +1, and +3 of CAR-T
treatment. Additional dosing can be contemplated going out to at
least day 7. Tumor assessment to be performed at baseline and
months 1, 3, 6, 9, 12, 18, and 24. Blood samples (PBMC and serum)
days -5, -1, 0, 1, 3, 5, 7, 9, 11, 13, 21, 28, 90, 180, 270, and
360. (See Example 12).
[0056] FIGS. 23A-23B illustrate that GM-CSF depletion increases
CAR-T cell expansion. FIG. 23A illustrates an increased ex-vivo
expansion of GM-CSF.sup.k/o CAR-T cells compared to control CAR-T
cells. FIG. 23B illustrates a more robust CAR-T cell proliferation
after treatment with a GM-CSF neutralizing antibody in accordance
with embodiments described herein. (See Example 13).
[0057] FIG. 24 illustrates a safety profile of GM-CSF neutralizing
antibody in accordance with embodiments described herein. (See
Example 14).
[0058] FIGS. 25A-25D illustrate that GM-CSF neutralizing antibody
when added to CAR-T cell therapy demonstrates a 90% reduction in
neuroinflammation in mouse preclinical model. FIG. 25A illustrates
MRI data (T1 hyperintensity indicative of BBB disruption and
neuroinflammation) in which mice brains are protected from
neuroinflammation after administration of CAR-T cells and GM-CSF
neutralizing antibody in accordance with embodiments described
herein compared to mice brains showing signs of neurotoxicity after
administration of CAR-T cells and a control antibody (top row) and
compared to untreated (baseline) mice brains (bottom row). FIG. 25B
quantitatively illustrates the percent increase of T1
hyperintensity from baseline: there was an approximately 10%
percent increase in brain T1 hyperintensity from baseline in mice
administered CAR-T and GM-CSF neutralizing antibody in accordance
with embodiments described herein compared to the slightly over
100% increase in mice that had been administered CAR-T cells and
control antibody. As shown in the comparative graph, the .about.10%
increase in brain T1 hyperintensity from baseline in mice
administered the CAR-T and GM-CSF neutralizing antibody is a 90%
reduction in neuroinflammation, as measured by brain T1
hyperintensity from baseline, compared to the quantity of
neuroinflammation present in mice that received CAR-T cells and
control antibody. FIGS. 25C-25D show that compared to untreated
mice (which had 500,000 to 1.5M leukemic cells) and CAR-T plus
control antibody (which had between 15,000 and 100,000 leukemic
cells), treatment with CAR-T plus GM-CSF neutralizing antibody in
accordance with embodiments described herein led to a significant
reduction in the number of leukemic cells (decreased to between 500
and 5,000 cells) with improved overall disease control (See Example
15).
[0059] FIGS. 26A-26I show that GM-CSF blockade helps control CART19
toxicities and does improve efficacy. FIG. 26A shows CART19 and
lenzilumab treated CART19 are equally effective in survival
outcomes in a high tumor burden NALM6 relapse model compared to UTD
(untransduced T cells) (7-8 mice per group, n=2). FIGS. 26B-26D
show Lenzilumab & anti-mouse GM-CSF antibody-controlled CRS
induced weight loss, neutralized serum human GM-CSF, and reduced
expression of serum mouse MCP-1 (monocyte chemoattractant
protein-1) in a primary ALL xenograft CART19 CRS/NT model (3 mice
per group, * p<0.05). FIG. 26E shows Lenzilumab & anti-mouse
GM-CSF antibody reduced brain inflammation as shown by MRI in a
primary ALL xenograft CART19 CRS/NT model (3 mice per group, *
p<0.05, ** p<0.01). FIGS. 26F-26G show an improved efficacy
of CART19+Lenzilumab treated mice compared to anti-mouse GM-CSF
antibody treated mice, i.e., CART19+ anti-hGM-CSF antibody, showed
reduced CD19+ brain leukemic burden and reduced percentage of brain
macrophages in a primary ALL xenograft CART19 CRS/NT model (3 mice
per group). FIG. 26H shows CRISPR Cas9 K/O of GM-CSF reduces its
expression via intracellular staining in CART19 and UTD with NALM6
stimulation. (Representative experiment, n=2) FIG. 26I shows CART19
and GM-CSF K/O CART19 control tumor burden better than UTD, and an
improved efficacy of GM/CSF K/O CART19 cells controlling tumor
burden slightly better than CART19 in a high tumor burden NALM6
relapse model (6 mice per group, * p<0.05, **** p<0.0001).
Error bars SEM.
[0060] FIGS. 27A-27D show GM-CSF neutralization in vitro enhances
CAR-T cell proliferation in the presence of monocytes and does not
impair CAR-T cell effector function. FIG. 27A graphically depicts
Lenzilumab (a hGM-CSF neutralizing antibody) neutralization of
CAR-T cell produced hGM-CSF in vitro compared to isotype control
treatment as assayed by multiplex after 3 days of culture with
CART19 in media alone or CART19 co-cultured with NALM6, n=2
experiments, 2 replicates per experiment, representative experiment
depicted, *** p<0.001 between lenzilumab and isotype control
treatment, t test, mean.+-.SEM. FIG. 27B graphically shows that
hGM-CSF neutralizing antibody treatment did not inhibit the ability
of CAR-T cells to proliferate as assayed by CSFE flow cytometry
proliferation assay of live CD3 cells, n=3 donors, 2 replicates per
donor, representative experiment at 3-day time point depicted, ns
p>0.05 between lenzilumab and isotype control treatment, t test,
mean.+-.SEM. Alone: CART19 in media alone, MOLM13: CART19+MOLM13,
PMA/ION: CART19 plus 5 ng/mL PMA and 0.1 ug/mL ION, NALM6:
CART19+NALM6. FIG. 27C graphically depicts Lenzilumab enhancing the
proliferation of CART19 by neutralization of hGM-CSF compared to
isotype control treated with CART19 when co-cultured with human
monocytes, n=3 donors at 3-day time point, 2 replicates per donor,
**** p<0.0001, mean.+-.SEM. FIG. 27D graphically shows that
Lenzilumab treatment did not inhibit cytotoxicity of CART19 or
untransduced T cells (UTD) when cultured with NALM6, n=3 donors, 2
replicates per donor, representative experiment at 48 hr time point
depicted, ns p>0.05 between lenzilumab and isotype control
treatment, t test, mean.+-.SEM.
[0061] FIGS. 28A-28F demonstrate that GM-CSF neutralization in vivo
enhances CAR-T cell anti-tumor activity (i.e., tumor cell killing)
in xenograft models. FIG. 28 A illustrates the experimental schema:
NSG mice were injected with the CD19+ luciferase+ cell line NALM6
(1.times.106 cells per mouse I.V). 4-6 days later, mice were
imaged, randomized, and received 1-1.5.times.106CAR-T19 or
equivalent number of total cells of control UTD cells the following
day with either lenzilumab or control IgG (10 mg/Kg, given IP daily
for 10 days, starting on the day of CAR-T injection). Mice were
followed with serial bioluminescence imaging to assess disease
burden beginning day 7 post CAR-T cell injection and were followed
for overall survival. Tail vein bleeding was performed 7-8 days
after CAR-T cell injection. FIG. 28B depicts Lenzilumab
neutralization of CAR-T produced serum hGMCSF in vivo compared to
isotype control treatment as assayed by hGM-CSF singleplex, n=2
experiments, 7-8 mice per group, representative experiment, serum
from day 8 post CAR-T cell/UTD injection, *** p<0.001 between
lenzilumab and isotype control treatment, t test, mean.+-.SEM. FIG.
28C graphically depicts Lenzilumab treated CAR-T in vivo are
equally effective at controlling tumor burden compared to isotype
control treated CAR-T in a high tumor burden relapse xenograft
model of ALL, day 7 post CAR-T injection, n=2 experiments, 7-8 mice
per group, representative experiment depicted, *** p<0.001, *
p<0.05, ns p>0.05, t test, mean.+-.SEM. FIG. 28 D depicts
mouse images from FIG. 28C. FIG. 28E illustrates the experimental
schema: NSG mice were injected with the blasts derived from
patients with ALL (1.times.106 cells per mouse I.V). Mice were bled
serially and when the CD19+ cells >1/uL, mice were randomized to
receive 2.5.times.106 CART19 with either lenzilumab or control IgG
(10 mg/Kg, given IP daily for 10 days, starting on the day of CAR-T
injection). Mice were followed with serial tail vein bleeding to
assess disease burden beginning day 14 post CAR-T cell injection
and were followed for overall survival. FIG. 28F graphically
depicts that Lenzilumab treatment with CAR-T therapy results in
more sustained control of tumor burden over time in a primary acute
lymphoblastic leukemia (ALL) xenograft model compared to isotype
control treatment with CAR-T therapy, 6 mice per group, **
p<0.01, * p<0.05, ns p>0.05, t test, mean.+-.SEM.
[0062] FIGS. 29A-29E demonstrate that GM-CSF CRISPR knockout CAR-T
cells exhibit reduced expression of GM-CSF, similar levels of key
cytokines and chemokines, and enhanced anti-tumor activity. FIG.
29A illustrates that the CRISPR Cas9 GM-CSF.sup.k/o CART19 exhibit
reduced GM-CSF production compared to wild type CART19, but other
cytokine production and degranulation are not inhibited by the
GM-CSF gene disruption, CART19 and GM-CSF.sup.k/o CART19 stimulated
with NALM6, n=3 experiments, 2 replicates per experiment, ***
p<0.001, * p<0.05, ns p>0.05 comparing GM-CSF.sup.k/o
CART19 and CAR19, t test, mean.+-.SEM. FIG. 29B illustrates that
GM-CSF.sup.k/o CAR-T have reduced serum human GM-CSF in vivo
compared to CAR-T treatment as assayed by multiplex, 5-6 mice per
group (4-6 at time of bleed, 8 days post CAR-T cell injection),
**** p<0.0001, *** p<0.001 between GM-CSFk/o CART19 and wild
type CART19, t test, mean.+-.SEM. FIG. 29C illustrates that
GM-CSF.sup.k/o CART19 in vivo enhances overall survival compared to
wild type CART19 in a high tumor burden relapse xenograft model of
ALL utilizing a NALM6 cell line, 5-6 mice per group, ** p<0.01,
log-rank. FIGS. 29D-29E show human (FIG. 29D) and mouse (FIG. 29E)
cytokines and chemokines from multiplex of serum, other than
hGM-CSF, show no statistical differences between the GM-CSF.sup.k/o
CART19 and wild type CART19, further implicating critical T-cell
cytokines and chemokines are not adversely depleted by reducing
GM-CSF expression, 5-6 mice per group (4-6 at time of bleed), ****
p<0.0001, t test.
[0063] FIGS. 30A-30D illustrate a patient derived xenograft model
for neuro-inflammation and cytokine release syndrome. FIG. 30A
shows the experimental schema: Mice received 1-3.times.106 primary
blasts derived from the peripheral blood of patients with primary
ALL. Mice were monitored for engraftment for .about.10-13 weeks via
tail vein bleeding. When serum CD19+ cells were .gtoreq.10
cells/uL, the mice received CART19 (2-5.times.106 cells) and
commenced antibody therapy for a total of 10 days, as indicated.
Mice were weighed on a daily basis as a measure of their wellbeing.
Mouse brain MRIs were performed 5-6 days post CART19 injection and
tail vein bleeding for cytokine/chemokine and T cell analysis was
performed 4-11 days post CART19 injection, 2 independent
experiments. FIG. 30B illustrates that a combination of GM-CSF
neutralization with CART19 is equally effective as isotype control
antibodies combined with CART19 in controlling CD19+ burden of ALL
cells, representative experiment, 3 mice per group, 11 days post
CART19 injection, * p<0.05 between GM-CSF neutralization+CART19
and isotype control+CART19, t test, mean.+-.SEM. FIG. 30C
illustrates brain MRI data showing CART19 therapy exhibits T1
enhancement, suggestive of brain blood-brain barrier disruption and
possible edema. 3 mice per group, 5-6 days post CART19 injection,
representative image. FIG. 30D illustrates high tumor burden
primary ALL xenografts treated with CART19 show human CD3 cell
infiltration of the brain compared to untreated PDX controls. 3
mice per group, representative image.
[0064] FIG. 31 shows the canonical pathways altered in brains from
patient derived xenografts after treatment with CART19 cells. Red
boxes indicate upregulation of genes in CART19 plus isotype control
treated mice compared to the untreated patient derived
xenografts.
[0065] FIGS. 32A-32D demonstrate GM-CSF neutralization in vivo
ameliorates CRS after CART19 therapy in a xenograft model. FIG. 32A
shows Lenzilumab and anti-mouse GM-CSF antibody prevent CRS induced
weight loss compared to mice treated with CART19 and isotype
control antibodies, 3 mice per group, 2-way anova, mean.+-.SEM.
FIG. 32B shows human GM-CSF was neutralized in patient derived
xenografts treated with lenzilumab and mouse GM-CSF neutralizing
antibody, 3 mice per group, *** p<0.001, * p<0.05, t test,
mean.+-.SEM. FIG. 32C shows human cytokine/chemokine heat map
(serum collected 11 days after CART19 injection) exhibits increases
in cytokines and chemokines typical of CRS after CART19 treatment.
GMCSF neutralization results in a significant decrease in several
cytokines and chemokines compared to mice treated with CART19 and
isotype control antibodies, including several myeloid associated
cytokines and chemokines, as indicated in the panel, 3 mice per
group, serum from day 11 post CART19 injection, *** p<0.001, **
p<0.01, * p<0.05, comparing GM-CSF neutralizing antibody
treated and isotype control treated mice that received CAR-T cell
therapy, t test. FIG. 32D shows mouse cytokine/chemokine heat map
(serum collected 11 days after CART19 injection) exhibit increase
in mouse cytokines and chemokines typical of CRS after CART19
treatment. GM-CSF neutralization results in a significant decrease
in several cytokines and chemokines compared to treatment with
CART19 with control antibodies, including several myeloid
differentiating cytokines and chemokines, as indicated in the
panel, 3 mice per group, serum from day 11 post CART19 injection, *
p<0.05, comparing GM-CSF neutralizing antibody treated and
isotype control treated mice that received CAR-T cell therapy, t
test.
[0066] FIGS. 33A-33D demonstrate GM-CSF neutralization in vivo
ameliorates neuro-inflammation after CART19 therapy in a xenograft
model. FIGS. 33A-33B depict gadolinium enhanced T1-hyperintensity
(cubic mm) MRI showed that GM-CSF neutralization helped reduced
brain inflammation, blood-brain barrier disruption, and possible
edema compared to isotype control (A) representative images, (33B)
3 mice per group, ** p<0.01, * p<0.05, 1-way ANOVA, mean+SD.
FIG. 33C shows human CD3 T cells were present in the brain after
treatment with CART19 therapy. GM-CSF neutralization resulted in a
trend toward decreased CD3 infiltration in the brain as assayed by
flow cytometry in brain hemispheres, 3 mice per group, mean.+-.SEM.
FIG. 33D depicts CD11b+ bright macrophages were decreased in the
brains of mice receiving GM-CSF neutralization during CAR-T therapy
compared to isotype control during CAR-T therapy as assayed by flow
cytometry in brain hemispheres, 3 mice per group, mean.+-.SEM.
[0067] FIGS. 34A(i)-34B illustrate the generation of GM-CSF.sup.k/o
CART19 cells. FIGS. 34A(i)-34A(iv) show the experimental schema;
FIG. 34B shows the gRNA sequence and primer sequences for
generation of GM-CSF.sup.k/o CART19. To generate GM-CSF.sup.k/o
CART19 cells, gRNA was cloned into a Cas9 lentivirus vector under
the control of a U6 promotor and used for lentivirus production. T
cells derived from normal donors were stimulated with CD3/CD28
beads and dual transduced with CAR19 virus and CRISPR/Cas9 virus 24
hours later. CD3/CD28 magnetic bead removal was performed on Day
+6, and GM-CSF.sup.k/o CART19 cells or control CART19 cells were
cryopreserved on Day 8.
[0068] FIG. 35 shows a flow chart for procedures used in RNA
sequencing. The binary base call data was converted to fastq using
Illumina bcl2fastq software. The adapter sequences were removed
using Trimmomatic, and FastQC was used to check for quality. The
latest human (GRCh38) and mouse (GRCm38) reference genomes were
downloaded from NCBI. Genome index files were generated using STAR,
and the paired end reads were mapped to the genome for each
condition. HTSeq was used to generate expression counts for each
gene, and DeSeq2 was used to calculate differential expression.
Gene ontology was assessed using Enrichr.
[0069] FIG. 36 shows that Lenzilumab plus CAR-T cell treated mice
have comparable survival compared to isotype control antibody plus
CAR-T cell treated mice in a high tumor burden relapse xenograft
model of ALL. n=2 experiments, 7-8 mice per group, representative
experiment depicted, **** p<0.0001, *** p<0.001, * p<0.05,
log-rank.
[0070] FIG. 37 shows a representative TIDE sequence to verify
genome alteration in the GM-CSF CRISPR Cas9 knockout CAR-T cells.
n=2 experiments, representative experiment depicted.
[0071] FIG. 38 shows GM-CSF knockout CAR-T cells in vivo show
slightly enhanced control of tumor burden compared to wild type
CAR-T cells in a high tumor burden relapse xenograft model of ALL.
Days post CAR-T cell injection listed on x-axis, 5-6 mice per group
(2 remained in UTD group at day 13), representative experiment
depicted, **** p<0.0001, * p<0.05, 2-way ANOVA,
mean.+-.SEM.
[0072] FIG. 39 demonstrates a patient derived xenograft model for
neuro-inflammation and CRS with CART19+ anti-hGM-CSF antibody
treatment. High tumor burden primary ALL xenografts treated CART19+
anti-hGM-CSF antibody treatment show human CD3 cell infiltration of
the brain (FIG. 39) compared to untreated PDX controls (FIG. 30D).
3 mice per group, representative image.
[0073] FIGS. 40A-40B show that BBB integrity is preserved and
neuro-inflammation is significantly reduced following CAR-T and
Lenzilumab therapy. FIG. 40A shows confocal microscopy distinctly
showing in high resolution images that following CAR-T therapy, the
BBB is significantly impaired and shows maintenance of the
integrity of the BBB with CAR-T and Lenzilumab therapy. FIG. 40B is
adapted from Santomasso, B D, et al., published OnlineFirst on Jun.
7, 2018; DOI: 10.1158/2159-8290.CD-17-1319, and is incorporated by
reference in its entirety, shows high levels of protein in the CSF
(as shown in Santomasso's data) is an indication of BBB disruption
and protein leak into the CNS.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The present subject matter may be understood more readily by
reference to the following detailed description which forms a part
of this disclosure. It is to be understood that this disclosure is
not limited to the specific products, methods, conditions or
parameters described and/or shown herein, and that the terminology
used herein is for the purpose of describing particular embodiments
by way of example only and is not intended to be limiting of the
claimed disclosure.
Immunotherapy-Related Toxicity
[0075] A skilled artisan would appreciate that the term
"immunotherapy-related toxicity" refers to a spectrum of
inflammatory symptoms resulting from high levels of immune
activation. Different types of toxicity are associated with
different immunotherapy approaches. In some embodiments,
immunotherapy-related toxicity comprises capillary leak syndrome,
cardiac disease, respiratory disease, CAR-T-cell-related
encephalopathy syndrome (CRES), neurotoxicity, colitis,
convulsions, cytokine release syndrome (CRS), cytokine storm,
decreased left ventricular ejection fraction, diarrhea,
disseminated intravascular coagulation, edema, encephalopathy,
exanthema, gastrointestinal bleeding, gastrointestinal perforation,
hemophagocytic lymphohistiocytosis (HLH), hepatosis, hypertension,
hypophysitis, immune related adverse events, immunohepatitis,
immunodeficiencies, ischemia, liver toxicity, macrophage-activation
syndrome (MAS), pleural effusions, pericardial effusions,
pneumonitis, polyarthritis, posterior reversible encephalopathy
syndrome (PRES), pulmonary hypertension, thromboembolism, and
transaminitis.
[0076] While different types of toxicities differ in their
pathophysiology and clinical manifestations, they are usually
associated with an increase in inflammation-associated factors,
such as C-reactive protein, GM-CSF, IL-1, IL-2, sIL-2R.alpha.,
IL-5, IL-6, IL-8, IL-10, IP10, IL-15, MCP-1 (AKA CCL2), MIG,
MIP-1.beta., IFN.gamma., CX3CR1, or TNF.alpha.. A skilled artisan
would appreciate that, in some embodiments, the term
"inflammation-associated factor" comprises molecules, small
molecules, peptides, gene transcripts, oligonucleotides, proteins,
hormones, and biomarkers that are affected during inflammation. A
skilled artisan would appreciate that systems affected during
inflammation comprises upregulation, downregulation, activation,
de-activation, or any kind of molecular modification. The serum
concentration of inflammation-associated factors, such as
cytokines, can be used as an indicator of immunotherapy-related
toxicities, and may be expressed as -fold increase, percent (%)
increase, net increase or rate of change in cytokine levels or
concentration. The concentration of inflammation-associated factors
in body fluids other than serum can also be used as indicators of
immunotherapy-related toxicities. In some embodiments, absolute
cytokine levels or concentrations above a certain level or
concentration may be an indication of a subject undergoing or about
to experience an immunotherapy-related toxicity. In another
embodiment, absolute cytokine levels or concentration at a certain
level, for example a level or concentration normally found in a
control subject, may be an indication of a method for inhibiting or
reducing the incidence of an immunotherapy-related toxicity in a
subject. A skilled artisan would appreciate that the term "cytokine
level" may encompass a measure of concentration, a measure of fold
change, a measure of percent (%) change, or a measure of rate
change. Further, the methods for measuring cytokines in blood,
cerebrospinal fluid (CSF), saliva, serum, urine, and plasma are
well known in the art.
[0077] A number of approaches have been elaborated to classify the
type of neurotoxicity and manage it accordingly. These
classifications are based on clinical and biological symptoms, as
fever, hypotension, hypoxia, organ toxicity, cardiac dysfunction,
respiratory dysfunction, gastrointestinal dysfunction, hepatic
dysfunction, renal dysfunction, coagulopathy, seizure presence,
intracranial pressure, muscle tone, motor performance, ferritin
levels, and haemagophagocytosis. Similarly, each type of
neurotoxicity can be graded according to its severity. Table 1A
(taken from Cellular Therapy Implementation: the MDACC Approach, P.
Kebriaei, Feb. 24, 2017) discloses a method for grading
neurotoxicity according to its severity into Grade 1, Grade 2,
Grade 3, and Grade 4. However, some of the foregoing symptoms are
not typically associated with neurotoxicity. (Lee, et al., Blood
2014; 124:188-195, which is incorporated in its entirety herein by
reference.).
TABLE-US-00001 TABLE 1A Method for Grading Neurotoxicity--Criteria
for Adverse Events (CTCAE) Symptom or sign Grade 1 Grade 2 Grade 3
Grade 4 Level of Mild Moderate somnolence, Obtundation
Life-threatening consciousness drowsiness/ limiting instrumental or
stupor needing urgent sleepiness ADL intervention or mechanical
ventilation Orientation/ Mild Moderate disorientation, Severe
Life-threatening Confusion disorientation/ limiting instrumental
disorientation, needing urgent confusion ADL limiting self-
intervention or care ADL mechanical ventilation ADL/ Mild limiting
Limiting instrumental Limiting self- Life-threatening
Encephalopathy of ADL ADL care ADL needing urgent intervention or
mechanical ventilation Speech Dysphasia Dysphasia with moderate
Severe -- not impairing impairment in ability receptive or ability
to to communicate expressive communicate spontaneously dysphasia,
impairing ability to read, write or communicate intelligibly
Seizure Brief partial Brief generalized seizure Multiple
Life-threatening; seizure; no seizures prolonged repetitive loss of
despite seizures consciousness medical intervention Incontinent
Bowel/bladder or motor incontinence; weakness Weakness limiting
selfcare ADL, disabling MD Anderson Mild (7-9) Moderate (3-6)
Severe (1-2), Critical (Obtunded; Cancer Center grade 1 and 2
convulsive status (MDACC) papilledema epilepticus; motor 10-point
with CSF weakness, grade 3, Neurotoxicity opening 4 & 5
papilledema, grade pressure (op) CSF op .gtoreq. 20 mm <20 mm Hg
Hg, cerebral edema)
[0078] Patients with body temperature above 38.9.degree. C., JL-6
serum concentration above 16 pg/ml, or MCP-1 (AKA CCL2) serum
concentration above 1,343.5 pg/ml in the first 36 hours after
immunotherapy infusion had higher probabilities of developing
severe neurotoxicity (Gust, et al. Cancer Discov. 2017 Oct.
12).
[0079] CRS is a serious condition and life-threatening adverse
effect because of abnormal cytokine regulation and thus, severe
inflammation. Symptoms can include, without limitation, fever,
disordered heartbeat and breathing, nausea, vomiting, and seizures.
CRS can be graded by assessing symptoms and their severities, such
as, for example: Grade 1 CRS: Fever, constitutional symptoms; Grade
2 CRS: Hypotension--responds to fluids or one low dose pressor,
Hypoxia--responds to <40% O.sub.2, Organ toxicity; grade 2;
Grade 3 CRS: Hypotension--requires multiple pressors or high dose
pressors, Hypoxia--requires .gtoreq.40% O.sub.2, Organ
toxicity--grade 3, grade 4 transaminitis; Grade 4 CRS: Mechanical
ventilation, Organ toxicity--grade 4, excluding transaminitis.
(Lee, et al., Blood 2014; 124:188-195, which is incorporated in its
entirety herein by reference.).
[0080] CRES can be graded, for example, by combining neurological
assessment with other parameters as papilloedema, CSF opening
pressure, imaging assessment, and the presence of seizures and
motor weakness. A method for grading CRES is described in Neelapu
et al., Nat Rev Clin Oncol. 15(1):47-62 (2018) (Epub 2017 Sep. 19),
which is incorporated in its entirety herein by reference. Table 1B
(taken from Neelapu et al., Nat Rev Clin Oncol. 15(1):47-62 (2018))
discloses a method for grading CRES according to its severity into
Grade 1, Grade 2, Grade 3, and Grade 4.
TABLE-US-00002 TABLE 1B Method for grading CRES. In CARTOX-10, a
point is assigned for each of the following tasks performed
correctly: orientation to year, month, city, hospital, and
President/Prime Minister of country of residence (1 point for
each); naming three objects (1 point for each); writing a standard
sentence counting backwards from 100 in tens. Symptom or sign Grade
1 Grade 2 Grade 3 Grade 4 Neurological 7-9 (mild 3-6 0-2 (severe
Patient in assessment impairment) (moderate impairment) critical
score (by impairment) condition, CARTOX- and/or 10) obtunded and
cannot perform assessment of tasks Raised NA NA Stage 1-2 Stage 3-5
intracranial papilloedema, papilloedema, pressure or CSF or CSF
opening opening pressure pressure .gtoreq.20 <20 mmHg mmHg, or
cerebral oedema Seizures or NA NA Partial seizure, Generalized
motor or non- seizures, or weakness convulsive convulsive or
seizures on non- EEG with convulsive response to status
benzodiazepine epilepticus, or new motor weakness
[0081] NT, CRS, and CRES manifestations can include encephalopathy,
headaches, delirium, anxiety, tremor, seizure activity, confusion,
alterations in wakefulness, decreased level of consciousness,
hallucinations, dysphasia, aphasia, ataxia, apraxia, facial nerve
palsy, motor weakness, seizures, nonconvulsive EEG seizures,
cerebral edema, and coma. CRES is associated with elevated
concentrations of circulating cytokines, as C-reactive protein,
GM-CSF, IL-1, IL-2, sIL2R.alpha., IL-5, IL-6, IL-8, IL-10, IP10,
IL-15, MCP-1, MIG, MIP1.beta., IFN.gamma., CX3CR1, and
TNF.alpha..
[0082] The cytokine concentration gradient between serum and CSF
observed in normal conditions is reduced or lost during CRES.
Additionally, CAR T-cells and high protein concentrations are
observed in the CSF of patients and is correlated with the severity
of the condition. All this indicates a blood-brain barrier
dysfunction following immunotherapy. Increased vascular
permeability can be partially explained by increased concentrations
of ANG2 and increased ANG2:ANG1 ratio in patients with
neurotoxicity. While ANG1 induces endothelial cell quiescence, ANG2
causes endothelial cell activation and microvascular permeability.
Patients with increased endothelial activation before immunotherapy
were reported to have higher probability of suffering neurotoxicity
(Gust, et al. Cancer Discov. 2017 Oct. 12).
[0083] Hemophagocytic lymphohistiocytosis (HLH) comprises severe
hyperinflammation caused by uncontrolled proliferation of benign
lymphocytes and macrophages that secrete high amounts of
inflammatory cytokines. In some embodiments, HLH can be classified
as one of the cytokine storm syndromes. In some embodiments, HLH
occurs after strong immunologic activation, such as systemic
infections, immunodeficiency, malignancies. or immunotherapy. In
some embodiments, the term "HLH" may be used interchangeably with
the terms "hemophagocytic lymphohistiocytosis", "hemophagocytic
syndrome", or "hemophagocytic syndrome" having all the same
qualities and meanings.
[0084] Primary HLH comprises a heterogeneous autosomal recessive
disorder. Patients with homozygous mutations in one of several
genes, exhibit loss of function of proteins involved in cytolytic
granule exocytosis. In some embodiments, HLH can present in infancy
with minimal or no trigger. Secondary HLH, or acquired HLH, occurs
after strong immunologic activation, such as that which occurs with
systemic infection, immunodeficiency, an underlying malignancy, or
immunotherapies. Both forms of HLH are characterized by an
overwhelming activation of normal T lymphocytes and macrophages,
invariably leading to clinical and haematologic alterations and
death in the absence of treatment.
[0085] In some embodiments, HLH can be initiated by viral
infections, EBV, CMV, parvovirus, HSV, VZV, HHV8, HIV, influenza,
hepatitis A, hepatitis B, hepatitis C, bacterial infections,
gram-negative rods, Mycoplasma species and Mycobacterium
tuberculosis, parasitic infections, Plasmodium species, Leishmania
species, Toxoplasma species, fungal infections, Cryptococcal
species, Candidal species and Pneumocystis species, among
others.
[0086] Macrophage-activation syndrome (MAS) comprises a condition
comprising uncontrolled activation and proliferation of
macrophages, and T lymphocytes, with a marked increase in
circulating cytokine levels, such as IFN.gamma., and GM-CSF. MAS is
closely related to secondary HLH. MAS manifestations include high
fever, hepatosplenomegaly, lymphadenopathy, pancytopenia, liver
dysfunction, disseminated intravascular coagulation,
hemophagocytosis, hypofibrinogenemia, hyperferritinemia, and
hypertriglyceridemia.
[0087] CRS comprises a non-antigen-specific immune response similar
to that found in severe infection. CRS is characterized by any or
all of the following symptoms: fever with or without rigors,
malaise, fatigue, anorexia, myalgias, arthalgias, nausea, vomiting,
headache, skin rash, diarrhea, tachypnea, hypoxemia, hypoxia,
shock, cardiovascular tachycardia, widened pulse pressure,
hypotension, capillary leak, increased cardiac output (early),
potentially diminished cardiac output (late), elevated D-dimer,
hypofibrinogenemia with or without bleeding, azotemia,
transaminitis, hyperbilirubinemia, headache, mental status changes,
confusion, delirium, word finding difficulty or frank aphasia,
hallucinations, tremor, dysmetria, altered gait, seizures, organ
failure, multi-organ failure. Deaths have also been reported.
Severe CRS has been reported to occur in up to 60% of patients
receiving CAR-T19.
[0088] Cytokine storm comprises an immune reaction consisting of a
positive feedback loop between cytokines and white blood cells,
with highly elevated levels of various cytokines. The term
"cytokine storm" may be used interchangeably with the terms
"cytokine cascade" and "hypercytokinemia" having all the same
qualities and meanings. In some embodiments, a cytokine storm is
characterized by IL-2 release and lymphoproliferation. Cytokine
storm leads to potentially life-threatening complications including
cardiac dysfunction, adult respiratory distress syndrome,
neurologic toxicity, renal and/or hepatic failure, and disseminated
intravascular coagulation.
[0089] As noted, CAR-T cell therapy is currently limited by the
risk of life-threatening neurotoxicity and CRS. Despite active
management, all CAR-T responders experience some degree of CRS. Up
to 50% of patients treated with CD19 CAR-T have at least Grade 3
CRS or neurotoxicity. GM-CSF levels and T-cell expansion are the
factors most associated with grade 3 or higher CRS and
neurotoxicity.
[0090] Reducing or eliminating CRS and neurotoxicity in
immunotherapies such as CAR-T cell therapy is of great value and it
is crucial to determine what is driving or exacerbating the
signature CAR-T inflammatory response. Although many cytokines,
signaling molecules, and cell types are involved in this pathway,
GM-CSF is the one cytokine that appears to be at the center of the
pathway. Normally undetectable in human serum, it is central to the
cyclical positive feedback loop that drives inflammation to the
extremes of cytokine storms and endothelial cell activation.
Neurotoxicity and cytokine storms are not the result of a
simultaneous release of cytokines, but rather a cascade of
inflammation initiated by GM-CSF resulting in the trafficking and
recruitment of myeloid cells to the tumor site. These myeloid cells
produce the cytokines observed in CRS and neurotoxicity,
perpetuating the inflammatory cascade.
Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF)
[0091] As used herein, "Granulocyte Macrophage-Colony Stimulating
Factor" (GM-CSF) refers to a small, naturally occurring
glycoprotein with internal disulfide bonds having a molecular
weight of approximately 23 kDa. In some embodiments, GM-CSF refers
to human GM-CSF. In some embodiments, GM-CSF refers to non-human
GM-CSF. In humans, it is encoded by a gene located within the
cytokine cluster on human chromosome 5. The sequence of the human
gene and protein are known. The protein has an N-terminal signal
sequence, and a C-terminal receptor binding domain (Rasko and Gough
In: The Cytokine Handbook, A. Thomson, et al, Academic Press, New
York (1994) pages 349-369). Its three-dimensional structure is
similar to that of the interleukins, although the amino acid
sequences are not similar. GM-CSF is produced in response to a
number of inflammatory mediators by mesenchymal cells present in
the hemopoietic environment and at peripheral sites of
inflammation. GM-CSF is able to stimulate the production of
neutrophilic granulocytes, macrophages, and mixed
granulocyte-macrophage colonies from bone marrow cells and can
stimulate the formation of eosinophil colonies from fetal liver
progenitor cells. GM-CSF can also stimulate some functional
activities in mature granulocytes and macrophages. GM-CSF, a
cytokine present in the bone marrow microenvironment, recruits
inflammatory monocyte-derived dendritic cells, stimulates the
secretion of high levels of IL-6 and CCL2/MCP-1, and leads to a
feedback loop, recruiting more monocytes, inflammatory dendritic
cells to inflammatory sites.
[0092] As noted, CRS involves the increase of several cytokines and
chemokines, including IFN-.gamma., IL-6, IL-8, CCL2 (MCP-1), CCL3
(MIP1.alpha.), and GM-CSF. (Teachey, D. et al. (June 2016), Cancer
Discovery, CD-16-0040; Morgan R., et al., (April 2010), Molecular
Therapy.). IL-6, one of the key inflammatory cytokines, is not
produced by CAR-T cells. (Barrett, D. et al. (2016), Blood).
Instead, it is produced by myeloid cells, which are recruited to
the tumor site. GM-CSF mediates this recruitment, which induces
chemokine production that activates myeloid cells and causes them
to traffic to the tumor site. Elevated GM-CSF levels serve as both
a predictive biomarker for CRS and an indicator of its severity.
More than a critical component of the inflammation cascade, GM-CSF
is the key initiator, responsible for both CRS and NT. As described
herein, in vivo studies using murine models indicate that genetic
silencing of GM-CSF prevents cytokine storm--while still
maintaining CAR-T efficacy. GM-CSF knockout mice have normal levels
of INF-7, IL-6, IL-10, CCL2 (MCP1), CCL3/4 (MIG-1) in vivo and do
not develop CRS. (Sentman, M.-L., et al (2016), The Journal of
Immunology, 197(12), 4674-4685.). GM-CSF knockout CAR-T models
recruit fewer NK cells, CD8 cells, myeloid cells, and neutrophils
to the tumor site in comparison to GM-CSF+ CAR-T.
