U.S. patent application number 16/310738 was filed with the patent office on 2019-10-31 for compositions and methods for the depletion of cells.
This patent application is currently assigned to Magenta Therapeutics, Inc.. The applicant listed for this patent is MAGENTA THERAPEUTICS, INC.. Invention is credited to Adam HARTIGAN, Dwight MORROW, Andrew NIXON.
Application Number | 20190328901 16/310738 |
Document ID | / |
Family ID | 60664586 |
Filed Date | 2019-10-31 |
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United States Patent
Application |
20190328901 |
Kind Code |
A1 |
NIXON; Andrew ; et
al. |
October 31, 2019 |
COMPOSITIONS AND METHODS FOR THE DEPLETION OF CELLS
Abstract
The invention provides compositions and methods useful for the
depletion of cells, such as CD45+, CD135+, CD34+, CD90+, and/or
CD110+ cells, and for the treatment of various hematopoietic
diseases, metabolic disorders, cancers, and autoimmune diseases,
among others. Described herein are antibodies, antigen-binding
fragments, ligands, and conjugates thereof that can be applied to
effect the treatment of these conditions, for instance, by
depleting a population of CD45+, CD135+, CD34+, CD90+, or CD110+
cells in a patient, such as a human. The compositions and methods
described herein can be used to treat a disorder directly, for
instance, by depleting a population of CD45+, CD135+, CD34+, CD90+,
or CD110+ cancer cells or autoimmune cells. The compositions and
methods described herein can also be used to prepare a patient for
hematopoietic stem cell transplant therapy and to improve the
engraftment of hematopoietic stem cell transplants by selectively
depleting endogenous hematopoietic stem cells prior to the
transplant procedure.
Inventors: |
NIXON; Andrew; (Hanover,
MA) ; MORROW; Dwight; (West Chester, PA) ;
HARTIGAN; Adam; (Brookline, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MAGENTA THERAPEUTICS, INC. |
Cambridge |
MA |
US |
|
|
Assignee: |
Magenta Therapeutics, Inc.
Cambridge
MA
|
Family ID: |
60664586 |
Appl. No.: |
16/310738 |
Filed: |
June 19, 2017 |
PCT Filed: |
June 19, 2017 |
PCT NO: |
PCT/US2017/038151 |
371 Date: |
December 17, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62448782 |
Jan 20, 2017 |
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62448768 |
Jan 20, 2017 |
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62437756 |
Dec 22, 2016 |
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62437729 |
Dec 22, 2016 |
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62351778 |
Jun 17, 2016 |
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62351725 |
Jun 17, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2863 20130101;
A61K 2039/505 20130101; A61K 47/6897 20170801; C07K 16/2803
20130101; A61K 31/5517 20130101; A61K 35/12 20130101; C07K 16/289
20130101; A61K 2035/124 20130101; C07K 14/37 20130101; A61K 38/12
20130101; A61K 47/6803 20170801; A61P 35/02 20180101; C07K 16/2896
20130101; C07K 2317/73 20130101; C07K 2317/24 20130101; A61K
39/3955 20130101; A61K 47/6809 20170801; C12N 2501/599 20130101;
A61K 47/6811 20170801; A61K 47/6817 20170801; A61K 35/28 20130101;
A61P 37/06 20180101; A61K 31/4745 20130101; A61K 31/704 20130101;
C07K 16/2866 20130101; A61K 38/07 20130101; A61K 47/6831 20170801;
C12N 5/0087 20130101; A61K 47/6829 20170801; A61K 47/6849 20170801;
A61K 39/395 20130101; A61K 47/6825 20170801; C12N 5/0647
20130101 |
International
Class: |
A61K 47/68 20060101
A61K047/68; C07K 16/28 20060101 C07K016/28; A61K 35/28 20060101
A61K035/28; A61K 39/395 20060101 A61K039/395; A61K 38/12 20060101
A61K038/12; A61K 31/704 20060101 A61K031/704; A61K 38/07 20060101
A61K038/07; A61K 31/4745 20060101 A61K031/4745; A61K 31/5517
20060101 A61K031/5517 |
Claims
1. A method of depleting a population of CD45RO+ cells in a human
patient, the method comprising administering to the patient an
effective amount of an antibody or antigen-binding fragment thereof
capable of binding CD45RO.
2. A method of depleting a population of CD45RO+ cells in a human
patient in need of a hematopoietic stem cell transplant, the method
comprising administering to the patient an effective amount of an
antibody or antigen-binding fragment thereof capable of binding
CD45RO prior to the patient receiving a transplant comprising
hematopoietic stem cells.
3. A method comprising administering to a human patient a
transplant comprising hematopoietic stem cells, wherein the patient
has been previously administered an antibody or antigen-binding
fragment thereof capable of binding CD45RO in an amount sufficient
to deplete a population of CD45RO+ cells in the patient.
4. A method comprising: a. administering to a human patient an
antibody or antigen-binding fragment thereof capable of binding
CD45RO in an amount sufficient to deplete a population of CD45RO+
cells in the patient; and b. subsequently administering to the
patient a transplant comprising hematopoietic stem cells.
5. The method of any one of claims 1-4, wherein the antibody or
antigen-binding fragment thereof is conjugated to a cytotoxin.
6. A method of depleting a population of CD135+ cells in a human
patient, the method comprising administering to the patient an
effective amount of an antibody or antigen-binding fragment thereof
capable of binding CD135, wherein the antibody or antigen-binding
fragment thereof is conjugated to a cytotoxin.
7. A method of depleting a population of CD135+ cells in a human
patient in need of a hematopoietic stem cell transplant, the method
comprising administering to the patient an effective amount of an
antibody or antigen-binding fragment thereof capable of binding
CD135 prior to the patient receiving a transplant comprising
hematopoietic stem cells, wherein the antibody or antigen-binding
fragment thereof is conjugated to a cytotoxin.
8. A method comprising administering to a human patient a
transplant comprising hematopoietic stem cells, wherein the patient
has been previously administered an antibody or antigen-binding
fragment thereof capable of binding CD135 in an amount sufficient
to deplete a population of CD135+ cells in the patient, wherein the
antibody or antigen-binding fragment thereof is conjugated to a
cytotoxin.
9. A method comprising: a. administering to a human patient an
antibody or antigen-binding fragment thereof capable of binding
CD135 in an amount sufficient to deplete a population of CD135+
cells in the patient; and b. subsequently administering to the
patient a transplant comprising hematopoietic stem cells, wherein
the antibody or antigen-binding fragment thereof is conjugated to a
cytotoxin.
10. The method of any one of claims 6-9, wherein the antibody or
antigen-binding fragment thereof comprises the following
complementarity determining regions (CDRs): TABLE-US-00014 (SEQ ID
NO: 1) a. a CDR-H1 having the amino acid sequence SYYMH; (SEQ ID
NO: 2) b. a CDR-H2 having the amino acid sequence
IINPSGGSTSYAQKFQG; (SEQ ID NO: 3) c. a CDR-H3 having the amino acid
sequence GVGAHDAFDI or (SEQ ID NO: 4) VVAAAVADY; (SEQ ID NO: 5) d.
a CDR-L1 having the amino acid sequence RSSQSLLHSNGNNYLD or (SEQ ID
NO: 6) RSSQSLLHSNGYNYLD; (SEQ ID NO: 7) e. a CDR-L2 having the
amino acid sequence LGSNRAS; and (SEQ ID NO: 8) f. a CDR-L3 having
the amino acid sequence MQGTHPAIS or (SEQ ID NO: 9) MQSLQTPFT.
11. The method of any one of claims 6-9, wherein the antibody or
antigen-binding fragment thereof comprises the following CDRs:
TABLE-US-00015 (SEQ ID NO: 10) a. a CDR-H1 having the amino acid
sequence SYAIS; (SEQ ID NO: 11) b. a CDR-H2 having the amino acid
sequence GIIPIFGTANYAQKFQG; (SEQ ID NO: 12) c. a CDR-H3 having the
amino acid sequence FALFGFREQAFDI; (SEQ ID NO: 13) d. a CDR-L1
having the amino acid sequence RASQSISSYLN; (SEQ ID NO: 14) e. a
CDR-L2 having the amino acid sequence AASSLQS; and (SEQ ID NO: 15)
f. a CDR-L3 having the amino acid sequence QQSYSTPFT.
12. A method of depleting a population of CD135+ cells in a human
patient, the method comprising administering to the patient an
effective amount of human Flt3 ligand, or a fragment thereof
capable of binding CD135.
13. A method of depleting a population of CD135+ cells in a human
patient in need of a hematopoietic stem cell transplant, the method
comprising administering to the patient an effective amount of
human Flt3 ligand, or a fragment thereof capable of binding CD135,
prior to the patient receiving a transplant comprising
hematopoietic stem cells.
14. A method comprising administering to a human patient a
transplant comprising hematopoietic stem cells, wherein the patient
has been previously administered human Flt3 ligand, or a fragment
thereof capable of binding CD135, in an amount sufficient to
deplete a population of CD135+ cells in the patient.
15. A method comprising: a. administering to a human patient human
Flt3 ligand, or a fragment thereof capable of binding CD135, in an
amount sufficient to deplete a population of CD135+ cells in the
patient; and b. subsequently administering to the patient a
transplant comprising hematopoietic stem cells.
16. The method of any one of claims 12-15, wherein the human Flt3
ligand or fragment thereof is covalently bound to an Fc domain.
17. The method of claim 16, wherein the N-terminus of the human
Flt3 ligand or fragment thereof is covalently bound to the Fc
domain.
18. The method of claim 16, wherein the C-terminus of the human
Flt3 ligand or fragment thereof is covalently bound to the Fc
domain.
19. The method of any one of claims 16-18, wherein the Fc domain is
covalently bound to a cytotoxin.
20. The method of any one of claims 12-15, wherein the human Flt3
ligand or fragment thereof is covalently bound to a cytotoxin.
21. The method of claim 20, wherein the N-terminus of the human
Flt3 ligand or fragment thereof is covalently bound to the
cytotoxin.
22. The method of claim 20, wherein the C-terminus of the human
Flt3 ligand or fragment thereof is covalently bound to the
cytotoxin.
23. The method of any one of claims 20-22, wherein the cytotoxin is
covalently bound to an Fc domain.
24. The method of any claim 20, wherein the human Flt3 ligand or
fragment thereof is covalently bound to the cytotoxin at one site
and is covalently bound to an Fc domain at a different site.
25. The method of any one of claims 16-19, 23, and 24, wherein the
Fc domain is a human IgG1, IgG2, IgG3, or IgG4 isotype Fc
domain.
26. A method of depleting a population of CD34+ cells in a human
patient, the method comprising administering to the patient an
effective amount of an antibody or antigen-binding fragment thereof
capable of binding CD34, wherein the antibody or antigen-binding
fragment thereof is conjugated to a cytotoxin.
27. A method of depleting a population of CD34+ cells in a human
patient in need of a hematopoietic stem cell transplant, the method
comprising administering to the patient an effective amount of an
antibody or antigen-binding fragment thereof capable of binding
CD34 prior to the patient receiving a transplant comprising
hematopoietic stem cells, wherein the antibody or antigen-binding
fragment thereof is conjugated to a cytotoxin.
28. A method comprising administering to a human patient a
transplant comprising hematopoietic stem cells, wherein the patient
has been previously administered an antibody or antigen-binding
fragment thereof capable of binding CD34 in an amount sufficient to
deplete a population of CD34+ cells in the patient, wherein the
antibody or antigen-binding fragment thereof is conjugated to a
cytotoxin.
29. A method comprising: a. administering to a human patient an
antibody or antigen-binding fragment thereof capable of binding
CD34 in an amount sufficient to deplete a population of CD34+ cells
in the patient; and b. subsequently administering to the patient a
transplant comprising hematopoietic stem cells, wherein the
antibody or antigen-binding fragment thereof is conjugated to a
cytotoxin.
30. A method of depleting a population of CD90+ cells in a human
patient, the method comprising administering to the patient an
effective amount of an antibody or antigen-binding fragment thereof
capable of binding CD90, wherein the antibody or antigen-binding
fragment thereof is conjugated to a cytotoxin.
31. A method of depleting a population of CD90+ cells in a human
patient in need of a hematopoietic stem cell transplant, the method
comprising administering to the patient an effective amount of an
antibody or antigen-binding fragment thereof capable of binding
CD90 prior to the patient receiving a transplant comprising
hematopoietic stem cells, wherein the antibody or antigen-binding
fragment thereof is conjugated to a cytotoxin.
32. A method comprising administering to a human patient a
transplant comprising hematopoietic stem cells, wherein the patient
has been previously administered an antibody or antigen-binding
fragment thereof capable of binding CD90 in an amount sufficient to
deplete a population of CD90+ cells in the patient, wherein the
antibody or antigen-binding fragment thereof is conjugated to a
cytotoxin.
33. A method comprising: a. administering to a human patient an
antibody or antigen-binding fragment thereof capable of binding
CD90 in an amount sufficient to deplete a population of CD90+ cells
in the patient; and b. subsequently administering to the patient a
transplant comprising hematopoietic stem cells, wherein the
antibody or antigen-binding fragment thereof is conjugated to a
cytotoxin.
34. A method of depleting a population of CD110+ cells in a human
patient, the method comprising administering to the patient an
effective amount of an antibody or antigen-binding fragment thereof
capable of binding CD110, wherein the antibody or antigen-binding
fragment thereof is conjugated to a cytotoxin.
35. A method of depleting a population of CD110+ cells in a human
patient in need of a hematopoietic stem cell transplant, the method
comprising administering to the patient an effective amount of an
antibody or antigen-binding fragment thereof capable of binding
CD110 prior to the patient receiving a transplant comprising
hematopoietic stem cells, wherein the antibody or antigen-binding
fragment thereof is conjugated to a cytotoxin.
36. A method comprising administering to a human patient a
transplant comprising hematopoietic stem cells, wherein the patient
has been previously administered an antibody or antigen-binding
fragment thereof capable of binding CD110 in an amount sufficient
to deplete a population of CD110+ cells in the patient, wherein the
antibody or antigen-binding fragment thereof is conjugated to a
cytotoxin.
37. A method comprising: a. administering to a human patient an
antibody or antigen-binding fragment thereof capable of binding
CD110 in an amount sufficient to deplete a population of CD110+
cells in the patient; and b. subsequently administering to the
patient a transplant comprising hematopoietic stem cells, wherein
the antibody or antigen-binding fragment thereof is conjugated to a
cytotoxin.
38. The method of any one of claims 5-11 and 19-37, wherein the
cytotoxin is selected from the group consisting of an amatoxin,
pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin,
maytansine, a maytansinoid, an auristatin, an anthracycline, a
calicheamicin, irinotecan, SN-38, a duocarmycin, a
pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an
indolinobenzodiazepine, and an indolinobenzodiazepine dimer, or a
variant thereof.
39. A method of depleting a population of CD45+ cells in a human
patient, the method comprising administering to the patient an
effective amount of an antibody or antigen-binding fragment thereof
capable of binding CD45, wherein the antibody or antigen-binding
fragment thereof is conjugated to a cytotoxin selected from the
group consisting of an amatoxin, pseudomonas exotoxin A,
deBouganin, diphtheria toxin, saporin, maytansine, a maytansinoid,
an auristatin, an anthracycline, a calicheamicin, irinotecan,
SN-38, a duocarmycin, a pyrrolobenzodiazepine, a
pyrrolobenzodiazepine dimer, an indolinobenzodiazepine, and an
indolinobenzodiazepine dimer, or a variant thereof.
40. A method of depleting a population of CD45+ cells in a human
patient in need of a hematopoietic stem cell transplant, the method
comprising administering to the patient an effective amount of an
antibody or antigen-binding fragment thereof capable of binding
CD45 prior to the patient receiving a transplant comprising
hematopoietic stem cells, wherein the antibody or antigen-binding
fragment thereof is conjugated to a cytotoxin selected from the
group consisting of an amatoxin, pseudomonas exotoxin A,
deBouganin, diphtheria toxin, saporin, maytansine, a maytansinoid,
an auristatin, an anthracycline, a calicheamicin, irinotecan,
SN-38, a duocarmycin, a pyrrolobenzodiazepine, a
pyrrolobenzodiazepine dimer, an indolinobenzodiazepine, and an
indolinobenzodiazepine dimer, or a variant thereof.
41. A method of comprising administering to a human patient a
transplant comprising hematopoietic stem cells, wherein the patient
has been previously administered an antibody or antigen-binding
fragment thereof capable of binding CD45 in an amount sufficient to
deplete a population of CD45+ cells in the patient, wherein the
antibody or antigen-binding fragment thereof is conjugated to a
cytotoxin selected from the group consisting of an amatoxin,
pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin,
maytansine, a maytansinoid, an auristatin, an anthracycline, a
calicheamicin, irinotecan, SN-38, a duocarmycin, a
pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an
indolinobenzodiazepine, and an indolinobenzodiazepine dimer, or a
variant thereof.
42. A method comprising: a. administering to a human patient an
antibody or antigen-binding fragment thereof capable of binding
CD45 in an amount sufficient to deplete a population of CD45+ cells
in the patient; and b. subsequently administering to the patient a
transplant comprising hematopoietic stem cells, wherein the
antibody or antigen-binding fragment thereof is conjugated to a
cytotoxin selected from the group consisting of an amatoxin,
pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin,
maytansine, a maytansinoid, an auristatin, an anthracycline, a
calicheamicin, irinotecan, SN-38, a duocarmycin, a
pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an
indolinobenzodiazepine, and an indolinobenzodiazepine dimer, or a
variant thereof.
43. The method of any one of claims 5-11 and 19-42, wherein the
antibody, antigen-binding fragment thereof, or ligand conjugated to
a cytotoxin is represented by the formula Ab-Am, wherein Ab is the
antibody, antigen-binding fragment thereof, or ligand and Am is an
amatoxin represented by formula (I) ##STR00048## wherein R.sub.1 is
H, OH, OR.sub.A, or OR.sub.C; R.sub.2 is H, OH, OR.sub.B, or
OR.sub.C; R.sub.A and R.sub.B, together with the oxygen atoms to
which they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group; R.sub.3 is H, R.sub.C, or
R.sub.D; R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each
independently H, OH, OR.sub.C, OR.sub.D, R.sub.C, or R.sub.D;
R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or
NR.sub.CR.sub.D; R.sub.9 is H, OH, OR.sub.C, or OR.sub.D; X is
--S--, --S(O)--, or --SO.sub.2--; R.sub.C is -L-Z; R.sub.D is
optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted
C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6
heteroalkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl,
optionally substituted C.sub.2-C.sub.6 heteroalkynyl, optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted heteroaryl;
L is optionally substituted C.sub.1-C.sub.6 alkylene, optionally
substituted C.sub.1-C.sub.6 heteroalkylene, optionally substituted
C.sub.2-C.sub.6 alkenylene, optionally substituted C.sub.2-C.sub.6
heteroalkenylene, optionally substituted C.sub.2-C.sub.6
alkynylene, optionally substituted C.sub.2-C.sub.6
heteroalkynylene, optionally substituted cycloalkylene, optionally
substituted heterocycloalkylene, optionally substituted arylene, or
optionally substituted heteroarylene; and Z is a chemical moiety
formed from a coupling reaction between a reactive substituent
present on L and a reactive substituent present within the antibody
or antigen-binding fragment thereof, wherein Am comprises exactly
one R.sub.C substituent.
44. The method of claim 43, wherein Am is an amatoxin represented
by formula (IA). ##STR00049##
45. The method of claim 43, wherein Am is an amatoxin represented
by formula (IB). ##STR00050##
46. The method of claim 44 or 45, wherein R.sub.A and R.sub.B,
together with the oxygen atoms to which they are bound, combine to
form: ##STR00051## wherein Y is selected from O, S, NR.sub.E, and
CR.sub.ER.sub.E', and R.sub.E and R.sub.E' are each independently
optionally substituted C.sub.1-C.sub.6 alkylene-R.sub.C, optionally
substituted C.sub.1-C.sub.6 heteroalkylene-R.sub.C, optionally
substituted C.sub.2-C.sub.6 alkenylene-R.sub.C, optionally
substituted C.sub.2-C.sub.6 heteroalkenylene-R.sub.C, optionally
substituted C.sub.2-C.sub.6 alkynylene-R.sub.C, optionally
substituted C.sub.2-C.sub.6 heteroalkynylene-R.sub.C, optionally
substituted cycloalkylene-R.sub.C, optionally substituted
heterocycloalkylene-R.sub.C, optionally substituted
arylene-R.sub.C, or optionally substituted
heteroarylene-R.sub.C.