[0093] The term "soluble granulocyte macrophage-colony stimulating
factor receptor" (sGM-CSFR) refers to a non-membrane bound receptor
that binds GM-CSF, but does not transduce a signal when bound to
the ligand.
[0094] As used herein, a "peptide GM-CSF antagonist" refers to a
peptide that interacts with GM-CSF, or its receptor, to reduce or
block (either partially or completely) signal transduction that
would otherwise result from the binding of GM-CSF to its cognate
receptor expressed on cells. GM-CSF antagonists may act by reducing
the amount of GM-CSF ligand available to bind the receptor (e.g.,
antibodies that once bound to GM-CSF increase the clearance rate of
GM-CSF) or prevent the ligand from binding to its receptor either
by binding to GM-CSF or the receptor (e.g., neutralizing
antibodies). GM-CSF antagonists may also include other peptide
inhibitors, which may include polypeptides that bind GM-CSF or its
receptor to partially or completely inhibit signaling. A peptide
GM-CSF antagonist can be, e.g., an antibody; a natural or synthetic
GM-CSF receptor ligand that antagonizes GM-CSF, or other
polypeptides. An exemplary assay to detect GM-CSF antagonist
activity is provided in Example 1. Typically, a peptide GM-CSF
antagonist, such as a neutralizing antibody, has an EC50 of 10 nM
or less.
[0095] A "purified" GM-CSF antagonist as used herein refers to a
GM-CSF antagonist that is substantially or essentially free from
components that normally accompany it as found in its native state.
For example, a GM-CSF antagonist such as an anti-GM-CSF antibody
that is purified from blood or plasma is substantially free of
other blood or plasma components such as other immunoglobulin
molecules. Purity and homogeneity are typically determined using
analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high-performance liquid chromatography. A
protein that is the predominant species present in a preparation is
substantially purified. Typically, "purified" means that the
protein is at least 85% pure, more preferably at least 95% pure,
and most preferably at least 99% pure relative to the components
with which the protein naturally occurs.
Antibodies
[0096] As used herein, an "antibody" refers to a protein
functionally defined as a binding protein and structurally defined
as comprising an amino acid sequence that is recognized by one of
skill as being derived from the framework region of an
immunoglobulin-encoding gene of an animal that produces antibodies.
An antibody can consist of one or more polypeptides substantially
encoded by immunoglobulin genes or fragments of immunoglobulin
genes. The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as well as myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0097] A typical immunoglobulin (antibody) structural unit is known
to comprise a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains, respectively.
[0098] The term "antibody" includes antibody fragments that retain
binding specificity. For example, there are a number of well
characterized antibody fragments. Thus, for example, pepsin digests
an antibody C-terminal to the disulfide linkages in the hinge
region to produce F(ab')2, a dimer of Fab which itself is a light
chain joined to VH-CH1 by a disulfide bond. The F(ab')2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region thereby converting the (Fab')2 dimer into a Fab'
monomer. The Fab' monomer is essentially a Fab with part of the
hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven
Press, N.Y. (1993), for a more detailed description of other
antibody fragments). While various antibody fragments are defined
in terms of the digestion of an intact antibody, one of skill will
appreciate that fragments can be synthesized de novo either
chemically or by utilizing recombinant DNA methodology. Thus, the
term antibody, as used herein also includes antibody fragments
either produced by the modification of whole antibodies or
synthesized using recombinant DNA methodologies.
[0099] Antibodies include dimers such as V.sub.H--V.sub.L dimers,
V.sub.H dimers, or V.sub.L dimers, including single chain
antibodies (antibodies that exist as a single polypeptide chain),
such as single chain Fv antibodies (sFv or scFv), in which a
variable heavy and a variable light region are joined together
(directly or through a peptide linker) to form a continuous
polypeptide. The single chain Fv antibody is a covalently linked
V.sub.H-V.sub.L heterodimer which may be expressed from a nucleic
acid including V.sub.H- and V.sub.L-encoding sequences either
joined directly or joined by a peptide-encoding linker (e.g.,
Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988).
While the V.sub.H and V.sub.L are connected to each as a single
polypeptide chain, the V.sub.H and V.sub.L domains associate
non-covalently. Alternatively, the antibody can be another
fragment, such as a disulfide-stabilized Fv (dsFv). Other fragments
can also be generated, including using recombinant techniques. The
scFv antibodies and a number of other structures converting the
naturally aggregated, but chemically separated light and heavy
polypeptide chains from an antibody V region into a molecule that
folds into a three-dimensional structure substantially similar to
the structure of an antigen-binding site and are known to those of
skill in the art (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405,
and 4,956,778). In some embodiments, antibodies include those that
have been displayed on phage or generated by recombinant technology
using vectors where the chains are secreted as soluble proteins,
e.g., scFv, Fv, Fab, (Fab')2 or generated by recombinant technology
using vectors where the chains are secreted as soluble proteins.
Antibodies for use in the invention can also include diantibodies
and miniantibodies.
[0100] Antibodies of the invention also include heavy chain dimers,
such as antibodies from camelids. Since the V.sub.H region of a
heavy chain dimer IgG in a camelid does not have to make
hydrophobic interactions with a light chain, the region in the
heavy chain that normally contacts a light chain is changed to
hydrophilic amino acid residues in a camelid. V.sub.H domains of
heavy-chain dimer IgGs are called VHH domains. Antibodies for use
in the current invention include single domain antibodies (dAbs)
and nanobodies (see, e.g., Cortez-Retamozo, et al., Cancer Res.
64:2853-2857, 2004).
[0101] As used herein, "V-region" refers to an antibody variable
region domain comprising the segments of Framework 1, CDR1,
Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4,
which segments are added to the V-segment as a consequence of
rearrangement of the heavy chain and light chain V-region genes
during B-cell differentiation. A "V-segment" as used herein refers
to the region of the V-region (heavy or light chain) that is
encoded by a V gene. The V-segment of the heavy chain variable
region encodes FR1-CDR1-FR2-CDR2 and FR3. For the purposes of this
invention, the V-segment of the light chain variable region is
defined as extending though FR3 up to CDR3.
[0102] As used herein, the term "J-segment" refers to a subsequence
of the variable region encoded comprising a C-terminal portion of a
CDR3 and the FR4. An endogenous J-segment is encoded by an
immunoglobulin J-gene.
[0103] As used herein, "complementarity-determining region (CDR)"
refers to the three hypervariable regions in each chain that
interrupt the four "framework" regions established by the light and
heavy chain variable regions. The CDRs are primarily responsible
for binding to an epitope of an antigen. The CDRs of each chain are
typically referred to as CDR1, CDR2, and CDR3, numbered
sequentially starting from the N-terminus, and are also typically
identified by the chain in which the particular CDR is located.
Thus, for example, a V.sub.H CDR3 is located in the variable domain
of the heavy chain of the antibody in which it is found, whereas a
V.sub.L CDR1 is the CDR1 from the variable domain of the light
chain of the antibody in which it is found.
[0104] The sequences of the framework regions of different light or
heavy chains are relatively conserved within a species. The
framework region of an antibody, that is the combined framework
regions of the constituent light and heavy chains, serves to
position and align the CDRs in three-dimensional space.
[0105] The amino acid sequences of the CDRs and framework regions
can be determined using various well-known definitions in the art,
e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT),
and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk,
1987, Canonical structures for the hypervariable regions of
immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al.,
1989, Conformations of immunoglobulin hypervariable regions. Nature
342, 877-883; Chothia C. et al., 1992, structural repertoire of the
human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al.,
J. Mol. Biol 1997, 273(4)). Definitions of antigen combining sites
are also described in the following: Ruiz et al., IMGT, the
international ImMunoGeneTics database. Nucleic Acids Res., 28,
219-221 (2000); and Lefranc, M.-P. IMGT, the international
ImMunoGeneTics database. Nucleic Acids Res. January 1; 29(1):207-9
(2001); MacCallum et al, Antibody-antigen interactions: Contact
analysis and binding site topography, J. Mol. Biol., 262 (5),
732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86,
9268-9272 (1989); Martin, et al, Methods Enzymol., 203, 121-153,
(1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et
al, In Sternberg M. J. E. (ed.), Protein Structure Prediction.
Oxford University Press, Oxford, 141-172 1996).
[0106] "Epitope" or "antigenic determinant" refers to a site on an
antigen to which an antibody binds. Epitopes can be formed both
from contiguous amino acids or noncontiguous amino acids juxtaposed
by tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing
solvents whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation. Methods of determining
spatial conformation of epitopes include, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See,
e.g., Epitope Mapping Protocols in Methods in Molecular Biology,
Vol. 66, Glenn E. Morris, Ed (1996).
[0107] The term "binding specificity determinant" or "BSD" as used
in the context of the current invention refers to the minimum
contiguous or non-contiguous amino acid sequence within a CDR
region necessary for determining the binding specificity of an
antibody. In the current invention, the minimum binding specificity
determinants reside within a portion or the full-length of the CDR3
sequences of the heavy and light chains of the antibody.
[0108] As used herein, "anti-GM-CSF antibody" or "GM-CSF antibody"
are used interchangeably to refer to an antibody that binds to
GM-CSF and inhibits GM-CSF receptor binding and activation. Such
antibodies may be identified using any number of art-recognized
assays that assess GM-CSF binding and/or function. For example,
binding assays such as ELISA assays that measure the inhibition of
GM-CSF binding to the alpha receptor subunit may be used.
Cell-based assays for GM-CSF receptor signaling, such as assays
which determine the rate of proliferation of a GM-CSF-dependent
cell line in response to a limiting amount of GM-CSF, are also
conveniently employed, as are assays that measure amounts of
cytokine production, e.g., IL-8 production, in response to GM-CSF
exposure.
[0109] As used herein, "neutralizing antibody" refers to an
antibody that binds to GM-CSF and inhibits signaling by the GM-CSF
receptor, or prevents binding of GM-CSF to its receptor.
[0110] As used herein, "human Granulocyte Macrophage-Colony
Stimulating Factor" (hGM-CSF) refers to a small naturally occurring
glycoprotein with internal disulfide bonds having a molecular
weight of approximately 23 kDa; the source and the target of the
GM-CSF are human; as such, anti-hGM-CSF antibody, as described in
embodiments herein, binds only human and primate GM-CSF, but not
mouse, rat, and other mammalian GM-CSF. The hGM-CSF antibodies, as
described in embodiments herein, neutralize human GM-CSF. In some
embodiments, the hGM-CSF in humans is encoded by a gene located
within the cytokine cluster on human chromosome 5. The sequences of
the human gene and protein are known. The protein has an N-terminal
signal sequence, and a C-terminal receptor binding domain (Rasko
and Gough In: The Cytokine Handbook, A. Thomson, et al., Academic
Press, New York (1994) pages 349-369). Its three-dimensional
structure is similar to that of the interleukins, although the
amino acid sequences are not similar. GM-CSF is produced in
response to a number of inflammatory mediators present in the
hemopoietic environment and at peripheral sites of inflammation.
GM-CSF is able to stimulate the production of neutrophilic
granulocytes, macrophages, and mixed granulocyte-macrophage
colonies from bone marrow cells and can stimulate the formation of
eosinophil colonies from fetal liver progenitor cells. GM-CSF can
also stimulate some functional activities in mature granulocytes
and macrophages and inhibits apoptosis of granulocytes and
macrophages.
[0111] The term "equilibrium dissociation constant" or "affinity"
abbreviated (K.sub.D), refers to the dissociation rate constant
(k.sub.d, time.sup.-1) divided by the association rate constant
(ka, time.sup.-1 M.sup.-1). Equilibrium dissociation constants can
be measured using any known method in the art. The antibodies of
the present invention are high affinity antibodies. Such antibodies
have a monovalent affinity better (less) than about 10 nM, and
often better than about 500 pM or better than about 50 pM as
determined by surface plasmon resonance analysis performed at
37.degree. C. Thus, in some embodiments, the antibodies of the
invention have an affinity (as measured using surface plasmon
resonance), of less than 50 pM, typically less than about 25 pM, or
even less than 10 pM.
[0112] In some embodiments, an anti-GM-CSF antibody of the
invention has a slow dissociation rate with a dissociation rate
constant (kd) determined by surface plasmon resonance analysis at
37.degree. C. for the monovalent interaction with GM-CSF less than
approximately 10.sup.-4 s.sup.-1, preferably less than
5.times.10.sup.-5 s.sup.-1 and most preferably less than 10.sup.-5
s.sup.-1.
[0113] As used herein, "chimeric antibody" refers to an
immunoglobulin molecule in which (a) the constant region, or a
portion thereof, is altered, replaced or exchanged so that the
antigen binding site (variable region) is linked to a constant
region of a different or altered class, effector function and/or
species, or an entirely different molecule that confers new
properties to the chimeric antibody, e.g., an enzyme, toxin,
hormone, growth factor, drug, etc.; or (b) the variable region, or
a portion thereof, is altered, replaced or exchanged with a
variable region, or portion thereof, having a different or altered
antigen specificity; or with corresponding sequences from another
species or from another antibody class or subclass.
[0114] As used herein, "humanized antibody" refers to an
immunoglobulin molecule in CDRs from a donor antibody are grafted
onto human framework sequences. Humanized antibodies may also
comprise residues of donor origin in the framework sequences. The
humanized antibody can also comprise at least a portion of a human
immunoglobulin constant region. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. Humanization can be
performed using methods known in the art (e.g., Jones et al.,
Nature 321:522-525; 1986; Riechmann et al., Nature 332:323-327,
1988; Verhoeyen et al., Science 239:1534-1536, 1988); Presta, Curr.
Op. Struct. Biol. 2:593-596, 1992; U.S. Pat. No. 4,816,567),
including techniques such as "superhumanizing" antibodies (Tan et
al., J. Immunol. 169: 1119, 2002) and "resurfacing" (e.g., Staelens
et al., Mol. Immunol. 43: 1243, 2006; and Roguska et al., Proc.
Natd. Acad. Sci USA 91: 969, 1994).
[0115] A "HUMANEERED.RTM." antibody in the context of this
invention refers to an engineered human antibody having a binding
specificity of a reference antibody. An engineered human antibody
for use in this invention has an immunoglobulin molecule that
contains minimal sequence derived from a donor immunoglobulin. In
some embodiments, the engineered human antibody may retain only the
minimal essential binding specificity determinant from the CDR3
regions of a reference antibody. Typically, an engineered human
antibody is engineered by joining a DNA sequence encoding a binding
specificity determinant (BSD) from the CDR3 region of the heavy
chain of the reference antibody to human V.sub.H segment sequence
and a light chain CDR3 BSD from the reference antibody to a human
V.sub.L segment sequence. A "BSD" refers to a CDR3-FR4 region, or a
portion of this region that mediates binding specificity. A binding
specificity determinant therefore can be a CDR3-FR4, a CDR3, a
minimal essential binding specificity determinant of a CDR3 (which
refers to any region smaller than the CDR3 that confers binding
specificity when present in the V region of an antibody), the D
segment (with regard to a heavy chain region), or other regions of
CDR3-FR4 that confer the binding specificity of a reference
antibody. Methods for engineering human antibodies are provided in
US patent application publication no. 20050255552 and US patent
application publication no. 20060134098.
[0116] The term "human antibody" as used herein refers to an
antibody that is substantially human, i.e., has FR regions, and
often CDR regions, from a human immune system. Accordingly, the
term includes humanized and humaneered antibodies as well as
antibodies isolated from mice reconstituted with a human immune
system and antibodies isolated from display libraries.
[0117] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not normally found in the same
relationship to each other in nature. For instance, the nucleic
acid is typically recombinantly produced, having two or more
sequences, e.g., from unrelated genes arranged to make a new
functional nucleic acid. Similarly, a heterologous protein will
often refer to two or more subsequences that are not found in the
same relationship to each other in nature.
[0118] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, e.g., recombinant cells
express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under-expressed or not expressed at
all. By the term "recombinant nucleic acid" herein is meant nucleic
acid, originally formed in vitro, in general, by the manipulation
of nucleic acid, e.g., using polymerases and endonucleases, in a
form not normally found in nature. In this manner, operable linkage
of different sequences is achieved. Thus, an isolated nucleic acid,
in a linear form, or an expression vector formed in vitro by
ligating DNA molecules that are not normally joined, are both
considered recombinant for the purposes of this invention. It is
understood that once a recombinant nucleic acid is made and
reintroduced into a host cell or organism, it will replicate
non-recombinantly, i.e., using the in vivo cellular machinery of
the host cell rather than in vitro manipulations; however, such
nucleic acids, once produced recombinantly, although subsequently
replicated non-recombinantly, are still considered recombinant for
the purposes of the invention. Similarly, a "recombinant protein"
is a protein made using recombinant techniques, i.e., through the
expression of a recombinant nucleic acid.
[0119] The phrase "specifically (or selectively) binds" to an
antibody or is "specifically (or selectively) immunoreactive with",
refers to a binding reaction where the antibody binds to the
antigen of interest. In the context of this invention, the antibody
typically binds to the antigen, e.g., GM-CSF, with an affinity of
500 nM or less, and has an affinity of 5000 nM or greater, for
other antigens.
[0120] The terms "identical" or percent "identity," in the context
of two or more polypeptide (or nucleic acid) sequences, refer to
two or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues (or nucleotides) that
are the same (i.e., about 60% identity, preferably 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
identity over a specified region, when compared and aligned for
maximum correspondence over a comparison window or designated
region) as measured using a BLAST or BLAST 2.0 sequence comparison
algorithms with default parameters described below, or by manual
alignment and visual inspection (see, e.g., NCBI web site). Such
sequences are then said to be "substantially identical."
"Substantially identical" sequences also includes sequences that
have deletions and/or additions, as well as those that have
substitutions, as well as naturally occurring, e.g., polymorphic or
allelic variants, and man-made variants. As described below, the
preferred algorithms can account for gaps and the like. Preferably,
protein sequence identity exists over a region that is at least
about 25 amino acids in length, or more preferably over a region
that is 50-100 amino acids=in length, or over the length of a
protein.
[0121] A "comparison window", as used herein, includes reference to
a segment of one of the number of contiguous positions selected
from the group consisting typically of from 20 to 600, usually
about 50 to about 200, more usually about 100 to about 150 in which
a sequence may be compared to a reference sequence of the same
number of contiguous positions after the two sequences are
optimally aligned. Methods of alignment of sequences for comparison
are well-known in the art. Optimal alignment of sequences for
comparison can be conducted, e.g., by the local homology algorithm
of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the
homology alignment algorithm of Needleman & Wunsch, J. Mol.
Biol. 48:443 (1970), by the search for similarity method of Pearson
& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
manual alignment and visual inspection (see, e.g., Current
Protocols in Molecular Biology (Ausubel et al., eds. 1995
supplement)).
[0122] An indication that two polypeptides are substantially
identical is that the first polypeptide is immunologically cross
reactive with the antibodies raised against the second polypeptide.
Thus, a polypeptide is typically substantially identical to a
second polypeptide, e.g., where the two peptides differ only by
conservative substitutions.
[0123] Preferred examples of algorithms that are suitable for
determining percent sequence identity and sequence similarity
include the BLAST and BLAST 2.0 algorithms, which are described in
Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul
et al., J. Mol. Biol. 215:403-410 (1990). BLAST and BLAST 2.0 are
used, with the parameters described herein, to determine percent
sequence identity for the nucleic acids and proteins of the
invention. The BLASTN program (for nucleotide sequences) uses as
defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,
N=-4 and a comparison of both strands. For amino acid sequences,
the BLASTP program uses as defaults a wordlength of 3, and
expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and
a comparison of both strands.
[0124] The terms "isolated," "purified," or "biologically pure"
refer to material that is substantially or essentially free from
components that normally accompany it as found in its native state.
Purity and homogeneity are typically determined using analytical
chemistry techniques such as polyacrylamide gel electrophoresis or
high-performance liquid chromatography. A protein that is the
predominant species present in a preparation is substantially
purified. The term "purified" in some embodiments denotes that a
protein gives rise to essentially one band in an electrophoretic
gel. Preferably, it means that the protein is at least 85% pure,
more preferably at least 95% pure, and most preferably at least 99%
pure.
[0125] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers, those containing modified
residues, and non-naturally occurring amino acid polymer.
[0126] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function similarly to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs may have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions
similarly to a naturally occurring amino acid.
[0127] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0128] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refer to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical or associated, e.g.,
naturally contiguous, sequences. Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode most proteins. For instance, the codons GCA, GCC, GCG
and GCU all encode the amino acid alanine. Thus, at every position
where an alanine is specified by a codon, the codon can be altered
to another of the corresponding codons described without altering
the encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of conservatively modified
variations. Every nucleic acid sequence herein which encodes a
polypeptide also describes silent variations of the nucleic acid.
One of skill will recognize that in certain contexts each codon in
a nucleic acid (except AUG, which is ordinarily the only codon for
methionine, and TGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, often silent variations of a nucleic acid
which encodes a polypeptide is implicit in a described sequence
with respect to the expression product, but not with respect to
actual probe sequences.
[0129] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables and
substitution matrices such as BLOSUM providing functionally similar
amino acids are well known in the art. Such conservatively modified
variants are in addition to and do not exclude polymorphic
variants, interspecies homologs, and alleles of the invention.
Typical conservative substitutions for one another include: 1)
Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)
Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),
Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,
Creighton, Proteins (1984)).
Methods for Preventing or Treating an Immunotherapy-Related
Toxicity
[0130] In some embodiments, disclosed herein are methods of
inhibiting immunotherapy-related toxicity in a subject. In some
embodiments, herein are methods of reducing the incidence of
immunotherapy-related toxicity in a subject. In some embodiments,
disclosed herein are methods of neutralizing hGM-CSF. In some
embodiment, the methods comprise a step of administering a
recombinant hGM-CSF antagonist to the subject. In some embodiments,
the method comprises hGM-CSF gene silencing. In some embodiments,
the method comprises hGM-CSF gene knockout. Methods of gene
silencing and gene knockout are well known to those of ordinary
skill in the art, and may include, without limitation, RNA
interference (RNAi), CRISPR, short interfering RNS (siRNA),
DNA-directed RNA interference (ddRNAi), targeted genome editing
with engineered transcription activator-like effector nucleases
(TALENs) or other suitable techniques.
[0131] In some embodiments, inhibiting or reducing the incidence or
the severity of immunotherapy-related toxicity comprises reducing
immune activation. In some embodiments, inhibiting or reducing the
incidence or the severity of immunotherapy-related toxicity
comprises ameliorating capillary leak syndrome. In some
embodiments, inhibiting or reducing the incidence or the severity
of immunotherapy-related toxicity comprises ameliorating a cardiac
dysfunction. In some embodiments, inhibiting or reducing the
incidence or the severity of immunotherapy-related toxicity
comprises ameliorating encephalopathy. In some embodiments,
inhibiting or reducing the incidence or the severity of
immunotherapy-related toxicity comprises alleviating colitis. In
some embodiments, inhibiting or reducing the incidence or the
severity of immunotherapy-related toxicity comprises inhibiting
convulsions. In some embodiments, inhibiting or reducing the
incidence or the severity of immunotherapy-related toxicity
comprises ameliorating CRS. In some embodiments, inhibiting or
reducing the incidence or the severity of immunotherapy-related
toxicity comprises ameliorating neurotoxicity. In various
embodiments, the CAR-T cell related neurotoxicity in a subject is
reduced by about 90% compared to a reduction in neurotoxicity in a
subject treated with CAR-T cells and a control antibody. In certain
embodiments, the recombinant GM-CSF antagonist is an antibody, in
particular, a GM-CSF neutralizing antibody in accordance with
embodiments described herein, including Example 15.
[0132] In some embodiments, inhibiting or reducing the incidence or
the severity of immunotherapy-related toxicity comprises reducing
cytokine storm symptoms. In some embodiments, inhibiting or
reducing the incidence or the severity of immunotherapy-related
toxicity comprises increasing impaired left ventricular ejection
fraction. In some embodiments, inhibiting or reducing the incidence
or the severity of immunotherapy-related toxicity comprises
ameliorating diarrhea. In some embodiments, inhibiting or reducing
the incidence or the severity of immunotherapy-related toxicity
comprises ameliorating disseminated intravascular coagulation.
[0133] In some embodiments, inhibiting or reducing the incidence or
the severity of immunotherapy-related toxicity comprises reducing
edema. In some embodiments, inhibiting or reducing the incidence or
the severity of immunotherapy-related toxicity comprises
alleviating exanthema. In some embodiments, inhibiting or reducing
the incidence or the severity of immunotherapy-related toxicity
comprises reducing gastrointestinal bleeding. In some embodiments,
inhibiting or reducing the incidence or the severity of
immunotherapy-related toxicity comprises treating a
gastrointestinal perforation. In some embodiments, inhibiting or
reducing the incidence or the severity of immunotherapy-related
toxicity comprises treating hemophagocytic lymphohistiocytosis
(HLH). In some embodiments, inhibiting or reducing the incidence or
the severity of immunotherapy-related toxicity comprises treating
hepatosis. In some embodiments, inhibiting or reducing the
incidence or the severity of immunotherapy-related toxicity
comprises reducing hypotension. In some embodiments, inhibiting or
reducing the incidence or the severity of immunotherapy-related
toxicity comprises reducing hypophysitis.
[0134] In some embodiments, inhibiting or reducing the incidence or
the severity of immunotherapy-related toxicity comprises inhibiting
immune related adverse events. In some embodiments, inhibiting or
reducing the incidence or the severity of immunotherapy-related
toxicity comprises reducing immunohepatitis. In some embodiments,
inhibiting or reducing the incidence or the severity of
immunotherapy-related toxicity comprises reducing
immunodeficiencies. In some embodiments, inhibiting or reducing the
incidence or the severity of immunotherapy-related toxicity
comprises treating ischemia. In some embodiments, inhibiting or
reducing the incidence or the severity of immunotherapy-related
toxicity comprises reducing liver toxicity. In some embodiments,
inhibiting or reducing the incidence or the severity of
immunotherapy-related toxicity comprises treating
macrophage-activation syndrome (MAS). In some embodiments,
inhibiting or reducing the incidence or the severity of
immunotherapy-related toxicity comprises reducing neurotoxicity
symptoms.
[0135] In some embodiments, inhibiting or reducing the incidence or
the severity of immunotherapy-related toxicity comprises reducing
pleural effusions. In some embodiments, inhibiting or reducing the
incidence or the severity of immunotherapy-related toxicity
comprises reducing pericardial effusions. In some embodiments,
inhibiting or reducing the incidence or the severity of
immunotherapy-related toxicity comprises reducing pneumonitis.
[0136] In some embodiments, inhibiting or reducing the incidence or
the severity of immunotherapy-related toxicity comprises reducing
polyarthritis. In some embodiments, inhibiting or reducing the
incidence or the severity of immunotherapy-related toxicity
comprises treating posterior reversible encephalopathy syndrome
(PRES). In some embodiments, inhibiting or reducing the incidence
or the severity of immunotherapy-related toxicity comprises
reducing pulmonary hypertension. In some embodiments, inhibiting or
reducing the incidence or the severity of immunotherapy-related
toxicity comprises treating thromboembolism. In some embodiments,
inhibiting or reducing the incidence or the severity of
immunotherapy-related toxicity comprises reducing transaminitis. In
some embodiments, inhibiting or reducing the incidence or the
severity of immunotherapy-related toxicity comprises reducing a
patient's CRES, neurotoxicity (NT), and/or cytokine release
syndrome (CRS) grade. In some embodiments, inhibiting or reducing
the incidence or the severity of immunotherapy-related toxicity
comprises improving a patient's CARTOX-10 score.
[0137] In one aspect, this invention further provides a method for
treating or preventing immunotherapy-related toxicity in a subject,
the method comprising administering to the subject chimeric antigen
receptor-expressing T-cells (CAR-T cells), the CAR-T cells having a
GM-CSF gene knockout (GM-CSF.sup.k/o CAR-T cells), and a
recombinant hGM-CSF antagonist, as demonstrated in Examples 6 and
20-21. In some embodiments, the GM-CSF.sup.k/o CAR-T cells express
a reduced level of GM-CSF compared to a level of GM-CSF expression
by wild-type CAR-T cells. In certain embodiments, the
GM-CSF.sup.k/o CAR-T cells express a level of one or more cytokine
and/or chemokine that is lower than or equivalent to a level of the
one or more cytokine and/or chemokine expressed by wild-type CAR-T
cells. In particular embodiments, the one or more cytokine is a
human cytokine selected from the group consisting of IFN-.gamma.,
GRO, MDC, IL-2, IL-3, IL-5, IL-7, IP-10, CD107a., TNF-.alpha. and
VEGF. In some embodiments the one or more cytokine is selected from
the group consisting of IFN-.gamma., IL-1a, IL-1b, IL-2, IL-4,
IL-5, IL-6, IL7, IL-9, IL-10, IL-12p40, IL-12p70, ILF, IL-13, LIX,
IL-15, IP-10, KC, MCP-1, MIP-1a, MIP-1b, M-CSF MIP-2, MIG, RANTES,
and TNF-.alpha., eotaxin, G-CSF and a combination thereof. In
various embodiments, the recombinant GM-CSF antagonist is an
hGM-CSF antagonist. In some embodiments, the recombinant GM-CSF
antagonist is an anti-GM-CSF antibody. In particular embodiments,
the anti-GM-CSF antibody binds a human GM-CSF. In other
embodiments, the anti-GM-CSF antibody binds a primate GM-CSF. In
various embodiments, the anti-GM-CSF antibody binds a mammalian
GM-CSF. In some embodiments, the anti-GM-CSF antibody is an
anti-hGM-CSF antibody. In certain embodiments, the anti-hGM-CSF
antibody is a monoclonal antibody. In various embodiments, the
anti-hGM-CSF antibody is an antibody fragment that is a Fab, a
Fab', a F(ab')2, a scFv, or a dAB. In some embodiments, the
anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. In
certain embodiments, the anti-hGM-CSF antibody is a recombinant or
chimeric antibody. In various embodiments, the anti-hGM-CSF
antibody is a human antibody. In some embodiments, the CAR-T cells
are CD19 CAR-T cells. In particular embodiments, the GM-CSF.sup.k/o
CAR-T cells enhance anti-tumor activity of the recombinant hGM-CSF
antagonist. In specific embodiments, the GM-CSF.sup.k/o CAR-T cells
improve overall survival of the subject compared to survival in a
subject treated by administration of wild-type CAR-T cells. In
particular embodiments, administering to the subject the CAR-T
cells having a GM-CSF gene knockout (GM-CSF.sup.k/o CAR-T cells)
and a recombinant hGM-CSF antagonist is a durable treatment for
preventing or treating an immunotherapy-related toxicity, such as
CRS, neurotoxicity and neuroinflammation. In some embodiments, the
subject has cancer. In various embodiments, the cancer is acute
lymphoblastic leukemia.
Methods for Reducing Blood Brain Barrier Disruption and for
Preserving/Maintaining the Integrity of the BBB
[0138] In one aspect, this invention provides a method for reducing
blood-brain barrier disruption in a subject treated with
immunotherapy, the method comprising administering a recombinant
GM-CSF antagonist to the subject. In some embodiments, the subject
has an incidence of immunotherapy-related toxicity.
[0139] In certain embodiments, the immunotherapy comprises adoptive
cell transfer, administration of monoclonal antibodies,
administration of cytokines, administration of a cancer vaccine, T
cell engaging therapies, or any combination thereof. In various
embodiments, the adoptive cell transfer comprises administering
chimeric antigen receptor-expressing T-cells (CAR T-cells), T-cell
receptor (TCR) modified T-cells, tumor-infiltrating lymphocytes
(TIL), chimeric antigen receptor (CAR)-modified natural killer
cells, or dendritic cells, or any combination thereof. In
particular embodiments, the CAR T-cells are CD19 CAR-T cells.
[0140] In further embodiments, the recombinant GM-CSF antagonist is
an hGM-CSF antagonist. In some embodiments, the recombinant GM-CSF
antagonist is an anti-GM-CSF antibody. In various embodiments, the
anti-GM-CSF antibody binds mammalian GM-CSF. In certain
embodiments, the anti-GM-CSF antibody binds primate GM-CSF. In some
embodiments, the primate is a monkey, a baboon, a macaque, a
chimpanzee, a gorilla, a lemur, a lorise, a tarsier, a galago, a
potto, a sifaka, an indri, an aye-ayes an ape or a human.
[0141] In particular embodiments, the anti-GM-CSF antibody is an
anti-hGM-CSF antibody. In various embodiments, the anti-hGM-CSF
antibody binds human GM-CSF. In certain embodiments, the
anti-hGM-CSF antibody is a monoclonal antibody. In various
embodiments, the anti-hGM-CSF antibody is an antibody fragment that
is a Fab, a Fab', a F(ab')2, a scFv, or a dAB. In some embodiments,
the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody.
In further embodiments, the anti-hGM-CSF antibody is a recombinant
or chimeric antibody. In further embodiments, the anti-hGM-CSF
antibody is a human antibody. In particular embodiments, the
anti-hGM-CSF antibody binds to the same epitope as chimeric 19/2
antibody. In more particular embodiments, the anti-hGM-CSF antibody
comprises the VH region CDR3 and VL region CDR3 of chimeric 19/2
antibody. In some embodiments, the anti-hGM-CSF antibody is
administered prior to, concurrent with, following immunotherapy or
a combination thereof
[0142] In various embodiments, the anti-hGM-CSF antibody comprises
the VH region and VL region CDR1, CDR2, and CDR3 of chimeric 19/2
antibody. In certain embodiments, the anti-hGM-CSF antibody
comprises a VH region that comprises a CDR3 binding specificity
determinant RQRFPY (SEQ ID NO: 12) or RDRFPY (SEQ ID NO: 13), a J
segment, and a V-segment, wherein the J-segment comprises at least
95% identity to human JH4 (YFD YWGQGTL VTVSS) and the V-segment
comprises at least 90% identity to a human germ line VH1 1-02 or
VH1 1-03 sequence; or a VH region that comprises a CDR3 binding
specificity determinant RQRFPY (SEQ ID NO: 12). In some
embodiments, the J segment comprises YFDYWGQGTLVTVSS (SEQ ID NO:
14). In certain embodiments, the CDR3 comprises RQRFPYYFDY (SEQ ID
NO: 15) or RDRFPYYFDY (SEQ ID NO: 16). In further embodiments, the
VH region CDR1 is a human germline VH1 CDR1; the VH region CDR2 is
a human germline VH1 CDR2; or both the CDR1 and CDR2 are from a
human germline VH1 sequence. In still further embodiments, the
anti-hGM-CSF antibody comprises a VH CDR1, or a VH CDR2, or both a
VH CDR1 and a VH CDR2 as shown in a VH region set forth in FIG. 1.
In some embodiments, the V-segment sequence has a VH V segment
sequence shown in FIG. 1. In various embodiments, the VH has the
sequence of VH #1, VH #2, VH #3, VH #4, or VH #5 set forth in FIG.
1. In certain embodiments, the anti-hGM-CSF antibody comprises a
VL-region that comprises a CDR3 comprising the amino acid sequence
FNK or FNR.
[0143] In further embodiments, the anti-hGM-CSF antibody comprises
a human germline JK4 region. In particular embodiments, the VL
region CDR3 comprises QQFN(K/R)SPL. In some embodiments, the
anti-hGM-CSF antibody comprises a VL region that comprises a CDR3
comprising QQFNKSPLT (SEQ ID NO: 18). In particular embodiments,
the VL region comprises a CDR1, or a CDR2, or both a CDR1 and CDR2
of a VL region shown in FIG. 1. In certain embodiments, the VL
region comprises a V segment that has at least 95% identity to the
VKIII A27 V-segment sequence as shown in FIG. 1. In various
embodiments, the VL region has the sequence of VK #1, VK #2, VK #3,
or VK #4 set forth in FIG. 1. In some embodiments, the anti-hGM-CSF
antibody has a VH region CDR3 binding specificity determinant
RQRFPY (SEQ ID NO: 12) or RDRFPY (SEQ ID NO: 13) and a VL region
that has a CDR3 comprising QQFNKSPLT (SEQ ID NO: 18). In further
embodiments, the anti-hGM-CSF antibody has a VH region sequence set
forth in FIG. 1 and a VL region sequence set forth in FIG. 1. In
still further embodiments, the VH region or the VL region, or both
the VH and VL region amino acid sequences comprise a methionine at
the N-terminus.