47. The method of claim 46, wherein R.sub.A and R.sub.B, together
with the oxygen atoms to which they are bound, combine to form:
##STR00052##
48. The method of claim 44 or 45, wherein R.sub.1 is H, OH, or
OR.sub.A; R.sub.2 is H, OH, or OR.sub.B; R.sub.A and R.sub.B,
together with the oxygen atoms to which they are bound, combine to
form: ##STR00053## R.sub.3, R.sub.4, R.sub.6, and R.sub.7 are each
H; R.sub.5 is OR.sub.C; R.sub.5 is OH or NH.sub.2; and R.sub.9 is H
or OH.
49. The method of claim 44 or 45, wherein R.sub.1 and R.sub.2 are
each independently H or OH; R.sub.3 is R.sub.C; R.sub.4, R.sub.6,
and R.sub.7 are each H; R.sub.5 is H, OH, or OC.sub.1-C.sub.6
alkyl; R.sub.5 is OH or NH.sub.2; and R.sub.9 is H or OH.
50. The method of claim 44 or 45, wherein R.sub.1 and R.sub.2 are
each independently H or OH; R.sub.3, R.sub.6, and R.sub.7 are each
H; R.sub.4 and R.sub.5 are each independently H, OH, OR.sub.C, or
R.sub.C; R.sub.5 is OH or NH.sub.2; and R.sub.9 is H or OH.
51. The method of claim 44 or 45, wherein R.sub.1 and R.sub.2 are
each independently H or OH; R.sub.3, R.sub.6, and R.sub.7 are each
H; R.sub.4 and R.sub.5 are each independently H or OH; R.sub.8 is
OR.sub.C or NHR.sub.C; and R.sub.9 is H or OH.
52. The method of any one of claims 5-11 and 19-42, wherein the
antibody, antigen-binding fragment thereof, or ligand conjugated to
a cytotoxin is represented by the formula Ab-Am, wherein Ab is the
antibody, antigen-binding fragment thereof, or ligand and Am is an
amatoxin represented by formula (II) ##STR00054## wherein X is S,
SO, or SO.sub.2; R.sub.1 is H or a linker covalently bound to the
antibody or antigen-binding fragment thereof; and R.sub.2 is H or a
linker covalently bound to the antibody or antigen-binding fragment
thereof; wherein when R.sub.1 is H, R.sub.2 is the linker, and when
R.sub.2 is H, R.sub.1 is the linker.
53. The method of any one of claims 5-11 and 19-42, wherein the
cytotoxin is a maytansinoid selected from the group consisting of
DM1 and DM4.
54. The method of any one of claims 5-11 and 19-42, wherein the
cytotoxin is an auristatin selected from the group consisting of
monomethyl auristatin E and monomethyl auristatin F.
55. The method of any one of claims 5-11 and 19-42, wherein the
cytotoxin is an anthracycline selected from the group consisting of
daunorubicin, doxorubicin, epirubicin, and idarubicin.
56. The method of any one of claims 1-11 and 26-55, wherein the
antibody or antigen-binding fragment thereof is selected from the
group consisting of a monoclonal antibody or antigen-binding
fragment thereof, a polyclonal antibody or antigen-binding fragment
thereof, a humanized antibody or antigen-binding fragment thereof,
a bispecific antibody or antigen-binding fragment thereof, a
dual-variable immunoglobulin domain, a single-chain Fv molecule
(scFv), a diabody, a triabody, a nanobody, an antibody-like protein
scaffold, a Fv fragment, a Fab fragment, a F(ab').sub.2 molecule,
and a tandem di-scFv.
57. The method of any one of claims 1-11 and 26-56, wherein the
antibody has an isotype selected from the group consisting of IgG,
IgA, IgM, IgD, and IgE.
58. The method of any one of claims 1-57, wherein the antibody,
antigen-binding fragment thereof, or ligand is internalized by a
cancer cell, autoimmune cell, or hematopoietic stem cell following
administration to the patient.
59. The method of any one of claims 1-58, wherein the antibody,
antigen-binding fragment thereof, or ligand is capable of promoting
necrosis of a cancer cell, autoimmune cell, or hematopoietic stem
cell.
60. The method of any one of claims 2-5, 7-11, 13-25, 27-29, 31-33,
35-38, and 40-59, wherein the antibody, antigen-binding fragment
thereof, or ligand is capable of recruiting one or more complement
proteins to the hematopoietic stem cell upon administration to the
patient.
61. The method of any one of claims 2-5, 7-11, 13-25, 27-29, 31-33,
35-38, and 40-60, wherein the transplant comprising hematopoietic
stem cells is administered to the patient after the concentration
of the antibody, antigen-binding fragment thereof, or ligand has
substantially cleared from the blood of the patient.
62. The method of any one of claims 2-5, 7-11, 13-25, 27-29, 31-33,
35-38, and 40-61, wherein the hematopoietic stem cells or progeny
thereof maintain hematopoietic stem cell functional potential after
two or more days following transplantation of the hematopoietic
stem cells into the patient.
63. The method of any one of claims 2-5, 7-11, 13-25, 27-29, 31-33,
35-38, and 40-62, wherein the hematopoietic stem cells or progeny
thereof are capable of localizing to hematopoietic tissue and/or
reestablishing hematopoiesis following transplantation of the
hematopoietic stem cells into the patient.
64. The method of any one of claims 2-5, 7-11, 13-25, 27-29, 31-33,
35-38, and 40-63, wherein upon transplantation into the patient,
the hematopoietic stem cells give rise to recovery of a population
of cells selected from the group consisting of megakaryocytes,
thrombocytes, platelets, erythrocytes, mast cells, myeoblasts,
basophils, neutrophils, eosinophils, microglia, granulocytes,
monocytes, osteoclasts, antigen-presenting cells, macrophages,
dendritic cells, natural killer cells, T-lymphocytes, and
B-lymphocytes.
65. The method of any one of claims 1-64, wherein the patient is
suffering from a stem cell disorder.
66. The method of any one of claims 1-65, wherein the patient is
suffering from a hemoglobinopathy disorder.
67. The method of claim 66, wherein the hemoglobinopathy disorder
is selected from the group consisting of sickle cell anemia,
thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich
syndrome.
68. The method of claim 66, wherein the hemoglobinopathy disorder
is Fanconi anemia.
69. The method of claim 66, wherein the hemoglobinopathy disorder
is aplastic anemia.
70. The method of claim 66, wherein the hemoglobinopathy disorder
is sickle cell anemia.
71. The method of claim 66, wherein the hemoglobinopathy disorder
is thalassemia.
72. The method of any one of claims 1-71, wherein the patient is
suffering from a myelodysplastic disorder.
73. The method of any one of claims 1-72, wherein the patient is
suffering from an immunodeficiency disorder.
74. The method of claim 73, wherein the immunodeficiency disorder
is a congenital immunodeficiency.
75. The method of claim 73, wherein the immunodeficiency disorder
is an acquired immunodeficiency.
76. The method of claim 75, wherein the acquired immunodeficiency
is human immunodeficiency virus or acquired immune deficiency
syndrome.
77. The method of any one of claims 1-76, wherein the patient is
suffering from a metabolic disorder.
78. The method of claim 77, wherein the metabolic disorder is
selected from the group consisting of glycogen storage diseases,
mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease,
sphingolipidoses, and metachromatic leukodystrophy.
79. The method of any one of claims 1-78, wherein the patient is
suffering from cancer.
80. The method of claim 79, wherein the cancer is selected from the
group consisting of leukemia, lymphoma, multiple myeloma, and
neuroblastoma.
81. The method of claim 79, wherein the cancer is a hematological
cancer.
82. The method of claim 79, wherein the cancer is acute myeloid
leukemia.
83. The method of claim 79, wherein the cancer is acute lymphoid
leukemia.
84. The method of claim 79, wherein the cancer is chronic myeloid
leukemia.
85. The method of claim 79, wherein the cancer is chronic lymphoid
leukemia.
86. The method of claim 79, wherein the cancer is diffuse large
B-cell lymphoma.
87. The method of claim 79, wherein the cancer is non-Hodgkin's
lymphoma.
88. The method of any one of claims 1-87, wherein the patient is
suffering from a disorder selected from the group consisting of
adenosine deaminase deficiency and severe combined
immunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashi
disease, hereditary lymphohistiocytosis, osteopetrosis,
osteogenesis imperfecta, storage diseases, thalassemia major,
systemic sclerosis, systemic lupus erythematosus, multiple
sclerosis, and juvenile rheumatoid arthritis.
89. The method of any one of claims 1-88, wherein the patient is
suffering from an autoimmune disorder.
90. The method of claim 89, wherein the autoimmune disorder is
selected from the group consisting of multiple sclerosis, human
systemic lupus, rheumatoid arthritis, inflammatory bowel disease,
treating psoriasis, Type 1 diabetes mellitus, acute disseminated
encephalomyelitis, Addison's disease, alopecia universalis,
ankylosing spondylitisis, antiphospholipid antibody syndrome,
aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis,
autoimmune inner ear disease, autoimmune lymphoproliferative
syndrome, autoimmune oophoritis, Balo disease, Behcet's disease,
bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic
fatigue immune dysfunction syndrome, chronic inflammatory
demyelinating polyneuropathy, Crohn's disease, cicatrical
pemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinin
disease, CREST syndrome, Degos disease, discoid lupus,
dysautonomia, endometriosis, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, Goodpasture's syndrome, Grave's
disease, Guillain-Barre syndrome, Hashimoto's thyroiditis,
Hidradenitis suppurativa, idiopathic and/or acute thrombocytopenic
purpura, idiopathic pulmonary fibrosis, IgA neuropathy,
interstitial cystitis, juvenile arthritis, Kawasaki's disease,
lichen planus, Lyme disease, Meniere disease, mixed connective
tissue disease, myasthenia gravis, neuromyotonia, opsoclonus
myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus
vulgaris, pernicious anemia, polychondritis, polymyositis and
dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa,
polyglandular syndromes, polymyalgia rheumatica, primary
agammaglobulinemia, Raynaud phenomenon, Reiter's syndrome,
rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome,
stiff person syndrome, Takayasu's arteritis, temporal arteritis,
ulcerative colitis, uveitis, vasculitis, vitiligo, vulvodynia, and
Wegener's granulomatosis.