[0144] In various embodiments, the hGM-CSF antagonist is selected
from the group comprising of an anti-hGM-CSF receptor antibody or a
soluble hGM-CSF receptor, a cytochrome b562 antibody mimetic, a
hGM-CSF peptide analog, an adnectin, a lipocalin scaffold antibody
mimetic, a calixarene antibody mimetic, and an antibody like
binding peptidomimetic. In particular embodiments, the
immunotherapy-related toxicity is CAR-T related toxicity. In more
particular embodiments, the CAR-T related toxicity is cytokine
release syndrome, neurotoxicity, neuro-inflammation or a
combination thereof.
[0145] In another aspect, this invention provides a method for
preserving blood-brain barrier integrity in a subject treated with
immunotherapy, the method comprising administering a recombinant
hGM-CSF antagonist to the subject.
[0146] In a further aspect, this invention provides methods for
preventing or reducing blood-brain barrier in a subject treated
with immunotherapy, the method comprising administering CAR-T cells
having a GM-CSF gene knockout (GM-CSF.sup.k/o CAR-T cells) to the
subject.
[0147] In an embodiment of the provided methods, the recombinant
hGM-CSF antagonist is an anti-GM-CSF antibody. In another
embodiment, the anti-GM-CSF antibody binds mammalian GM-CSF. In yet
another embodiment, the anti-GM-CSF antibody binds primate GM-CSF.
In a further embodiment, the primate is a monkey, a baboon, a
macaque, a chimpanzee, a gorilla, a lemur, a lorise, a tarsier, a
galago, a potto, a sifaka, an indri, an aye-ayes an ape or a
human.
[0148] In a particular embodiment of the provided methods, the
anti-GM-CSF antibody is an anti-hGM-CSF antibody. In a further
particular embodiment, the anti-hGM-CSF antibody binds human
GM-CSF. In a still further embodiment, the anti-hGM-CSF antibody is
a monoclonal antibody. In a particular embodiment, the anti-hGM-CSF
antibody is an antibody fragment that is a Fab, a Fab', a F(ab')2,
a scFv, or a dAB. In an embodiment, the anti-hGM-CSF antibody is a
human GM-CSF neutralizing antibody. In another embodiment, the
anti-hGM-CSF antibody is a recombinant or chimeric antibody. In
some embodiments, the anti-hGM-CSF antibody is a human antibody. In
certain embodiments, the anti-hGM-CSF antibody binds to the same
epitope as chimeric 19/2 antibody. In various embodiments, the
anti-hGM-CSF antibody comprises the VH region CDR3 and VL region
CDR3 of chimeric 19/2 antibody. In some embodiments, the
anti-hGM-CSF antibody is administered prior to, concurrent with,
following immunotherapy or a combination thereof
[0149] In an embodiment of the provided methods, the anti-hGM-CSF
antibody comprises the VH region and VL region CDR1, CDR2, and CDR3
of chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF
antibody comprises a VH region that comprises a CDR3 binding
specificity determinant RQRFPY (SEQ ID NO: 12) or RDRFPY (SEQ ID
NO: 13), a J segment, and a V-segment, wherein the J-segment
comprises at least 95% identity to human JH4 (YFD YWGQGTL VTVSS)
and the V-segment comprises at least 90% identity to a human germ
line VH1 1-02 or VH1 1-03 sequence; or a VH region that comprises a
CDR3 binding specificity determinant RQRFPY (SEQ ID NO: 12). In
certain embodiments, the J segment comprises YFDYWGQGTLVTVSS (SEQ
ID NO: 14). In particular embodiments, the CDR3 comprises
RQRFPYYFDY (SEQ ID NO: 15) or RDRFPYYFDY (SEQ ID NO: 16). In some
embodiments, the VH region CDR1 is a human germline VH1 CDR1; the
VH region CDR2 is a human germline VH1 CDR2; or both the CDR1 and
CDR2 are from a human germline VH1 sequence. In a particular
embodiment, the anti-hGM-CSF antibody comprises a VH CDR1, or a VH
CDR2, or both a VH CDR1 and a VH CDR2 as shown in a VH region set
forth in FIG. 1. In an embodiment, the V-segment sequence has a VH
V segment sequence shown in FIG. 1. In some embodiments, the VH has
the sequence of VH #1, VH #2, VH #3, VH #4, or VH #5 set forth in
FIG. 1. In various embodiments, the anti-hGM-CSF antibody comprises
a VL-region that comprises a CDR3 comprising the amino acid
sequence FNK or FNR. In certain embodiments, the anti-hGM-CSF
antibody comprises a human germline JK4 region. In an embodiment,
the VL region CDR3 comprises QQFN(K/R)SPL. In certain embodiments,
the anti-hGM-CSF antibody comprises a VL region that comprises a
CDR3 comprising QQFNKSPLT (SEQ ID NO: 18). In some embodiments, the
VL region comprises a CDR1, or a CDR2, or both a CDR1 and CDR2 of a
VL region shown in FIG. 1. In various embodiments, the VL region
comprises a V segment that has at least 95% identity to the VKIII
A27 V-segment sequence as shown in FIG. 1. In particular
embodiments, the VL region has the sequence of VK #1, VK #2, VK #3,
or VK #4 set forth in Figure In particular embodiments, the
anti-hGM-CSF antibody has a VH region CDR3 binding specificity
determinant RQRFPY (SEQ ID NO: 12) or RDRFPY (SEQ ID NO: 13) and a
VL region that has a CDR3 comprising QQFNKSPLT (SEQ ID NO: 18). In
further embodiments, the anti-hGM-CSF antibody has a VH region
sequence set forth in FIG. 1 and a VL region sequence set forth in
FIG. 1. In some embodiments, the VH region or the VL region, or
both the VH and VL region amino acid sequences comprise a
methionine at the N-terminus. In certain embodiments, the hGM-CSF
antagonist is selected from the group comprising of an anti-hGM-CSF
receptor antibody or a soluble hGM-CSF receptor, a cytochrome b562
antibody mimetic, a hGM-CSF peptide analog, an adnectin, a
lipocalin scaffold antibody mimetic, a calixarene antibody mimetic,
and an antibody like binding peptidomimetic. In a particular
embodiment of the provided methods, the subject has an
immunotherapy-related toxicity. In a further embodiment, the
immunotherapy-related toxicity is CAR-T related toxicity. In
further embodiments, the CAR-T related toxicity is cytokine release
syndrome, neurotoxicity, neuro-inflammation or a combination
thereof.
[0150] In a further aspect, this invention provides a method for
decreasing or preventing CAR-T cell therapy-induced
neuroinflammation in a subject in need thereof, the method
comprising administering a recombinant GM-CSF antagonist to the
subject.
[0151] In some embodiments, administering the recombinant GM-CSF
antagonist reduces disruption of the blood brain barrier, thereby
maintaining integrity thereof. In particular embodiments, reducing
the disruption of the blood brain barrier decreases or prevents an
influx of pro-inflammatory cytokines into the central nervous
system. In various embodiments, the pro-inflammatory cytokines are
selected from the group consisting of IP-10, IL-2, IL-3, IL-5,
IL-1Ra, VEGF, TNF-a, FGF-2, IFN-.gamma., IL-12p40, IL-12p70,
sCD40L, MDC, MCP-1, MIP-1a, MIP-1b or a combination thereof. In
certain embodiments, the pro-inflammatory cytokines are selected
from the group consisting of IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-9,
IL-10, IP-10, KC, MCP-1, MIP or a combination thereof. In
particular embodiments, the neuroinflammation in the subject is
decreased by 75% to 95% compared to a subject treated with CAR-T
cell therapy and a control antibody. In various embodiments, the
75% to 95% decrease in neuroinflammation is similar to
neuroinflammation in an untreated control subject. In some
embodiments, the subject is administered chimeric antigen
receptor-expressing T-cells (CAR T-cells). In certain embodiments,
the subject is administered T-cell receptor (TCR) modified T-cells,
tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor
(CAR)-modified natural killer cells, or dendritic cells, or any
combination thereof. In particular embodiments, the CAR T-cells are
CD19 CAR-T cells. In various embodiments, the recombinant GM-CSF
antagonist is an hGM-CSF antagonist. In certain embodiments, the
recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In some
embodiments, the anti-GM-CSF antibody binds mammalian GM-CSF. In
further embodiments, the anti-GM-CSF antibody binds primate GM-CSF.
In some embodiments, the primate is a monkey, a baboon, a macaque,
a chimpanzee, a gorilla, a lemur, a lorise, a tarsier, a galago, a
potto, a sifaka, an indri, an aye-ayes an ape or a human. In an
embodiment, the anti-GM-CSF antibody is an anti-hGM-CSF antibody.
In another embodiment, the anti-hGM-CSF antibody binds human
GM-CSF. In a further embodiment, the anti-hGM-CSF antibody is a
monoclonal antibody. In a still further embodiment, the
anti-hGM-CSF antibody is an antibody fragment that is a Fab, a
Fab', a F(ab')2, a scFv, or a dAB. In particular embodiments,
anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody.
[0152] In various embodiments, the anti-hGM-CSF antibody is a
recombinant or chimeric antibody. In some embodiments, the
anti-hGM-CSF antibody is a human antibody. In particular
embodiments, the anti-hGM-CSF antibody binds to the same epitope as
chimeric 19/2 antibody. In further embodiments, the anti-hGM-CSF
antibody comprises the VH region CDR3 and VL region CDR3 of
chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF
antibody is administered prior to, concurrent with, following
immunotherapy or a combination thereof
[0153] In still further embodiments, the anti-hGM-CSF antibody
comprises the VH region and VL region CDR1, CDR2, and CDR3 of
chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF
antibody comprises a VH region that comprises a CDR3 binding
specificity determinant RQRFPY (SEQ ID NO: 12) or RDRFPY (SEQ ID
NO: 13), a J segment, and a V-segment, wherein the J-segment
comprises at least 95% identity to human JH4 (YFD YWGQGTL VTVSS)
and the V-segment comprises at least 90% identity to a human germ
line VH1 1-02 or VH1 1-03 sequence; or a VH region that comprises a
CDR3 binding specificity determinant RQRFPY (SEQ ID NO: 12). In
various embodiments, the J segment comprises YFDYWGQGTLVTVSS (SEQ
ID NO: 14).
[0154] In particular embodiments, the CDR3 comprises RQRFPYYFDY
(SEQ ID NO: 15) or RDRFPYYFDY (SEQ ID NO: 16). In some embodiments,
the VH region CDR1 is a human germline VH1 CDR1; the VH region CDR2
is a human germline VH1 CDR2; or both the CDR1 and CDR2 are from a
human germline VH1 sequence. In various embodiments, the
anti-hGM-CSF antibody comprises a VH CDR1, or a VH CDR2, or both a
VH CDR1 and a VH CDR2 as shown in a VH region set forth in FIG. 1.
In certain embodiments, the V-segment sequence has a VH V segment
sequence shown in FIG. 1. In particular embodiments, the VH has the
sequence of VH #1, VH #2, VH #3, VH #4, or VH #5 set forth in FIG.
1. In further embodiments, the anti-hGM-CSF antibody comprises a
VL-region that comprises a CDR3 comprising the amino acid sequence
FNK or FNR. In still further embodiments, the anti-hGM-CSF antibody
comprises a human germline JK4 region. In some embodiments, the VL
region CDR3 comprises QQFN(K/R)SPL In certain embodiments, the
anti-hGM-CSF antibody comprises a VL region that comprises a CDR3
comprising QQFNKSPLT (SEQ ID NO: 18). In various embodiments, the
VL region comprises a CDR1, or a CDR2, or both a CDR1 and CDR2 of a
VL region shown in FIG. 1. In further embodiments, the VL region
comprises a V segment that has at least 95% identity to the VKIII
A27 V-segment sequence as shown in FIG. 1. In still further
embodiments, the VL region has the sequence of VK #1, VK #2, VK #3,
or VK #4 set forth in FIG. 1.
[0155] In certain embodiments, the anti-hGM-CSF antibody has a VH
region CDR3 binding specificity determinant RQRFPY (SEQ ID NO: 12)
or RDRFPY (SEQ ID NO: 13) and a VL region that has a CDR3
comprising QQFNKSPLT (SEQ ID NO: 18). In some embodiments, the
anti-hGM-CSF antibody has a VH region sequence set forth in FIG. 1
and a VL region sequence set forth in FIG. 1. In various
embodiments, the VH region or the VL region, or both the VH and VL
region amino acid sequences comprise a methionine at the
N-terminus.
[0156] In further embodiments, the hGM-CSF antagonist is selected
from the group comprising of an anti-hGM-CSF receptor antibody or a
soluble hGM-CSF receptor, a cytochrome b562 antibody mimetic, a
hGM-CSF peptide analog, an adnectin, a lipocalin scaffold antibody
mimetic, a calixarene antibody mimetic, and an antibody like
binding peptidomimetic. In some embodiments. the subject further
has a CAR-T related toxicity selected from cytokine release
syndrome, neurotoxicity, or a combination thereof.
Methods for Reducing Relapse Rate or Preventing Occurrence
[0157] In one aspect, this invention provides a method for reducing
relapse rate or preventing occurrence of tumor relapse in a subject
treated with immunotherapy, the method comprising administering to
the subject a recombinant GM-CSF antagonist. In some embodiments,
the reducing relapse rate or preventing occurrence of tumor relapse
in the subject occurs in an absence of an incidence of
immunotherapy-related toxicity. In certain embodiments, the
reducing relapse rate or preventing occurrence of tumor relapse in
the subject occurs in a presence of an incidence of
immunotherapy-related toxicity. In some embodiments, the
recombinant GM-CSF antagonist is an hGM-CSF antagonist. In various
embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF
antibody. In some embodiments, the anti-GM-CSF antibody binds a
human GM-CSF. In certain embodiments, the anti-GM-CSF antibody
binds a primate GM-CSF. In various embodiments, the primate is
selected from a monkey, a baboon, a macaque, a chimpanzee, a
gorilla, a lemur, a lorise, a tarsier, a galago, a potto, a sifaka,
an indri, an aye-ayes or an ape. In some embodiments, the
anti-GM-CSF antibody binds a mammalian GM-CSF.
[0158] In particular embodiments, the anti-GM-CSF antibody is an
anti-hGM-CSF antibody. As described above, the anti-GM-CSF antibody
is a monoclonal antibody. In another embodiment, the anti-hGM-CSF
antibody is an antibody fragment that is a Fab, a Fab', a F(ab')2,
a scFv, or a dAB. In some embodiments, the anti-hGM-CSF antibody is
a human GM-CSF neutralizing antibody. In certain embodiments, the
anti-hGM-CSF antibody is a recombinant or chimeric antibody. In
various embodiments, the anti-hGM-CSF antibody is a human antibody.
In some embodiments, the anti-hGM-CSF antibody binds to the same
epitope as chimeric 19/2 antibody. In certain embodiments, the
anti-hGM-CSF antibody comprises the VH region CDR3 and VL region
CDR3 of chimeric 19/2 antibody. In various embodiments, the
anti-hGM-CSF antibody comprises the VH region and VL region CDR1,
CDR2, and CDR3 of chimeric 19/2 antibody. In some embodiments, the
anti-hGM-CSF antibody comprises a VH region that comprises a CDR3
binding specificity determinant RQRFPY (SEQ ID NO: 12) or RDRFPY
(SEQ ID NO: 13), a J segment, and a V-segment, wherein the
J-segment comprises at least 95% identity to human JH4 (YFD YWGQGTL
VTVSS) and the V-segment comprises at least 90% identity to a human
germ line VH1 1-02 or VH1 1-03 sequence; or a VH region that
comprises a CDR3 binding specificity determinant RQRFPY (SEQ ID NO:
12). In particular embodiments, the J segment comprises
YFDYWGQGTLVTVSS (SEQ ID NO: 14). In additional embodiments, the
CDR3 comprises RQRFPYYFDY (SEQ ID NO: 15)_or RDRFPYYFDY (SEQ ID NO:
16). In some embodiments, the VH region CDR1 is a human germline
VH1 CDR1; the VH region CDR2 is a human germline VH1 CDR2; or both
the CDR1 and CDR2 are from a human germline VH1 sequence.
[0159] In certain embodiments, the anti-hGM-CSF antibody comprises
a VH CDR1, or a VH CDR2, or both a VH CDR1 and a VH CDR2 as shown
in a VH region set forth in FIG. 1. In various embodiments, the
V-segment sequence has a VH V segment sequence shown in FIG. 1. In
certain embodiments, the VH has the sequence of VH #1, VH #2, VH
#3, VH #4, or VH #5 set forth in FIG. 1. In some embodiments, the
anti-hGM-CSF antibody comprises a VL-region that comprises a CDR3
comprising the amino acid sequence FNK or FNR. In some embodiments,
the anti-hGM-CSF antibody comprises a human germline JK4 region. In
certain embodiments, the VL region CDR3 comprises QQFN(K/R)SPLT
(SEQ ID NO: 17). In various embodiments, the anti-hGM-CSF antibody
comprises a VL region that comprises a CDR3 comprising QQFNKSPLT
(SEQ ID NO: 18). In some embodiments, the VL region comprises a
CDR1, or a CDR2, or both a CDR1 and CDR2 of a VL region shown in
FIG. 1. In particular embodiments, the VL region comprises a V
segment that has at least 95% identity to the VKIII A27 V-segment
sequence as shown in FIG. 1. In some embodiments, the VL region has
the sequence of VK #1, VK #2, VK #3, or VK #4 set forth in FIG. 1.
In certain embodiments, the anti-hGM-CSF antibody has a VH region
CDR3 binding specificity determinant RQRFPY (SEQ ID NO: 12) or
RDRFPY (SEQ ID NO: 13) and a VL region that has a CDR3 comprising
QQFNKSPLT (SEQ ID NO: 18). In some embodiments, the anti-hGM-CSF
antibody has a VH region sequence set forth in FIG. 1 and a VL
region sequence set forth in FIG. 1. In other embodiments, the VH
region or the VL region, or both the VH and VL region amino acid
sequences comprise a methionine at the N-terminus.
[0160] In some embodiments, the hGM-CSF antagonist is selected from
the group comprising of an anti-hGM-CSF receptor antibody or a
soluble hGM-CSF receptor, a cytochrome b562 antibody mimetic, a
hGM-CSF peptide analog, an adnectin, a lipocalin scaffold antibody
mimetic, a calixarene antibody mimetic, and an antibody like
binding peptidomimetic. In certain embodiments, the CAR-T cells are
CD19 CAR-T cells. In particular embodiments, the
immunotherapy-related toxicity is CAR-T related toxicity. In some
embodiments, the CAR-T related toxicity is CRS, NT or
neuro-inflammation.
[0161] In particular embodiments, the tumor relapse occurrence is
reduced by from 50% to 100% in the first one-quarter of a year
after administering the recombinant GM-CSF antagonist compared to
tumor relapse occurrence in a subject treated with immunotherapy
and not administered a recombinant GM-CSF antagonist. In certain
embodiments, the tumor relapse occurrence is reduced by from 50% to
95% in the first half-year after administering the recombinant
GM-CSF antagonist. In various embodiments, the tumor relapse
occurrence is reduced by from 50% to 90% in the first year after
administering the recombinant GM-CSF antagonist. In some
embodiments, the tumor relapse occurrence is prevented long-term.
As used herein the term "long-term" means during an extended period
of time of at least a year, i.e. 12 months, from the last date of
treatment with a recombinant hGM-CSF antagonist. In some
embodiments, the recombinant hGM-CSF antagonist is a hGM-CSF
neutralizing antibody. In various embodiments, the recombinant
hGM-CSF antagonist is an anti-hGM-CSF antibody, e.g., Lenzilumab.
In certain embodiments, the tumor relapse occurrence is prevented
by 12-36 months. In some embodiments, the tumor relapse occurrence
is prevented "completely" (100%), which as used herein means that
there is no recurrence of the tumor for at least 12 months, from
the last date of treatment with a recombinant hGM-CSF antagonist.
In certain embodiments, the subject has acute lymphoblastic
leukemia.
[0162] In various embodiments of the herein provided methods for
reducing relapse rate or preventing occurrence of tumor relapse in
a subject treated with immunotherapy, the subject has a "refractory
cancer", which as used herein is (a) a malignancy (also called
"cancer" or a "tumor" herein) for which surgery is ineffective and
is (b) either initially unresponsive or resistant to treatment,
wherein the treatment is chemotherapy, radiation or a combination
thereof, or is (b) a malignancy which becomes or has become
unresponsive to the aforementioned treatment(s). In some
embodiments, the subject has a "relapsed" cancer, which as used
herein is a cancer that responded but to treatment, but has
returned. In particular embodiments, the refractory cancer or the
relapsed cancer is non-Hodgkin lymphoma (NHL). In various
embodiments, the refractory cancer or the relapsed cancer is
non-Hodgkin lymphoma (NHL). In certain embodiments, the refractory
cancer is refractory aggressive B cell non-Hodgkin lymphoma. In
some embodiments, the refractory cancer or the relapsed cancer is
chemo-refractory B cell lymphoma. In various embodiments, the
refractory cancer or the relapsed cancer is hormone-refractory
prostate cancer. In certain embodiments, the refractory cancer or
the relapsed cancer is a pediatric cancer. In some embodiments, the
refractory pediatric cancer or the relapsed pediatric cancer is
neuroblastoma. In particular embodiments, the refractory pediatric
cancer or the relapsed pediatric cancer is a pediatric leukemia
selected from the group consisting of acute lymphoblastic leukemia
(ALL), acute myelogenous leukemia (AML) or an uncommon pediatric
leukemia which is juvenile myelomonocytic leukemia or chronic
myeloid leukemia. In certain embodiments, the refractory cancer or
the relapsed cancer is a pediatric bone cancer. In some
embodiments, the refractory cancer or the relapsed cancer is an
adrenal cancer. In various embodiments, the refractory cancer or
the relapsed cancer is a breast cancer. In certain embodiments, the
refractory cancer or the relapsed cancer is a colon cancer, rectal
cancer or colorectal cancer. In particular embodiments, the
refractory cancer or the relapsed cancer is a T-cell lymphoma. In
some embodiments, the refractory cancer or the relapsed cancer is a
head and neck cancer. In some embodiments, the refractory cancer or
the relapsed cancer is a brain and/or spinal cord cancer, including
but not limited to glioma an glioblastoma. In additional
embodiments, the refractory cancer or the relapsed cancer is a
tumor of bone or soft tissue, including but not limited to a
chondrosarcoma. In various embodiments, the refractory cancer or
the relapsed cancer is a bone cancer. In some embodiments, the
refractory cancer or the relapsed cancer is esophageal cancer. In
certain embodiments, the refractory cancer or the relapsed cancer
is a gall bladder cancer. In some embodiments, the refractory
cancer or the relapsed cancer is a kidney cancer. In various
embodiments, the refractory cancer or the relapsed cancer is
melanoma. In some embodiments, the refractory cancer or the
relapsed cancer is an ovary cancer. In certain embodiments, the
refractory cancer or the relapsed cancer is a pancreatic cancer. In
some embodiments, the refractory cancer or the relapsed cancer is a
skin cancer selected from a basal cell carcinoma, a squamous cell
carcinoma or a melanoma. In various embodiments, the refractory
cancer or the relapsed cancer is a lung cancer. In some
embodiments, the refractory cancer or the relapsed cancer is a
salivary gland cancer. In additional embodiments, the refractory
cancer or the relapsed cancer is a uterine smooth muscle cancer. In
some embodiments, the refractory cancer or the relapsed cancer is a
testicular cancer. In various embodiments, the refractory cancer or
the relapsed cancer is a stomach cancer or a gastrointestinal
cancer. In certain embodiments, the refractory cancer or the
relapsed cancer is a bladder cancer. In additional embodiments, the
refractory cancer or the relapsed cancer is an adipose tissue
neoplasm. In some embodiments, the refractory pediatric cancer or
the relapsed pediatric cancer is an adenocarcinoma. In certain
embodiments, the refractory cancer or the relapsed cancer is a
thymoma. In various embodiments, the refractory cancer or the
relapsed cancer is an angiosarcoma, i.e., a cancer of the lining of
blood vessels, which can occur in any part of the body, including
but not limited to skin, breast, liver, spleen and deep tissue,
i.e., deep-seated tumors. In some embodiments, the refractory
cancer or the relapsed cancer is a metastasis of any one of the
aforementioned refractory cancer or the relapsed cancer.
[0163] In some embodiments, the immunotherapy is an activation
immunotherapy. In some embodiments, immunotherapy is provided as a
cancer treatment. In some embodiments, immunotherapy comprises
adoptive cell transfer.
[0164] In some embodiments, adoptive cell transfer comprises
administration of a chimeric antigen receptor-expressing T-cell
(CAR T-cell). A skilled artisan would appreciate that CARs are a
type of antigen-targeted receptor composed of intracellular T-cell
signaling domains fused to extracellular tumor-binding moieties,
most commonly single-chain variable fragments (scFvs) from
monoclonal antibodies. CARs directly recognize cell surface
antigens, independent of MHC-mediated presentation, permitting the
use of a single receptor construct specific for any given antigen
in all patients. Initial CARs fused antigen-recognition domains to
the CD3.zeta. activation chain of the T-cell receptor (TCR)
complex. While these first-generation CARs induced T-cell effector
function in vitro, they were largely limited by poor antitumor
efficacy in vivo. Subsequent CAR iterations have included secondary
costimulatory signals in tandem with CD3.zeta., including
intracellular domains from CD28 or a variety of TNF receptor family
molecules such as 4-1BB (CD137) and OX40 (CD134). Further, third
generation receptors include two costimulatory signals in addition
to CD3.zeta., most commonly from CD28 and 4-1BB. Second and third
generation CARs dramatically improve antitumor efficacy, in some
cases inducing complete remissions in patients with advanced
cancer. In one embodiment, a CAR T-cell is an immunoresponsive cell
modified to express CARs, which is activated when CARs bind to its
antigen.
[0165] In one embodiment, a CAR T-cell is an immunoresponsive cell
comprising an antigen receptor, which is activated when its
receptor binds to its antigen. In one embodiment, the CAR T-cells
used in the compositions and methods as disclosed herein are first
generation CAR T-cells. In another embodiment, the CAR T-cells used
in the compositions and methods as disclosed herein are second
generation CAR T-cells. In another embodiment, the CAR T-cells used
in the compositions and methods as disclosed herein are third
generation CAR T-cells. In another embodiment, the CAR T-cells used
in the compositions and methods as disclosed herein are fourth
generation CAR T-cells.
[0166] In some embodiments, adoptive cell transfer comprises
administering T-cell receptor (TCR) modified T-cells. A skilled
artisan would appreciate that TCR modified T-cells are manufactured
by isolating T-cells from tumor tissue and isolating their TCRa and
TCRP chains. These TCRa and TCRP are later cloned and transfected
into T cells isolated from peripheral blood, which then express
TCRa and TCRP from T-cells recognizing the tumor.
[0167] In some embodiments, adoptive cell transfer comprises
administering tumor infiltrating lymphocytes (TIL). In some
embodiments, adoptive cell transfer comprises administering
chimeric antigen receptor (CAR)-modified NK cells. A skilled
artisan would appreciate that CAR-modified NK cells comprise NK
cells isolated from the patient or commercially available NK
engineered to express a CAR that recognizes a tumor-specific
protein.
[0168] In some embodiments, adoptive cell transfer comprises
administering dendritic cells.
[0169] In some embodiments, immunotherapy comprises administering
monoclonal antibodies. In some embodiments, monoclonal antibodies
attach to specific proteins on cancer cells, thus flagging the
cells for the immune system finding and destroying them. In some
embodiments, monoclonal antibodies work by inhibiting immune
checkpoints, thus hindering the inhibition of the immune system by
cancer cells. In some embodiments, monoclonal antibodies improve
utility of CAR-T to synergize with checkpoint inhibitors.
[0170] In some embodiments, the antibody targets a protein selected
from the group comprising: 5AC, 5T4, activin receptor-like kinase
1, AGS-22M6, alpha-fetoprotein, angiopoietin 2, angiopoietin 3,
B7-H3, BAFF, BCMA, C242 antigen, CA-125, carbonic anhydrase 9,
CCR4, CD125, CD152, CD184, CD19, CD2, CD20, CD200, CD22, CD221,
CD23, CD25, CD27, CD274, CD276, CD28, CD3, CD30, CD33, CD37, CD38,
CD4, CD40, CD41, CD44 v6, CD49b, CD5, CD51, CD52, CD54, CD56, CD6,
CD70, CD74, CD79B, CD80, CEA, CFD, CGRP, ch4D5, CLDN18.2, clumping
factor A, CSF1R, CSF2, CTGF, CTLA-4, DLL3, DLL4, DPP4, DR5, EGFL7,
EGFR, endoglin, EpCAM, ephrin receptor A3, episialin, ERBB3 (HER3),
FAP, FGF 23, fibrin II, beta chain, fibronectin extra domain-B,
folate hydrolase, folate receptor, Frizzled receptor, GCGR, GD2
ganglioside, GD3 ganglioside, GDF-8, glypican 3, GM-CSF, GM-CSF
receptor .alpha.-chain, GPNMB, GUCY2C, HER1, HER2/neu, HGF, HHGFR,
histone complex, human scatter factor receptor kinase, human TNF,
ICOSL, IFN-.alpha., IGF1, IGF2, IGHE, IL-17A, IL-13, IL1A, IL-2,
IL-6, IL-6 receptor, IL-8, IL-9, ILGF2, integrin .alpha.4, integrin
.alpha.5.beta.1, integrin .alpha.7 .beta.7, integrin
.alpha.v.beta., IP10, KIR2D, KLRC1, Lewis-Y antigen, MAGE-A, MCP-1,
mesothelin, MIF, MIG, MIP10, MS4A1, MSLN, MUC1, mucin CanAg,
N-glycolylneuraminic acid, NOGO-A, Notch 1, Notch receptor, NRP1,
OX-40, PD-1, PDCD1, PDGF-R.alpha., phosphate-sodium co-transporter,
phosphatidylserine, platelet-derived growth factor receptor beta,
prostatic carcinoma cells, RHD, RON, RTN4, SDC1, sIL2R.alpha.,
SLAMF7, SOST, sphingosine-1-phosphate, Staphylococcus aureus,
STEAP1, TAG-72, T-cell receptor, TEM1, tenascin C, TFPI, TGF beta
1, TGF beta 2, TGF-.beta., TNFR superfamily member 4, TNF-.alpha.,
TRAIL-R1, TRAIL-R2, TRP-1, TRP-2, TSLP, tumor antigen CTAA16.88,
tumor specific glycosylation of MUC1, tumor-associated calcium
signal transducer 2, TWEAK receptor, TYRP1(glycoprotein 75), VEGFA,
VEGFR-1, VEGFR2, vimentin, and VWF.
[0171] In some embodiments, the antibody is a bi-specific antibody.
In some embodiments, the antibody is a bispecific T-cell engager
(BiTE) antibody. In some embodiments, the antibody is selected from
a group comprising: ipilimumab, nivolumab, pembrolizumab,
atezolizumab, avelumab, durvalumab, rituximab, TGN1412,
alemtuzumab, OKT3 or any combination thereof.
[0172] In some embodiments, immunotherapy comprises administering
cytokines. A skilled artisan would appreciate that cytokines can be
administered in order to enhance the immune system to attack the
tumor by increasing its recognition and killing by immune cytotoxic
cells. In some embodiments, the cytokine is selected from a group
comprising: IFN.alpha., IFN.beta., IFN.gamma., IFN.lamda., IL-1,
IL-2, IL-6, IL-7, IL-15, IL-21, IL-11, IL-12, IL-18, GM-CSF,
TNF.alpha., or any combination thereof.
[0173] In some embodiments, immunotherapy comprises administering
immune checkpoint inhibitors. A skilled artisan would appreciate
that immune checkpoints are membranal proteins that keep T cells
from attacking the cells that express it. Immune checkpoints are
often expressed by cancer cells, thus preventing T cells from
attacking them. In some embodiments, checkpoint proteins comprise
PD-1/PD-L1 and CTLA-4/B7-1/B7-2. Blocking checkpoint proteins was
shown to disengage the inhibition of T cells to attack and kill
cancer cells. In some embodiments, checkpoint inhibitors are
selected from a group comprising molecules blocking CTLA-4, PD-1,
or PD-L1. In some embodiments, the checkpoint inhibitors are
antibodies or parts thereof.
[0174] In some embodiments, immunotherapy comprises administering
polysaccharides. A skilled artisan would appreciate that certain
polysaccharides found in mushroom enhance the immune system and its
anti-cancer properties. In some embodiments, polysaccharides are
beta-glucans or lentinan.
[0175] In some embodiments, immunotherapy comprises administering
or a cancer vaccine. A skilled artisan would appreciate that a
cancer vaccine exposes the immune system to a cancer-specific
antigen and an adjuvant. In some embodiments, the cancer vaccine is
selected from a group comprising: sipuleucel-T, GVAX, ADXS11-001,
ADXS31-001, ADXS31-164, ALVAC-CEA vaccine, AC Vaccine, talimogene
laherparepvec, BiovaxID, Prostvac, CDX110, CDX1307, CDX1401,
CimaVax-EGF, CV9104, DNDN, NeuVax, Ae-37, GRNVAC, tarmogens,
GI-4000, GI-6207, GI-6301, ImPACT Therapy, IMA901,
hepcortespenlisimut-L, Stimuvax, DCVax-L, DCVax-Direct, DCVax
Prostate, CBLI, Cvac, RGSH4K, SCIB1, NCT01758328, and PVX-410.
[0176] Methods for Reducing a Level of a Cytokine or Chemokine
Other than GM-CSF
[0177] In some embodiments, inhibiting or reducing the incidence or
the severity of immunotherapy-related toxicity comprises decreasing
the concentration of at least one inflammation-associated factor in
a body fluid. In some embodiments, inhibiting or reducing the
incidence or the severity of immunotherapy-related toxicity
comprises decreasing the concentration of at least one
inflammation-associated factor in the serum. In some embodiments,
inhibiting or reducing the incidence or the severity of
immunotherapy-related toxicity comprises decreasing the
concentration of at least one inflammation-associated factor in the
cerebrospinal fluid (CSF). In some embodiments, disclosed herein
are methods for decreasing the concentration of at least one
inflammation-associated factor in serum. In some embodiments,
disclosed herein are methods for decreasing the concentration of at
least one inflammation-associated factor in a tissue fluid. In some
embodiments, disclosed herein are methods for decreasing the
concentration of at least one inflammation-associated factor in
CSF. In some embodiments, the concentration of at least one
inflammation-associated factor in serum is decreased. In some
embodiments, the concentration of at least one
inflammation-associated factor in a tissue fluid is decreased. In
some embodiments, the concentration of at least one
inflammation-associated factor in CSF is decreased. A skilled
artisan would appreciate that decreasing the concentration of an
inflammation-associated factor comprises decreasing or inhibiting
the production of said inflammation-associated factor in a subject,
or inhibiting or reducing the incidence or the severity of
immunotherapy-related toxicity in a subject. In another embodiment,
decreasing or inhibiting the production of an
inflammation-associated factor comprises treating
immunotherapy-related toxicity. In another embodiment, decreasing
or inhibiting the production of an inflammation-associated factor
comprises preventing immunotherapy-related toxicity. In another
embodiment, decreasing or inhibiting the production of an
inflammation-associated factor levels comprises alleviating
immunotherapy-related toxicity. In another embodiment, decreasing
or inhibiting the production of an inflammation-associated factor
comprises ameliorating immunotherapy-related toxicity.
[0178] In some embodiments, the inflammation-associated factor is a
cytokine. In some embodiments, inhibiting or reducing the incidence
or the severity of immunotherapy-related toxicity comprises
decreasing the concentration of at least one cytokine in the serum.
In some embodiments, inhibiting or reducing the incidence or the
severity of immunotherapy-related toxicity comprises decreasing the
concentration of at least one cytokine in the CSF.
[0179] In some embodiments, the cytokine is hGM-CSF. In some
embodiments, the cytokine is interleukin (IL)-1.beta.. In some
embodiments, the cytokine is IL-2. In some embodiments, the
cytokine is sIL2R.alpha.. In some embodiments, the cytokine is
IL-5. In some embodiments, the cytokine is IL-6. In some
embodiments, the cytokine is IL-8. In some embodiments, the
cytokine is IL-10. In some embodiments, the cytokine is IP10. In
some embodiments, the cytokine is IL-13. In some embodiments, the
cytokine is IL-15. In some embodiments, the cytokine is tumor
necrosis factor .alpha. (TNF.alpha.). In some embodiments, the
cytokine is interferon .gamma. (IFN.gamma.). In some embodiments,
the cytokine is monokine induced by gamma interferon (MIG). In some
embodiments, the cytokine is macrophage inflammatory protein (MIP)
10. In some embodiments, the cytokine is C-reactive protein. In
some embodiments, decreasing or inhibiting the production of
cytokine levels comprises decreasing or inhibiting the production
of one cytokine. In some embodiments, decreasing or inhibiting the
production of cytokine levels comprises decreasing or inhibiting
the production of at least one cytokine. In some embodiments,
decreasing or inhibiting the production of cytokine levels
comprises decreasing or inhibiting the production of a number of
cytokines.
[0180] In one aspect, this invention provides a method reducing a
level of a cytokine or chemokine other than GM-CSF in a subject
having an incidence of immunotherapy-related toxicity, the method
comprising administering to the subject a recombinant hGM-CSF
antagonist, wherein the level of the cytokine or chemokine is
reduced compared the level thereof in a subject administered an
isotype control antibody during the incidence of
immunotherapy-related toxicity. In some embodiments, the
immunotherapy comprises adoptive cell transfer, administration of
monoclonal antibodies, administration of a cancer vaccine, T cell
engaging therapies, or any combination thereof. In certain
embodiments, the adoptive cell transfer comprises administering
chimeric antigen receptor-expressing T-cells (CAR T-cells), T-cell
receptor (TCR) modified T-cells, tumor-infiltrating lymphocytes
(TIL), chimeric antigen receptor (CAR)-modified natural killer
cells, or dendritic cells, or any combination thereof. In some
embodiments, the CAR-T cells are CD19 CAR-T cells. In certain
embodiments, the recombinant GM-CSF antagonist is an hGM-CSF
antagonist. In various embodiments, the recombinant GM-CSF
antagonist is an anti-GM-CSF antibody. In particular embodiments,
the anti-GM-CSF antibody binds a human GM-CSF. In other
embodiments, the anti-GM-CSF antibody binds a primate GM-CSF, as
described above. In some embodiments, the anti-GM-CSF antibody
binds a mammalian GM-CSF. In certain embodiments, the anti-GM-CSF
antibody is an anti-hGM-CSF antibody. In some embodiments, the
anti-hGM-CSF antibody is a monoclonal antibody. In various
embodiments, the anti-hGM-CSF antibody is an antibody fragment that
is a Fab, a Fab', a F(ab')2, a scFv, or a dAB. In some embodiments,
the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody.
In certain embodiments, the anti-hGM-CSF antibody is a recombinant
or chimeric antibody. In some embodiments, the anti-hGM-CSF
antibody is a human antibody. In particular embodiments, the
cytokine or chemokine is a human cytokine or chemokine selected
from the group consisting of IP-10, IL-2, IL-3, IL-5, IL-1Ra, VEGF,
TNF-.alpha., FGF-2, IFN-.gamma., IL-12.beta.40, IL-12.beta.70,
sCD40L, MDC, MCP-1, MIP-1a, MIP-1b or a combination thereof, as
demonstrated in Example 22.
In some embodiments, the cytokine or chemokine is selected from the
group consisting of IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-9, IL-10,
IP-10, KC, MCP-1, MIP or a combination thereof (see Example 22). In
certain embodiments, the subject has acute lymphoblastic
leukemia.
[0181] In one embodiment, the methods disclosed herein do not
affect the efficacy of the immunotherapy. In another embodiment,
the methods disclosed herein reduce the efficacy of the
immunotherapy by less than about 5%. In another embodiment, the
methods disclosed herein reduce the efficacy of the immunotherapy
by less than about 10%. In another embodiment, the methods
disclosed herein reduce the efficacy of the immunotherapy by less
than about 15%. In another embodiment, the methods disclosed herein
reduce the efficacy of the immunotherapy by less than about 20%. In
another embodiment, the methods disclosed herein reduce the
efficacy of the immunotherapy by less than about 50%.
[0182] In one embodiment, the methods described herein increase the
efficacy of the immunotherapy. In one embodiment, increasing the
efficacy allows for improvement of the clinical management, patient
outcomes, and therapeutic index of the immunotherapy. In another
embodiment, the increased efficacy enables administration of higher
immunotherapy doses. In another embodiment, the increased efficacy
reduces hospitalization stay and additional treatments and
monitoring. In an embodiment, the immunotherapy comprises
CAR-T.
[0183] Any appropriate method of quantifying cytotoxicity can be
used to determine whether the immunotherapy efficacy remains
substantially unchanged. For example, cytotoxicity can be
quantified using a cell culture-based assay such as the cytotoxic
assays described in the Examples. Cytotoxicity assays can employ
dyes that preferentially stain the DNA of dead cells. In other
cases, fluorescent and luminescent assays that measure the relative
number of live and dead cells in a cell population can be used. For
such assays, protease activities serve as markers for cell
viability and cell toxicity, and a labeled cell permeable peptide
generates fluorescent signals that are proportional to the number
of viable cells in the sample. In another embodiment, a measure of
cytotoxicity may be qualitative. In another embodiment, a measure
of cytotoxicity may be quantitative.
[0184] In an embodiment, said increased efficacy comprises
increased CAR-T cell expansion, reduced number and/or activity of
myeloid-derived suppressor cells (MDSC) that inhibit T-cell
function, synergy with a checkpoint inhibitor, or any combination
thereof. In another embodiment, said increased CAR-T cell expansion
comprises at least a 50% increase compared to a control. In another
embodiment, said increased CAR-T cell expansion comprises at least
a one quarter log expansion compared to a control. In another
embodiment, said increased cell expansion comprises at least a
one-half log expansion compared to a control. In another
embodiment, said increased cell expansion comprises at least a one
log expansion compared to a control. In another embodiment, said
increased cell expansion comprises a greater than one log expansion
compared to a control.
[0185] In one embodiment, immunotherapy-related toxicity appears
between 2 days to 4 weeks after administration of immunotherapy. In
one embodiment, immunotherapy-related toxicity appears between 0 to
2 days after administration of immunotherapy. In some embodiments,
the hGM-CSF antagonist is administered to subjects at the same time
as immunotherapy as prophylaxis. In another embodiment, the hGM-CSF
antagonist is administered to subjects 0-2 days after
administration of immunotherapy. In another embodiment, the hGM-CSF
antagonist is administered to subjects 2-3 days after
administration of immunotherapy. In another embodiment, the hGM-CSF
antagonist is administered to subjects 7 days after administration
of immunotherapy. In another embodiment, the hGM-CSF antagonist is
administered to subjects 10 days after administration of
immunotherapy. In another embodiment, the hGM-CSF antagonist is
administered to subjects 14 days after administration of
immunotherapy. In another embodiment, the hGM-CSF antagonist is
administered to subjects 2-14 days after administration of
immunotherapy.
[0186] In another embodiment, the hGM-CSF antagonist is
administered to subjects 2-3 hours after administration of
immunotherapy. In another embodiment, the hGM-CSF antagonist is
administered to subjects 7 hours after administration of
immunotherapy. In another embodiment, the hGM-CSF antagonist is
administered to subjects 10 hours after administration of
immunotherapy. In another embodiment, the GM-CSF antagonist is
administered to subjects 14 hours after administration of
immunotherapy. In another embodiment, the hGM-CSF antagonist is
administered to subjects 2-14 hours after administration of
immunotherapy.
[0187] In an alternative embodiment, the hGM-CSF antagonist is
administered to subjects prior to immunotherapy as prophylaxis. In
another embodiment, the hGM-CSF antagonist is administered to
subjects 1 day before administration of immunotherapy. In another
embodiment, the hGM-CSF antagonist is administered to subjects 2-3
days before administration of immunotherapy. In another embodiment,
the hGM-CSF antagonist is administered to subjects 7 days before
administration of immunotherapy. In another embodiment, the hGM-CSF
antagonist is administered to subjects 10 days before
administration of immunotherapy. In another embodiment, the hGM-CSF
antagonist is administered to subjects 14 days before
administration of immunotherapy. In another embodiment, the hGM-CSF
antagonist is administered to subjects 2-14 days before
administration of immunotherapy.
[0188] In another embodiment, the hGM-CSF antagonist is
administered to subjects 2-3 hours before administration of
immunotherapy. In another embodiment, the hGM-CSF antagonist is
administered to subjects 7 hours before administration of
immunotherapy. In another embodiment, the hGM-CSF antagonist is
administered to subjects 10 hours before administration of
immunotherapy. In another embodiment, the hGM-CSF antagonist is
administered to subjects 14 hours before administration of
immunotherapy. In another embodiment, the hGM-CSF antagonist is
administered to subjects 2-14 hours before administration of
immunotherapy.
[0189] In another embodiment, the hGM-CSF antagonist may be
administered therapeutically, once immunotherapy-related toxicity
has occurred. In one embodiment, the hGM-CSF antagonist may be
administered once pathophysiological processes leading up to or
attesting to the beginning of immunotherapy-related toxicity are
detected. In one embodiment, the hGM-CSF antagonist can terminate
the pathophysiological processes and avoid its sequelae. In some
embodiments, the pathophysiological processes comprise at least one
of the following: increased cytokine concentrations in serum,
increased cytokine concentrations in CSF, increased C-reactive
protein (CRP) in serum, increased ferritin in the serum, increased
IL-6 in serum, endothelial activation, disseminated intravascular
coagulation (DIC), increased ANG2 serum concentration, increased
ANG2:ANG1 ratio in serum, CAR T-cell presence in CSF, increased Von
Willebrand factor (VWF) serum concentration, blood-brain-barrier
(BBB) leakage, or any combination thereof.
[0190] In another embodiment, the hGM-CSF antagonist may be
administered therapeutically, at multiple time points. In another
embodiment, administration of the hGM-CSF antagonist is at least at
two time points. In another embodiment, administration of the
hGM-CSF antagonist is at least at three time points.
[0191] In one embodiment, the hGM-CSF antagonist is administered
once. In another embodiment, the hGM-CSF antagonist is administered
twice. In another embodiment, the hGM-CSF antagonist is
administered three times. In another embodiment, the hGM-CSF
antagonist is administered four times. In another embodiment, the
hGM-CSF antagonist is administered at least four times. In another
embodiment, the hGM-CSF antagonist is administered more than four
times.
[0192] A skilled artisan would appreciate that
immunotherapy-related toxicity is managed by different treatments.
In some embodiments, the hGM-CSF antagonist is co-administered with
other treatments. In some embodiments, other treatments are
selected from a group comprising: cytokine-directed therapy,
anti-IL-6 therapy, corticosteroids, tocilizumab, siltuximab,
low-dose vasopressors, inotropic agents, supplemental oxygen,
diuresis, thoracentesis, antiepileptics, benzodiazepines,
levetiracetam, phenobarbital, hyperventilation, hyperosmolar
therapy, and standard therapies for specific organ toxicities.
[0193] In some embodiments, immunotherapy-related toxicity
comprises a brain disease, damage or malfunction. In some
embodiments, immunotherapy-related toxicity comprises CAR T-cell
related NT. In some embodiments, immunotherapy-related toxicity
comprises CAR T-cell-related encephalopathy syndrome (CRES). In
some embodiments, provided herein methods for inhibiting or
reducing the incidence of a brain disease, damage or
malfunction.
[0194] In some embodiments, inhibiting or reducing the incidence of
CRES comprises ameliorating headaches. In some embodiments,
inhibiting or reducing the incidence of CRES comprises alleviating
delirium. In some embodiments, inhibiting or reducing the incidence
of CRES comprises reducing anxiety. In some embodiments, inhibiting
or reducing the incidence of CRES comprises reducing tremors. In
some embodiments, inhibiting or reducing the incidence of CRES
comprises decreasing seizure activity. In some embodiments,
inhibiting or reducing the incidence of CRES comprises decreasing
confusion. In some embodiments, inhibiting or reducing the
incidence of CRES comprises reducing alterations in
wakefulness.
[0195] In some embodiments, inhibiting or reducing the incidence of
CRES comprises reducing hallucinations. In some embodiments,
inhibiting or reducing the incidence of CRES comprises reducing
dysphasia. In some embodiments, inhibiting or reducing the
incidence of CRES comprises reducing ataxia. In some embodiments,
inhibiting or reducing the incidence of CRES comprises reducing
apraxia. In some embodiments, inhibiting or reducing the incidence
of CRES comprises ameliorating facial nerve palsy. In some
embodiments, inhibiting or reducing the incidence of CRES comprises
reducing motor weakness. In some embodiments, inhibiting or
reducing the incidence of CRES comprises reducing seizures. In some
embodiments, inhibiting or reducing the incidence of CRES comprises
reducing non-convulsive EEG seizures. In some embodiments,
inhibiting or reducing the incidence or severity of CRES comprises
improving coma recovery.
[0196] In some embodiments, inhibiting or reducing the incidence or
severity of CRES comprises reducing endothelial activation. A
skilled artisan would appreciate that endothelial activation is an
inflammatory and procoagulant state of endothelial cells
characterized by increased interactions with leukocytes.
[0197] In some embodiments, inhibiting or reducing the incidence of
CRES comprises reducing vascular leak. The term "vascular leak" may
be used interchangeably with the terms "vascular leak syndrome" and
"capillary leak syndrome" having all the same qualities and
meanings. A skilled artisan would appreciate that vascular leak is
associated with endothelial cells are separated allowing a leakage
of plasma and transendothelial migration of inflammatory cells into
body tissues, resulting in tissue and organ damage. In addition,
neutrophils can cause microcirculatory occlusion, leading to
decreased tissue perfusion. In some embodiments reducing the
incidence of CRES comprises reducing intravascular coagulation.
[0198] In some embodiments, inhibiting or reducing the incidence of
CRES comprises reducing the concentration of at least one
circulating cytokine. In some embodiments, the cytokine is selected
from a group comprising: hGM-CSF, IFN.gamma., IL-1, IL-15, IL-6,
IL-8, IL-10, and IL-2. In some embodiments, inhibiting or reducing
the incidence of CRES comprises reducing serum concentration of
ANG2. In some embodiments, inhibiting or reducing the incidence of
CRES comprises reducing ANG2:ANG1 ratio in serum.
[0199] In some embodiments, inhibiting or reducing the incidence of
CRES comprises reducing the CRES grade. In some embodiments,
inhibiting or reducing the incidence of CRES comprises improving
CARTOX-10 score. In some embodiments, inhibiting or reducing the
incidence of CRES comprises reducing a raise in intracranial
pressure. In some embodiments, inhibiting or reducing the incidence
of CRES comprises reducing seizures. In some embodiments,
inhibiting or reducing the incidence of CRES comprises reducing
motor weakness.
[0200] In some embodiments, immunotherapy-related toxicity
comprises CAR T-cell related CRS. In some embodiments, provided
herein are methods for inhibiting or reducing the incidence or
severity of CRS and/or NT.
[0201] In some embodiments, inhibiting or reducing the incidence of
CRS or NT comprises, without limitation, ameliorating fever (with
or without rigors, malaise, fatigue, anorexia, myalgia, arthralgia,
nausea, vomiting, headache, skin rash, diarrhea, tachypnea,
hypoxemia, hypoxia, shock, cardiovascular tachycardia, widened
pulse pressure, hypotension, capillary leak, increased early
cardiac output, diminished late cardiac output, elevated D-dimer,
hypofibrinogenemia with or without bleeding, azotemia,
transaminitis, hyperbilirubinemia, mental status changes,
confusion, delirium, frank aphasia, hallucinations, tremor,
dysmetria, altered gait, seizures, organ failure, or any
combination thereof, or any other symptom or characteristic known
in the art to be associated with CRS.
[0202] In some embodiments, inhibiting or reducing the incidence of
CRS comprises reducing the concentration of at least one
circulating cytokine. In some embodiments, the cytokine is selected
from a group comprising: GM-CSF, IFN.gamma., IL-1, IL-15, IL-6,
IL-8, IL-10, and IL-2.
[0203] In some embodiments, inhibiting or reducing the incidence of
CRS comprises reducing the CRS grade. In some embodiments,
inhibiting or reducing the incidence of NT comprises reducing the
NT grade. In some embodiments, inhibiting or reducing the incidence
of CRS comprises improving CARTOX-10 score. In some embodiments,
inhibiting or reducing the incidence of NT comprises improving
CARTOX-10 score. In some embodiments, inhibiting or reducing the
incidence of CRS comprises reducing raised intracranial pressure.
In some embodiments, inhibiting or reducing the incidence of CRS
comprises reducing seizures. In some embodiments, inhibiting or
reducing the incidence of CRS comprises reducing motor weakness. In
some embodiments, inhibiting or reducing the incidence of NT or CRS
comprises inhibiting or reducing the incidence to less than 60%. In
some embodiments, inhibiting or reducing the incidence of NT or CRS
comprises inhibiting or reducing the incidence to less than 50%. In
some embodiments, inhibiting or reducing the incidence of NT or CRS
comprises inhibiting or reducing the incidence to less than 40%. In
some embodiments, inhibiting or reducing the incidence of NT or CRS
comprises inhibiting or reducing the incidence to less than 30%. In
some embodiments, inhibiting or reducing the incidence of NT or CRS
comprises inhibiting or reducing the incidence to less than 20% of
patients. In some embodiments, inhibiting or reducing the incidence
of NT or CRS comprises eliminating NT or CRS.
[0204] In some embodiments, the subject has Grade 1 CRS and/or NT.
In some embodiments, the subject has Grade 2 CRS and or NT. In some
embodiments, the subject has Grade 3 CRS and/or NT. In some
embodiments, the subject has Grade 4 CRS and/or NT. In some
embodiments, the subject has any combination of the above.
[0205] In some embodiments, inhibiting or reducing the incidence of
NT or CRS comprises reducing the CRS grade, the NT grade, or both.
In some embodiments, the grade is reduced to .ltoreq.3 NT and/or
CRS in 95% of patients.
[0206] In some embodiments, the subject has a body temperature
above 37.degree. C. following immunotherapy administration. In some
embodiments, the subject has a body temperature above 38.degree. C.
following immunotherapy administration. In some embodiments, the
subject has a body temperature above 39.degree. C. following
immunotherapy administration. In some embodiments, the subject has
a body temperature above 40.degree. C. following immunotherapy
administration. In some embodiments, the subject has a body
temperature above 41.degree. C. following immunotherapy
administration. In some embodiments, the subject has a body
temperature above 42.degree. C. following immunotherapy
administration.
[0207] In some embodiments, the subject has IL-6 serum
concentration above 10 pg/mL following immunotherapy
administration. In some embodiments, the subject has IL-6 serum
concentration above 12 pg/mL following immunotherapy
administration. In some embodiments, the subject has IL-6 serum
concentration above 14 pg/mL following immunotherapy
administration. In some embodiments, the subject has IL-6 serum
concentration above 16 pg/mL following immunotherapy
administration. In some embodiments, the subject has IL-6 serum
concentration above 18 pg/mL following immunotherapy
administration. In some embodiments, the subject has IL-6 serum
concentration above 20 pg/mL following immunotherapy
administration. In some embodiments, the subject has IL-6 serum
concentration above 22 pg/mL following immunotherapy
administration.
[0208] In some embodiments, the subject has an MCP-1 serum
concentration above 200 pg/ml following immunotherapy
administration. In some embodiments, the subject has an MCP-1 serum
concentration above 400 pg/ml following immunotherapy
administration. In some embodiments, the subject has an MCP-1 serum
concentration above 600 pg/ml following immunotherapy
administration. In some embodiments, the subject has an MCP-1 serum
concentration above 800 pg/ml following immunotherapy
administration. In some embodiments, the subject has an MCP-1 serum
concentration above 1000 pg/ml following immunotherapy
administration. In some embodiments, the subject has an MCP-1 serum
concentration above 1200 pg/ml following immunotherapy
administration. In some embodiments, the subject has an MCP-1 serum
concentration above 1400 pg/ml following immunotherapy
administration. In some embodiments, the subject has an MCP-1 serum
concentration above 1600 pg/ml following immunotherapy
administration. In some embodiments, the subject has an MCP-1 serum
concentration above 1800 pg/ml following immunotherapy
administration. In some embodiments, the subject has an MCP-1 serum
concentration above 2000 pg/ml following immunotherapy
administration.
[0209] In some embodiments, the subject has Grade 1 CRES. In some
embodiments, the subject has Grade 2 CRES. In some embodiments, the
subject has Grade 3 CRES. In some embodiments, the subject has
Grade 4 CRES.
[0210] In some embodiments, the subject is predisposed to have a
brain disease, damage or malfunction prior to immunotherapy. In
some embodiments, the predisposition is genetic. In some
embodiments, the predisposition is acquired. In some embodiments,
the predisposition regards an existing medical condition. In some
embodiments, the predisposition is diagnosed prior to
immunotherapy. In some embodiments, the predisposition is not
diagnosed. In some embodiments, the subject goes through medical
evaluations in order to determine predisposition to acquire an
immunotherapy-related brain disease, damage or malfunction prior to
immunotherapy.
[0211] In some embodiments, medical evaluations comprise
determining ANG1 concentration in a body fluid. In some
embodiments, medical evaluations comprise determining ANG1
concentration in serum. In some embodiments, medical evaluations
comprise determining ANG2 concentration in a body fluid. In some
embodiments, medical evaluations comprise determining ANG2
concentration in serum. In some embodiments, medical evaluations
comprise calculating the ANG2:ANG1 ratio in serum. In some
embodiments, subjects with serum ANG2:ANG1 ratio above 0.5 prior to
immunotherapy are predisposed to CRES. In some embodiments,
subjects with serum ANG2:ANG1 ratio above 0.7 prior to
immunotherapy are predisposed to CRES. In some embodiments,
subjects with serum ANG2:ANG1 ratio above 0.9 prior to
immunotherapy are predisposed to CRES. In some embodiments,
subjects with serum ANG2:ANG1 ratio above 1 prior to immunotherapy
are predisposed to CRES. In some embodiments, subjects with serum
ANG2:ANG1 ratio above 1.1 prior to immunotherapy are predisposed to
CRES. In some embodiments, subjects with serum ANG2:ANG1 ratio
above 1.3 prior to immunotherapy are predisposed to CRES. In some
embodiments, subjects with serum ANG2:ANG1 ratio above 1.5 prior to
immunotherapy are predisposed to CRES.
[0212] In some embodiments, immunotherapy-related toxicity
comprises hemophagocytic lymphohistiocytosis (HLH). In some
embodiments, immunotherapy-related toxicity comprises
macrophage-activation syndrome (MAS). In some embodiments, provided
herein methods for inhibiting or reducing the incidence of HLH. In
some embodiments, provided herein methods for inhibiting or
reducing the incidence of MAS.
[0213] In some embodiments, inhibiting or reducing the incidence of
HLH comprises increasing survival of the subject. In some
embodiments, inhibiting reducing the incidence of HLH comprises
increasing time to relapse. In some embodiments, inhibiting or
reducing the incidence of MAS comprises increasing survival of the
subject. In some embodiments, inhibiting reducing the incidence of
MAS comprises increasing time to relapse.
[0214] In some embodiments, inhibiting or reducing the incidence of
HLH or MAS comprises inhibiting macrophage activation and/or
proliferation. In some embodiments, inhibiting or reducing the
incidence of HLH or MAS comprises inhibiting T lymphocytes
activation and/or proliferation. In some embodiments, inhibiting or
reducing the incidence of HLH or MAS comprises reducing the
concentration of circulating IFN.gamma.. In some embodiments,
inhibiting or reducing the incidence of HLH or MAS comprises
reducing the concentration of circulating of GM-CSF.
[0215] In some embodiments the subject presents with fever
following immunotherapy. In some embodiments the subject presents
with splenomegaly following immunotherapy. In some embodiments the
subject presents with cytopenia following immunotherapy. In some
embodiments the subject presents with cytopenia in two or more cell
lines following immunotherapy. In some embodiments the subject
presents with hypertriglyceridemia following immunotherapy. In some
embodiments the subject presents with hypofibrinogenemia following
immunotherapy. In some embodiments the subject presents with
hemophagocytosis following immunotherapy. In some embodiments
hemophagocytosis is observed in bone marrow. In some embodiments
the subject presents with low NK-cell activity following
immunotherapy. In some embodiments the subject presents with absent
NK activity following immunotherapy.
[0216] In some embodiments the subject presents with ferritin serum
concentrations above 100 U/ml following immunotherapy. In some
embodiments the subject presents with ferritin serum concentrations
above 300 U/ml following immunotherapy. In some embodiments the
subject presents with ferritin serum concentrations above 500 U/ml
following immunotherapy. In some embodiments the subject presents
with ferritin serum concentrations above 700 U/ml following
immunotherapy. In some embodiments the subject presents with
ferritin serum concentrations above 900 U/ml following
immunotherapy.
[0217] In some embodiments the subject presents with soluble CD25
serum concentration above 1200 U/ml following immunotherapy. In
some embodiments the subject presents with soluble CD25 serum
concentration above 1500 U/ml following immunotherapy. In some
embodiments the subject presents with soluble CD25 serum
concentration above 1800 U/ml following immunotherapy. In some
embodiments the subject presents with soluble CD25 serum
concentration above 2000 U/ml following immunotherapy. In some
embodiments the subject presents with soluble CD25 serum
concentration above 2200 U/ml following immunotherapy. In some
embodiments the subject presents with soluble CD25 serum
concentration above 2400 U/ml following immunotherapy. In some
embodiments the subject presents with soluble CD25 serum
concentration above 2700 U/ml following immunotherapy. In some
embodiments the subject presents with soluble CD25 serum
concentration above 3000 U/ml following immunotherapy.
[0218] In some embodiments, the subject is predisposed to have HLH.
In some embodiments, the predisposition is genetic. In some
embodiments, the predisposition regards an existing medical
condition. A skilled artisan would appreciate that sporadic HLH has
been associated with a number of genetic mutations. In some
embodiments, the subject carries a mutation in a gene selected from
PRF1, UNC13D, STX11, STXBP2, or RAB27A, or any combination thereof.
In some embodiments, the subject has reduced or absent expression
of perforin.
hGM-CSF Antagonists
[0219] hGM-CSF antagonists suitable for use selectively interfere
with the induction of signaling by the hGM-CSF receptor by causing
a reduction in the binding of hGM-CSF to the receptor. Such
antagonists may include antibodies that bind the hGM-CSF receptor,
antibodies that bind to hGM-CSF, GM-CSF analogs such as E21R, and
other proteins or small molecules that compete for binding of
hGM-CSF to its receptor or inhibit signaling that normally results
from the binding of the ligand to the receptor.
[0220] In many embodiments, the hGM-CSF antagonist used in the
invention is a polypeptide e.g., an anti-hGM-CSF antibody, an
anti-hGM-CSF receptor antibody, a soluble hGM-CSF receptor, or a
modified GM-CSF polypeptide that competes for binding with hGM-CSF
to a receptor, but is inactive. Such proteins are often produced
using recombinant expression technology. Such methods are widely
known in the art. General molecular biology methods, including
expression methods, can be found, e.g., in instruction manuals,
such as, Sambrook and Russell (2001) Molecular Cloning: A
laboratory manual 3rd ed. Cold Spring Harbor Laboratory Press;
Current Protocols in Molecular Biology (2006) John Wiley and Sons
ISBN: 0-471-50338-X.
[0221] A variety of prokaryotic and/or eukaryotic based protein
expression systems may be employed to produce a hGM-CSF antagonist
protein. Many such systems are widely available from commercial
suppliers.
hGM-CSF Antibodies
[0222] The hGM-CSF antibodies of the present invention are
antibodies that bind with high affinity to hGM-CSF and are
antagonists of hGM-CSF. The antibodies comprise variable regions
with a high degree of identity to human germ-line V.sub.H and
V.sub.L sequences. In preferred embodiments, the BSD sequence in
CDRH3 of an antibody of the invention comprises the amino acid
sequence RQRFPY (SEQ ID NO: 12) or RDRFPY (SEQ ID NO: 13). The BSD
in CDRL3 comprises FNK or FNR.
[0223] Complete V-regions are generated in which the BSD forms part
of the CDR3 and additional sequences are used to complete the CDR3
and add a FR4 sequence. Typically, the portion of the CDR3
excluding the BSD and the complete FR4 are comprised of human
germ-line sequences. In some embodiments, the CDR3-FR4 sequence
excluding the BSD differs from human germ-line sequences by not
more than 2 amino acids on each chain. In some embodiments, the
J-segment comprises a human germline J-segment. Human germline
sequences can be determined, for example, through the publicly
available international ImMunoGeneTics database (IMGT) and V-base
(on the worldwide web at vbase.mrc-cpe.cam.ac.uk).
[0224] The human germline V-segment repertoire consists of 51 heavy
chain V-regions, 40 K light chain V-segments, and 31.lamda. light
chain V-segments, making a total of 3,621 germline V-region pairs,
in addition, there are stable allelic variants for most of these
V-segments, but the contribution of these variants to the
structural diversity of the germline repertoire is limited. The
sequences of all human germ-line V-segment genes are known and can
be accessed in the V-base database, provided by the MRC Centre for
Protein Engineering, Cambridge, United Kingdom (see, also Chothia
et al., 1992, J Mol Biol 227:776-798; Tomlinson et al., 1995, EMBO
J 14:4628-4638; and Williams et al., 1996, J Mol Biol
264:220-232).
[0225] Antibodies or antibodies fragments as described herein can
be expressed in prokaryotic or eukaryotic microbial systems or in
the cells of higher eukaryotes such as mammalian cells.
[0226] An antibody that is employed in the invention can be in any
format. For example, in some embodiments, the antibody can be a
complete antibody including a constant region, e.g., a human
constant region, or can be a fragment or derivative of a complete
antibody, e.g., an Fd, a Fab, Fab', F(ab')2, scFv, Fv, an Fv
fragment, or a single domain antibody, such as a nanobody or a
camelid antibody. Such antibodies may additionally be recombinantly
engineered by methods well known to persons of skill in the art. As
noted above, such antibodies can be produced using known
techniques.
[0227] In some embodiments, the hGM-CSF antagonist is an antibody
that binds to hGM-CSF or an antibody that binds to the hGM-CSF
receptor .alpha. or .beta. subunit. The antibodies can be raised
against hGM-CSF (or hGM-CSF receptor) proteins, or fragments, or
produced recombinantly. Antibodies to GM-CSF for use in the
invention can be neutralizing or can be non-neutralizing antibodies
that bind GM-CSF and increase the rate of in vivo clearance of
hGM-CSF such that the hGM-CSF level in the circulation is reduced.
Often, the hGM-CSF antibody is a neutralizing antibody.
[0228] Methods of preparing polyclonal antibodies are known to the
skilled artisan (e.g., Harlow & Lane, Antibodies, A Laboratory
Manual (1988); Methods in Immunology). Polyclonal antibodies can be
raised in a mammal by one or more injections of an immunizing agent
and, if desired, an adjuvant. The immunizing agent includes a
GM-CSF or GM-CSF receptor protein, e.g., a human GM-CSF or GM-CSF
receptor protein, or fragment thereof.
[0229] In some embodiment, a GM-CSF antibody for use in the
invention is purified from human plasma. In such embodiments, the
GM-CSF antibody is typically a polyclonal antibody that is isolated
from other antibodies present in human plasma. Such an isolation
procedure can be performed, e.g., using known techniques, such as
affinity chromatography.
[0230] In some embodiments, the GM-CSF antagonist is a monoclonal
antibody. Monoclonal antibodies may be prepared using hybridoma
methods, such as those described by Kohler & Milstein, Nature
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent, such as human GM-CSF, to elicit lymphocytes that produce or
are capable of producing antibodies that will specifically bind to
the immunizing agent. Alternatively, the lymphocytes may be
immunized in vitro. The immunizing agent preferably includes human
GM-CSF protein, fragments thereof, or fusion protein thereof.
[0231] Human monoclonal antibodies can be produced using various
techniques known in the art, including phage display libraries
(Hoogenboom & Winter, J. MoI. Biol. 227:381 (1991); Marks et
al, J. MoI. Biol. 222:581 (1991)). The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, p. 77 (1985) and Boerner et al., J. Immunol.
147(1):86-95 (1991)). Similarly, human antibodies can be made by
introducing of human immunoglobulin loci into transgenic animals,
e.g., mice in which the endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody
production is observed, which closely resembles that seen in humans
in all respects, including gene rearrangement, assembly, and
antibody repertoire. This approach is described, e.g., in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
5,661,016, and in the following scientific publications: Marks et
al., Bio/Technology 10:779-783 (1992); Lonberg et al, Nature
368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et
al, Nature Biotechnology 14:845-51 (1996); Neuberger, Nature
Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev.
Immunol. 13:65-93 (1995).
[0232] In some embodiments the anti-GM-CSF antibodies are chimeric
or humanized monoclonal antibodies. As noted supra, humanized forms
of antibodies are chimeric immunoglobulins in which residues from a
complementary determining region (CDR) of human antibody are
replaced by residues from a CDR of a non-human species such as
mouse, rat or rabbit having the desired specificity, affinity and
capacity.
[0233] In some embodiments of the invention, the antibody is
additionally engineered to reduced immunogenicity, e.g., so that
the antibody is suitable for repeat administration. Methods for
generating antibodies with reduced immunogenicity include
humanization/humaneering procedures and modification techniques
such as de-immunization, in which an antibody is further
engineered, e.g., in one or more framework regions, to remove T
cell epitopes.
[0234] In some embodiments, the antibody is a humaneered antibody.
A humaneered antibody is an engineered human antibody having a
binding specificity of a reference antibody, obtained by joining a
DNA sequence encoding a binding specificity determinant (BSD) from
the CDR3 region of the heavy chain of the reference antibody to
human VH segment sequence and a light chain CDR3 BSD from the
reference antibody to a human VL segment sequence. Methods for
Humaneering are provided in US patent application publication no.
20050255552 and US patent application publication no. 20060134098.
Methods for signal-less secretion of antibody fragments from E.
coli are described in US patent application 20070020685.
[0235] An antibody can further be de-immunized to remove one or
more predicted T-cell epitopes from the V-region of an antibody.
Such procedures are described, for example, in WO 00/34317.
[0236] The heavy chain constant region is often a gamma chain
constant region, for example, a gamma-1, gamma-2, gamma-3, or
gamma-4 constant region. In some embodiments, e.g., where the
antibody is a fragment, the antibody can be conjugated to another
molecule, e.g., to provide an extended half-life in vivo such as a
polyethylene glycol (pegylation) or serum albumin. Examples of
PEGylation of antibody fragments are provided in Knight et al
(2004) Platelets 15: 409 (for abciximab); Pedley et al (1994) Br.
J. Cancer 70: 1126 (for an anti-CEA antibody) Chapman et al (1999)
Nature Biotech. 17: 780.
[0237] An antibody for use in the invention binds to hGM-CSF or
hGM-CSF receptor. Any number of techniques can be used to determine
antibody binding specificity. See, e.g., Harlow & Lane,
Antibodies, A Laboratory Manual (1988) for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity of an antibody.
[0238] An exemplary antibody suitable for use with the present
invention is c19/2 (a mouse/human chimeric anti-hGM-CSF antibody).