91. The method of claim 89, wherein the autoimmune disorder is
scleroderma.
92. The method of claim 89, wherein the autoimmune disorder is
multiple sclerosis.
93. The method of claim 89, wherein the autoimmune disorder is
ulcerative colitis.
94. The method of claim 89, wherein the autoimmune disorder is
Chrohn's disease.
95. The method of claim 89, wherein the autoimmune disorder is Type
1 diabetes.
96. The method of any one of claims 65-95, wherein the methods
treats the disorder or cancer.
97. A method of depleting a population of CD45+ cells, the method
comprising contacting the population with an effective amount of a
conjugate represented by the formula Ab-Am, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD45 and Am
is an amatoxin.
98. The method of claim 97, wherein Am is represented by formula
(IA) ##STR00055## wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
R.sub.2 is H, OH, OR.sub.B, or OR.sub.C; R.sub.A and R.sub.B,
together with the oxygen atoms to which they are bound, combine to
form an optionally substituted 5-membered heterocyclolalkyl group;
R.sub.3 is H, R.sub.C, or R.sub.D; R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 are each independently H, OH, OR.sub.C, OR.sub.D, R.sub.C,
or R.sub.D; R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C,
or NR.sub.CR.sub.D; R.sub.9 is H, OH, OR.sub.C, or OR.sub.D; X is
--S--, --S(O)--, or --SO.sub.2--; R.sub.C is -L-Z; R.sub.D is
optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted
C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6
heteroalkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl,
optionally substituted C.sub.2-C.sub.6 heteroalkynyl, optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted heteroaryl;
L is optionally substituted C.sub.1-C.sub.6 alkylene, optionally
substituted C.sub.1-C.sub.6 heteroalkylene, optionally substituted
C.sub.2-C.sub.6 alkenylene, optionally substituted C.sub.2-C.sub.6
heteroalkenylene, optionally substituted C.sub.2-C.sub.6
alkynylene, optionally substituted C.sub.2-C.sub.6
heteroalkynylene, optionally substituted cycloalkylene, optionally
substituted heterocycloalkylene, optionally substituted arylene, or
optionally substituted heteroarylene; and Z is a chemical moiety
formed from a coupling reaction between a reactive substituent
present on L and a reactive substituent present within the antibody
or antigen-binding fragment thereof, wherein Am comprises exactly
one R.sub.C substituent.
99. The method of claim 97, wherein Am is represented by formula
(IB) ##STR00056## wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
R.sub.2 is H, OH, OR.sub.B, or OR.sub.C; R.sub.A and R.sub.B,
together with the oxygen atoms to which they are bound, combine to
form an optionally substituted 5-membered heterocyclolalkyl group;
R.sub.3 is H, R.sub.C, or R.sub.D; R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 are each independently H, OH, OR.sub.C, OR.sub.D, R.sub.C,
or R.sub.D; R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C,
or NR.sub.CR.sub.D; R.sub.9 is H, OH, OR.sub.C, or OR.sub.D; X is
--S--, --S(O)--, or --SO.sub.2--; R.sub.C is -L-Z; R.sub.D is
optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted
C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6
heteroalkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl,
optionally substituted C.sub.2-C.sub.6 heteroalkynyl, optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted heteroaryl;
L is optionally substituted C.sub.1-C.sub.6 alkylene, optionally
substituted C.sub.1-C.sub.6 heteroalkylene, optionally substituted
C.sub.2-C.sub.6 alkenylene, optionally substituted C.sub.2-C.sub.6
heteroalkenylene, optionally substituted C.sub.2-C.sub.6
alkynylene, optionally substituted C.sub.2-C.sub.6
heteroalkynylene, optionally substituted cycloalkylene, optionally
substituted heterocycloalkylene, optionally substituted arylene, or
optionally substituted heteroarylene; and Z is a chemical moiety
formed from a coupling reaction between a reactive substituent
present on L and a reactive substituent present within the antibody
or antigen-binding fragment thereof, wherein Am comprises exactly
one R.sub.C substituent
100. The method of claim 97, wherein the wherein the antibody or
antigen-binding fragment thereof is conjugated to the amatoxin by
way of a cysteine residue in the Fc domain of the antibody or
antigen-binding fragment thereof.
101. The method of claim 100, wherein the cysteine residue is
introduced by way of a mutation in the Fc domain of the antibody or
antigen-binding fragment thereof.
102. The method of claim 101, wherein the cysteine residue is
selected from the group consisting of Cys118, Cys239, and
Cys265.
103. The method of claim 100, wherein the cysteine residue is
naturally occurring in the Fc domain of the antibody or
antigen-binding fragment thereof.
104. The method of claim 103, wherein the Fc domain is an IgG Fc
domain and the cysteine residue is selected from the group
consisting of Cys261, Csy321, Cys367, and Cys425.
105. The method of claim 98 or 99, wherein R.sub.1 is H, OH, or
OR.sub.A; R.sub.2 is H, OH, or OR.sub.B; R.sub.A and R.sub.B,
together with the oxygen atoms to which they are bound, combine to
form: ##STR00057## R.sub.3, R.sub.4, R.sub.6, and R.sub.7 are each
H; R.sub.5 is OR.sub.C; R.sub.8 is OH or NH.sub.2; and R.sub.9 is H
or OH.
106. The method of claim 98 or 99, wherein R.sub.1 and R.sub.2 are
each independently H or OH; R.sub.3 is R.sub.C; R.sub.4, R.sub.6,
and R.sub.7 are each H; R.sub.5 is H, OH, or OC.sub.1-C.sub.6
alkyl; R.sub.8 is OH or NH.sub.2; and R.sub.9 is H or OH.
107. The method of claim 98 or 99, wherein R.sub.1 and R.sub.2 are
each independently H or OH; R.sub.3, R.sub.6, and R.sub.7 are each
H; R.sub.4 is OR.sub.C, or R.sub.C; R.sub.5 is H, OH, or
OC.sub.1-C.sub.6 alkyl; R.sub.8 is OH or NH.sub.2; and R.sub.9 is H
or OH.
108. The method of claim 98 or 99, wherein R.sub.1 and R.sub.2 are
each independently H or OH; R.sub.3, R.sub.6, and R.sub.7 are each
H; R.sub.4 and R.sub.5 are each independently H or OH; R.sub.8 is
OR.sub.C or NHR.sub.C; and R.sub.9 is H or OH.
109. The method of claim 97, wherein the antibody or
antigen-binding fragment thereof is internalized by a CD45+
cell.
110. The method of claim 97, wherein the antibody or
antigen-binding fragment thereof binds CD45 with a K.sub.d of from
about 0.1 pM to about 1 .mu.M.
111. The method of claim 97, wherein the antibody or
antigen-binding fragment thereof binds CD45 with a k.sub.on of from
about 9.times.10.sup.-2 M.sup.-1 s.sup.-1 to about 1.times.10.sup.2
M.sup.-1s.sup.-1.
112. The method of claim 97, wherein the antibody or
antigen-binding fragment thereof competitively inhibits the binding
of CD45 to a second antibody or antigen binding fragment thereof,
wherein the second antibody or antigen-binding fragment thereof
comprises the following complementarity determining regions (CDRs):
TABLE-US-00016 (SEQ ID NO: 16) a. a CDR-H1 having the amino acid
sequence SYAMS; (SEQ ID NO: 17) b. a CDR-H2 having the amino acid
sequence AISGSGGSTFYADSVRG; (SEQ ID NO: 18) c. a CDR-H3 having the
amino acid sequence EVMGPIFFDY; (SEQ ID NO: 19) d. a CDR-L1 having
the amino acid sequence RASQSIISSALA; (SEQ ID NO: 20) e. a CDR-L2
having the amino acid sequence GASSRAT; and (SEQ ID NO: 21) f. a
CDR-L3 having the amino acid sequence QQYGSTPLT.
113. The method of claim 97, wherein the antibody or
antigen-binding fragment thereof is selected from the group
consisting of a monoclonal antibody or antigen-binding fragment
thereof, a polyclonal antibody or antigen-binding fragment thereof,
a humanized antibody or antigen-binding fragment thereof, a
bispecific antibody or antigen-binding fragment thereof, a
dual-variable immunoglobulin domain, a single-chain Fv molecule
(scFv), a diabody, a triabody, a nanobody, an antibody-like protein
scaffold, a Fv fragment, a Fab fragment, a F(ab').sub.2 molecule,
and a tandem di-scFV.
114. A conjugate represented by the formula Ab-Am, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD45 and Am
is an amatoxin.
115. The conjugate of claim 114, wherein Am is represented by
formula (IA) ##STR00058## wherein R.sub.1 is H, OH, OR.sub.A, or
OR.sub.C; R.sub.2 is H, OH, OR.sub.B, or OR.sub.C; R.sub.A and
R.sub.B, together with the oxygen atoms to which they are bound,
combine to form an optionally substituted 5-membered
heterocyclolalkyl group; R.sub.3 is H, R.sub.C, or R.sub.D;
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each independently H,
OH, OR.sub.C, OR.sub.D, R.sub.C, or R.sub.D; R.sub.8 is OH,
NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or NR.sub.CR.sub.D;
R.sub.9 is H, OH, OR.sub.C, or OR.sub.D; X is --S--, --S(O)--, or
--SO.sub.2--; R.sub.C is -L-Z; R.sub.D is optionally substituted
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6
heteroalkyl, optionally substituted C.sub.2-C.sub.6 alkenyl,
optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, optionally substituted
C.sub.2-C.sub.6 heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted heterocycloalkyl, optionally substituted
aryl, or optionally substituted heteroaryl; L is optionally
substituted C.sub.1-C.sub.6 alkylene, optionally substituted
C.sub.1-C.sub.6 heteroalkylene, optionally substituted
C.sub.2-C.sub.6 alkenylene, optionally substituted C.sub.2-C.sub.6
heteroalkenylene, optionally substituted C.sub.2-C.sub.6
alkynylene, optionally substituted C.sub.2-C.sub.6
heteroalkynylene, optionally substituted cycloalkylene, optionally
substituted heterocycloalkylene, optionally substituted arylene, or
optionally substituted heteroarylene; and Z is a chemical moiety
formed from a coupling reaction between a reactive substituent
present on L and a reactive substituent present within the antibody
or antigen-binding fragment thereof, wherein Am comprises exactly
one R.sub.C substituent.
116. The conjugate of claim 114, wherein Am is represented by
formula (IB) ##STR00059## wherein R.sub.1 is H, OH, OR.sub.A, or
OR.sub.C; R.sub.2 is H, OH, OR.sub.B, or OR.sub.C; R.sub.A and
R.sub.B, together with the oxygen atoms to which they are bound,
combine to form an optionally substituted 5-membered
heterocyclolalkyl group; R.sub.3 is H, R.sub.C, or R.sub.D;
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each independently H,
OH, OR.sub.C, OR.sub.D, R.sub.C, or R.sub.D; R.sub.8 is OH,
NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or NR.sub.CR.sub.D;
R.sub.9 is H, OH, OR.sub.C, or OR.sub.D; X is --S--, --S(O)--, or
--SO.sub.2--; R.sub.C is -L-Z; R.sub.D is optionally substituted
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6
heteroalkyl, optionally substituted C.sub.2-C.sub.6 alkenyl,
optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, optionally substituted
C.sub.2-C.sub.6 heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted heterocycloalkyl, optionally substituted
aryl, or optionally substituted heteroaryl; L is optionally
substituted C.sub.1-C.sub.6 alkylene, optionally substituted
C.sub.1-C.sub.6 heteroalkylene, optionally substituted
C.sub.2-C.sub.6 alkenylene, optionally substituted C.sub.2-C.sub.6
heteroalkenylene, optionally substituted C.sub.2-C.sub.6
alkynylene, optionally substituted C.sub.2-C.sub.6
heteroalkynylene, optionally substituted cycloalkylene, optionally
substituted heterocycloalkylene, optionally substituted arylene, or
optionally substituted heteroarylene; and Z is a chemical moiety
formed from a coupling reaction between a reactive substituent
present on L and a reactive substituent present within the antibody
or antigen-binding fragment thereof, wherein Am comprises exactly
one R.sub.C substituent
117. The conjugate of claim 114, wherein the antibody or
antigen-binding fragment thereof is conjugated to the amatoxin by
way of a cysteine residue in the Fc domain of the antibody or
antigen-binding fragment thereof.
118. The conjugate of claim 117, wherein the cysteine residue is
introduced by way of a mutation in the Fc domain of the antibody or
antigen-binding fragment thereof.
119. The conjugate of claim 118, wherein the cysteine residue is
selected from the group consisting of Cys118, Cys239, and
Cys265.
120. The conjugate of claim 117, wherein the cysteine residue is
naturally occurring in the Fc domain of the antibody or
antigen-binding fragment thereof.
121. The conjugate of claim 120, wherein the Fc domain is an IgG Fc
domain and the cysteine residue is selected from the group
consisting of Cys261, Csy321, Cys367, and Cys425.
122. The conjugate of claim 115 or 116, wherein R.sub.1 is H, OH,
or OR.sub.A; R.sub.2 is H, OH, or OR.sub.B; R.sub.A and R.sub.B,
together with the oxygen atoms to which they are bound, combine to
form: ##STR00060## R.sub.3, R.sub.4, R.sub.6, and R.sub.7 are each
H; R.sub.5 is OR.sub.C; R.sub.8 is OH or NH.sub.2; and R.sub.9 is H
or OH.
123. The conjugate of claim 115 or 116, wherein R.sub.1 and R.sub.2
are each independently H or OH; R.sub.3 is R.sub.C; R.sub.4,
R.sub.6, and R.sub.7 are each H; R.sub.5 is H, OH, or
OC.sub.1-C.sub.6 alkyl; R.sub.8 is OH or NH.sub.2; and R.sub.9 is H
or OH.
124. The conjugate of claim 115 or 116, wherein R.sub.1 and R.sub.2
are each independently H or OH; R.sub.3, R.sub.6, and R.sub.7 are
each H; R.sub.4 is OR.sub.C, or R.sub.C; R.sub.5 is H, OH, or
OC.sub.1-C.sub.6 alkyl; R.sub.8 is OH or NH.sub.2; and R.sub.9 is H
or OH.
125. The conjugate of claim 115 or 116, wherein R.sub.1 and R.sub.2
are each independently H or OH; R.sub.3, R.sub.6, and R.sub.7 are
each H; R.sub.4 and R.sub.5 are each independently H or OH; R.sub.8
is OR.sub.C or NHR.sub.C; and R.sub.9 is H or OH.
126. The conjugate of claim 114, wherein the antibody or
antigen-binding fragment thereof is internalized by a CD45+
cell.
127. The conjugate of claim 114, wherein the antibody or
antigen-binding fragment thereof binds CD45 with a K.sub.d of from
about 0.1 pM to about 1 .mu.M.
128. The conjugate of claim 114, wherein the antibody or
antigen-binding fragment thereof binds CD45 with a k.sub.on of from
about 9.times.10.sup.-2 M.sup.-1 s.sup.-1 to about 1.times.10.sup.2
M.sup.-1s.sup.-1.
129. The conjugate of claim 114, wherein the antibody or
antigen-binding fragment thereof competitively inhibits the binding
of CD45 to a second antibody or antigen binding fragment thereof,
wherein the second antibody or antigen-binding fragment thereof
comprises the following complementarity determining regions (CDRs):
TABLE-US-00017 (SEQ ID NO: 16) a. a CDR-H1 having the amino acid
sequence SYAMS; (SEQ ID NO: 17) b. a CDR-H2 having the amino acid
sequence AISGSGGSTFYADSVRG; (SEQ ID NO: 18) c. a CDR-H3 having the
amino acid sequence EVMGPIFFDY; (SEQ ID NO: 19) d. a CDR-L1 having
the amino acid sequence RASQSIISSALA; (SEQ ID NO: 20) e. a CDR-L2
having the amino acid sequence GASSRAT; and (SEQ ID NO: 21) f. a
CDR-L3 having the amino acid sequence QQYGSTPLT.
130. The conjugate of claim 114, wherein the antibody or
antigen-binding fragment thereof is selected from the group
consisting of a monoclonal antibody or antigen-binding fragment
thereof, a polyclonal antibody or antigen-binding fragment thereof,
a humanized antibody or antigen-binding fragment thereof, a
bispecific antibody or antigen-binding fragment thereof, a
dual-variable immunoglobulin domain, a single-chain Fv molecule
(scFv), a diabody, a triabody, a nanobody, an antibody-like protein
scaffold, a Fv fragment, a Fab fragment, a F(ab').sub.2 molecule,
and a tandem di-scFV.
131. A method of depleting a population of CD135+ cells, the method
comprising contacting the population with an effective amount of a
conjugate represented by the formula Ab-Am, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD135 and
Am is an amatoxin.
132. The method of claim 131, wherein Am is represented by formula
(IA) ##STR00061## wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
R.sub.2 is H, OH, OR.sub.B, or OR.sub.C; R.sub.A and R.sub.B,
together with the oxygen atoms to which they are bound, combine to
form an optionally substituted 5-membered heterocyclolalkyl group;
R.sub.3 is H, R.sub.C, or R.sub.D; R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 are each independently H, OH, OR.sub.C, OR.sub.D, R.sub.C,
or R.sub.D; R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C,
or NR.sub.CR.sub.D; R.sub.9 is H, OH, OR.sub.C, or OR.sub.D; X is
--S--, --S(O)--, or --SO.sub.2--; R.sub.C is -L-Z; R.sub.D is
optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted
C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6
heteroalkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl,
optionally substituted C.sub.2-C.sub.6 heteroalkynyl, optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted heteroaryl;
L is optionally substituted C.sub.1-C.sub.6 alkylene, optionally
substituted C.sub.1-C.sub.6 heteroalkylene, optionally substituted
C.sub.2-C.sub.6 alkenylene, optionally substituted C.sub.2-C.sub.6
heteroalkenylene, optionally substituted C.sub.2-C.sub.6
alkynylene, optionally substituted C.sub.2-C.sub.6
heteroalkynylene, optionally substituted cycloalkylene, optionally
substituted heterocycloalkylene, optionally substituted arylene, or
optionally substituted heteroarylene; and Z is a chemical moiety
formed from a coupling reaction between a reactive substituent
present on L and a reactive substituent present within the antibody
or antigen-binding fragment thereof, wherein Am comprises exactly
one R.sub.C substituent.
133. The method of claim 131, wherein Am is represented by formula
(IB) ##STR00062## wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
R.sub.2 is H, OH, OR.sub.B, or OR.sub.C; R.sub.A and R.sub.B,
together with the oxygen atoms to which they are bound, combine to
form an optionally substituted 5-membered heterocyclolalkyl group;
R.sub.3 is H, R.sub.C, or R.sub.D; R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 are each independently H, OH, OR.sub.C, OR.sub.D, R.sub.C,
or R.sub.D; R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C,
or NR.sub.CR.sub.D; R.sub.9 is H, OH, OR.sub.C, or OR.sub.D; X is
--S--, --S(O)--, or --SO.sub.2--; R.sub.C is -L-Z; R.sub.D is
optionally substituted C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted
C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6
heteroalkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl,
optionally substituted C.sub.2-C.sub.6 heteroalkynyl, optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted heteroaryl;
L is optionally substituted C.sub.1-C.sub.6 alkylene, optionally
substituted C.sub.1-C.sub.6 heteroalkylene, optionally substituted
C.sub.2-C.sub.6 alkenylene, optionally substituted C.sub.2-C.sub.6
heteroalkenylene, optionally substituted C.sub.2-C.sub.6
alkynylene, optionally substituted C.sub.2-C.sub.6
heteroalkynylene, optionally substituted cycloalkylene, optionally
substituted heterocycloalkylene, optionally substituted arylene, or
optionally substituted heteroarylene; and Z is a chemical moiety
formed from a coupling reaction between a reactive substituent
present on L and a reactive substituent present within the antibody
or antigen-binding fragment thereof, wherein Am comprises exactly
one R.sub.C substituent
134. The method of claim 131, wherein the wherein the antibody or
antigen-binding fragment thereof is conjugated to the amatoxin by
way of a cysteine residue in the Fc domain of the antibody or
antigen-binding fragment thereof.