In some embodiments, a monoclonal antibody that competes for
binding to the same epitope as c19/2, or that binds the same
epitope as c19/2, is used. The ability of a particular antibody to
recognize the same epitope as another antibody is typically
determined by the ability of the first antibody to competitively
inhibit binding of the second antibody to the antigen. Any of a
number of competitive binding assays can be used to measure
competition between two antibodies to the same antigen. For
example, a sandwich ELISA assay can be used for this purpose. This
is carried out by using a capture antibody to coat the surface of a
well. A subsaturating concentration of tagged-antigen is then added
to the capture surface. This protein will be bound to the antibody
through a specific antibody-epitope interaction. After washing a
second antibody, which has been covalently linked to a detectable
moiety (e.g., HRP, with the labeled antibody being defined as the
detection antibody) is added to the ELISA. If this antibody
recognizes the same epitope as the capture antibody it will be
unable to bind to the target protein as that particular epitope
will no longer be available for binding. If, however this second
antibody recognizes a different epitope on the target protein it
will be able to bind and this binding can be detected by
quantifying the level of activity (and hence antibody bound) using
a relevant substrate. The background is defined by using a single
antibody as both capture and detection antibody, whereas the
maximal signal can be established by capturing with an antigen
specific antibody and detecting with an antibody to the tag on the
antigen. By using the background and maximal signals as references,
antibodies can be assessed in a pair-wise manner to determine
epitope specificity.
[0239] A first antibody is considered to competitively inhibit
binding of a second antibody, if binding of the second antibody to
the antigen is reduced by at least 30%, usually at least about 40%,
50%, 60% or 75%, and often by at least about 90%, in the presence
of the first antibody using any of the assays described above.
[0240] In some embodiments of the invention, an antibody is
employed that competes with binding, or bind, to the same epitope
as a known antibody, e.g., c19/2. Method of mapping epitopes are
well known in the art. For example, one approach to the
localization of functionally active regions of human
granulocyte-macrophage colony-stimulating factor (hGM-CSF) is to
map the epitopes recognized by neutralizing anti-hGM-CSF monoclonal
antibodies. For example, the epitope to which c19/2 (which has the
same variable regions as the neutralizing antibody LMM 102) binds
has been defined using proteolytic fragments obtained by enzymic
digestion of bacterially synthesized hGM-CSF (Dempsey, et al,
Hybridoma 9:545-558, 1990). RP-HPLC fractionation of a tryptic
digest resulted in the identification of an immunoreactive "tryptic
core" peptide containing 66 amino acids (52% of the protein).
Further digestion of this "tryptic core" with S. aureus V8 protease
produced a unique immunoreactive hGM-CSF product comprising two
peptides, residues 86-93 and 112-127, linked by a disulfide bond
between residues 88 and 121. The individual peptides were not
recognized by the antibody.
[0241] In some embodiments, the antibodies suitable for use with
the present invention have a high affinity binding for human GM-CSF
or hGM-CSF receptor. High affinity binding between an antibody and
an antigen exists if the dissociation constant (KD) of the antibody
is <about 10 nM, typically <1 nM, and preferably <100 pM.
In some embodiments, the antibody has a dissociation rate of about
10.sup.-4 per second or better.
[0242] A variety of methods can be used to determine the binding
affinity of an antibody for its target antigen such as surface
plasmon resonance assays, saturation assays, or immunoassays such
as ELISA or RIA, as are well known to persons of skill in the art.
An exemplary method for determining binding affinity is by surface
plasmon resonance analysis on a BIAcore.TM. 2000 instrument
(Biacore AB, Freiburg, Germany) using CM5 sensor chips, as
described by Krinner et al, (2007) MoI. Immunol. February;
44(5):916-25. (Epub 2006 May H)).
[0243] In some embodiments, the hGM-CSF antagonists are
neutralizing antibodies to hGM-CSF, its receptor or its receptor
subunit, which bind in a manner that interferes with the binding of
hGM-CSF to its receptor or receptor subunit. In some embodiments,
an anti-hGM-CSF antibody for use in the invention inhibits binding
to the alpha subunit of the hGM-CSF receptor. Such an antibody can,
for example, bind to hGM-CSF at the region where hGM-CSF binds to
the receptor and thereby inhibit binding. In another embodiments,
the anti-hGM-CSF antibody inhibits hGM-CSF functioning without
blocking its binding to the alpha subunit of the hGM-CSF
receptor.
[0244] II. Heavy Chains
[0245] A heavy chain of an anti-hGM-CSF antibody of the invention
comprises a heavy-chain V-region that comprises the following
elements:
[0246] 1) human heavy-chain V-segment sequences comprising
FR1-CDR1-FR2-CDR2-FR3
[0247] 2) a CDRH3 region comprising the amino acid sequence
R(Q/D)RFPY (SEQ ID NO: 22).
[0248] 3) a FR4 contributed by a human germ-line J-gene
segment.
[0249] Examples of V-segment sequences that support binding to
hGM-CSF in combination with a CDR3-FR4 segment described above
together with a complementary V.sub.L region are shown in FIG. 1.
The V-segments can be, e.g., from the human VH1 subclass. In some
embodiments, the V-segment is a human V.sub.H1 sub-class segment
that has a high degree of amino-acid sequence identity, e.g., at
least 80%, 85%, or 90% or greater identity, to the germ-line
segment VH1 1-02 (SEQ ID NO: 19) or VH1 1-03 (SEQ ID NO: 20). In
some embodiments, the V-segment differs by not more than 15
residues from VH1 1-02 or VH1 1-03 and preferably not more than 7
residues.
[0250] The FR4 sequence of the antibodies of the invention is
provided by a human JH1, JH3, JH4, JH5 or JH6 gene germline
segment, or a sequence that has a high degree of amino-acid
sequence identity to a human germline JH segment. In some
embodiments, the J segment is a human germline JH4 sequence.
[0251] The CDRH3 also comprises sequences that are derived from a
human J-segment. Typically, the CDRH3-FR4 sequence excluding the
BSD differs by not more than 2 amino acids from a human germ-line
J-segment. In typical embodiments, the J-segment sequences in CDRH3
are from the same J-segment used for the FR4 sequences. Thus, in
some embodiments, the CDRH3-FR4 region comprises the BSD and a
complete human JH4 germ-line gene segment. An exemplary combination
of CDRH3 and FR4 sequences is shown below, in which the BSD is in
bold and human germ-line J-segment JH4 residues are underlined:
[0252] CDR3.
[0253] R(Q/D)RFPYYFDYWGOGTLVTVSS (SEQ ID NO: 23)
[0254] In some embodiments, an antibody of the invention comprises
a V-segment that has at least 90% identity, or at least 91%, 92%
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the
germ-line segment VH 1-02 or VH1-03; or to one of the V-segments of
the V.sub.H regions shown in FIG. 1, such as a V-segment portion of
VH #1, VH #2, VH #3, VH #4, or VH #5.
[0255] In some embodiments, the V-segment of the V.sub.H region has
a CDR1 and/or CDR2 as shown in FIG. 1. For example, an antibody of
the invention may have a CDR1 that has the sequence GYYMH (SEQ ID
NO: 24) or NYYIH (SEQ ID NO: 25); or a CDR2 that has the sequence
WINPNSGGTNYAQKFQG (SEQ ID NO:26) or WINAGNGNTKYSQKFQG (SEQ ID NO:
27).
[0256] In particular embodiments, an antibody has both a CDR1 and a
CDR2 from one of the V.sub.H region V-segments shown in FIG. 1 and
a CDR3 that comprises R(Q/D)RFPY (SEQ ID NO: 22), e.g., RDRFPYYFDY
(SEQ ID NO: 16) or RQRFPYYFDY (SEQ ID NO: 15). Thus, in some
embodiments, an anti-GM-CSF antibody of the invention, may for
example, have a CDR3-FR4 that has the sequence
R(Q/D)RFPYYFDYWGQGTLVTVSS (SEQ ID NO: 23) and a CDR1 and/or CDR2 as
shown in FIG. 1.
[0257] In some embodiments, a V.sub.H region of an antibody of the
invention has a CDR3 that has a binding specificity determinant
R(Q/D)RFPY (SEQ ID NO: 22), a CDR2 from a human germline VH1
segment or a CDR1 from a human germline VH1. In some embodiments,
both the CDR1 and CDR2 are from human germline VH1 segments.
[0258] III. Light Chains
[0259] A light chain of an anti-hGM-CSF antibody of the invention
comprises at light-chain V-region that comprises the following
elements:
[0260] 1) human light-chain V-segment sequences comprising
FR1-CDR1-FR2-CDR2-FR3
[0261] 2) a CDRL3 region comprising the sequence FNK or FNR, e.g.,
QQFNRSPLT (SEQ ID NO: 28)_or QQFNKSPLT (SEQ ID NO: 18).
[0262] 3) a FR4 contributed by a human germ-line J-gene
segment.
[0263] The V.sub.L region comprises either a Vlambda or a Vkappa
V-segment. An example of a Vkappa sequence that supports binding in
combination with a complementary VH-region is provided in FIG.
1.
[0264] The V.sub.L region CDR3 sequence comprises a J-segment
derived sequence. In typical embodiments, the J-segment sequences
in CDRL3 are from the same J-segment used for FR4. Thus, the
sequence in some embodiments may differ by not more than 2 amino
acids from human kappa germ-line V-segment and J-segment sequences.
In some embodiments, the CDRL3-FR4 region comprises the BSD and the
complete human JK4 germline gene segment. Exemplary CDRL3-FR4
combinations for kappa chains are shown below in which the minimal
essential binding specificity determinant is shown in bold and JK4
sequences are underlined:
TABLE-US-00003 CDR3 (SEQ ID NO: 29) QQFNRSPLTFGGGTKVEIK (SEQ ID NO:
30) QQFNKSPLTFGGGTKVEIK
[0265] The Vkappa segments are typically of the VKIII sub-class. In
some embodiments, the segments have at least 80% sequence identity
to a human germline VKIII subclass, e.g., at least 80% identity to
the human germ-line VKIIIA27 (SEQ ID NO: 21) sequence. In some
embodiments, the Vkappa segment may differ by not more than 18
residues from VKIIIA27. In other embodiments, the V.sub.L region
V-segment of an antibody of the invention has at least 85%
identity, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100% identity to the human kappa V-segment sequence of a
V.sub.L region shown in FIG. 1, for example, the V-segment sequence
of VK #1, VK #2, VK #3, or VK #4.
[0266] In some embodiments, the variable region is comprised of
human V-gene sequences. For example, a variable region sequence can
have at least 80% identity, or at least 85% identity, at least 90%
identity, at least 95% identity, at least 96% identity, at least
97% identity, at least 98% identity, or at least 99% identity, or
greater, with a human germ-line V-gene sequence.
[0267] In some embodiments, the V-segment of the V.sub.L region has
a CDR1 and/or CDR2 as shown in FIG. 1. For example, an antibody of
the invention may have a CDR1 sequence of RASQSVGTNVA (SEQ ID NO:
31) or RASQSIGSNLA (SEQ ID NO: 32) RASQS(V/I)G(T/S)N(V/L)A (SEQ ID
NO: 39); or a CDR2 sequence STSSRAT (SEQ ID NO: 33).
[0268] In particular embodiments, an anti-GM-CSF antibody of the
invention may have a CDR1 and a CDR2 in a combination as shown in
one of the V-segments of the V.sub.L regions set forth in FIG. 1
and a CDR3 sequence that comprises FNK or FNR, e.g., the CDR3 may
be QQFNKSPLT (SEQ ID NO: 18) or QQFNRSPLT (SEQ ID NO: 28). In some
embodiments, such a GM-CSF antibody may comprise an FR4 region that
is FGGGTKVEIK (SEQ ID NO: 34). Thus, an anti-GM-CSF antibody of the
invention, can comprise, e.g., both the CDR1 and CDR2 from one of
the V.sub.L regions shown in FIG. 1 and a CDR3-FR4 region that is
FGGGTKVEIK (SEQ ID NO: 34).
[0269] IV. Preparation of hGM-CSF Antibodies
[0270] An antibody of the invention may comprise any of the V.sub.H
regions VH #1, VH #2, VH #3, VH #4, or VH #5 as shown in FIG. 1. In
some embodiment, an antibody of the invention may comprise any of
the V.sub.L regions VK #1, VK #2, VK #3, or VK #4 as shown in FIG.
1. In some embodiments, the antibody has a V.sub.H region VH #1, VH
#2, VH #3, VH #4, or VH #5 as shown in FIG. 1; and a V.sub.L region
VK #1, VK #2, VK #3, or VK #4 as shown in FIG. 1, as described,
e.g., in U.S. Pat. Nos. 8,168,183 and 9,017,674, each of which is
incorporated herein by reference in its entirety.
[0271] An antibody may be tested to confirm that the antibody
retains the activity of antagonizing hGM-CSF activity. The
antagonist activity can be determined using any number of
endpoints, including proliferation assays. Neutralizing antibodies
and other hGM-CSF antagonists may be identified or evaluated using
any number of assays that assess hGM-CSF function. For example,
cell-based assays for hGM-CSF receptor signaling, such as assays
which determine the rate of proliferation of a hGM-CSF-dependent
cell line in response to a limiting amount of hGM-CSF, are
conveniently used. The human TF-1 cell line is suitable for use in
such an assay. See, Krinner et al., (2007) Mol. Immunol. In some
embodiments, the neutralizing antibodies of the invention inhibit
hGM-CSF stimulated TF-I cell proliferation by at least 50%, when a
hGM-CSF concentration is used which stimulates 90% maximal TF-I
cell proliferation. Thus, typically, a neutralizing antibody, or
other hGM-CSF antagonist for use in the invention, has an EC50 of
less than 10 nM (e.g., Table 2). Additional assays suitable for use
in identifying neutralizing antibodies suitable for use with the
present invention will be well known to persons of skill in the
art. In other embodiments, the neutralizing antibodies inhibit
hGM-CSF stimulated proliferation by at least about 75%, 80%, 90%,
95%, or 100%, of the antagonist activity of the antibody chimeric
c19/2, e.g., WO03/068920, which has the variable regions of the
mouse monoclonal antibody LMM102 and the CDRs.
[0272] An exemplary chimeric antibody suitable for use as a hGM-CSF
antagonist is c19/2. The c19/2 antibody binds hGM-CSF with a
monovalent binding affinity of about 10 pM as determined by surface
plasmon resonance analysis. The heavy and light chain variable
region sequences of c19/2 antibody are known (e.g., WO03/068920).
The CDRs, as defined according to Kabat, are:
TABLE-US-00004 (SEQ ID NO: 35) CDRH1 DYNIH (SEQ ID NO: 36) CDRH2
YIAPYSGGTGYNQEFKN (SEQ ID NO: 16) CDRH3 RDRFPYYFDY (SEQ ID NO: 37)
CDRL1 KASQNVGSNVA (SEQ ID NO: 38) CDRL2 SASYRSG (SEQ ID NO: 28)
CDRL3 QQFNRSPLT
[0273] The CDRs can also be determined using other well-known
definitions in the art, e.g., Chothia, international ImMunoGeneTics
database (IMGT), and AbM.
[0274] In some embodiments, an antibody used in the invention
competes for binding to, or binds to, the same epitope as c19/2.
The GM-CSF epitope recognized by c19/2 has been identified as a
product that has two peptides, residues 86-93 and residues 112-127,
linked by a disulfide bond between residues 88 and 121. The c19/2
antibody inhibits the GM-CSF-dependent proliferation of a human
TF-I leukemia cell line with an EC50 of 30 pM when the cells are
stimulated with 0.5 ng/ml GM-CSF. In some embodiments, the antibody
used in the invention binds to the same epitope as c19/2.
[0275] An antibody for administration, such as c19/2, can be
additionally Humaneered. For example, the c19/2 antibody can be
further engineered to contain human V gene segments.
[0276] A high-affinity antibody may be identified using well known
assays to determine binding activity and affinity. Such techniques
include ELISA assays as well as binding determinations that employ
surface plasmon resonance or interferometry. For example,
affinities can be determined by biolayer interferometry using a
ForteBio (Mountain View, Calif.) Octet biosensor. An antibody of
the invention typically binds with similar affinity to both
glycosylated and non-glycosylated form of hGM-CSF.
[0277] Antibodies of the invention compete with c19/2 antibody for
binding to hGM-CSF. The ability of an antibody described herein to
block or compete with c19/2 antibody for binding to hGM-CSF
indicates that the antibody binds to the same epitope c19/2
antibody or to an epitope that is close to, e.g., overlapping, with
the epitope that is bound by c19/2 antibody. In other embodiments
an antibody described herein, e.g., an antibody comprising a
V.sub.H and V.sub.L region combination as shown in the table
provided in FIG. 1, can be used as a reference antibody for
assessing whether another antibody competes for binding to hGM-CSF.
A test antibody is considered to competitively inhibit binding of a
reference antibody, if binding of the reference antibody to the
antigen is reduced by at least 30%, usually at least about 40%,
50%, 60% or 75%, and often by at least about 90%, in the presence
of the test antibody. Many assays can be employed to assess
binding, including ELISA, as well as other assays, such as
immunoblots. In some embodiments, an antibody of the invention has
a dissociation rate that is at least 2 to 3-fold slower than a
reference chimeric c19/2 monoclonal antibody assayed under the same
conditions, but has a potency that is at least 6-10 times greater
than that of the reference antibody in neutralizing hGM-CSF
activity in a cell-based assay that measures hGM-CSF activity.
[0278] Methods for the isolation of antibodies with V-region
sequences close to human germ-line sequences have previously been
described (US patent application publication nos. 20050255552 and
20060134098). Antibody libraries may be expressed in a suitable
host cell including mammalian cells, yeast cells or prokaryotic
cells. For expression in some cell systems, a signal peptide can be
introduced at the N-terminus to direct secretion to the
extracellular medium. Antibodies may be secreted from bacterial
cells such as E. coli with or without a signal peptide. Methods for
signal-less secretion of antibody fragments from E. coli are
described in US patent application 20070020685.
[0279] In some embodiments, an hGM-CSF-binding antibody of the
invention is generated where, an antibody that has a CDR from one
of the VH-regions of the invention shown in FIG. 1, is combined
with an antibody having a CDR of one of the V.sub.L-regions shown
in FIG. 1, and expressed in any of a number of formats in a
suitable expression system. Thus, the antibody may be expressed as
a scFv, Fab, Fab' (containing an immunoglobulin hinge sequence),
F(ab')2, (formed by di-sulfide bond formation between the hinge
sequences of two Fab' molecules), whole immunoglobulin or truncated
immunoglobulin or as a fusion protein in a prokaryotic or
eukaryotic host cell, either inside the host cell or by secretion.
A methionine residue may optionally be present at the N-terminus,
for example, in polypeptides produced in signal-less expression
systems. Each of the VH-regions described herein may be paired with
each of the V.sub.L regions to generate an anti-hGM-CSF antibody.
In an embodiment, a fusion protein comprises an
anti-hGM-CSF-binding antibody of the invention or a fragment
thereof (in non-limiting examples, an anti-hGM-CSF antibody
fragment is a Fab, a Fab', a F(ab')2, a scFv, or a dAB), and human
transferrin, wherein the human transferrin is fused to the antibody
at the end of the heavy chain constant region 1 (CH1), after the
hinge, or after C.sub.H3, as described in Shin, S-U., et al. Proc.
Natl. Acad. Sci. USA, Vol. 92, pp. 2820-2824, 1995, which is
incorporated herein by reference in its entirety.
[0280] Exemplary combinations of heavy and light chains are shown
in the table provided in FIG. 1. In some embodiment, the antibody
VL region, e.g., VK #1, VK #2, VK #3, or VK #4 of FIG. 1, is
combined with a human kappa constant region to form the complete
light-chain. Further, in some embodiments, the VH region is
combined a human gamma-1 constant regions. Any suitable gamma-1
allotype can be chose, such as the f-allotype. Thus, in some
embodiments, the antibody is an IgG, e.g., having an f-allotype,
that has a VH selected from VH #1, VH #2, VH #3, VH #4, or VH #5
(FIG. 1), and a VL selected from VK #1, VK #2, VK #3, or VK #4
(FIG. 1).
[0281] The antibodies of the invention inhibit hGM-CSF receptor
activation, e.g., by inhibiting hGM-CSF binding to the receptor,
and exhibit high affinity binding to hGM-CSF, e.g., 500 pM. In some
embodiments, the antibody has a dissociation constant of about 10-4
per sec or less. Not to be bound by theory, an antibody with a
slower dissociation constant provides improved therapeutic benefit.
For example, an antibody of the invention that has a three-fold
slower off-rate than c19/2 antibody, produced a 10-fold more potent
hGM-CSF neutralizing activity, e.g., in a cell-based assay such as
IL-8 production (see, e.g., Example 2).
[0282] Antibodies may be produced using any number of expression
systems, including both prokaryotic and eukaryotic expression
systems. In some embodiments, the expression system is a mammalian
cell expression, such as a CHO cell expression system. Many such
systems are widely available from commercial suppliers. In
embodiments in which an antibody comprises both a V.sub.H and
V.sub.L region, the V.sub.H and V.sub.L regions may be expressed
using a single vector, e.g., in a dicistronic expression unit, or
under the control of different promoters. In other embodiments, the
V.sub.H and V.sub.L region may be expressed using separate vectors.
A V.sub.H or V.sub.L region as described herein may optionally
comprise a methionine at the N-terminus.
[0283] An antibody of the invention may be produced in any number
of formats, including as a Fab, a Fab', a F(ab')2, a scFv, or a
dAB. An antibody of the invention can also include a human constant
region. The constant region of the light chain may be a human kappa
or lambda constant region. The heavy chain constant region is often
a gamma chain constant region, for example, a gamma-1, gamma-2,
gamma-3, or gamma-4 constant region. In other embodiments, the
antibody may be an IgA.
[0284] In some embodiments of the invention, the antibody V.sub.L
region, e.g., VK #1, VK #2, VK #3, or VK #4 of FIG. 1, is combined
with a human kappa constant region (e.g., SEQ ID NO:10) to form the
complete light-chain.
[0285] In some embodiments of the invention, the V.sub.H region is
combined a human gamma-1 constant region. Any suitable gamma-1 f
allotype can be chosen, such as the f-allotype. Thus, in some
embodiments, the antibody is an IgG having an f-allotype constant
region, e.g., SEQ ID NO:11, that has a V.sub.H selected from VH #1,
VH #2, VH #3, VH #4, or VH #5 (FIG. 1). In some embodiments, the
antibody has a V.sub.L selected from VK #1, VK #2, VK #3, or VK #4
(FIG. 1.) In particular embodiments, the antibody has a kappa
constant region as set forth in SEQ ID NO:10, and a heavy chain
constant region as set forth in SEQ ID NO:11, where the heavy and
light chain variable regions comprise one of the following
combinations from the sequences set forth in FIG. 1: a) VH #2, VK
#3; b) VH #1, VK #3; c) VH #3, VK #1; d) VH #3, VL #3; e) VH #4, VK
#4; f) VH #4, VK #2; g) VH #5, VK #1; h) VH #5, VK #2; i) VH #3, VK
#4; or j) VH #3, VL #3).
[0286] In some embodiments, e.g., where the antibody is a fragment,
the antibody can be conjugated to another molecule, e.g.,
polyethylene glycol (PEGylation) or serum albumin, to provide an
extended half-life in vivo. Examples of PEGylation of antibody
fragments are provided in Knight et al. Platelets 15:409, 2004 (for
abciximab); Pedley et al., Br. J. Cancer 70:1126, 1994 (for an
anti-CEA antibody); Chapman et al., Nature Biotech. 17:780, 1999;
and Humphreys, et al., Protein Eng. Des. 20: 227, 2007).
[0287] In some embodiments, the antibodies of the invention are in
the form of a Fab' fragment. A full-length light chain is generated
by fusion of a V.sub.L-region to human kappa or lambda constant
region. Either constant region may be used for any light chain;
however, in typical embodiments, a kappa constant region is used in
combination with a Vkappa variable region and a lambda constant
region is used with a Vlambda variable region.
[0288] The heavy chain of the Fab' is a Fd' fragment generated by
fusion of a V.sub.H-region of the invention to human heavy chain
constant region sequences, the first constant (CH1) domain and
hinge region. The heavy chain constant region sequences can be from
any of the immunoglobulin classes, but is often from an IgG, and
may be from an IgG1, IgG2, IgG3 or IgG4. The Fab' antibodies of the
invention may also be hybrid sequences, e.g., a hinge sequence may
be from one immunoglobulin sub-class and the CH1 domain may be from
a different sub-class.
[0289] V. Administration of Anti-hGM-CSF Antibodies for the
Treatment of Diseases in which GM-CSF is a Target.
[0290] The invention also provides methods of treating a patient
that has a disease involving hGM-CSF in which it is desirable to
inhibit hGM-CSF activity, i.e., in which hGM-CSF is a therapeutic
target. In some embodiments, such a patient has a chronic
inflammatory disease, e.g., arthritis, e.g., rheumatoid arthritis,
psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic
arthritis, systemic-onset Still's disease and other inflammatory
diseases of the joints; inflammatory bowel diseases, e.g.,
ulcerative colitis, Crohn's disease, Barrett's syndrome, ileitis,
enteritis, eosinophilic esophagitis and gluten-sensitive
enteropathy; inflammatory disorders of the respiratory system, such
as asthma, eosinophilic asthma, adult respiratory distress
syndrome, allergic rhinitis, silicosis, chronic obstructive
pulmonary disease, hypersensitivity lung diseases, interstitial
lung disease, diffuse parenchymal lung disease, bronchiectasis;
inflammatory diseases of the skin, including psoriasis,
scleroderma, and inflammatory dermatoses such as eczema, atopic
dermatitis, urticaria, and pruritis; disorders involving
inflammation of the central and peripheral nervous system,
including multiple sclerosis, idiopathic demyelinating
polyneuropathy, Guillain-Barre syndrome, chronic inflammatory
demyelinating polyneuropathy, neurofibromatosis and
neurodegenerative diseases such as Alzheimer's disease. Various
other inflammatory diseases can be treated using the methods of the
invention. These include systemic lupus erythematosis,
immune-mediated renal disease, e.g., glomerulonephritis, and
spondyloarthropathies; and diseases with an undesirable chronic
inflammatory component such as systemic sclerosis, idiopathic
inflammatory myopathies, Sjogren's syndrome, vasculitis,
sarcoidosis, thyroiditis, gout, otitis, conjunctivitis, sinusitis,
sarcoidosis, Behcet's syndrome, autoimmune lymphoproliferative
syndrome (or ALPS, also known as Canale-Smith syndrome),
Ras-associated autoimmune leukoproliferative disorder (or RALD),
Noonan syndrome, hepatobiliary diseases such as hepatitis, primary
biliary cirrhosis, granulomatous hepatitis, and sclerosing
cholangitis. In some embodiments, the patient has inflammation
following injury to the cardiovascular system. Various other
inflammatory diseases include Kawasaki's disease, Multicentric
Castleman's Disease, tuberculosis and chronic cholecystitis.
Additional chronic inflammatory diseases are described, e.g., in
Harrison's Principles of Internal Medicine, 12th Edition, Wilson,
et al., eds., McGraw-Hill, Inc.). In some embodiments, a patient
treated with an antibody has a cancer in which GM-CSF contributes
to tumor or cancer cell growth, including but not limited to, e.g.,
acute myeloid leukemia, plexiform neurofibromatosis, autoimmune
lymphoproliferative syndrome (or ALPS, also known as Canale-Smith
syndrome), Ras-associated autoimmune leukoproliferative disorder
(or RALD), Noonan syndrome, chronic myelomonocytic leukemia,
juvenile myelomonocytic leukemia, and acute myeloid leukemia. In
some embodiments, a patient treated with an antibody of the
invention has, or is at risk of heart failure, e.g., due to
ischemic injury to the cardiovascular system such as ischemic heart
disease, stroke, and atherosclerosis. In some embodiments, a
patient treated with an antibody of the invention has asthma. In
some embodiments, a patient treated with an antibody of the
invention has Alzheimer's disease. In some embodiments, a patient
treated with an antibody of the invention has osteopenia, e.g.,
osteoporosis. In some embodiments, a patient treated with an
antibody of the invention has thrombocytopenia purpura. In some
embodiments, the patient has Type I or Type II diabetes. In some
embodiments, a patient may have more than one disease in which
GM-CSF is a therapeutic target, e.g., a patient may have rheumatoid
arthritis and heart failure, or osteoporosis and rheumatoid
arthritis, etc.
[0291] Two other examples of neutralizing anti-GM-CSF antibody are
the human E1O antibody and human G9 antibody described in Li et al,
(2006) PNAS 103(10):3557-3562. E1O and G9 are IgG class antibodies.
E1O has an 870 pM binding affinity for GM-CSF and G9 has a 14 pM
affinity for GM-CSF. Both antibodies are specific for binding to
human GM-CSF and show strong neutralizing activity as assessed with
a TFl cell proliferation assay.
[0292] An additional exemplary neutralizing anti-GM-CSF antibody is
the MT203 antibody described by Krinner et al, (MoI Immunol.
44:916-25, 2007; Epub 2006 May 112006). MT203 is an IgG1 class
antibody that binds GM-CSF with picomolar affinity. The antibody
shows potent inhibitory activity as assessed by TF-I cell
proliferation assay and its ability to block IL-8 production in
U937 cells.
[0293] Additional antibodies suitable for use with the present
invention will be known to persons of skill in the art.
[0294] hGM-CSF antagonists that are anti-hGM-CSF receptor
antibodies can also be employed with the methods of the present
disclosure. Such hGM-CSF antagonists include antibodies to the
hGM-CSF receptor alpha chain or beta chain. An anti-hGM-CSF
receptor antibody employed in the invention can be in any antibody
format as explained above, e.g., intact, chimeric, monoclonal,
polyclonal, antibody fragment, humanized, Humaneered, and the like.
Examples of anti-hGM-CSF receptor antibodies, e.g., neutralizing,
high-affinity antibodies, suitable for use in the invention are
known (see, e.g., U.S. Pat. No. 5,747,032 and Nicola et al., Blood
82: 1724, 1993).
Non-Antibody GM-CSF Antagonists
[0295] Other proteins that may interfere with the productive
interaction of hGM-CSF with its receptor include mutant hGM-CSF
proteins and secreted proteins comprising at least part of the
extracellular portion of one or both of the hGM-CSF receptor chains
that bind to hGM-CSF and compete with binding to cell-surface
receptor. For example, a soluble hGM-CSF receptor antagonist can be
prepared by fusing the coding region of the sGM-CSFR alpha with the
CH2-CH3 regions of murine IgG2a. An exemplary soluble hGM-CSF
receptor is described by Raines et al. (1991) Proc. Natl. Acad. Sci
USA 88: 8203. An example of a GM-CSFR alpha-Fc fusion protein is
provided, e.g., in Brown et al (1995) Blood 85: 1488. In some
embodiments, the Fc component of such a fusion can be engineered to
modulate binding, e.g., to increase binding, to the Fc
receptor.
[0296] Other hGM-CSF antagonists include hGM-CSF mutants. For
example, hGM-CSF having a mutation of amino acid residue 21 of
hGM-CSF to Arginine or Lysine (E21R or E21K) described by Hercus et
al. Proc. Natl. Acad. Sci USA 91:5838, 1994 has been shown to have
in vivo activity in preventing dissemination of hGM-CSF-dependent
leukemia cells in mouse xenograft models (Iversen et al. Blood
90:4910, 1997). As appreciated by one of skill in the art, such
antagonists can include conservatively modified variants of hGM-CSF
that have substitutions, such as the substitution noted at amino
acid residue 21, or hGM-CSF variants that have, e.g., amino acid
analogs to prolong half-life.
[0297] In some embodiments, the hGM-CSF antagonist may be a
peptide. For example, an hGM-CSF peptide antagonist may be a
peptide designed to structurally mimic the positions of specific
residues on the B and C helices of human GM-CSF that are implicated
in receptor binding and bioactivity (e.g., Monfardini et al, J.
Biol. Chem 271:2966-2971, 1996).
[0298] In other embodiments, the hGM-CSF antagonist is an "antibody
mimetic" that targets and binds to the antigen in a manner similar
to antibodies. Certain of these "antibody mimics" use
non-immunoglobulin protein scaffolds as alternative protein
frameworks for the variable regions of antibodies. For example, Ku
et al. (Proc. Natl. Acad. Sci. U.S.A. 92(14):6552-6556 (1995))
discloses an alternative to antibodies based on cytochrome b562 in
which two of the loops of cytochrome b562 were randomized and
selected for binding against bovine serum albumin. The individual
mutants were found to bind selectively with BSA similarly with
anti-BSA antibodies. U.S. Pat. Nos. 6,818,418 and 7,115,396
disclose an antibody mimic featuring a fibronectin or
fibronectin-like protein scaffold and at least one variable loop.
Known as Adnectins, these fibronectin-based antibody mimics exhibit
many of the same characteristics of natural or engineered
antibodies, including high affinity and specificity for any
targeted ligand. The structure of these fibronectin-based antibody
mimics is similar to the structure of the variable region of the
IgG heavy chain. Therefore, these mimics display antigen binding
properties similar in nature and affinity to those of native
antibodies. Further, these fibronectin-based antibody mimics
exhibit certain benefits over antibodies and antibody fragments.
For example, these antibody mimics do not rely on disulfide bonds
for native fold stability, and are, therefore, stable under
conditions which would normally break down antibodies. In addition,
since the structure of these fibronectin-based antibody mimics is
similar to that of the IgG heavy chain, the process for loop
randomization and shuffling may be employed in vitro that is
similar to the process of affinity maturation of antibodies in
vivo.
[0299] Beste et al. (Proc. Natl. Acad. Sci. U.S.A. 96(5):1898-1903
(1999)) disclose an antibody mimic based on a lipocalin scaffold
(Anticalin.RTM.). Lipocalins are composed of a .beta.-barrel with
four hypervariable loops at the terminus of the protein. The loops
were subjected to random mutagenesis and selected for binding with,
for example, fluorescein. Three variants exhibited specific binding
with fluorescein, with one variant showing binding similar to that
of an anti-fluorescein antibody. Further analysis revealed that all
of the randomized positions are variable, indicating that Anticalin
would be suitable to be used as an alternative to antibodies. Thus,
Anticalins are small, single chain peptides, typically between 160
and 180 residues, which provides several advantages over
antibodies, including decreased cost of production, increased
stability in storage and decreased immunological reaction.
[0300] U.S. Pat. No. 5,770,380 discloses a synthetic antibody
mimetic using the rigid, non-peptide organic scaffold of
calixarene, attached with multiple variable peptide loops used as
binding sites. The peptide loops all project from the same side
geometrically from the calixarene, with respect to each other.
Because of this geometric confirmation, all of the loops are
available for binding, increasing the binding affinity to a ligand.
However, in comparison to other antibody mimics, the
calixarene-based antibody mimic does not consist exclusively of a
peptide, and therefore it is less vulnerable to attack by protease
enzymes. Neither does the scaffold consist purely of a peptide, DNA
or RNA, meaning this antibody mimic is relatively stable in extreme
environmental conditions and has a long life-span. Further, since
the calixarene-based antibody mimic is relatively small, it is less
likely to produce an immunogenic response.
[0301] Murali et al. (Cell MoI Biol 49(2):209-216 (2003)) describe
a methodology for reducing antibodies into smaller peptidomimetics,
they term "antibody-like binding peptidomimetics" (ABiP) which may
also be useful as an alternative to antibodies.
[0302] In addition to non-immunoglobulin protein frameworks,
antibody properties have also been mimicked in compounds comprising
RNA molecules and unnatural oligomers (e.g., protease inhibitors,
benzodiazepines, purine derivatives and beta-turn mimics).
Accordingly, non-antibody GM-CSF antagonists can also include such
compounds.
Therapeutic Administration
[0303] In some embodiments, the methods of the present disclosure
comprise administering a hGM-CSF antagonist, (e.g., an anti-hGM-CSF
antibody) as a pharmaceutical composition to a subject having a CRS
or a cytokine storm. In some embodiments, the hGM-CSF antagonist is
administered in a therapeutically effective amount using a dosing
regimen suitable for treatment of the disease.
[0304] In some embodiments, a therapeutically effective amount is
an amount that at least partially arrests the condition or its
symptoms. For example, a therapeutically effective amount may
arrest immune activation, may decrease the levels of circulating
cytokines, may decrease T-cell activation, or may ameliorate fever,
malaise, fatigue, anorexia, myalgias, arthalgias, nausea, vomiting,
headache, skin rash, nausea, vomiting, diarrhea, tachypnea,
hypoxemia, cardiovascular tachycardia, widened pulse pressure,
hypotension, increased cardiac output (early), potentially
diminished cardiac output (late), elevated D-dimer,
hypofibrinogenemia with or without bleeding, azotemia,
transaminitis, hyperbilirubinemia, headache, mental status changes,
confusion, delirium, word finding difficulty or frank aphasia,
hallucinations, tremor, dysmetria, altered gait, or seizures.