135. The method of claim 134, wherein the cysteine residue is
introduced by way of a mutation in the Fc domain of the antibody or
antigen-binding fragment thereof.
136. The method of claim 135, wherein the cysteine residue is
selected from the group consisting of Cys118, Cys239, and
Cys265.
137. The method of claim 134, wherein the cysteine residue is
naturally occurring in the Fc domain of the antibody or
antigen-binding fragment thereof.
138. The method of claim 137, wherein the Fc domain is an IgG Fc
domain and the cysteine residue is selected from the group
consisting of Cys261, Csy321, Cys367, and Cys425.
139. The method of claim 132 or 133, wherein R.sub.1 is H, OH, or
OR.sub.A; R.sub.2 is H, OH, or OR.sub.B; R.sub.A and R.sub.B,
together with the oxygen atoms to which they are bound, combine to
form: ##STR00063## R.sub.3, R.sub.4, R.sub.6, and R.sub.7 are each
H; R.sub.5 is OR.sub.C; R.sub.8 is OH or NH.sub.2; and R.sub.9 is H
or OH.
140. The method of claim 132 or 133, wherein R.sub.1 and R.sub.2
are each independently H or OH; R.sub.3 is R.sub.C; R.sub.4,
R.sub.6, and R.sub.7 are each H; R.sub.5 is H, OH, or
OC.sub.1-C.sub.6 alkyl; R.sub.8 is OH or NH.sub.2; and R.sub.9 is H
or OH.
141. The method of claim 132 or 133, wherein R.sub.1 and R.sub.2
are each independently H or OH; R.sub.3, R.sub.6, and R.sub.7 are
each H; R.sub.4 is OR.sub.C, or R.sub.C; R.sub.5 is H, OH, or
OC.sub.1-C.sub.6 alkyl; R.sub.8 is OH or NH.sub.2; and R.sub.9 is H
or OH.
142. The method of claim 132 or 133, wherein R.sub.1 and R.sub.2
are each independently H or OH; R.sub.3, R.sub.6, and R.sub.7 are
each H; R.sub.4 and R.sub.5 are each independently H or OH; R.sub.8
is OR.sub.C or NHR.sub.C; and R.sub.9 is H or OH.
143. The method of claim 131, wherein the antibody or
antigen-binding fragment thereof is internalized by a CD135+
cell.
144. The method of claim 131, wherein the antibody or
antigen-binding fragment thereof binds CD135 with a K.sub.d of from
about 0.1 pM to about 1 .mu.M.
145. The method of claim 131, wherein the antibody or
antigen-binding fragment thereof binds CD135 with a k.sub.on of
from about 9.times.10.sup.-2 M.sup.-1 s.sup.-1 to about
1.times.10.sup.2 M.sup.-1s.sup.-1.
146. The method of claim 131, wherein the antibody or
antigen-binding fragment thereof competitively inhibits the binding
of CD135 to a second antibody or antigen binding fragment thereof,
wherein the second antibody or antigen-binding fragment thereof
comprises the following complementarity determining regions (CDRs):
TABLE-US-00018 (SEQ ID NO: 1) a. a CDR-H1 having the amino acid
sequence SYYMH; (SEQ ID NO: 2) b. a CDR-H2 having the amino acid
sequence IINPSGGSTSYAQKFQG; (SEQ ID NO: 3) c. a CDR-H3 having the
amino acid sequence GVGAHDAFDI or (SEQ ID NO: 4) VVAAAVADY; (SEQ ID
NO: 5) d. a CDR-L1 having the amino acid sequence RSSQSLLHSNGNNYLD
or (SEQ ID NO: 6) RSSQSLLHSNGYNYLD; (SEQ ID NO: 7) e. a CDR-L2
having the amino acid sequence LGSNRAS; and (SEQ ID NO: 8) f. a
CDR-L3 having the amino acid sequence MQGTHPAIS or (SEQ ID NO: 9)
MQSLQTPFT.
147. The method of claim 131, wherein the antibody or
antigen-binding fragment thereof competitively inhibits the binding
of CD135 to a second antibody or antigen binding fragment thereof,
wherein the second antibody or antigen-binding fragment thereof
comprises the following complementarity determining regions (CDRs):
TABLE-US-00019 (SEQ ID NO: 10) a. a CDR-H1 having the amino acid
sequence SYAIS; (SEQ ID NO: 11) b. a CDR-H2 having the amino acid
sequence GIIPIFGTANYAQKFQG; (SEQ ID NO: 12) c. a CDR-H3 having the
amino acid sequence FALFGFREQAFDI; (SEQ ID NO: 13) d. a CDR-L1
having the amino acid sequence RASQSISSYLN; (SEQ ID NO: 14) e. a
CDR-L2 having the amino acid sequence AASSLQS; and (SEQ ID NO: 15)
f. a CDR-L3 having the amino acid sequence QQSYSTPFT.
148. The method of claim 131, wherein the antibody or
antigen-binding fragment thereof is selected from the group
consisting of a monoclonal antibody or antigen-binding fragment
thereof, a polyclonal antibody or antigen-binding fragment thereof,
a humanized antibody or antigen-binding fragment thereof, a
bispecific antibody or antigen-binding fragment thereof, a
dual-variable immunoglobulin domain, a single-chain Fv molecule
(scFv), a diabody, a triabody, a nanobody, an antibody-like protein
scaffold, a Fv fragment, a Fab fragment, a F(ab').sub.2 molecule,
and a tandem di-scFV.
149. A conjugate represented by the formula Ab-Am, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD135 and
Am is an amatoxin.
150. The conjugate of claim 149, wherein Am is represented by
formula (IA) ##STR00064## wherein R.sub.1 is H, OH, OR.sub.A, or
OR.sub.C; R.sub.2 is H, OH, OR.sub.B, or OR.sub.C; R.sub.A and
R.sub.B, together with the oxygen atoms to which they are bound,
combine to form an optionally substituted 5-membered
heterocyclolalkyl group; R.sub.3 is H, R.sub.C, or R.sub.D;
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each independently H,
OH, OR.sub.C, OR.sub.D, R.sub.C, or R.sub.D; R.sub.8 is OH,
NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or NR.sub.CR.sub.D;
R.sub.9 is H, OH, OR.sub.C, or OR.sub.D; X is --S--, --S(O)--, or
--SO.sub.2--; R.sub.C is -L-Z; R.sub.D is optionally substituted
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6
heteroalkyl, optionally substituted C.sub.2-C.sub.6 alkenyl,
optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, optionally substituted
C.sub.2-C.sub.6 heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted heterocycloalkyl, optionally substituted
aryl, or optionally substituted heteroaryl; L is optionally
substituted C.sub.1-C.sub.6 alkylene, optionally substituted
C.sub.1-C.sub.6 heteroalkylene, optionally substituted
C.sub.2-C.sub.6 alkenylene, optionally substituted C.sub.2-C.sub.6
heteroalkenylene, optionally substituted C.sub.2-C.sub.6
alkynylene, optionally substituted C.sub.2-C.sub.6
heteroalkynylene, optionally substituted cycloalkylene, optionally
substituted heterocycloalkylene, optionally substituted arylene, or
optionally substituted heteroarylene; and Z is a chemical moiety
formed from a coupling reaction between a reactive substituent
present on L and a reactive substituent present within the antibody
or antigen-binding fragment thereof, wherein Am comprises exactly
one R.sub.C substituent.
151. The conjugate of claim 149, wherein Am is represented by
formula (IB) ##STR00065## wherein R.sub.1 is H, OH, OR.sub.A, or
OR.sub.C; R.sub.2 is H, OH, OR.sub.B, or OR.sub.C; R.sub.A and
R.sub.B, together with the oxygen atoms to which they are bound,
combine to form an optionally substituted 5-membered
heterocyclolalkyl group; R.sub.3 is H, R.sub.C, or R.sub.D;
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each independently H,
OH, OR.sub.C, OR.sub.D, R.sub.C, or R.sub.D; R.sub.8 is OH,
NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or NR.sub.CR.sub.D;
R.sub.9 is H, OH, OR.sub.C, or OR.sub.D; X is --S--, --S(O)--, or
--SO.sub.2--; R.sub.C is -L-Z; R.sub.D is optionally substituted
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6
heteroalkyl, optionally substituted C.sub.2-C.sub.6 alkenyl,
optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, optionally substituted
C.sub.2-C.sub.6 heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted heterocycloalkyl, optionally substituted
aryl, or optionally substituted heteroaryl; L is optionally
substituted C.sub.1-C.sub.6 alkylene, optionally substituted
C.sub.1-C.sub.6 heteroalkylene, optionally substituted
C.sub.2-C.sub.6 alkenylene, optionally substituted C.sub.2-C.sub.6
heteroalkenylene, optionally substituted C.sub.2-C.sub.6
alkynylene, optionally substituted C.sub.2-C.sub.6
heteroalkynylene, optionally substituted cycloalkylene, optionally
substituted heterocycloalkylene, optionally substituted arylene, or
optionally substituted heteroarylene; and Z is a chemical moiety
formed from a coupling reaction between a reactive substituent
present on L and a reactive substituent present within the antibody
or antigen-binding fragment thereof, wherein Am comprises exactly
one R.sub.C substituent
152. The conjugate of claim 149, wherein the wherein the antibody
or antigen-binding fragment thereof is conjugated to the amatoxin
by way of a cysteine residue in the Fc domain of the antibody or
antigen-binding fragment thereof.
153. The conjugate of claim 152, wherein the cysteine residue is
introduced by way of a mutation in the Fc domain of the antibody or
antigen-binding fragment thereof.
154. The conjugate of claim 153, wherein the cysteine residue is
selected from the group consisting of Cys118, Cys239, and
Cys265.