[0305] The methods of the invention comprise administering an
anti-hGM-CSF antibody as a pharmaceutical composition to a patient
in a therapeutically effective amount using a dosing regimen
suitable for treatment of the disease. The composition can be
formulated for use in a variety of drug delivery systems. One or
more physiologically acceptable excipients or carriers can also be
included in the compositions for proper formulation. Suitable
formulations for use in the present invention are found in
Remington: The Science and Practice of Pharmacy, 21st Edition,
Philadelphia, Pa. Lippincott Williams & Wilkins, 2005. For a
brief review of methods for drug delivery, see, Langer, Science
249: 1527-1533 (1990).
[0306] The anti-hGM-CSF antibody for use in the methods of the
invention is provided in a solution suitable for injection into the
patient such as a sterile isotonic aqueous solution for injection.
The antibody is dissolved or suspended at a suitable concentration
in an acceptable carrier. In some embodiments the carrier is
aqueous, e.g., water, saline, phosphate buffered saline, and the
like. The compositions may contain auxiliary pharmaceutical
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting
agents, and the like.
[0307] The pharmaceutical compositions of the invention are
administered to a patient, e.g., a patient that has osteopenia,
rheumatoid arthritis, juvenile idiopathic arthritis, systemic-onset
Still's disease, asthma, eosinophilic asthma, eosinophilic
esophagitis, multiple sclerosis, psoriasis, atopic dermatitis,
plexiform neurofibromatosis, autoimmune lymphoproliferative
syndrome (or ALPS, also known as Canale-Smith syndrome),
Ras-associated autoimmune leukoproliferative disorder (or RALD),
Noonan syndrome, chronic myelomonocytic leukemia, juvenile
myelomonocytic leukemia, acute myeloid leukemia, Multicentric
Castleman's Disease, chronic obstructive pulmonary disease,
interstitial lung disease, diffuse parenchymal lung disease,
idiopathic thrombocytopenia purpura, Alzheimer's disease, heart
failure, Kawasaki's Disease, cardiac damage due to an ischemic
event, or diabetes, in an amount sufficient to cure or at least
partially arrest the disease or symptoms of the disease and its
complications. An amount adequate to accomplish this is defined as
a "therapeutically effective dose." A therapeutically effective
dose is determined by monitoring a patient's response to therapy.
Typical benchmarks indicative of a therapeutically effective dose
includes amelioration of symptoms of the disease in the patient.
Amounts effective for this use will depend upon the severity of the
disease and the general state of the patient's health, including
other factors such as age, weight, gender, administration route,
etc. Single or multiple administrations of the antibody may be
administered depending on the dosage and frequency as required and
tolerated by the patient. In any event, the methods provide a
sufficient quantity of anti-hGM-CSF antibody to effectively treat
the patient.
[0308] The antibody may be administered alone, or in combination
with other therapies to treat the disease of interest.
[0309] The antibody can be administered by injection or infusion
through any suitable route including but not limited to
intravenous, sub-cutaneous, intramuscular or intraperitoneal
routes. In some embodiments, the antibody may be administered by
insufflation. In an exemplary embodiment, the antibody may be
stored at 10 mg/ml in sterile isotonic aqueous saline solution for
injection at 4.degree. C. and is diluted in either 100 ml or 200 ml
0.9% sodium chloride for injection prior to administration to the
patient. The antibody is administered by intravenous infusion over
the course of 1 hour at a dose of between 0.2 and 10 mg/kg. In
other embodiments, the antibody is administered, for example, by
intravenous infusion over a period of between 15 minutes and 2
hours. In still other embodiments, the administration procedure is
via sub-cutaneous or intramuscular injection.
[0310] In some embodiments, the hGM-CSF antagonist, e.g., an
anti-hGM-CSF antibody, is administered by a perispinal route.
Perispinal administration involves anatomically localized delivery
performed so as to place the therapeutic molecule directly in the
vicinity of the spine at the time of initial administration.
Perispinal administration is described, e.g., in U.S. Pat. No.
7,214,658 and in Tobinick & Gross, J. Neuroinflammation 5:2,
2008.
[0311] The dose of hGM-CSF antagonist is chosen in order to provide
effective therapy for a subject that has been diagnosed with CRS or
cytokine storm. The dose is typically in the range of about 0.1
mg/kg body weight to about 50 mg/kg body weight or in the range of
about 1 mg to about 2 g per patient. The dose is often in the range
of about 1 to about 20 mg/kg or approximately about 50 mg to about
2000 mg/patient. The dose may be repeated at an appropriate
frequency which may be in the range once per day to once every
three months, depending on the pharmacokinetics of the antagonist
(e.g. half-life of the antibody in the circulation) and the
pharmacodynamic response (e.g. the duration of the therapeutic
effect of the antibody). In some embodiments where the antagonist
is an antibody or modified antibody fragment, the in vivo half-life
of between about 7 and about 25 days and antibody dosing is
repeated between once per week and once every 3 months. In other
embodiments, the antibody is administered approximately once per
month.
[0312] A V.sub.H region and/or V.sub.L region of the invention may
also be used for diagnostic purposes. For example, the V.sub.H
and/or V.sub.L region may be used for clinical analysis, such as
detection of GM-CSF levels in a patient. A V.sub.H or V.sub.L
region of the invention may also be used, e.g., to produce anti-Id
antibodies.
[0313] Unless otherwise defined herein, scientific and technical
terms used in connection with the present application shall have
the meanings that are commonly understood by those of skill in the
art. Further, unless otherwise required by context, singular terms
shall include pluralities and plural terms shall include the
singular.
[0314] In one embodiment, "treating" comprises therapeutic
treatment and "preventing" comprises prophylactic or preventative
measures, wherein the object is to prevent or lessen the targeted
pathologic condition or disorder as described hereinabove. Thus, in
one embodiment, treating may include directly affecting or curing,
suppressing, inhibiting, preventing, reducing the severity of,
delaying the onset of, reducing symptoms associated with the
disease, disorder or condition, or a combination thereof. Thus, in
one embodiment, "treating," "ameliorating," and "alleviating" refer
inter alia to delaying progression, expediting remission, inducing
remission, augmenting remission, speeding recovery, increasing
efficacy of or decreasing resistance to alternative therapeutics,
or a combination thereof. In one embodiment, "preventing" refers,
inter alia, to delaying the onset of symptoms, preventing relapse
to a disease, decreasing the number or frequency of relapse
episodes, increasing latency between symptomatic episodes, or a
combination thereof. In one embodiment, "suppressing" or
"inhibiting", refers inter alia to reducing the severity of
symptoms, reducing the severity of an acute episode, reducing the
number of symptoms, reducing the incidence of disease-related
symptoms, reducing the latency of symptoms, ameliorating symptoms,
reducing secondary symptoms, reducing secondary infections,
prolonging patient survival, or a combination thereof.
[0315] In the present disclosure the singular forms "a," "an," and
"the" include the plural reference, and reference to a particular
numerical value includes at least that particular value, unless the
context clearly indicates otherwise. The term "plurality", as used
herein, means more than one. When a range of values is expressed,
another embodiment includes from the one particular and/or to the
other particular value.
[0316] Similarly, when values are expressed as approximations, by
use of the antecedent "about," it is understood that the particular
value forms another embodiment. All ranges are inclusive and
combinable. In some embodiments, the term "about", refers to a
deviance of between 0.0001-5% from the indicated number or range of
numbers. In some embodiments, the term "about", refers to a
deviance of between 1-10% from the indicated number or range of
numbers. In some embodiments, the term "about", refers to a
deviance of up to 25% from the indicated number or range of
numbers. The term "comprises" means encompasses all the elements
listed, but may also include additional, unnamed elements, and it
may be used interchangeably with the terms "encompasses",
"includes", or "contains" having all the same qualities and
meanings. The term "consisting of" means being composed of the
recited elements or steps, and it may be used interchangeably with
the terms "composed of" having all the same qualities and
meanings.
EXAMPLES
Example 1--Exemplary Humaneered Antibodies to GM-CSF
[0317] A panel of engineered Fab' molecules with the specificity of
c19/2 were generated from epitope-focused human V-segment libraries
as described in US patent application publication nos. 20060134098
and 20050255552. Epitope-focused libraries were constructed from
human V-segment library sequences linked to a CDR3-FR4 region
containing BSD sequences in CDRH3 and CDRL3 together with human
germ-line J-segment sequences. For the heavy chain, human germ-line
JH4 sequence was used and for the light chain, human germ-line JK4
sequence was used.
[0318] Full-length Humaneered V-regions from a Vh1-restricted
library were selected that supported binding to recombinant human
GM-CSF. The "full-length" V-kappa library was used as a base for
construction of "cassette" libraries as described in US patent
application publication no. 20060134098, in which only part of the
murine c19/2 antibody V-segment was initially replaced by a library
of human sequences. Two types of cassettes were constructed.
Cassettes for the V-kappa chains were made by bridge PCR with
overlapping common sequences within the framework 2 region. In this
way "front-end" and "middle" human cassette libraries were
constructed for the human V-kappa III isotype. Human V-kappa III
cassettes which supported binding to GM-CSF were identified by
colony-lift binding assay and ranked according to affinity in
ELISA. The V-kappa human "front-end" and "middle" cassettes were
fused together by bridge PCR to reconstruct a fully human V-kappa
region that supported GM-CSF binding activity. The Humaneered Fabs
thus consist of Humaneered V-heavy and V-kappa regions that support
binding to human GM-CSF.
[0319] Binding activity was determined by surface plasmon resonance
(spr) analysis. Biotinylated GM-CSF was captured on a
streptavidin-coated CM5 biosensor chip. Humaneered Fab fragments
expressed from E. coli were diluted to a starting concentration of
30 nM in 10 mM HEPES, 150 mM NaCl, 0.1 mg/ml BSA and 0.005% P20 at
pH 7.4. Each Fab was diluted 4 times using a 3-fold dilution series
and each concentration was tested twice at 37 degrees C. to
determine the binding kinetics with the different density antigen
surfaces. The data from all three surfaces were fit globally to
extract the dissociation constants.
[0320] Binding kinetics were analyzed by Biacore 3000 surface
plasmon resonance (SPR). Recombinant human GM-CSF antigen was
biotinylated and immobilized on a streptavidin CM5 sensor chip. Fab
samples were diluted to a starting concentration of 3 nM and run in
a 3-fold dilution series. Assays were run in 10 mM HEPES, 150 mM
NaCl, 0.1 mg/mL BSA and 0.005% p20 at pH 7.4 and 37.degree. C. Each
concentration was tested twice. Fab' binding assays were run on two
antigen density surfaces providing duplicate data sets. The mean
affinity (KD) for each of 6 various humaneered anti-GM-CSF Fab
clones, calculated using a 1:1 Langmuir binding model, is shown in
Table 2.
[0321] Fabs were tested for GM-CSF neutralization using a TF-I cell
proliferation assay. GM-CSF-dependent proliferation of human TF-I
cells was measured after incubation for 4 days with 0.5 ng/ml
GM-CSF using a MTS assay (Cell titer 96, Promega) to determine
viable cells. All Fabs inhibited cell proliferation in this assay
indicating that these are neutralizing antibodies. There is a good
correlation between relative affinities of the anti-GM-CSF Fabs and
EC50 in the cell-based assay. Anti-GM-CSF antibodies with
monovalent affinities in the range 18 pM-104 pM demonstrate
effective neutralization of GM-CSF in the cell-based assay.
[0322] Exemplary engineered anti-GM-CSF V region sequences are
shown in FIG. 1.
TABLE-US-00005 TABLE 2 Affinity of anti-GM-CSF Fabs determined by
surface plasmon resonance analysis in comparison with activity
(EC50) in a GM-CSF dependent TF-I cell proliferation assay
Monovalent EC.sub.50(pM) in binding affinity TF-1 cell determined
by proliferation Fab SPR (pM) assay 94 18 165 104 19 239 77 29 404
92 58 539 42 104 3200 44 81 7000
Example 2--Evaluation of a Humaneered GM-CSF Antibody
[0323] This example evaluates the binding activity and biological
potency of a humaneered anti-GM-CSF antibody in a cell-based assay
in comparison to a chimeric IgG1k antibody (Ab2) having variable
regions from the mouse antibody LMM102 (Nice et al., Growth Factors
3:159, 1990). Ab1 is a humaneered IgG1k antibody against GM-CSF
having identical constant regions to Ab2.
Surface Plasmon Resonance Analysis of Binding of Human GM-CSF to
Ab1 and Ab2
[0324] Surface Plasmon resonance analysis was used to compare
binding kinetics and monovalent affinities for the interaction of
Ab and Ab2 with glycosylated human GM-CSF using a Biacore 3000
instrument. Ab1 or Ab2 was captured onto the Biacore chip surface
using polyclonal anti-human F(ab')2. Glycosylated recombinant human
GM-CSF expressed from human 293 cells was used as the analyte.
Kinetic constants were determined in 2 independent experiments (see
FIGS. 2A-2B and Table 3). The results show that GM-CSF bound to Ab2
and Ab1 with comparable monovalent affinity in this experiment.
However, Ab1 had a two-fold slower "on-rate" than Ab2, but an
"off-rate" that was approximately three-fold slower.
TABLE-US-00006 TABLE 3 Kinetic constants at 37.degree. C.
determined from the surface plasmon resonance analysis in FIGS.
2A-2B; association constant (k.sub.a), dissociation constant
(k.sub.d) and calculated affinity (KD) are shown. k.sub.a
(M.sup.-1s.sup.-1) k.sub.d (s.sup.-1) KD (pM) Ab2 7.20 .times.
10.sup.5 2.2 .times. 10.sup.-5 30.5 Ab1 2.86 .times. 10.sup.5 7.20
.times. 10.sup.-6 25.1
[0325] GM-CSF is naturally glycosylated at both N-linked and
O-linked glycosylation sites although glycosylation is not required
for biological activity. In order to determine whether GM-CSF
glycosylation affects the binding of Ab or Ab2, the antibodies were
compared in an ELISA using recombinant GM-CSF from two different
sources; GM-CSF expressed in E. coli (non-glycosylated) and GM-CSF
expressed from human 293 cells (glycosylated). The results in FIGS.
3A-3B and Table 4 showed that both antibodies bound glycosylated
and non-glycosylated GM-CSF with equivalent activities. The two
antibodies also demonstrated comparable ECso values in this
assay.
TABLE-US-00007 TABLE 4 Summary of EC.sub.50 for binding of Ab2 and
Ab1 to human GM-CSF from two different sources determined by ELISA.
Binding to recombinant GM-CSF from human 293 cells (glycosylated)
or from E. coli (non- glycosylated) was determined from two
independent experiments. Experiment 1 is shown in FIGS. 3A-3B.
Non-glycosylated Non-glycosylated Glycosylated (exp 1) (exp 2) (exp
1) Ab2 400 pM 433 pM 387 pM Ab1 373 pM 440 pM 413 pM
[0326] Ab1 is a Humaneered antibody that was derived from the mouse
variable regions present in Ab2. Ab1 was tested for overlapping
epitope specificity (Ab2) by competition ELISA.
[0327] Biotinylated Ab2 was prepared using known techniques.
Biotinylation did not affect binding of Ab2 to GM-CSF as determined
by ELISA. In the assay, Ab2 or Ab1 was added in varying
concentrations with a fixed amount of biotinylated Ab2. Detection
of biotinylated Ab2 was assayed in the presence of unlabeled Ab or
Ab1 competitor (FIGS. 4A-4B). Both Ab1 and Ab2 competed with
biotinylated Ab2 for binding to GM-CSF, thus indicating binding to
the same epitope. Ab1 competed more effectively for binding to
GM-CSF than Ab2, consistent with the slower dissociation kinetics
for Ab1 when compared with Ab2 by surface plasmon resonance
analysis.
Neutralization of GM-CSF Activity by Ab1 and Ab2
[0328] A cell-based assay for neutralization of GM-CSF activity was
employed to evaluate biological potency. The assay measures IL-8
secretion from U937 cells induced with GM-CSF. IL-8 secreted into
the culture supernatant is determined by ELISA after 16 hours
induction with 0.5 ng/ml E. coli-derived GM-CSF.
[0329] A comparison of the neutralizing activity of Ab1 and Ab2 in
this assay is shown in a representative assay in FIG. 5. In three
independent experiments, Ab1 inhibited GM-CSF activity more
effectively than Ab2 when comparing IC50 (Table 5).
TABLE-US-00008 TABLE 5 Comparison of IC50 for inhibition of GM-CSF
induced IL-8 expression. Data from three independent experiments
shown in FIG. 5 and mean IC.sub.50 are expressed in ng/ml and nM.
Experiment Ab2 (ng/ml) Ab2 (nM) Ab1 (ng/ml) Ab1 (nM) A 363 2.4 31.3
0.21 B 514 3.4 92.5 0.62 C 343 2.2 20.7 0.14 Mean 407 2.7 48.2
0.32
SUMMARY
[0330] The Humaneered Ab1 bound to GM-CSF with a calculated
equilibrium binding constant (KD) of 25 pM. Ab2 bound to GM-CSF
with a KD of 30.5 pM. Ab2 showed a two-fold higher association
constant (k.sub.a) than Ab1 for GM-CSF while Ab1 showed three-fold
slower dissociation kinetics (k.sub.d) than Ab2. Ab2 and Ab1 showed
similar binding activity for glycosylated and non-glycosylated
GM-CSF in an antigen-binding ELISA. A competition ELISA confirmed
that both antibodies competed for the same epitope; Ab1 showed
higher competitive binding activity than Ab2. In addition, Ab1
showed higher GM-CSF neutralization activity than Ab2 in a
GM-CSF-induced IL-8 induction assay.
Example 3--Administration of a Neutralizing Anti-GM-CSF Antibody in
a Mouse Model of Immunotherapy-Related Toxicity
[0331] A mouse model of immunotherapy-related toxicity can be used
to show the efficacy of an anti-GM-CSF antibody for preventing and
treating immunotherapy-related toxicity. In one model of
immunotherapy-related toxicity, mice are injected with CAR T-cells
in doses provoking toxicity. For example, van der Stegen et al. (J.
Immunol 191:4589-4598 (2013)), incorporated herein by reference,
describe a CRS model induced by the i.p. injection of a single dose
of 30.times.10.sup.6 cells termed T4.sup.+ T cells. T4.sup.+ T
cells are engineered T cells expressing the chimeric Ag receptor
(CAR) T1E28z. T cells engineered to express T1E28z are activated by
cells expressing ErbB1- and ErbB4-based dimers and ErbB2/3
heterodimer.
[0332] To evaluate the efficacy of anti-GM-CSF antibodies for
preventing and treating CRS, mice will be divided in groups (n=10),
each group receiving either: a) a single i.p. saline injection; b)
an i.p. injection of 30.times.10.sup.6 T4.sup.+ T cells; c) an i.p.
injection of 30.times.10.sup.6 T4.sup.+ T cells and 0.25 mg
intravenous (i.v.) anti-GM-CSF monoclonal antibody 22E9 (a
recombinant rat anti-mouse-GM-CSF antibody) co-administered with
T4.sup.+ T cells; d) an i.p. injection of 30.times.10.sup.6
T4.sup.+ T cells and 0.25 mg intranasal (i.n.) anti-GM-CSF antibody
22E9 co-administered with T4.sup.+ T cells; e) an i.p. injection of
30.times.10.sup.6 T4.sup.+ T cells and 0.25 mg i.v. anti-GM-CSF
antibody 22E9 6 hours before T4.sup.+ T cells administration; f) an
i.p. injection of 30.times.10.sup.6 T4.sup.+ T cells and 0.25 mg
i.n. anti-GM-CSF antibody 22E9 6 hours before T4.sup.+ T cells
administration; g) an i.p. injection of 30.times.10.sup.6 T4.sup.+
T cells and 0.25 mg i.v. anti-GM-CSF antibody 22E9 2 hours after
T4.sup.+ T cells administration; or h) an i.p. injection of
30.times.10.sup.6 T4.sup.+ T cells and 0.25 mg i.n. anti-GM-CSF
antibody 22E9 2 hours after T4.sup.+ T cells administration.
Further doses, administration times, and administration routes will
be evaluated.
[0333] In order to assess anti-GM-CSF antibody 22E9 effect, organs
will be collected from mice, formalin fixed, and subjected to
histopathologic analysis. Blood will be collected and
concentrations of human IFN.gamma., human IL-2, and mouse IL-6,
IL-2, IL-4, IL-6, IL-10, IL-17, IFN.gamma., and TNF.alpha. will be
assessed by well methods described in the literature, such as ELISA
assay. Mice weight, behavior, and clinical manifestations will be
observed.
Example 4--Anti-GM-CSF Antibody Effect on Immunotherapy
[0334] A mouse model can be used to show that GM-CSF antagonists do
not negatively affect the efficacy of cancer immunotherapy. SCID
beige mice can be inoculated with a cancer cell line and treated
with an immunotherapeutic agent known to induce CRS, as T4.sup.+ T
cells, with or without an anti-GM-CSF antibody.
[0335] To evaluate whether anti-GM-CSF antibodies affect the
efficacy of immunotherapy, mice will be divided in groups (n=10),
each group receiving either: a) a subcutaneous (s.c.) injection of
30.times.10.sup.6 SKOV3 cells; b) a s.c. injection of
30.times.10.sup.6 SKOV3 cells and an i.p. injection of
30.times.10.sup.6 T4.sup.+ T cells; or c) a s.c. implant of
30.times.10.sup.6 SKOV3 cells, an i.p. injection of
30.times.10.sup.6 T4.sup.+ T cells, and an i.v. injection of 0.25
mg of anti-GM-CSF antibody 22E9.
[0336] In order to assess anti-GM-CSF antibody 22E9 effect on
T4.sup.+ T cells efficacy, tumor size will be measured every four
days by caliper, and tumor volume calculated by the formula:
0.5.times.(larger diameter).times.(smaller diameter).sup.2. Mice
weight, behavior, and clinical manifestations will be observed. At
the end of the experiment, the animals will be sacrificed, and the
tumor tissues harvested and weighted.
Example 5--Mouse Model of Human CRS
[0337] A mouse model for CRS for investigating the effects of a
humanized anti-GM-CSF monoclonal antibody in treating or preventing
CRS was developed. (FIGS. 17a.-17b.).
[0338] Method: The model used is a primary AML model.
Immunocompromised NSG-S mice that were additionally transgenic for
human SCF, IL-3, and GM-CSF were engrafted with AML blasts derived
from AML patients that were CD123 positive. After 2-4 weeks, they
were bled to confirm engraftment and achievement of high disease
burden. The mice were then treated with high doses of CAR-T123 at
1.times.10.sup.6 cells, which is 10 times higher than doses
previously studied.
[0339] Results: It was observed that within 1-2 weeks after CAR-T
cell injection, these mice developed an illness characterized by
weakness, emaciation, hunched bodies, withdrawal, and poor motor
response. The mice eventually died of their disease within 7-10
days. The symptoms correlate with massive T-cell expansion in the
mice and with elevation of multiple human cytokines, such as IL-6,
MIP 1.alpha., IFN-.gamma., TNF.alpha., GM-CSF, MIP10, and IL-2, and
in a pattern that resembles what is seen in human CRS after CAR-T
cell therapy. GM-CSF fold change was significantly greater than
other cytokines. (FIG. 17 a-b).
Example 6--Generation of GM-CSF Knockout CAR-Ts
[0340] GM-CSF CRISPR knockout T cells were generated and shown to
exhibit reduced expression of GM-CSF but similar levels of other
cytokines and degranulation, which showed immune cell
functionality. (See FIGS. 15A-15G).
Example 7--Anti-GM-CSF Neutralizing Antibody does not Inhibit CAR-T
Mediated Killing, Proliferation, or Cytokine Production but
Neutralizes GM-CSF
[0341] Anti-GM-CSF neutralizing antibody does not inhibit CAR-T
mediated killing, proliferation, or cytokine production but
successfully neutralizes GM-CSF. (See FIGS. 16A-16J).
Example 8--Anti-GM-CSF Neutralizing Antibody does not Inhibit CAR-T
Efficacy In Vivo
[0342] Humanized anti-GM-CSF monoclonal antibody, a neutralizing
hGM-CSF antibody, does not inhibit CAR-T efficacy in vivo (FIG.
18a-18c). CAR-T efficacy in a xenograft model in combination with
an anti-GM-CSF neutralizing antibody in accordance with embodiments
described herein. As shown in FIG. 18A, NSG mice were injected with
NALM-6-GFP/Luciferase cells (human, peripheral blood leukemia pre-B
cell), and bioluminescent imaging (BLI0 was performed to confirm
tumor growth. Mice were treated with either (1) anti-GM-CSF
antibody (10 mg/Kg daily for ten days) and (a) CART19 or (b)
untransduced human T cells (UTD) 1.times.10.sup.6 cells or (2) IgG
control antibody (10 mg/Kg daily for ten days) and (a) CART19 or
(b) untransduced human T cells (UTD) 1.times.10.sup.6 cells. FIGS.
18B and 18C demonstrate that the anti-GM-CSF neutralizing antibody
did not inhibit CAR-T efficacy in vivo.
Example 9--Anti-GM-CSF Neutralizing Antibody does not Impair CAR-T
Impact on Survival
[0343] In vitro and in vivo preclinical data show anti-GM-CSF
neutralizing antibody (a humanized anti-GM-CSF monoclonal antibody)
does not impair CAR-T impact on survival in mouse models. (FIG.
19).
[0344] The anti-GM-CSF neutralizing antibody does not impede CAR-T
cell function in vivo in the absence of PBMCs. Survival shown to be
similar for CAR-T+ control and CAR-T+ anti-GM-CSF neutralizing
antibody.
Example 10--Anti-GM-CSF Neutralizing Antibody May Increase CAR-T
Expansion
[0345] In vitro and In vivo preclinical data show anti-GM-CSF
neutralizing antibody (a humanized anti-GM-CSF monoclonal antibody)
may increase CAR-T Expansion (FIG. 20). The anti-GM-CSF
neutralizing antibody may increase in vitro CAR-T cancer cell
killing. The antibody increases proliferation of CAR-T cells and
could improve efficacy. CAR-T proliferation increased by the GM-CSF
neutralizing antibody in presence of PBMCs. (It was not affected
without PBMCs). The antibody did not inhibit degranulation,
intracellular GM-CSF production, or IL-2 production.
Example 11--CAR-T Expansion Associated with Improved Overall
Response Rate
[0346] CAR-T expansion associated with improved overall response
rate. (FIG. 21). CAR AUC (area under the curve) defined as
cumulative levels of CAR+ cells/.mu.L of blood over the first 28
days post CAR-T administration. P values calculated by Wilcoxon
rank sum test. (Neelapu, et al ICML 2017 Abstract 8).
Example 12--Study Protocol for an Anti-GM-CSF Neutralizing Antibody
in Accordance with Embodiments Described Herein
[0347] Study protocol for an anti-GM-CSF neutralizing antibody (a
humaneered anti-GM-CSF monoclonal antibody) in accordance with
embodiments described herein. (See FIG. 22). CRS and NT to be
assessed daily while hospitalized and at clinic visit for first 30
days. Eligible subjects to receive GM-CSF neutralizing antibody on
days -1, +1, and +3 of CAR-T treatment. Tumor assessment to be
performed at baseline and months 1, 3, 6, 9, 12, 18, and 24. Blood
samples (PBMC and serum) days -5, -1, 0, 1, 3, 5, 7, 9, 11, 13, 21,
28, 90, 180, 270, and 360.
Example 13--GM-CSF Depletion Increases CAR-T Cell Expansion
[0348] GM-CSF depletion increases CAR-T cell expansion. (FIG.
23A-23B) FIG. 23A shows increased ex-vivo expansion of
GM-CSF.sup.k/o CAR-T cells compared to control CAR-T cells. FIG.
23b demonstrates more robust proliferation after in vivo treatment
with an anti-GM-CSF neutralizing antibody (a humaneered anti-GM-CSF
monoclonal antibody) in accordance with embodiments described
herein.
Example 14--Safety Profile of an Anti-GM-CSF Neutralizing Ab in
>100 Human Patients*
[0349] Phase I: Single-dose, dose escalation in healthy adult
volunteers. Objectives were to analyze Safety/tolerability, PK, and
Immunogenicity.
[0350] Enrollment/dose:
[0351] (n=12)
[0352] 3/1 mg/kg
[0353] 3/3 mg/kg
[0354] 3/10 mg/kg
[0355] 3/placebo
[0356] Safety Results: [0357] Clean Safety Profile: [0358] No drug
related serious adverse effects (SAE) [0359] Non-immunogenic Phase
II: 1) Dose at weeks 0, 2, 4, 8, 12 in rheumatoid arthritis
patients. Objectives were to analyze Efficacy, Safety/tolerability,
PK, and Immunogenicity.
[0360] Enrollment/dose:
[0361] (n=9)
[0362] 7/600 mg
[0363] 2/placebo
[0364] Safety Results: [0365] Clean Safety Profile: [0366] No drug
related serious adverse effects (SAE) [0367] Non-immunogenic [0368]
2) Dose at weeks 0, 2, 4, 8, 12, 16 20 in severe asthma patients.
Objectives were to analyze Efficacy, Safety/tolerability, PK, and
Immunogenicity.
[0369] Enrollment/dose:
[0370] (n=160)
[0371] 78/400 mg
[0372] 82/placebo
[0373] Safety Results: [0374] Clean Safety Profile: [0375] No drug
related serious adverse effects (SAE) [0376] Non-immunogenic * 94
patients in studies depicted above, plus 12 patients in ongoing
CMML Phase I trial, where drug is well tolerated; an additional 76
patients received a chimeric version of a GM-CSF neutralizing Ab
(KB002) and showed a similar safety profile.
[0377] All studies randomized double-blind placebo-controlled, IV
administration. (See FIG. 24.)
Example 15--Effect of Anti-GM-CSF Antibody on CART Activity and
Toxicity
[0378] The study will investigate the effect of GMCSF blockade with
anti-GM-CSF antibody on chimeric antigen receptor T cells (CART)
activity and toxicity. This can be accomplished through these two
AIMS:
AIM #1: to investigate the effect of GMCSF blockade with
anti-GM-CSF antibody on CART cell effector functions AIM #2: To
study the effect of GMCSF blockade with anti-GM-CSF antibody on
reducing cytokine release syndrome after CART cell therapy Research
strategy. The following experiments are proposed:
[0379] In vitro studies of the combination of four different doses
of GMCSF blockade with anti-GM-CSF antibody with CART cells
(cytokine production (30 plex Lumiex, including GM-CSF, IL-2, INFg,
IL-6, IL-8, MCP-1), antigen specific killing, degranulation,
proliferation and exhaustion), in the presence or absence of
myeloid cells using the model: CART19 against ALL.
[0380] In vivo studies of the combination of different doses of
GMCSF blockade with anti-GM-CSF antibody (with and without murine
GMCSF blockade) with CART cells, using two models: CD19 positive
cell line (NALM6) engrafted xenografts, treated with CART19 with or
without anti-GM-CSF antibody; and
Patient derived xenografts with primary ALL, and then treated with
CART19 with or without anti-GM-CSF antibody.
[0381] Mice will be dosed i.p with anti-GM-CSF antibody 10 mg/kg
immediately prior to CART cell implantation and 10 mg/kg/day for 10
days. Mice will be followed for tumor response and survival.
Retro-orbital bleedings will be obtained starting one week after
CART cell therapy and weekly afterwards. Disease burden, T cell
expansion kinetics, expression of exhaustion markers and cytokine
levels (30 Plex) will be analyzed. At the completion of the
experiment, spleens and bone marrows will be harvested and analyzed
for tumor characteristics and CAR-T cell numbers.
[0382] In vivo studies of the combination of GMCSF blockade with
anti-GM-CSF antibody (with or without murine GMCSF blockade) with
CART cells in CRS models (in this model, high doses of CART cells
will be used to elicit CRS), in the presence of PBMCs, using the
following model:
[0383] Primary ALL patient derived xenografts, then treated with
CART19 with or without anti-GM-CSF antibody.
[0384] Mice will be dosed i.p. with anti-GM-CSF antibody 10 mg/kg
immediately prior to CART cell implantation and 10 mg/kg/day for 10
days. Mice will be followed for tumor response, CRS toxicity
symptoms and survival. Retro-orbital bleedings will be obtained at
baseline, 2 days post, one week-post CART cell therapy and weekly
afterwards. Disease burden, T cell expansion kinetics, expression
of exhaustion markers and cytokine levels (30 Plex) will be
analyzed. At the completion of the experiment, spleens and bone
marrows will be harvested and analyzed for tumor characteristics
and CAR-T cell numbers
In Vivo Neurotoxicity Assays
[0385] Using models discussed in #3 above, mice will be imaged with
MRI while sick to assess for development of neurotoxicity after
CART cell therapy. Images will be compared between mice that
received CART cells and anti-GM-CSF antibody vs control antibody.
Repeat experiments will be performed. Mice will be euthanized 14
days after CART cells in these repeat experiments. Brain tissue
will be analyzed for cytokines with multiplex assays, for the
presence of monocytes, human T cells, and for integrity of blood
brain barrier by IHC, flow and microscopy.
Example 16
Anti-hGM-CSF Neutralizing Antibody Reduces Neuroinflammation in
CAR-T Cell Related Neurotoxicity (NT)
[0386] There is extensive scientific rationale implicating GM-CSF
as essential to the initiation of cytokine release syndrome (CRS),
neurotoxicity (NT) and the inflammatory cascade seen following
initiation of CAR-T cell therapy. The hypothesis studied is that
blocking soluble GM-CSF with the neutralizing antibody (lenzilumab)
will abrogate or prevent the onset and severity of both CRS and NT
observed with CAR-T cell therapy. Importantly, CAR-T cell activity
should be preserved or improved if possible. The experimental
design tests the effects of GM-CSF blockade with anti-GM-CSF
antibody (lenzilumab) on CAR-T cell effector functions, CAR-T
efficacy in a tumor xenograft model, development of CRS in a CRS
xenograft model and the development of NT using MRI imaging and
volumetric analysis to quantify the neuro-inflammation seen with
CAR-T cell therapy. In vitro and in vivo experiments with CAR-T+/-
lenzilumab both in the presence and absence of human PBMCs were
studied. (see Examples 9 and 10, FIGS. 19 and 20A-20B).
Methods
[0387] In vitro studies were conducted to evaluate the combination
of GM-CSF neutralizing antibody lenzilumab with human CD19+ CAR-T
cells on antigen-specific killing, degranulation, proliferation and
exhaustion in the presence or absence of human PBMCs.
[0388] To assess the impact of anti-GM-CSF antibody (lenzilumab) on
CAR-T cell proliferation and efficacy, in vivo studies were
subsequently conducted using the following model (with and without
murine GM-CSF blockade):
Effector/Target Control Experiments: CD19 positive cell line
(NALM6) engrafted xenografts, treated with CART19 with or without
anti-GM-CSF antibody (lenzilumab) in the absence of human
PBMCs.
[0389] NSG mice were dosed i.p. with anti-GM-CSF antibody
(lenzilumab) 10 mg/kg immediately prior to CAR-T cell implantation
and at the same dose every day thereafter for 10 days and followed
to assess tumor response and survival. Retro-orbital bleedings were
obtained starting one week after CAR-T cell therapy and weekly
afterwards. Disease burden, T cell expansion kinetics, expression
of exhaustion markers and cytokine levels (30 Plex) were also
analyzed. At the completion of the experiment, spleens and bone
marrows were harvested and analyzed for tumor characteristics and
CAR-T cell numbers.
CRS/NT Experiments: Patient Derived Xenografts with Primary ALL,
Subsequently Treated with CART19 with or without Lenzilumab in the
Presence of Human PBMCs:
[0390] To assess the impact of lenzilumab on abrogating or
preventing the onset and severity of CAR-T induced CRS and NT, in
vivo studies were conducted with human CAR-T cells (with and
without murine GM-CSF blockade) in a CRS model (where high doses of
CAR-T cells were used to illicit CRS) in the presence of PBMCs
using primary ALL patient derived xenografts, treated with CART19
with and without lenzilumab. NSG mice were dosed i.p. with
lenzilumab 10 mg/kg immediately prior to CAR-T cell implantation
and every day thereafter for 10 days. Mice were followed for tumor
response, survival, CRS and NT symptoms. Brain MRI scans were taken
at baseline, during and at the end of CAR-T cell therapy and
volumetric analysis was conducted to assess and quantify
neuro-inflammation and MRI T1 hyperintensity across treatment arms.
Body weight and retro-orbital bleedings were obtained at baseline,
2 days post, one week-post CAR-T cell therapy and weekly
afterwards. Disease burden, T cell expansion kinetics, expression
of exhaustion markers and cytokine levels (30 Plex) were analyzed.
At the completion of the experiment, spleens and bone marrows were
harvested and analyzed for tumor characteristics and CAR-T cell
numbers.
Results
In Vitro Model
[0391] In this experiment, the impact of GM-CSF neutralization with
lenzilumab on CAR-T cell effector functions was investigated. It
was demonstrated that GM-CSF is secreted by CAR-T cells at very
high levels (over 1,500 pg/ml) and the use of lenzilumab completely
neutralized GM-CSF but did not inhibit CAR-T degranulation,
intracellular GM-CSF production or IL2 production. Moreover,
lenzilumab did not inhibit CAR-T antigen specific proliferation or
CAR-T killing. Effector-to-target rations (E:T) were similar with
CAR-T+ lenzilumab vs. CAR-T+ control antibody, p=ns (FIGS. 16A-16D
and 16J).
In Vivo Models:
Effector/Target Control Experiments:
[0392] To study the effect of lenzilumab on CART19 cell function in
vivo, we engrafted immunocompromised NOD-SCID-g-/- with the CD19+
ALL cell line NALM6 in the absence of human PBMCs. Treatment with
CART19 combined with lenzilumab resulted in potent anti-tumor
activity and improved overall survival, similar to CART19 with
control antibody despite complete neutralization of GM-CSF levels
in these mice, indicating that GM-CSF does not impair CAR-T cell
activity in vivo in the absence of PBMCs (FIGS. 16F and 16G).
CRS and NT Experiments:
[0393] Using human ALL blasts, human CD19 CAR-T, and human PBMCs,
lenzilumab in combination with CAR-T cell therapy was found to
reduce neuro-inflammation by .about.90% compared to CAR-T alone as
assessed by quantitative MRI T1 hyperintensity. This is a landmark
finding and the first time it has been demonstrated in vivo that
the neuroinflammation caused by CAR-T cell therapy can be
effectively abrogated. MRI images following lenzilumab plus CAR-T
cell therapy were similar to baseline pre-treatment scans, in sharp
contrast to MRI images following control antibody plus CAR-T cell
therapy which showed marked increased inflammation. Moreover, a
decrease in myeloid cells was seen in the brains of mice treated
with lenzilumab plus CAR-T compared to mice treated with CAR-T and
control antibody. This finding is consistent with data reported in
clinical trials with CD19 CAR-T cell therapy where an increase in
myeloid cells was observed in the CSF of patients with severe grade
>3 neurotoxicity. In addition, lenzilumab in combination with
CAR-T cell therapy was found to reduce the onset and severity of
CRS as compared to CAR-T plus control antibody. This finding is
supported by the statistically significant reduction in body weight
seen in mice treated with CAR-T plus control, the most objective
marker and hallmark symptom of CRS seen in vivo. In mice treated
with lenzilumab plus CAR-T, body weight was maintained at baseline
levels as compared to CAR-T plus control (p<0.05). Moreover,
mice treated with CAR-T plus control antibody displayed physical
symptoms consistent with CRS including hunched posture, withdrawal,
and weakness while mice treated with CAR-T plus lenzilumab appeared
healthy. Importantly, lenzilumab plus CAR-T also demonstrates a
significant 5-fold increase in the proliferation of CAR-T cells
compared to CAR-T plus control in these CRS/NT experiments that
included PBMCs. It has been previously shown in clinical trials
with various CD19 CAR-T cell therapies that improved CAR-T
proliferation or expansion correlates with improved efficacy
(including ORR, CR), suggesting that lenzilumab may potentially
improve anti-tumor response. This finding may be in part explained
by a decrease in MDSC expansion and trafficking which is known to
be promulgated by GM-CSF. Lastly, the combination of lenzilumab
plus CAR-T results in significantly better leukemic control as
quantified by flow cytometry compared to CAR-T and control
antibody. Compared to untreated mice (which had 500,000 to 1.5M
leukemic cells) and CAR-T plus control antibody (which had between
15,000 and 100,000 leukemic cells), treatment with CAR-T plus
lenzilumab led to a significant reduction in the number of leukemic
cells (decreased to between 500 and 5,000 cells) with improved
overall disease control (see FIGS. 25A-25D).
[0394] The MRI images in FIG. 25A shows a clear improvement in
neurotoxicity (NT) (neuroinflammation) in the brains of mice
administered CAR-T cells and anti-GM-CSF neutralizing antibody in
accordance with embodiments described herein. In contrast, the
brains of mice administered CAR-T cells and a control antibody
showed signs of neurotoxicity in the MRI images. FIG. 25B
graphically illustrates that the NT was reduced by 90% in the mice
of Group 1 compared to the NT increased in Group 2 mice. The extent
of quantitative improvement (90% reduction in NT) after
administration of CAR-T cells and anti-GM-CSF neutralizing antibody
in accordance with embodiments described herein was an unexpected
finding.
Conclusions
[0395] Anti-GM-CSF antibody (Lenzilumab), when combined with CAR-T
cell therapy demonstrates the potential to prevent the onset and
severity of CRS and NT, while improving CAR-T
expansion/proliferation and overall leukemic control in-vivo using
human ALL blasts, human CD19 CAR-T and human PBMCs. This is the
first time it has been demonstrated that CAR-T induced
neurotoxicity can be abrogated in-vivo. Pivotal clinical trials
with lenzilumab in combination with CAR-T cell therapy are planned
to validate these findings of improved safety and efficacy.
Example 17
GM-CSF Blockade During Chimeric Antigen Receptor T Cell Therapy
Reduces Cytokine Release Syndrome and Neurotoxicity and May Enhance
their Effector Functions
[0396] Despite its efficacy, chimeric antigen receptor T-cell
therapy (CART) is limited by the development of cytokine release
syndrome (CRS) and neurotoxicity (NT). While CRS is related to
extreme elevation of cytokines and massive T cell expansion, the
exact mechanisms for NT have not yet been elucidated. Preliminary
studies suggest that NT might be mediated by myeloid cells that
cross the blood brain barrier. This is supported by correlative
analysis from CART19 pivotal trials where CD14+ cell numbers were
increased in the cerebrospinal fluid of patients that developed
severe NT (Locke et al, ASH 2017). Therefore, the aimed of this
study was to investigate the role of GM-CSF neutralization in
preventing CRS and NT after CART cell therapy via monocyte
control.
[0397] First, the effect of GM-CSF blockade on CART cell effector
functions was investigated. Here, the human GM-CSF neutralizing
antibody (lenzilumab, Humanigen, Burlingame, Calif.) was used that
has been shown to be safe in phase II clinical trials. Lenzilumab
(10 ug/kg) neutralizes GM-CSF when CART19 cells are stimulated with
the CD19+ Luciferase+ acute lymphoblastic leukemia (ALL) cell line
NALM6, but does not impair CART cell function in vitro. It was
found that malignancy associated macrophages reduce CART
proliferation. GM-CSF neutralization with lenzilumab results in
enhanced CART cell antigen specific proliferation in the presence
of monocytes. To confirm this in vivo, NOD-SCID-g-/- mice were
engrafted with high disease burdens of NALM6 and treated with low
doses of CART19 or control T cells (to induce tumor relapse), in
combination with lenzilumab or isotype control antibody. The
combination of CART19 and lenzilumab resulted in significant
anti-tumor activity and overall survival benefit compared to
control T cells (FIG. 26A), similar to mice treated with CART19
combined with isotype control antibody, indicating that GM-CSF
neutralization does not impair CART cell activity in vivo. This
anti-tumor activity was validated in an ALL patient derived
xenograft model.
[0398] Next, explored was the impact of GM-CSF neutralization on
CART cell related toxicities in a novel patient derived xenograft
model. Here, NOD-SCID-g-/- mice were engrafted with leukemic blasts
(1-3.times.106 cells) derived from patients with high risk relapsed
ALL. Mice were then treated with high doses of CART19 cells
(2-5.times.106 intravenously). Five days after CART19 treatment,
mice began to develop progressive motor weakness, hunched bodies,
and weight loss that correlated with massive elevation of
circulating human cytokine levels. Magnetic Resonance Imaging (MRI)
of the brain during this syndrome showed diffuse enhancement and
edema, associated with central nervous system (CNS) infiltration of
CART cells and murine activated myeloid cells. This is similar to
what has been reported in CART19 clinical trials in patients with
severe NT. The combination of CART19, lenzilumab (to neutralize
human GM-CS) and murine GM-CSF blocking antibody (to neutralize
mouse GM-CSF) resulted in prevention of weight loss (FIG. 26B),
decrease in critical myeloid cytokines (FIGS. 26C-26D), reduction
of cerebral edema (FIG. 26E), enhanced leukemic disease control in
the brain (FIG. 26F), and reduction in brain macrophages (FIG.
26G).
[0399] Finally, it was hypothesized that disrupting GM-CSF through
CRISPR/Cas9 gene editing during the process of CART cell
manufacture would result in functional CART cells with reduced
secretion of GM-CSF. Guide RNA targeting exon 3 of the GM-CSF gene
was designed and GM-CSF.sup.k/o CART19 cells were generated. The
preliminary data suggest that these CARTs produce significantly
less GM-CSF upon activation but continue to exhibit similar
production of other cytokines and exhibit normal effector functions
in vitro (FIG. 26H). Using the NALM6 high tumor burden relapse
xenograft model as described above, GM-CSF.sup.k/o CART19 cells
resulted in slightly enhanced disease control compared to CART19
cells (FIG. 26I).
[0400] Thus, modulating myeloid cell behavior through GM-CSF
blockade can help control CART mediated toxicities and may reduce
their immunosuppressive features to improve leukemic control. These
studies illuminate a novel approach to abrogate NT and CRS through
GM-CSF neutralization that also potentially enhances CART cell
functions. Based on these results, a phase II clinical trial has
been designed using lenzilumab as a modality to prevent CART
related toxicities in patients with diffuse large B cell
lymphoma.
Example 18
GM-CSF Neutralization In Vitro Enhances CAR-T Cell Proliferation in
the Presence of Monocytes and does not Impair CAR-T Cell Effector
Function
Cells Lines and Primary Cells
[0401] The NALM6 cell line was purchased from ATCC, Manassas, Va.,
USA, and the MOLM13 cell line was a gift from the Jelinek
Laboratory at the Mayo Clinic (purchased from DSMZ, Braunschweig,
Germany). These cell lines were transduced with a
luciferase-ZsGreen lentivirus (Addgene, Cambridge, Mass., USA) and
sorted to 100% purity. Cell lines were cultured in R10 (made with
RPMI 1640 (Gibco, Gaithersburg, Md., US), 10% Fetal Bovine Serum
(FBS, Millipore Sigma, Ontaria, Canada), and 1%
Penicillin-Streptomycin-Glutamine (Gibco, Gaithersburg, Md., US).
Primary cells were obtained from the Mayo Clinic Biobank for
patients with acute leukemia under a Mayo Clinic Institutional
Review Board (IRB) approved protocol. The use of recombinant DNA in
the laboratory was approved by the Mayo Clinic Institutional
Biosafety Committee (IBC).
Primary Cells and CAR-T Cells
[0402] Peripheral blood mononuclear cells (PBMC) were isolated from
de-identified normal donor blood apheresis cones obtained under a
Mayo Clinic IRB approved protocol, using SepMate tubes (STEMCELL
Technologies, Vancouver, Canada). T cells were separated with
negative selection magnetic beads using EasySep.TM. Human T Cell
Isolation Kit (STEMCELL Technologies, Vancouver, Canada). Monocytes
were isolated using a Human Monocyte Isolation Kit from Miltenyi
Biotec, Bergisch Gladbach, Germany, which isolates CD14+ monocytes.
Primary cells were cultured in T Cell Medium made with X-Vivo 15
(Lonza, Walkersville, Md., USA) supplemented with 10% human serum
albumin (Corning, N.Y., USA) and 1%
Penicillin-Streptomycin-Glutamine (Gibco, Gaithersburg, Md., USA).
CART19 cells were generated through the lentiviral transduction of
normal donor T cells as described herein below. Second generation
CART19 constructs were de novo synthesized (IDT) and cloned into a
third-generation lentivirus under the control of the EF-1.alpha.
promotor. The CD19 directed single chain variable region fragment
was derived from the clone FMC63. A second generation 4-1BB
co-stimulated (FMC63-41BBz) CAR construct was synthesized and used
for these experiments. Lentiviral particles were generated through
the transient transfection of plasmid into 293T virus producing
cells (gift from the Ikeda lab, Mayo Clinic), in the presence of
Lipofectamine 3000 (Invitrogen, Carlsbad, Calif., USA), VSV-G and
packaging plasmids (Addgene, Cambridge, Mass., USA). T cells
isolated from normal donors were stimulated using Cell Therapy
Systems Dynabeads CD3/CD28 (Life Technologies, Oslo, Norway) at a
1:3 ratio and then transduced with lentivirus particles 24 hours
after stimulation at a multiplicity of infection (MOI) of 3.0. To
determine titers and subsequently MOI, after lentivirus particles
were concentrated, titers were determined by transducing
1.times.10.sup.5 primary T cells in 100 ul of T cell medium with 50
ul of lentivirus. First, T cells were stimulated with CD3/CD28
beads and then transduced with lentivirus particles 24 hours later.
Transduction was performed in triplicates and at serial dilutions.
Fresh T cell medium was added one day later. Two days later, cells
were harvested, washed twice with PBS, and CAR expression on T
cells was determined by flow cytometry. Titers were determined
based on the percentage of CAR positive cells (percentage of CAR+
cells.times.T cell count at transduction.times.the specific
dilution/volume) and expressed as transducing units/mL (TU/mL).
Magnetic bead removal was performed on Day 6 and CAR-T cells were
harvested and cryopreserved on Day 8 for future experiments. CAR-T
cells were thawed and rested in T cell medium 12 hours prior to
their use in experiments.
GM-CSF Neutralizing Antibody and Isotype Controls
[0403] Lenzilumab (Humanigen, Burlingame, Calif.), an hGM-CSF
neutralizing antibody in accordance with embodiments described
herein and as described in U.S. Pat. Nos. 8,168,183 and 9,017,674,
each of which is incorporated herein by reference in its entirety,
is a novel, first in class Humaneered.RTM. monoclonal antibody that
neutralizes human GM-CSF. For in vitro experiments, lenzilumab or
InVivoMAb human IgG1 isotype control (BioXCell, West Lebanon, N.H.,
USA), 10 ug/mL was used. For in vivo experiments, 10 mg/kg of
lenzilumab or isotype control were intraperitoneally injected daily
for 10 days beginning on the day of CART19 injection. In some
experiments, anti-mouse GM-CSF neutralizing antibody (InVivoMAb
anti-mouse GM-CSF, BioXCel, West Lebanon, N.H., USA) or the
corresponding isotype control (InVivoMAb rat IgG2a isotype control
BioXCel, West Lebanon, N.H., USA) was also used, as indicated in
the experimental schema.
T Cell Functional Experiments
[0404] Cytokine assays were performed 24 or 72 hours after a
co-culture of CAR-T cells with their targets at a 1:1 ratio as
indicated. Human High Sensitivity T Cell Magnetic Bead Panel
(Millipore Sigma, Ontario, Canada), Milliplex Human
Cytokine/Chemokine MAGNETIC BEAD Premixed 38 Plex Kit (Millipore
Sigma, Ontario, Canada), or Milliplex Mouse Cytokine/Chemokine
MAGNETIC BEAD Premixed 32 Plex Kit (Millipore Sigma, Ontario,
Canada) were performed on supernatants collected from these
experiments or serum, as indicated. This was analyzed using Luminex
(Millipore Sigma, Ontario, Canada). Intracellular cytokine analysis
and T cell degranulation assays were performed following incubation
of CAR-T cells with targets at a 1:5 ratio for 4 hours, in the
presence of monensin (BioLegend, San Diego, Calif., USA), hCD49d
(BD Biosciences, San Diego, Calif., USA), and hCD28 (BD
Biosciences, San Diego, Calif., USA). After 4 hours, cells were
harvested and intracellular staining was performed after surface
staining, followed by fixation and permeabilization with fixation
medium A and B (Life Technologies, Oslo, Norway). For proliferation
assays, CFSE (Life Technologies, Oslo, Norway) labeled effector
cells (CART19), and irradiated target cells were co cultured at a
1:1 ratio. In some experiments, CD14+ monocytes were added to the
co-culture at a 1:1:1 ratio as indicated. Cells were co-cultured
for 3-5 days, as indicated in the specific experiment and then
cells were harvested and surface staining with anti-hCD3
(eBioscience, San Diego, Calif., USA) and LIVE/DEAD.TM. Fixable
Aqua Dead Cell Stain Kit (Invitrogen, Carlsbad, Calif., USA) was
performed. PMA/ionomycin (Millipore Sigma, Ontario, Canada) was
used as a positive non-specific stimulant of T cells, at different
concentrations as indicated in the specific experiments. For
killing assays, the CD19+ Luciferase+ ALL cell line NALM6 or the
CD19-Luciferase+ control MOLM13 cells were incubated at the
indicated ratios with effector T cells for 24, 48, or 72 hours as
listed in the specific experiment. Killing was calculated by
bioluminescence imaging on a Xenogen IVIS-200 Spectrum camera
(PerkinElmer, Hopkinton, Mass., USA) as a measure of residual live
cells. Samples were treated with 1 ul D-luciferin (30 ug/mL) per
100 ul sample volume (Gold Biotechnology, St. Louis, Mo., USA), for
10 minutes prior to imaging.
Multi-Parametric Flow Cytometry
[0405] Anti-human and anti-mouse antibodies were purchased from
Biolegend, eBioscience, or BD Biosciences (San Diego, Calif., USA).
Cells were isolated from in vitro culture or from peripheral blood
of animals. After BD FACS lyse (BD Biosciences, San Diego, Calif.,
USA), they were washed twice in phosphate-buffered saline
supplemented with 2% FBS (Millipore Sigma, Ontario, Canada) and 1%
sodium azide (Ricca Chemical, Arlington, Tex., USA) and stained at
4.degree. C. For cell number quantitation, Countbright beads
(Invitrogen, Carlsbad, Calif., USA) were used according to the
manufacturer's instructions (Invitrogen, Carlsbad, Calif., USA). In
all analyses, the population of interest was gated based on forward
vs side scatter characteristics, followed by singlet gating, and
live cells were gated following staining with LIVE/DEAD.TM. Fixable
Aqua Dead Cell Stain Kit (Invitrogen, Carlsbad, Calif., USA).
Surface expression of CAR was detected by staining with a goat
anti-mouse F(ab')2 antibody (Invitrogen, Carlsbad, Calif., USA).
Flow cytometry was performed on three-laser cytometers, Canto II
(BD Biosciences, San Diego, Calif., USA) and CytoFLEX (Beckman
Coulter, Chaska, Minn., USA). Analyses were performed using FlowJo
X10.0.7r2 software (Ashland, Oreg., USA) and Kaluza 2.0 software
(Beckman Coulter, Chaska, Minn., USA).
Results
[0406] If GM-CSF neutralization after CAR-T cell therapy is to be
utilized as a strategy to prevent CRS and neurotoxicity, it must
not inhibit CAR-T cell efficacy. Therefore, the initial experiments
aimed to investigate the impact of GM-CSF neutralization on CAR-T
cell effector functions. CART19 cells were co-cultured with or
without the CD19+ ALL cell line NALM6 in the presence of lenzilumab
(hGM-CSF neutralizing antibody) or an isotype control (IgG). It was
established that lenzilumab, but not IgG control antibody, was
indeed able to completely neutralize hGM-CSF (FIG. 27A) but did not
inhibit CAR-T cell antigen specific proliferation (FIG. 27B). When
CART19 cells were co-cultured with the CD19+ cell line NALM6 in the
presence of monocytes, lenzilumab in combination with CART19
demonstrated an exponential increase in antigen specific CART19
proliferation compared to CART19 plus isotype control IgG
(P<0.0001, FIG. 27C). To investigate CAR-T specific
cytotoxicity, either CART19 or control UTD T cells were cultured
with the luciferase+CD19+NALM6 cell line and treated with either
isotype control antibody or GM-CSF neutralizing antibody (FIG.
27D). GM-CSF neutralizing antibody treatment did not inhibit the
ability of CAR-T cells to kill NALM6 target cells (FIG. 27D).
Overall, these results indicate that lenzilumab does not inhibit
CAR-T cell function in vitro and enhances CART19 cell proliferation
in the presence of monocytes, suggesting that GM-CSF neutralization
may improve CAR-T cell mediated efficacy.
Example 19
GM-CSF Neutralization In Vivo Enhances CAR-T Cell Anti-Tumor
Activity in Xenograft Models
Xenograft Mouse Models
[0407] Male and female 8-12 week old NOD-SCID-IL2r.gamma..sup.-/-
(NSG) mice were bred and cared for within the Department of
Comparative Medicine at the Mayo Clinic under a breeding protocol
approved by the Mayo Clinic Institutional Animal Care and Use
Committee (IACUC). Mice were maintained in an animal barrier space
that is approved by the IBC for BSL2+ level experiments.
NALM6 Cell Line Xenografts
[0408] The CD19.sup.+, luciferase.sup.+ ALL NALM6 cell line was
used to establish ALL xenografts, under an IACUC approved protocol.
Here, 1.times.10.sup.6 cells were injected intravenously (IV) via a
tail vein injection. 4-6 days after injection, mice underwent
bioluminescent imaging using a Xenogen IVIS-200 Spectrum camera
(PerkinElmer, Hopkinton, Mass., USA), to confirm engraftment.
Imaging was performed 10 minutes after the intraperitoneal (IP)
injection of 10 ul/g D-luciferin (15 mg/mL, Gold Biotechnology, St.
Louis, Mo., USA). Mice were then randomized based on their
bioluminescent imaging to receive different treatments as outlined
in the specific experiments. Typically, 1-1.5.times.10.sup.6 CAR-T
cells (and an equivalent of total T cell number of untransduced
(UTD) T cells) were injected IV per mouse. Transduction efficiency
of CAR-T cells was typically approximately 50%. For example, with a
50% transduction efficiency of CAR-T cells, mice that received
1.5.times.10.sup.6 CAR-T cells received 3 million total T cells,
and the corresponding UTD mice received 3.times.10.sup.6 UTD.
Weekly imaging was performed to assess and follow disease burden.
Bioluminescent images were acquired using a Xenogen IVIS-200
Spectrum camera (PerkinElmer, Hopkinton, Mass., USA) and analyzed
using Living Image version 4.4 (Caliper LifeSciences, PerkinElmer).
Tail vein bleeding was done 7-8 days after injection of CAR-T cells
to assess T cell expansion and cytokines and chemokines, and
subsequently as needed. Mouse peripheral blood was subjected to red
blood cell lysis using BD FACS Lyse (BD Biosciences, San Diego,
Calif., USA) and then used for flow cytometric studies. Antibody
treated mice commenced daily antibody therapy (10 mg/kg lenzilumab
or isotype control) IP on the same day of CART cell therapy for a
total of 10 days.
RNA-Seq on Mouse Brain Tissue
[0409] RNA was isolated using miRNeasy Micro kit (Qiagen,
Gaithersburg, Md., USA) and treated with RNase-Free DNase Set
(Qiagen, Gaithersburg, Md., USA). RNA-seq was performed on an
Illumina HTSeq 4000 (Illumina, San Diego, Calif., USA) by the
Genome Analysis Core at Mayo Clinic. The binary base call data was
converted to fastq using Illumina bcl2fastq software. The adapter
sequences were removed using Trimmomatic, as described by Bolger, A
M, et al., Bioinformatics. 2014; 30(15):2114-2120.
10.1093/bioinformatics/btul70, which is hereby incorporated by
reference in its entirety, and FastQC as described by Leggett R M,
et al., Front Genet. 2013; 4:288. Prepublished on 2014 Jan. 2 as
DOI 10.3389/fgene.2013.00288, which is hereby incorporated by
reference in its entirety, was used to check for quality. The
latest human (GRCh38) and mouse (GRCm38) reference genomes were
downloaded from NCBI. Genome index files were generated using STAR,
as described by Dobin A, et al., Bioinformatics. 2013; 29(1):15-21.
10.1093/bioinformatics/bts635, which is hereby incorporated by
reference in its entirety, and the paired end reads were mapped to
the genome for each condition. HTSeq, as described by Anders S, et
al., Bioinformatics. 2015; 31(2):166-169. Prepublished on 2014 Sep.
28 as DOI 10.1093/bioinformatics/btu638, which is hereby
incorporated by reference in its entirety, was used to generate
expression counts for each gene, and DeSeq2.sup.38, as described by
Love M I, et al., Genome Biol. 2014; 15(12):550. Prepublished on
2014 Dec. 18 as DOI 10.1186/s13059-014-0550-8, which is hereby
incorporated by reference in its entirety was used to calculate
differential expression. Gene ontology was assessed using Enrichr,
as described by Kuleshov M V et al., Nucleic Acids Research 2016;
44(W1):W90-W97. 10.1093/nar/gkw377, which is hereby incorporated by
reference in its entirety. FIG. 35 summarizes the steps detailed
above. RNA sequencing data are available at the Gene Expression
Omnibus under accession number GSE121591.
[0410] Statistics
[0411] Prism Graph Pad (La Jolla, Calif., USA) and Microsoft Excel
(Microsoft, Redmond, Wash., USA) were used to analyze data. The
high cytokine concentrations in the heat map were normalized to "1"
and low concentrations normalized to "0" via Prism. Statistical
tests are described in the figure legends.
Results
[0412] To confirm that GM-CSF depletion does not inhibit CART19
effector functions, the role of GM-CSF neutralization with
lenzilumab on CART19 antitumor activity was investigated in
xenograft models. First, a relapse model intended to vigorously
investigate whether the antitumor activity of CART19 cells was
impacted by GM-CSF neutralization was used. NOD/SCID/interleukin-2
receptor gamma null (NSG) mice were injected with 1.times.10.sup.6
luciferase+NALM6 cells and then imaged 6 days later, allowing
sufficient time for mice to achieve very high tumor burdens. Mice
were randomized to receive a single injection of either CART19 or
UTD cells and 10 days of either isotype control antibody or
lenzilumab (FIG. 28A). GM-CSF assay on serum collected 8 days after
CART19 injection revealed that lenzilumab successfully neutralizes
GM-CSF in the context of CART19 therapy (FIG. 28B). Bioluminescence
imaging one week after CART19 injection showed that CART19 in
combination with lenzilumab effectively controlled leukemia in this
high tumor burden relapse model and significantly better than
control UTD cells (FIGS. 28C-28D). Treatment with CART19 in
combination with lenzilumab resulted in potent anti-tumor activity
and improved overall survival, similar to CART19 with control
antibody despite neutralization of GM-CSF levels, indicating that
GM-CSF does not impair CAR-T cell activity in vivo (FIG. 36).
Second, these experiments were performed in a primary ALL patient
derived xenograft model, in the presence of human PBMCs as this
represents a more relevant heterogeneous model. After conditioning
chemotherapy with busulfan, mice were injected with blasts derived
from patients with relapsed ALL. Mice were monitored for
engraftment for several weeks through serial tail vein bleedings
and when the CD19.sup.+ blasts in the blood were approximately
1/uL, mice were randomized to receive CART19 treatment in
combination with PBMCs with either lenzilumab plus an anti-mouse
GM-CSF neutralization antibody or isotype control IgG antibodies
starting on the day of CART19 injection for 10 days (FIG. 28E). In
this primary ALL xenograft model, GM-CSF neutralization in
combination with CART19 therapy resulted in a significant
improvement in leukemic disease control sustained over time for at
least 35 days post CART19 administration as compared to CART19 plus
isotype control (FIG. 28F). This suggests that GM-CSF
neutralization may play a role in reducing relapses and increasing
durable complete responses after CART19 cell therapy.
Example 20
Generation of GM-CSF.sup.k/o CART19
[0413] A guide RNA (gRNA) targeting exon 3 of human GM-CSF was
selected via screening gRNAs previously reported to have high
efficiency for human GM-CSF, as described in Sanjana N E et al.,
Improved vectors and genome-wide libraries for CRISPR screening.
Nature Methods. 2014; 11(8):783-784. Prepublished on 2014 Jul. 31
as DOI 10.1038/nmeth.3047, which is hereby incorporated by
reference in its entirety. This gRNA was ordered in a CAS9 third
generation lentivirus construct (lentiCRISPRv2), controlled under a
U6 promotor (GenScript, Township, N.J., USA). Lentiviral particles
encoding this construct were produced as described above. T cells
were dual transduced with CAR19 and GM-CSFgRNA-lentiCRISPRv2
lentiviruses, 24 hours after stimulation with CD3/CD28 beads. CAR-T
cell expansion was then continued as described above. To analyze
efficiency of targeting GM-CSF, genomic DNA was extracted from the
GM-CSF.sup.k/o CART19 cells using PureLink Genomic DNA Mini Kit
(Invitrogen, Carlsbad, Calif., USA). The DNA of interest was PCR
amplified using Choice Taq Blue Mastermix (Thomas Scientific,
Minneapolis, Minn., USA) and gel extracted using QIAquick Gel
Extraction Kit (Qiagen, Germantown, Md., USA) to determine editing.
PCR amplicons were sent for Eurofins sequencing (Louisville, Ky.,
USA) and allele modification frequency was calculated using TIDE
(Tracking of Indels by Decomposition) a method that requires only
two parallel PCR reactions followed by a pair of standard capillary
sequencing analyses; the two resulting sequencing traces are then
analyzed using specially designed software that is provided as a
simple web tool and as R code available at tide.nki.nl, as
described by Brinkman E K, et al., Easy quantitative assessment of
genome editing by sequence trace decomposition. Nucleic Acids
Research. 2014; 42(22):e168. Prepublished on 2014 Oct. 11 as DOI
10.1093/nar/gku936, which is incorporated herein by reference in
its entirety. FIG. 34B describes the gRNA sequence and primer
sequences, and FIGS. 34A(i)-34A(iv) depict the schema for
generation of GM-CSF.sup.k/o CART19.
Example 21
GM-CSF CRISPR Knockout CAR-T Cells Exhibit Reduced Expression of
GM-CSF, Similar Levels of Key Cytokines and Chemokines, and
Enhanced Anti-Tumor Activity
[0414] To confidently exclude any role for GM-CSF critical in CAR-T
cell function, the GM-CSF gene was disrupted during CAR-T cell
manufacturing using a gRNA that has been reported to yield high
efficiency knockout and is cloned into a CRISPR lentivirus
backbone, as described by Sanjana N E, et al., Nature Methods.
2014; 11(8):783-784. Prepublished on 2014 Jul. 31 as DOI
10.1038/nmeth.3047, which is hereby incorporated by reference in
its entirety. Using this gRNA, we achieved around 60% knockout
efficiency in CART19 cells (FIG. 37). When CAR-T cells were
stimulated with the CD19.sup.+ cell line NALM6, GM-CSF.sup.k/o
CAR-T cells produced statistically significantly less GM-CSF
compared to CART19 with a wild-type GM-CSF locus ("wild type CART19
cells"). GM-CSF knockout in CAR-T cells did not impair the
production of other key T cell cytokines, including IFN-.gamma.,
IL-2, or CAR-T cell antigen specific degranulation (CD107a) (FIG.
29A) but did exhibit reduced expression of GM-CSF (FIG. 29B). To
confirm that GM-CSF.sup.k/o CAR-T cells continue to exhibit normal
functions, their in vivo efficacy in the high tumor burden
relapsing xenograft model of ALL was tested (as described in FIG.
28A). In this xenograft model, utilization of GM-CSF.sup.k/o CART19
instead of wild type CART19 markedly reduced serum levels of human
GM-CSF at 7 days after CART19 treatment (FIG. 29B). Bioluminescence
imaging data implied that GM-CSF.sup.k/o CART19 cells show enhanced
leukemic control compared to CART19 in this model (FIG. 38).
Importantly, GM-CSF.sup.k/o CART19 cells demonstrated significant
improvement in overall survival compared to wild type CART19 cells
(FIG. 29C). Human GM-CSF was statistically significantly decreased
via t test in the GM-CSF/.degree. CART19 cells compared to wild
type CART19 (FIG. 29D). The mouse GM-CSF visually appears increased
although this is not statistically significant via t test
(P=0.472367) (FIG. 29E). This lack of mouse GM-CSF reduction is not
necessarily surprising as the GM-CSF.sup.k/o CART19 cells (which
are human) are the only cells within the mouse that possess the
knockout, thus mouse GMCSF would not likely be affected directly.
By visual inspection, mouse IP-10, a chemokine that attracts
numerous cell types including T cells and monocytes, appears
paradoxically increased in GM-CSF.sup.k/o CART19 compared to
CART19, but this is also not statistically significant, P=0.4877 by
t test (FIG. 29E). By visual inspection, mouse MIP1.alpha.(an
inflammatory cytokine important in neutrophil attraction) and mouse
M-CSF (a cytokine critical in macrophage differentiation) appear
reduced, although they are not statistically significant with
P=0.2437 and P=0.3619 (FIG. 29E). Mouse IL-1b, a critical
inflammatory cytokine produced by macrophages, and mouse IL-15, a
cytokine produced by macrophages that aids in NK cell
proliferation, appear reduced in GMCSF.sup.k/o CART19 compared to
CART19 (FIG. 29E) with P values of P=0.0741 and P=0.0900,
respectively (FIG. 29E). Critical human T cell cytokines were not
inhibited by GM-CSF.sup.k/o (FIG. 29D). It should be emphasized
that these xenografts were produced with high burdens of the NALM6
cell line, and our CRS/NI model (FIGS. 30A-30D, 31, 32A-32D and
33A-33D) require the use of primary ALL cells to be generated.
Thus, cytokine profiles unsurprisingly differ between the two
models as the NALM6 xenografts (FIGS. 29A-29E) do not develop CRS
or NI. Together, in the context of a NALM6 high tumor burden model
without CRS, results of FIGS. 29A-29E confirm FIGS. 27A-27D and
28A-28F, indicating that GM-CSF depletion does not impair normal
cytokines or chemokines that are critical to CAR-T efficacy
functions. In addition, the results in FIGS. 29A-29E indicate that
GM-CSF.sup.k/o CART may represent a therapeutic option for "built
in" GM-CSF control as a modification during CAR-T cell
manufacturing.
Example 22
Patient Derived Xenograft Model for Neuro-Inflammation (NI) and
Cytokine Release Syndrome/GM-CSF Neutralization In Vivo Ameliorates
Cytokine Release Syndrome and Neuroinflammation after CART19
Therapy in a Xenograft Model
Primary Patient-Derived ALL Xenografts
[0415] To establish primary ALL xenografts, NSG mice first received
30 mg/kg busulfan IP (Selleckchem, Houston, Tex., USA). The
following day, mice were injected with 2.5.times.10.sup.6 primary
blasts derived from the peripheral blood of patients with relapsed
or refractory ALL. Mice were monitored for engraftment for
.about.10-13 weeks. When CD19.sup.+ cells were consistently
observed in the blood (approximately 1 cell/uL), they were
randomized to receive different treatments of CART19
(2.5.times.10.sup.6 cells IV) and PBMCs derived from the same donor
(1.times.10.sup.5 cells IV) with or without antibody therapy (10
mg/kg lenzilumab or isotype control IP for a total of 10 days,
starting on the day they received CAR-T cell therapy). Mice were
periodically monitored for leukemic burden via tail vein
bleeding.