155. The conjugate of claim 152, wherein the cysteine residue is
naturally occurring in the Fc domain of the antibody or
antigen-binding fragment thereof.
156. The conjugate of claim 155, wherein the Fc domain is an IgG Fc
domain and the cysteine residue is selected from the group
consisting of Cys261, Csy321, Cys367, and Cys425.
157. The conjugate of claim 150 or 151, wherein R.sub.1 is H, OH,
or OR.sub.A; R.sub.2 is H, OH, or OR.sub.B; R.sub.A and R.sub.B,
together with the oxygen atoms to which they are bound, combine to
form: ##STR00066## R.sub.3, R.sub.4, R.sub.6, and R.sub.7 are each
H; R.sub.5 is OR.sub.C; R.sub.8 is OH or NH.sub.2; and R.sub.9 is H
or OH.
158. The conjugate of claim 150 or 151, wherein R.sub.1 and R.sub.2
are each independently H or OH; R.sub.3 is R.sub.C; R.sub.4,
R.sub.6, and R.sub.7 are each H; R.sub.5 is H, OH, or
OC.sub.1-C.sub.6 alkyl; R.sub.8 is OH or NH.sub.2; and R.sub.9 is H
or OH.
159. The conjugate of claim 150 or 151, wherein R.sub.1 and R.sub.2
are each independently H or OH; R.sub.3, R.sub.6, and R.sub.7 are
each H; R.sub.4 is OR.sub.C, or R.sub.C; R.sub.5 is H, OH, or
OC.sub.1-C.sub.6 alkyl; R.sub.8 is OH or NH.sub.2; and R.sub.9 is H
or OH.
160. The conjugate of claim 150 or 151, wherein R.sub.1 and R.sub.2
are each independently H or OH; R.sub.3, R.sub.6, and R.sub.7 are
each H; R.sub.4 and R.sub.5 are each independently H or OH; R.sub.8
is OR.sub.C or NHR.sub.C; and R.sub.9 is H or OH.
161. The conjugate of claim 149, wherein the antibody or
antigen-binding fragment thereof is internalized by a CD135+
cell.
162. The conjugate of claim 149, wherein the antibody or
antigen-binding fragment thereof binds CD135 with a K.sub.d of from
about 0.1 pM to about 1 .mu.M.
163. The conjugate of claim 149, wherein the antibody or
antigen-binding fragment thereof binds CD135 with a k.sub.on of
from about 9.times.10.sup.-2 M.sup.-1 s.sup.-1 to about
1.times.10.sup.2 M.sup.-1s.sup.-1.
164. The conjugate of claim 149, wherein the antibody or
antigen-binding fragment thereof competitively inhibits the binding
of CD135 to a second antibody or antigen binding fragment thereof,
wherein the second antibody or antigen-binding fragment thereof
comprises the following complementarity determining regions (CDRs):
TABLE-US-00020 (SEQ ID NO: 1) a. a CDR-H1 having the amino acid
sequence SYYMH; (SEQ ID NO: 2) b. a CDR-H2 having the amino acid
sequence IINPSGGSTSYAQKFQG; (SEQ ID NO: 3) c. a CDR-H3 having the
amino acid sequence GVGAHDAFDI or (SEQ ID NO: 4) VVAAAVADY; (SEQ ID
NO: 5) d. a CDR-L1 having the amino acid sequence RSSQSLLHSNGNNYLD
or (SEQ ID NO: 6) RSSQSLLHSNGYNYLD; (SEQ ID NO: 7) e. a CDR-L2
having the amino acid sequence LGSNRAS; and (SEQ ID NO: 8) f. a
CDR-L3 having the amino acid sequence MQGTHPAIS or (SEQ ID NO: 9)
MQSLQTPFT.
165. The conjugate of claim 149, wherein the antibody or
antigen-binding fragment thereof competitively inhibits the binding
of CD135 to a second antibody or antigen binding fragment thereof,
wherein the second antibody or antigen-binding fragment thereof
comprises the following complementarity determining regions (CDRs):
TABLE-US-00021 (SEQ ID NO: 10) a. a CDR-H1 having the amino acid
sequence SYAIS; (SEQ ID NO: 11) b. a CDR-H2 having the amino acid
sequence GIIPIFGTANYAQKFQG; (SEQ ID NO: 12) c. a CDR-H3 having the
amino acid sequence FALFGFREQAFDI; (SEQ ID NO: 13) d. a CDR-L1
having the amino acid sequence RASQSISSYLN; (SEQ ID NO: 14) e. a
CDR-L2 having the amino acid sequence AASSLQS; and (SEQ ID NO: 15)
f. a CDR-L3 having the amino acid sequence QQSYSTPFT.
166. The conjugate of claim 149, wherein the antibody or
antigen-binding fragment thereof is selected from the group
consisting of a monoclonal antibody or antigen-binding fragment
thereof, a polyclonal antibody or antigen-binding fragment thereof,
a humanized antibody or antigen-binding fragment thereof, a
bispecific antibody or antigen-binding fragment thereof, a
dual-variable immunoglobulin domain, a single-chain Fv molecule
(scFv), a diabody, a triabody, a nanobody, an antibody-like protein
scaffold, a Fv fragment, a Fab fragment, a F(ab').sub.2 molecule,
and a tandem di-scFV.
167. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD45 and Cy
is pseudomonas exotoxin A.
168. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD45 and Cy
is deBouganin.
169. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD45 and Cy
is diphtheria toxin.
170. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD45 and Cy
is saporin.
171. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD45 and Cy
is maytansine or a maytansinoid.
172. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD45 and Cy
is an auristatin.
173. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD45 and Cy
is an anthracycline.
174. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD45 and
Cy is a calicheamicin.
175. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD45 and
Cy is irinotecan.
176. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD45 and
Cy is SN-38.
177. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD45 and
Cy is a duocarmycin.
178. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD45 and
Cy is a pyrrolobenzodiazepine or a pyrrolobenzodiazepine dimer.
179. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD45 and
Cy is an indolinobenzodiazepine or an indolinobenzodiazepine
dimer.
180. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD135 and
Cy is pseudomonas exotoxin A.
181. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD135 and
Cy is deBouganin.
182. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD135 and
Cy is diphtheria toxin.
183. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD135 and
Cy is saporin.
184. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD135 and
Cy is maytansine or a maytansinoid.
185. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD135 and
Cy is an auristatin.
186. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD135 and
Cy is an anthracycline.
187. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD135
and Cy is a calicheamicin.
188. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD135
and Cy is irinotecan.
189. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD135
and Cy is SN-38.
190. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD135
and Cy is a duocarmycin.
191. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD135
and Cy is a pyrrolobenzodiazepine or a pyrrolobenzodiazepine
dimer.
192. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD135
and Cy is an indolinobenzodiazepine or an indolinobenzodiazepine
dimer.
193. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD34 and Cy
is pseudomonas exotoxin A.
194. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD34 and Cy
is deBouganin.
195. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD34 and Cy
is diphtheria toxin.
196. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD34 and Cy
is saporin.
197. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD34 and Cy
is maytansine or a maytansinoid.
198. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD34 and Cy
is an auristatin.
199. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD34 and Cy
is an anthracycline.
200. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD34 and
Cy is a calicheamicin.
201. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD34 and
Cy is irinotecan.
202. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD34 and
Cy is SN-38.
203. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD34 and
Cy is a duocarmycin.
204. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD34 and
Cy is a pyrrolobenzodiazepine or a pyrrolobenzodiazepine dimer.
205. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD34 and
Cy is an indolinobenzodiazepine or an indolinobenzodiazepine
dimer.
206. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD90 and Cy
is pseudomonas exotoxin A.
207. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD90 and Cy
is deBouganin.
208. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD90 and Cy
is diphtheria toxin.
209. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD90 and Cy
is saporin.
210. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD90 and Cy
is maytansine or a maytansinoid.
211. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD90 and Cy
is an auristatin.
212. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD90 and Cy
is an anthracycline.
213. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD90 and
Cy is a calicheamicin.
214. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD90 and
Cy is irinotecan.
215. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD90 and
Cy is SN-38.
216. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD90 and
Cy is a duocarmycin.
217. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD90 and
Cy is a pyrrolobenzodiazepine or a pyrrolobenzodiazepine dimer.
218. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD90 and
Cy is an indolinobenzodiazepine or an indolinobenzodiazepine
dimer.
219. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD110 and
Cy is pseudomonas exotoxin A.
220. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD110 and
Cy is deBouganin.
221. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD110 and
Cy is diphtheria toxin.
222. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD110 and
Cy is saporin.
223. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD110 and
Cy is maytansine or a maytansinoid.
224. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD110 and
Cy is an auristatin.
225. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or antigen-binding fragment thereof that binds CD110 and
Cy is an anthracycline.
226. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD110
and Cy is a calicheamicin.
227. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD110
and Cy is irinotecan.
228. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD110
and Cy is SN-38.
229. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD110
and Cy is a duocarmycin.
230. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD110
and Cy is a pyrrolobenzodiazepine or a pyrrolobenzodiazepine
dimer.
231. A conjugate represented by the formula Ab-Cy, wherein Ab is an
antibody or an antigen-binding fragment thereof that binds CD110
and Cy is an indolinobenzodiazepine or an indolinobenzodiazepine
dimer.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the treatment of patients suffering
from various pathologies, such as blood diseases, metabolic
disorders, cancers, and autoimmune diseases, among others, by
administration of an antibody, antigen-binding fragment thereof, or
ligand capable of binding an antigen expressed by a hematopoietic
cell, such as a hematopoietic stem cell.
BACKGROUND OF THE INVENTION
[0002] Despite advances in the medicinal arts, there remains a
demand for treating pathologies of the hematopoietic system, such
as diseases of a particular blood cell, metabolic disorders,
cancers, and autoimmune conditions, among others. While
hematopoietic stem cells have significant therapeutic potential, a
limitation that has hindered their use in the clinic has been the
difficulty associated with ensuring engraftment of hematopoietic
stem cell transplants in a host. There is currently a need for
compositions and methods for promoting the engraftment of exogenous
hematopoietic stem cell grafts such that the multi-potency and
hematopoietic functionality of these cells is preserved following
transplantation.