Primary Patient-Derived ALL Xenografts for CRS/NI
[0416] Similar to the experiments above, mice were IP injected with
30 mg/kg busulfan (Selleckchem, Houston, Tex., USA). The following
day, mice received 1-3.times.10.sup.6 primary blasts derived from
the peripheral blood of patients with relapsed ALL. Mice were
monitored for engraftment for .about.10-13 weeks via tail vein
bleeding. When serum CD19.sup.+ cells were .gtoreq.10 cells/ul, the
mice received CART19 (2-5.times.10.sup.6 cells IV) and commenced
antibody therapy for a total of 10 days, as indicated. Mice were
weighed on a daily basis as a measure of their well-being. Mouse
brain MRIs were performed 5-6 days post CART19 injection and tail
vein bleeding for cytokine/chemokine and T cell analysis was
performed 4-11 days post CART19 injection.
MRI Acquisition
[0417] A Bruker Avance II 7 Tesla vertical bore small animal MRI
system (Bruker Biospin) was used for image acquisition to evaluate
central nervous system (CNS) vascular permeability. Inhalation
anesthesia was induced and maintained via 3 to 4% isoflurane.
Respiratory rate was monitored during the acquisition sessions
using an MRI compatible vital sign monitoring system (Model 1030;
SA Instruments, Stony Brook, N.Y.). Mice were given an IP injection
of gadolinium using weight-based dosing of 100 mg/kg, and after a
standard delay of 15 min, a volume acquisition T1-weighted spin
echo sequence was used (repetition time=150 ms, echo time=8 ms,
field of view: 32 mm.times.19.2 mm.times.19.2 mm, matrix:
160.times.96.times.96; number of averages=1) to obtain T1-weighted
images. Gadolinium-enhanced MRI changes were indicative of
blood-brain-barrier disruption..sup.24 Volumetric analysis was
performed using Analyze Software package developed by the
Biomedical Imaging Resource at Mayo Clinic.
Results
[0418] In this model, conditioned NSG mice were engrafted with
primary ALL blasts and monitored for engraftment for several weeks
until they developed high disease burden (FIG. 30A). When the level
of CD19.sup.+ blasts in the peripheral blood was >10/uL, mice
were randomized to receive different treatments as indicated (FIG.
30A). Treatment with CART19 (with control IgG antibodies or with
GM-CSF neutralizing antibodies) successfully eradicated the disease
(FIG. 30B). Within 4-6 days after treatment with CART19, mice began
to develop motor weakness, hunched bodies, and progressive weight
loss; symptoms consistent with CRS and NI. This was associated with
elevation of key serum cytokines and chemokines 4-11 days post
CART19 injection similar to what is seen in human CRS after CAR-T
cell therapy (including human GM-CSF, TNF-.alpha., IFN-.gamma.,
IL-10, IL-12, IL-13, IL-2, IL-3, IP-10, MDC, MCP-1, MIP-1a, MIP-10,
and mouse IL-6, GM-CSF, IL-4, IL-9, IP-10, MCP-1, and MIG). These
mice treated with CART19 also developed NI as indicated by brain
MRI analyses revealing abnormal T1 enhancement, suggestive of
blood-brain barrier disruption and possibly brain edema (FIG. 30C),
together with flow cytometric analysis of harvested brains
revealing infiltration of human CART19 cells (FIG. 30D). In
addition, RNA-seq analyses of brain sections harvested from mice
that developed these signs of NI showed significant upregulation of
genes regulating the T cell receptor, cytokine receptors, T cell
immune activation, T cell trafficking, and T cell and myeloid cell
differentiation (FIG. 31, Table 6).
TABLE-US-00009 TABLE 6 Table of canonical pathways altered in
brains from patient derived xenografts after treatment with CART19
cells in tabular format. Conical Pathway Adj P-Value Genes
regulation of 9.45E-14 IFITM1, ITGB2, TRAC, ICAM3, immune response
CD3G, PTPN22, CD3E, ITGAL, (GO: 0050776) SAMHD1, SLA2, CD3D, ITGB7,
SLAMF6, B2M, NPDC1, CD96, BTN3A1, ITGA4, SH2D1A, HLA-B, HLA-C,
BTN3A2, HLA-A, CD8B, SELL, CD8A, CD226, CD247, CLEC2D, HCST, BIRC3
cytokine-mediated 1.36E-12 IFITM1, SP100, TRADD, ITGB2, signaling
pathway IL2RG, SAMHD1, IL27RA, OASL, (GO: 0019221) CNN2, IL18RAP,
RIPK1, CCR5, IL12RB1, B2M, GBP1, IL6R, JAK3, CCR2, IL32, ANXA1,
IL4R, TGEB1, IL10RB, IL10RA, STAT2, PRKCD, HLA-B, HLA-C, IL16,
HLA-A, TNERSF1B, CD4, IRF3, OA52, IL2RB, FAS, TNERSF25, LCP1, P4HB,
IL7R, MAP3K14, CD44, IL18R1, IRF9, MYD88, BIRC3 T cell receptor
1.30E-11 ZAP70, CD4, CD6, CD8B, CD8A, complex (GO: CD3G, CD247,
CD3E, CD3D, 0042101) CARD11 T cell activation 2.07E-11 ITK, RHOH,
CD3G, NLRC3, (GO: 0042110) PTPN22, CD3E, SLA2, CD3D, CD2, ZAP70,
CD4, PTPRC, CD8B, CD8A, LCK, CD28, LCP1, LAT regulation of T
2.46E-10 PTPN22, LAX1, CCDC88B, CD2, cell activation CD4, LCK,
SIT1, TBX21, TIGIT, (GO: 0050863) JAK3, LAT, PAG1, CCR2 T cell
receptor 4.35E-08 ITK, BTN3A1, TRAC, WAS, CD3G, signaling pathway
PTPN22, BTN3A2, CD3E, CD3D, (GO: 0050852) ZAP70, CD4, PTPRC, LCK,
GRAP2, LCP2, CD247, CARD11, LAT, PAG1 positive regulation
1.57502E-07 GBP5, ANXA1, TGFB1, CYBA, of cytokine PTPN22, PARK7,
TMEM173, production (GO: CCDC88B, MAVS, CD6, IRF3, 0001819) CD28,
RIPK1, SLAMF6, CD46, IL12RB1, TIGIT, IL6R, CARD11, MYD88, CCR2 T
cell 2.36E-07 ZAP70, CD4, ANXA1, PTPRC, differentiation CD8A, LCK,
CD28, RHOH, PTPN22, (GO: 0030217) CD3D cytokine receptor 2.43E-07
IL4R, IL10RB, IL10RA, IL2RG, activity (GO: CD4, CXCR3, IL2RB, CCR5,
0004896) IL12RB1, IL7R, IL6R, CD44, CCR2 type I interferon 3.27E-07
IFITM1, SP100, IRF3, OAS2, STAT2, signaling pathway HLA-B, HLA-C,
HLA-A, SAMHD1, (GO: 0060337) IRF9, MYD88, OASL response to
0.0004679 SIGIRR, IFITM1, SP100, HCLS1, cytokine (GO: RIPK1, PTPN7,
IKBKE, IL6R, JAK3, 0034097) IL18R1, MYD88, AES regulation of
0.001452 GBP5, GM, STAT2, ADAM8, innate immune NLRC3, PTPN22,
SAMHD1, BIRC3 response (GO: 0045088) regulation of 0.003843 CD2,
MAVS, CYBA, NLRC3, tumor necrosis PTPN22, RIPK1, SLAMF1 factor
production (GO: 0032680) T cell receptor 0.0102397 LCK, CD3G, CD3E
binding (GO: 0042608) regulation of 0.0124059 SHARPIN, TRADD,
CASP4, RIPK1, tumor necrosis TRAF1, BIRC3 factor-mediated signaling
pathway (GO: 0010803) Positive 0.0376647 CD4, HCLS1, RIPK1, EVI2B
regulation of myeloid Leukocyte differentiation (GO: 0002763)
[0419] Using the xenograft patient derived model for NI and CRS
shown in FIG. 30A, the effect of GM-CSF neutralization on CART19
toxicities was investigated. To rule out the cofounding effect of
mouse GM-CSF, mice received CART19 cells in combination with 10
days of GM-CSF antibody therapy (10 mg/kg lenzilumab and 10 mg/kg
antimouse GM-CSF neutralizing antibody) or isotype control
antibodies. GM-CSF neutralizing antibody therapy statistically
significantly reduced CRS induced weight loss after CART19 therapy
(FIG. 32A). Cytokine and chemokine analysis 11 days after CART19
cell therapy showed that human GM-CSF was neutralized by the
antibody (FIG. 32B). In addition, GM-CSF neutralization resulted in
significant reduction of several human (IP-10, IL-3, IL-2, IL-1Ra,
IL-12.beta.40, VEGF, GM-CSF) (FIG. 32C) and mouse (MIG, MCP-1, KC,
IP-10) (FIG. 32D) cytokines and chemokines. Interferon
gamma-induced protein (IP-10, CXCL10) is produced by monocytes
among other cell types and serves as a chemoattractant for numerous
cell types including monocytes, macrophages, and T cells. IL-3
plays a role in myeloid progenitor differentiation. IL-2 is a key T
cell cytokine. Interleukin-1 receptor antagonist (IL-1Ra) inhibits
IL-1. (IL-1 is produced by macrophages and is a family of critical
inflammatory cytokines.) IL-12.beta.40 is a subunit of IL-12, which
is produced by macrophages among other cell types and can encourage
Th1 differentiation. Vascular endothelial growth factor (VEGF)
encourages blood vessel formation. Monokine induced by gamma
interferon (MIG, CXCL9) is a T cell chemoattractant. Monocyte
chemoattractant protein 1 (MCP-1, CCL2) attracts monocytes, T
cells, and dendritic cells. KC (CXCL1) is produced by macrophages
among other cell types and attracts myeloid cells such as
neutrophils. There was also a non-statistically significant
reduction of several other human and moue cytokines and chemokines
after GM-CSF neutralization. This suggests that GMCSF plays a role
in the downstream activity of several cytokines and chemokines that
are instrumental in the cascade that results in CRS and NI.
[0420] Brain MRIs 5 days after CAR19 treatment showed that GM-CSF
neutralization reduced T1 enhancement as a measure of brain
inflammation, blood-brain barrier disruption, and possibly edema,
compared to CART19 plus control antibodies. The MRI images after
GM-CSF neutralization (with lenzilumab and anti-mouse GM-CSF
antibody) were similar to baseline pre-treatment scans, suggesting
that GM-CSF neutralization effectively helped abrogate the NI
associated with CART19 therapy (FIGS. 33A and 33B). Using human ALL
blasts and human CART19 in this patient-derived xenograft model,
GM-CSF neutralization after CART19 reduced neuro-inflammation by
75% compared to CART19 plus isotype controls (FIG. 33B). This is a
significant finding and the first time it has been demonstrated in
vivo that the NI caused by CART19 can be effectively abrogated.
Human CD3 T cells were present in the brain after CART19 therapy as
assayed by flow cytometry, and with GM-CSF neutralization, there
was a difference in raw average with reduction in brain CD3 T
cells, but it did not meet statistical significance (FIGS. 33C,
30D, and 39). Finally, a difference in raw average (although this
did not reach statistical significance) with reduction of CD11b+
bright macrophages was observed in the brains of mice receiving
GM-CSF neutralization during CAR-T cell therapy compared to isotype
control during CAR-T therapy (FIG. 33D), possibly implicating that
GM-CSF neutralization helps reduce macrophages within the
brain.
[0421] The results of Examples 18-19 and 22 demonstrate that
neutralization of GM-CSF abrogates toxicities after CAR-T cell
therapy and may enhance their therapeutic activity. Specifically,
it was shown that GM-CSF neutralization in combination with CART19
therapy prevents the development of CRS and significantly reduces
the severity of NI in a xenograft model using human ALL blasts and
human CART19. GM-CSF neutralization resulted in a reduction in
chemokines associated with myeloid trafficking, such as IP-10,
MCP-1, KC, and other inflammatory cytokines and chemokines, and is
associated with decreased raw averages (although not statistically
significant) of T cell infiltration and myeloid cell activation in
the brain. Intriguingly, the experiments herein also suggest that
GM-CSF inhibition enhances CART19 proliferation, anti-tumor
activity, and overall survival in vivo. Based on these results,
GM-CSF neutralization can be viewed as a potential next generation
strategy to enable routine CAR-T cellular immunotherapy.
[0422] In the studies described herein, GM-CSF neutralization with
lenzilumab did not impair any CART19 effector functions in vitro.
In two different xenograft models (NALM6 xenografts and patient
derived xenografts), CART19 combined with lenzilumab effectively
eradicated the tumor despite GM-CSF neutralization and
significantly improved leukemic disease control 35 days
post-treatment while CART19 plus isotype control could not maintain
disease control after 35 days. Lastly, in Examples 21-22,
GM-CSF.sup.k/o CART19 cells exhibited potent effector functions in
vitro and demonstrated significantly improved overall survival
compared to CART19 in vivo.
[0423] The herein described CRS and NI model is a unique and
relevant ALL patient derived xenograft model for the development of
therapies for toxicities after human CAR-T cell therapy. In the
model described here, the time interval between CAR-T cell infusion
to onset of symptoms, brain MRI changes, cytokine and chemokine
elevation, and infiltration of effector cells into the CNS are all
similar to what is reported in patients that develop toxicities
after CART19 therapy. Mice developed symptoms of CRS and NI (weight
loss, decline in motor function, and hunched bodies). Changes in
brain MRI were detected 4-6 days after infusion of CART19 cells.
Brain MRI T1 uptake is suggestive of blood-brain barrier disruption
and possibly brain edema and is comparable to changes noted on
human brain MRI in cases of severe neurotoxicity, as described by
Gust et al. 2017 Cancer Discovery. 2017; 7(12):1404-1419.
Prepublished on 2017 Oct. 14 as DOI 10.1158/2159-8290.CD-17-0698,
which is incorporated herein by reference in its entirety.
[0424] Interestingly, Gust et al. 2017 further describes that blood
brain barrier permeability prevented protection of the CSF from
systemic cytokines, which induced vascular pericyte stress and
secretion of endothelium-activating cytokines, and patients showed
evidence of endothelial activation. In the CRS/NI model described
herein, GM-CSF was found to be neutralized in the serum of mice
receiving CART19 therapy with GM-CSF neutralizing antibodies
compared to CART19 and isotype control antibodies. Thus, T cells
within the mouse brains themselves could provide GM-CSF production,
and serum GM-CSF among other cytokines and chemokines were possibly
able to reach the CSF. In addition, endothelium cells are able to
produce GM-CSF, which may result in a cycle of exacerbation. NI was
associated with infiltration of T cells and activation of myeloid
cells in the CNS, similar to CSF changes in patients with CAR-T
induced neurotoxicity, as well as in non-human primate models. The
herein described model is similar to previously reported patient
derived xenograft models where CRS developed after CAR-T cell
therapy. A recent report suggested that blockade of IL-1 prevents
NI through the depletion of myeloid cells. However, the development
of NI in that model was delayed and related to meningeal
thickening, unlike what was observed in the model described herein
and in patients receiving CART19 therapy. Therefore, the model
described herein is provided as a reliable way to investigate novel
interventions for the prevention and treatment of CRS and
neurotoxicity after CART19 cell therapy. The results described
herein show that GM-CSF neutralization results in a reduction in
key myeloid and several inflammatory cytokines and chemokines,
suggesting that GM-CSF is a critical cytokine in downstream
activation of several cytokines and chemokines; blockade
contributes to a decrease in raw averages in myeloid and T cell
infiltration in the brain/CNS (although statistical significance
was not reached); and blockade helps reduce neuro-inflammation of
apparent neurotoxicities.
[0425] Interestingly, an exponential increase in CART19 cell
proliferation was observed, enhanced anti-tumor activity, and
improved overall survival with GM-CSF blockade. For example, CART19
antigen specific proliferation in the presence of monocytes
increased in vitro after GM-CSF neutralization. Moreover, in ALL
patient derived xenografts, CART19 cells resulted in a more durable
disease control when combined with lenzilumab. In addition, it was
found that GM-CSF.sup.k/o CAR-T cells were more effective in
controlling leukemia in NALM6 xenografts and demonstrated improved
overall survival. While the mechanisms for enhanced CART effector
functions after GM-CSF depletion are currently unclear, the results
provided herein are consistent with previous reports indicating
that monocytes impair T cell expansion ex vivo and that M2
polarized macrophages inhibit CART19 antigen specific
proliferation. This is an important finding because across CAR-T
clinical trials, improved CAR-T cell proliferation was consistently
associated with improved efficacy and response (i.e., overall and
complete response rates).
[0426] It is known that activated T cells produce GM-CSF. T cells
do not possess all the subunits for the GM-CSF receptor, so in
ordinary circumstances, GM-CSF does not normally feedback on
T-cells directly, although it can under some circumstances at very
high levels. Instead, this GM-CSF affects the behaviors of numerous
other cell types including macrophages and dendritic cells. The
subsequent activation of these cells results in actions that work
to stimulate T cells such as cytokine production and antigen
presentation. T cell stimulation can further drive production of
GM-CSF and other cytokines to in turn act on the other cell types
like macrophages and dendritic cells, which drives the cycle. In
CAR-T cell therapy, it is likely that the large number of activated
T cells produced over a very short timeline pushes this cycle to an
extreme situation. The results described herein suggest that
blocking GM-CSF helps prevent this immune overstimulation without
impairing T cell functions, actually enhancing them. The exact
mechanisms for enhanced CAR-T cell effector functions after GM-CSF
blockade are unclear.
[0427] Finally, the results provided herein results additionally
suggest that the development of GM-CSF.sup.k/o CART19 cells may
represent a novel way to partially control GM-CSF production that
can be incorporated into current CAR-T cell manufacturing. These
results indicate that these cells function normally and could
represent an independent therapeutic approach to enhance the
therapeutic window after CAR-T cell therapy. An anti-GM-CSF
antibody, such as lenzilumab, is a clinical stage therapeutic
solution to neutralize GM-CSF, abrogate both CRS and
neuro-inflammation of apparent neurotoxicities, and potentially
improve CAR-T cell function.
[0428] The studies described herein represent a significant advance
in understanding and preventing toxicities after CAR-T cell
therapy. These results strongly suggest that modulating myeloid
cell behavior through GM-CSF blockade helps control CAR-T cell
mediated toxicities and reduce their immunosuppressive features to
improve leukemic control. These studies illuminate a novel approach
to abrogate neuro-inflammation of apparent neurotoxicities and CRS
through GM-CSF neutralization that also potentially enhances CAR-T
cell functions.
Example 23
Administration of an Anti-GM-CSF Monoclonal Antibody (Lenzilumab)
Significantly Reduced Neuro-Inflammation Caused by CAR-T Therapy
and Maintained the Integrity of the Blood-Brain-Barrier in a
Xenograft Model
[0429] This preclinical study was designed to closely replicate the
findings observed in CAR-T clinical trials and utilized human acute
lymphoblastic leukemia (ALL), human CD19 targeted CAR-T (CART19),
and human peripheral blood mononuclear cells (PBMCs) and conducted
in mice.
Primary Patient-Derived ALL Xenografts
[0430] ALL xenografts were established in mice essentially as
described in Example 22.
MRI Acquisition
[0431] The integrity of the BBB can be noninvasively monitored by
magnetic resonance imaging (MRI). Conventional MR contrast agents
(CAs) containing gadolinium are used in association with MRI to
detect and quantify BBB leakage. Under normal circumstances CAs do
not cross the intact BBB. However, due to their small size CAs
extravasate from the blood into the brain tissue even when the BBB
is partially compromised.
[0432] MRIs were acquired essentially as described in Example 22.
The gadolinium-enhanced MRI method based on T.sub.1-weighted images
taken prior to and after CA injection, as described by Ku, M C et
al., Methods Mol Biol. 2018; 1718:395-408. doi:
10.1007/978-1-4939-7531-0_23, which is incorporated by reference in
its entirety, is consistent with that used in the present
preclinical study of Lenzilumab and CART19. This
gadolinium-enhanced MRI method is useful for investigating BBB
permeability in in-vivo mouse models and can be easily applied in a
number of experimental disease conditions including
neuroinflammation disorders, or to assess (un)wanted drug
effects.
Confocal Microscopy
[0433] Confocal microscopy was used to assess impairment/disruption
of the blood brain barrier (also called BBB herein). This
microscopy technique uses spatial filtering to eliminate
out-of-focus light or flare in specimens that are thicker than the
plane of focus; as such confocal microscopy offers several
advantages over conventional optical microscopy, including
controllable depth of field, the elimination of image degrading
out-of-focus information, and the ability to collect serial optical
sections from thick specimens.
Results
BBB Integrity is Preserved and Neuro-Inflammation is Significantly
Reduced Following CAR-T and Lenzilumab Therapy
[0434] In the present study, MRI images taken on day 5
qualitatively revealed diffuse neuro-inflammation with CAR-T
therapy, while the MRI images taken following the combination of
lenzilumab+ CAR-T showed significantly less neuro-inflammation,
similar to the untreated control group (see FIG. 33A).
Quantification of MRI via gadolinium enhanced T1 hyperintensity
showed a significant 75% decrease in neuro-inflammation and BBB
impairment with lenzilumab+ CAR-T vs CAR-T+ control antibody (FIG.
33A). Moreover, confocal microscopy distinctly shows in high
resolution images that following CAR-T therapy, the BBB is
significantly impaired (FIG. 40A), which is consistent with the MRI
images that qualitatively showed diffuse neuro-inflammation with
CAR-T therapy (see FIG. 33A). In contrast, confocal microscopy
shows maintenance of the integrity of the BBB with lenzilumab in
combination with CAR-T (FIG. 40A), which is consistent with the
qualitative and quantitative MRI images taken following the
combination of lenzilumab+ CAR-T that showed a significant
reduction neuro-inflammation compared to CAR-T plus isotype
control. FIG. 40A shows confocal microscopy BBB data. FIG. 33A is a
demonstrative quantitative MRI using Gadolinium enhanced
T1-hyperintensity, showing three treatment groups: untreated vs
CART19+Lenzilumab vs CART19+isotype control. The confocal
micrsocopy results are critical as they help to explain the
pathology of CAR-T induced neuro-inflammation. This data suggests
that following CAR-T administration, the BBB becomes impaired
enabling a massive influx of pro-inflammatory cytokines into the
CNS, which is believed to propagate neuroinflammation. This data is
consistent with data reported in CAR-T clinical trials. In the
ZUMA-1 study with chimeric antigen receptor (CAR)-transduced
autologous T cells administered intravenously at a target dose of
2.times.10.sup.6 anti-CD19 CAR T cells/kg (Yescarta), a significant
increase in pro-inflammatory cytokines was seen in the CNS in
patients who developed grade 3+ neurotoxicity. The present confocal
microscopy data helps to explain why and how the addition of
lenzilumab significantly reduces CAR-T induced
neuro-inflammation.
[0435] MRI images qualitatively showed diffuse neuro-inflammation
and impairment of the BBB on day 5 following administration of
CAR-T therapy (CART19). When lenzilumab is co-administered in with
CAR-T, the integrity of the BBB was preserved/maintained and MRI
images taken on day 5 following the combination of lenzilumab+
CAR-T showed significantly less neuro-inflammation, similar to the
untreated control group (see FIG. 33A). Moreover, confocal
microscopy revealed this result is entirely consistent with MRI
imaging data showing a 75% reduction in neuro-inflammation and BBB
impairment following Lenzilumab and CAR-T compared to CAR-T and
control antibody (the Y-axis in this analysis is Gadolinium
Enhanced T1 Hyperintensity).
[0436] CART19 Cell Proliferation Exponentially Increased and
Leukemic Disease Control Significantly Improved Following CART and
Lenzilumab Therapy
[0437] Lenzilumab administration following CART19 therapy also
resulted in an exponential increase in CART19 cell proliferation
and significant improvement in leukemic disease control sustained
over time for at least 35 days post CART19 infusion compared to
CART19 plus control, as described in Example 22. These results
suggest that GM-CSF neutralization with an anti-GM-CSF monoclonal
antibody (Lenzilumab) may play a role in reducing relapses and
increasing durable complete responses after CART19 therapy. This is
a significant finding, given that more than 50% of adult lymphoma
patients who initially respond to CART19 therapy subsequently
relapse within the first year of follow-up.
FIG. 40B (adapted from Santomasso, B D, et al., published
OnlineFirst on Jun. 7, 2018; DOI: 10.1158/2159-8290.CD-17-1319,
which is incorporated by reference in its entirety), shows that
high levels of protein in the CSF (as shown in Santomasso's data)
are an indication of BBB disruption and protein leak into the CNS
(because of the increased blood-cerebrospinal fluid (CSF) barrier
permeability during neurotoxicity). This shows that BBB disruption
is central to the pathophysiology of NI and links the herein
provided xenograft model findings to the clinical findings of
Santomasso. In embodiments of the herein provided methods, the
methods further comprise performing a lumbar puncture (by a
clinician) and measuring CSF levels of protein/albumen that could
predict for subsequent clinical expectation of grade of NT and
preemptive measures.
[0438] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
[0439] All publications, accession numbers, patents, and patent
applications cited in this specification are herein incorporated by
reference as if each was specifically and individually indicated to
be incorporated by reference.
Exemplary V.sub.H Region Sequences of Anti-GM-CSF Antibodies of the
Invention:
TABLE-US-00010 [0440] SEQ ID NO: 1 (VH#1, FIG. 1)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGW
INPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRRD
RFPYYFDYWGQGTLVTVSS SEQ ID NO: 2 (VH#2, FIG. 1)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGW
INAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCARRD
RFPYYFDYWGQGTLVTVSS SEQ ID NO: 3 (VH#3, FIG. 1)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGW
INAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCARRQ
RFPYYFDYWGQGTLVTVSS SEQ ID NO: 4 (VH#4, FIG. 1)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGW
INAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCVRRQ
RFPYYFDYWGQGTLVTVSS SEQ ID NO: 5 (VH#5, FIG. 1)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGW
INAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCVRRQ
RFPYYFDYWGQGTLVTVSS
Exemplary V.sub.L Region Sequences of Anti-GM-CSF Antibodies of the
Invention:
TABLE-US-00011 [0441] SEQ ID NO: 6 (VK#1, FIG. 1)
EIVLTQSPATLSVSPGERATLSCRASQSVGTNVAWYQQKPGQAPRV
LIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFN RSPLTFGGGTKVEIK SEQ
ID NO: 7 (VK#2, FIG. 1)
EIVLTQSPATLSVSPGERATLSCRASQSVGTNVAWYQQKPGQAPRV
LIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFN KSPLTFGGGTKVEIK SEQ
ID NO: 8 (VK#3, FIG. 1)
EIVLTQSPATLSVSPGERATLSCRASQSIGSNLAWYQQKPGQAPRV
LIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFN RSPLTFGGGTKVEIK SEQ
ID NO: 9 (VK#4, FIG. 1)
EIVLTQSPATLSVSPGERATLSCRASQSIGSNLAWYQQKPGQAPRV
LIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFN KSPLTFGGGTKVEIK SEQ
ID NO: 10 Exemplary kappa constant region
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC SEQ
ID NO: 11 Exemplary heavy chain constant region, f-allotype:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
Sequence CWU 1
1
391119PRTArtificial Sequencesynthetic anti-granulocyte-macrophage
colony-stimulating factor (GM-CSF) antibody heavy chain variable
region (VH) VH#1 1Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Gly Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly
Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Arg Asp Arg
Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val
Thr Val Ser Ser 1152119PRTArtificial Sequencesynthetic
anti-granulocyte-macrophage colony-stimulating factor (GM-CSF)
antibody heavy chain variable region (VH) VH#2 2Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60Gln
Gly Arg Val Ala Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Asp Arg Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 1153119PRTArtificial
Sequencesynthetic anti-granulocyte-macrophage colony-stimulating
factor (GM-CSF) antibody heavy chain variable region (VH) VH#3 3Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Met 35 40 45Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln
Lys Phe 50 55 60Gln Gly Arg Val Ala Ile Thr Arg Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gln Arg Phe Pro Tyr Tyr Phe
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
1154119PRTArtificial Sequencesynthetic anti-granulocyte-macrophage
colony-stimulating factor (GM-CSF) antibody heavy chain variable
region (VH) VH#4 4Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Ser Phe Thr Asn Tyr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly
Gln Arg Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Ala Gly Asn Gly Asn
Thr Lys Tyr Ser Gln Lys Phe 50 55 60Gln Gly Arg Val Ala Ile Thr Arg
Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Arg Gln Arg
Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val
Thr Val Ser Ser 1155119PRTArtificial Sequencesynthetic
anti-granulocyte-macrophage colony-stimulating factor (GM-CSF)
antibody heavy chain variable region (VH) VH#5 5Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Val Arg Arg Gln Arg Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 1156107PRTArtificial
Sequencesynthetic anti-granulocyte-macrophage colony-stimulating
factor (GM-CSF) antibody kappa light chain variable region (VL)
VK#1 6Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro
Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly
Thr Asn 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Val Leu Ile 35 40 45Tyr Ser Thr Ser Ser Arg Ala Thr Gly Ile Thr Asp
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Phe Asn Arg Ser Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 1057107PRTArtificial Sequencesynthetic
anti-granulocyte-macrophage colony-stimulating factor (GM-CSF)
antibody kappa light chain variable region (VL) VK#2 7Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Thr Asn 20 25 30Val
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Val Leu Ile 35 40
45Tyr Ser Thr Ser Ser Arg Ala Thr Gly Ile Thr Asp Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Lys
Ser Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
1058107PRTArtificial Sequencesynthetic anti-granulocyte-macrophage
colony-stimulating factor (GM-CSF) antibody kappa light chain
variable region (VL) VK#3 8Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Ile Gly Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Val Leu Ile 35 40 45Tyr Ser Thr Ser Ser Arg Ala
Thr Gly Ile Thr Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Phe Asn Arg Ser Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 1059107PRTArtificial
Sequencesynthetic anti-granulocyte-macrophage colony-stimulating
factor (GM-CSF) antibody kappa light chain variable region (VL)
VK#4 9Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro
Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly
Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Val Leu Ile 35 40 45Tyr Ser Thr Ser Ser Arg Ala Thr Gly Ile Thr Asp
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Phe Asn Lys Ser Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 10510107PRTArtificial Sequencesynthetic kappa
constant region 10Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 100 10511330PRTArtificial Sequencesynthetic
f-allotype heavy chain constant region 11Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330126PRTArtificial
Sequencesynthetic heavy chain variable region (VH)
complementarity-determining region 3 (CDR3) binding specificity
determinant (BSD) 12Arg Gln Arg Phe Pro Tyr1 5136PRTArtificial
Sequencesynthetic heavy chain variable region (VH)
complementarity-determining region 3 (CDR3) binding specificity
determinant (BSD) 13Arg Asp Arg Phe Pro Tyr1 51415PRTArtificial
Sequencesynthetic heavy chain variable region (VH) human J-segment
JH4 14Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser1
5 10 151510PRTArtificial Sequencesynthetic heavy chain variable
region (VH) complementarity-determining region 3 (CDR3) 15Arg Gln
Arg Phe Pro Tyr Tyr Phe Asp Tyr1 5 101610PRTArtificial
Sequencesynthetic heavy chain variable region (VH)
complementarity-determining region 3 (CDR3) 16Arg Asp Arg Phe Pro
Tyr Tyr Phe Asp Tyr1 5 10179PRTArtificial Sequencesynthetic light
chain variable region (VL) complementarity-determining region 3
(CDR3)VARIANT(5)..(5)Xaa = Lys or Arg 17Gln Gln Phe Asn Xaa Ser Pro
Leu Thr1 5189PRTArtificial Sequencesynthetic light chain variable
region (VL) complementarity-determining region 3 (CDR3) 18Gln Gln
Phe Asn Lys Ser Pro Leu Thr1 51998PRTHomo
sapiensMISC_FEATUREgermline heavy chain variable region VH1 1-02
19Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly
Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg2098PRTHomo
sapiensMISC_FEATUREgermline heavy chain variable region VH1 1-03
20Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser
Gln Lys Pro 50 55 60Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg2196PRTHomo
sapiensMISC_FEATUREgermline kappa light chain variable region VKIII
A27 21Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro
Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Tyr Gly Ser Ser Pro 85 90 95226PRTArtificial
Sequencesynthetic heavy chain variable region (VH)
complementarity-determining region 3 (CDR3)VARIANT(2)..(2)Xaa = Gln
or Asp 22Arg Xaa Arg Phe Pro Tyr1 52321PRTArtificial
Sequencesynthetic combination of heavy chain variable region (VH)
complementarity-determining region 3 (CDR3) binding specificity
determinant (BSD) and human germline J-segment JH4 (CDRH3 and
FR4)VARIANT(2)..(2)Xaa = Gln or Asp 23Arg Xaa Arg Phe Pro Tyr Tyr
Phe Asp Tyr Trp Gly Gln Gly Thr Leu1 5 10 15Val Thr Val Ser Ser
20245PRTArtificial Sequencesynthetic heavy chain variable region
(VH) complementarity-determining region 1 (CDR1) 24Gly Tyr Tyr Met
His1 5255PRTArtificial Sequencesynthetic heavy chain variable
region (VH) complementarity-determining region 1 (CDR1) 25Asn Tyr
Tyr Ile His1 52617PRTArtificial Sequencesynthetic heavy chain
variable region (VH) complementarity-determining region 2 (CDR2)
26Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln1
5 10 15Gly2717PRTArtificial Sequencesynthetic heavy chain variable
region (VH) complementarity-determining region 2 (CDR2) 27Trp Ile
Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln1 5 10
15Gly289PRTArtificial Sequencesynthetic light chain variable region
(VL) complementarity-determining region 3 (CDR3) 28Gln Gln Phe Asn
Arg Ser Pro Leu Thr1 52919PRTArtificial Sequencesynthetic
combination of light chain variable region (VL)
complementarity-determining region 3 (CDR3) binding specificity
determinant (BSD) and human germline J-segment JK4 (CDRL3 and FR4)
29Gln Gln Phe Asn Arg Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val1
5 10 15Glu Ile Lys3019PRTArtificial Sequencesynthetic combination
of light chain variable region (VL) complementarity-determining
region 3 (CDR3)
binding specificity determinant (BSD) and human germline J-segment
JK4 (CDRL3 and FR4) 30Gln Gln Phe Asn Lys Ser Pro Leu Thr Phe Gly
Gly Gly Thr Lys Val1 5 10 15Glu Ile Lys3111PRTArtificial
Sequencesynthetic light chain variable region (VL)
complementarity-determining region 1 (CDR1) 31Arg Ala Ser Gln Ser
Val Gly Thr Asn Val Ala1 5 103211PRTArtificial Sequencesynthetic
light chain variable region (VL) complementarity-determining region
1 (CDR1) 32Arg Ala Ser Gln Ser Ile Gly Ser Asn Leu Ala1 5
10337PRTArtificial Sequencesynthetic light chain variable region
(VL) complementarity-determining region 2 (CDR2) 33Ser Thr Ser Ser
Arg Ala Thr1 53410PRTArtificial Sequencesynthetic light chain
variable region (VL) FR4 region 34Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys1 5 10355PRTArtificial Sequencesynthetic heavy chain
variable region (VH) complementarity-determining region 1 (CDRH1)
35Asp Tyr Asn Ile His1 53617PRTArtificial Sequencesynthetic heavy
chain variable region (VH) complementarity-determining region 2
(CDRH2) 36Tyr Ile Ala Pro Tyr Ser Gly Gly Thr Gly Tyr Asn Gln Glu
Phe Lys1 5 10 15Asn3711PRTArtificial Sequencesynthetic light chain
variable region (VL) complementarity-determining region 1 (CDRL1)
37Lys Ala Ser Gln Asn Val Gly Ser Asn Val Ala1 5 10387PRTArtificial
Sequencesynthetic light chain variable region (VL)
complementarity-determining region 2 (CDRL2) 38Ser Ala Ser Tyr Arg
Ser Gly1 53911PRTArtificial Sequencesynthetic light chain variable
region (VL) complementarity-determining region 1
(CDR1)VARIANT(6)..(6)Xaa = Val or IleVARIANT(8)..(8)Xaa = Thr or
SerVARIANT(10)..(10)Xaa = Val or Leu 39Arg Ala Ser Gln Ser Xaa Gly
Xaa Asn Xaa Ala1 5 10
* * * * *