SUMMARY OF THE INVENTION
[0003] The present invention provides compositions and methods for
the direct treatment of various disorders of the hematopoietic
system, metabolic disorders, cancers, and autoimmune diseases,
among others. The invention additionally features methods for
conditioning a patient, such as a human patient, prior to receiving
hematopoietic stem cell transplant therapy so as to promote the
engraftment of hematopoietic stem cell grafts. The patient may be
one that is suffering from one or more blood disorders, such as a
hemoglobinopathy or other hematopoietic pathology, and is thus in
need of hematopoietic stem cell transplantation. As described
herein, hematopoietic stem cells are capable of differentiating
into a multitude of cell types in the hematopoietic lineage, and
can be administered to a patient in order to populate or
re-populate a cell type that is deficient in the patient. The
invention features methods of treating a patient with antibodies,
antibody fragments, ligands, drug-antibody conjugates, and
drug-ligand conjugates capable of binding proteins expressed by
hematopoietic cells, such as cell-surface hematopoietic cell
antigens, so as to (i) directly treat a disease such as a blood
disorder, metabolic disease, cancer, or autoimmune disease, among
others described herein, by selectively depleting a population of
cells that express CD45, CD135, CD34, CD90, and/or CD110, such as
an aberrant blood cell, cancer cell, or autoimmune cell, and/or
(ii) deplete a population of endogenous hematopoietic stem cells
within the patient. The former activity enables the direct
treatment of a wide range of disorders associated with a cell of
the hematopoietic lineage, as CD45, CD135, CD34, CD90, and/or CD110
may be expressed by a cancerous cell, such as a leukemic cell, an
autoimmune lymphocyte, such as a T-cell that expresses a T-cell
receptor that cross-reacts with a self antigen, among other cell
types. The latter activity, the selective depletion of
hematopoietic stem cells, in turn creates a vacancy that can
subsequently be filled by transplantation of an exogenous (for
instance, an autologous, allogeneic, or syngeneic) hematopoietic
stem cell graft. The invention thus provides methods of treating a
variety of hematopoietic conditions, such as sickle cell anemia,
thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, adenosine
deaminase deficiency-severe combined immunodeficiency,
metachromatic leukodystrophy, Diamond-Blackfan anemia and
Schwachman-Diamond syndrome, human immunodeficiency virus
infection, and acquired immune deficiency syndrome, as well as
cancers and autoimmune diseases, among others.
[0004] In a first aspect, the invention provides a method of
depleting a population of CD135+ cells in a human patient by
administering an effective amount of an antibody or antigen-binding
fragment thereof capable of binding CD135 conjugated to a
cytotoxin.
[0005] In another aspect, the invention provides a method of
depleting a population of CD135+ cells in a human patient in need
of a hematopoietic stem cell transplant by administering, prior to
the patient receiving a transplant containing hematopoietic stem
cells, an effective amount of an antibody or antigen-binding
fragment thereof capable of binding CD135 conjugated to a
cytotoxin.
[0006] In an additional aspect, the invention features a method,
for example, of treating a human patient in need of a hematopoietic
stem cell transplant, including administering to a human patient a
transplant containing hematopoietic stem cells, wherein the patient
has been previously administered an antibody or antigen-binding
fragment thereof capable of binding CD135 conjugated to a cytotoxin
in an amount sufficient to deplete a population of CD135+ cells in
the patient.
[0007] In another aspect, the invention features a method, for
example, of treating a human patient in need of a hematopoietic
stem cell transplant, including: administering to a human patient
an antibody or antigen-binding fragment thereof capable of binding
CD135 conjugated to a cytotoxin in an amount sufficient to deplete
a population of CD135+ cells in the patient, and subsequently
administering to the patient a transplant containing hematopoietic
stem cells.
[0008] In some embodiments of the four preceding aspects, the
antibody or antigen-binding fragment thereof contains the following
complementarity determining regions (CDRs):
TABLE-US-00001 (SEQ ID NO: 1) a CDR-H1 having the amino acid
sequence SYYMH; (SEQ ID NO: 2) a CDR-H2 having the amino acid
sequence IINPSGGSTSYAQKFQG; (SEQ ID NO: 3) a CDR-H3 having the
amino acid sequence GVGAHDAFDI or (SEQ ID NO: 4) VVAAAVADY; (SEQ ID
NO: 5) a CDR-L1 having the amino acid sequence RSSQSLLHSNGNNYLD or
(SEQ ID NO: 6) RSSQSLLHSNGYNYLD; (SEQ ID NO: 7) a CDR-L2 having the
amino acid sequence LGSNRAS; and (SEQ ID NO: 8) a CDR-L3 having the
amino acid sequence MQGTHPAIS or (SEQ ID NO: 9) MQSLQTPFT.
[0009] In some embodiments of the four preceding aspects, the
antibody or antigen-binding fragment thereof contains the following
CDRs:
TABLE-US-00002 (SEQ ID NO: 10) a CDR-H1 having the amino acid
sequence SYAIS; (SEQ ID NO: 11) a CDR-H2 having the amino acid
sequence GIIPIFGTANYAQKFQG; (SEQ ID NO: 12) a CDR-H3 having the
amino acid sequence FALFGFREQAFDI; (SEQ ID NO: 13) a CDR-L1 having
the amino acid sequence RASQSISSYLN; (SEQ ID NO: 14) a CDR-L2
having the amino acid sequence AASSLQS; and (SEQ ID NO: 15) a
CDR-L3 having the amino acid sequence QQSYSTPFT.
[0010] In another aspect, the invention features a method of
depleting a population of CD135+ cells in a human patient by
administering to the patient an effective amount of human Flt3
ligand, or a fragment thereof capable of binding CD135.
[0011] In another aspect, the invention features a method of
depleting a population of CD135+ cells in a human patient in need
of a hematopoietic stem cell transplant by administering to the
patient an effective amount of human Flt3 ligand, or a fragment
thereof capable of binding CD135, prior to the patient receiving a
transplant containing hematopoietic stem cells.
[0012] In another aspect, the invention features a method, for
example, of treating a human patient in need of a hematopoietic
stem cell transplant, including administering to a human patient a
transplant containing hematopoietic stem cells, wherein the patient
has been previously administered human Flt3 ligand, or a fragment
thereof capable of binding CD135, in an amount sufficient to
deplete a population of CD135+ cells in the patient.
[0013] In an additional aspect, the invention features a method,
for example, of treating a human patient in need of a hematopoietic
stem cell transplant, including administering to the patient human
Flt3 ligand, or a fragment thereof capable of binding CD135, in an
amount sufficient to deplete a population of CD135+ cells in the
patient; and subsequently administering to the patient a transplant
containing hematopoietic stem cells.
[0014] In some embodiments of any of the preceding four aspects,
the human Flt3 ligand or fragment thereof is covalently bound to an
Fc domain, such as a dimeric Fc domain isolated from a human
antibody (for example, isolated from an IgG1, IgG2, IgG3, or IgG4
isotype human antibody). In some embodiments, the Fc domain is a
monomeric Fc domain containing a single polypeptide strand. In some
embodiments, the N-terminus of the human Flt3 ligand or fragment
thereof is bound to the Fc domain. In some embodiments, the
C-terminus of the human Flt3 ligand or fragment thereof is bound to
the Fc domain. The Fc domain may be conjugated to one or more
copies of the Flt3 ligand or fragment thereof. For instance,
conjugates that may be used with the methods described herein
include dimeric Fc domains in which each polypeptide strand of the
Fc domain is conjugated to a human Flt3 ligand or fragment thereof.
The Fc domain may in turn be conjugated to a cytotoxin, such as a
cytotoxin described herein (for example, pseudomonas exotoxin A,
deBouganin, diphtheria toxin, an amatoxin, such as
.alpha.-amanitin, saporin, maytansine, a maytansinoid, an
auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a
duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine
dimer, an indolinobenzodiazepine, and an indolinobenzodiazepine
dimer, or a variant thereof).
[0015] In some embodiments of any of the preceding four aspects,
the human Flt3 ligand or fragment thereof is covalently bound to a
cytotoxin, such as a cytotoxin described herein (for example,
pseudomonas exotoxin A, deBouganin, diphtheria toxin, an amatoxin,
such as .alpha.-amanitin, saporin, maytansine, a maytansinoid, an
auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a
duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine
dimer, an indolinobenzodiazepine, and an indolinobenzodiazepine
dimer, or a variant thereof). In some embodiments, the N-terminus
of the human Flt3 ligand or fragment thereof is bound to the
cytotoxin. In some embodiments, the C-terminus of the human Flt3
ligand or fragment thereof is bound to the cytotoxin. The cytotoxin
may in turn be conjugated to an Fc domain.
[0016] In some embodiments of any of the preceding four aspects,
the human Flt3 ligand or fragment thereof is covalently bound to
the cytotoxin at one site on the human Flt3 ligand or fragment
thereof (for example, the N- or C-terminus of the human Flt3 ligand
or fragment thereof) and is covalently bound to an Fc domain at
another site on the human Flt3 ligand or fragment thereof (for
example, the opposite terminus of the human Flt3 ligand or fragment
thereof).
[0017] In some embodiments of any of the preceding four aspects,
the Fc domain is a human IgG1 isotype Fc domain. In some
embodiments, the Fc domain is a human IgG2 isotype Fc domain. In
some embodiments, the Fc domain is a human IgG3 isotype Fc domain.
In some embodiments, the Fc domain is a human IgG4 isotype Fc
domain.
[0018] In another aspect, the invention features a method of
depleting a population of CD34+ cells in a human patient by
administering an effective amount of an antibody or antigen-binding
fragment thereof capable of binding CD34 conjugated to a
cytotoxin.
[0019] In another aspect, the invention features a method of
depleting a population of CD34+ cells in a human patient in need of
a hematopoietic stem cell transplant by administering, prior to the
patient receiving a transplant including hematopoietic stem cells,
an effective amount of an antibody or antigen-binding fragment
thereof capable of binding CD34 conjugated to a cytotoxin.
[0020] In another aspect, the invention provides a method, for
example, of treating a human patient in need of a hematopoietic
stem cell transplant, including administering to a human patient a
transplant including hematopoietic stem cells, wherein the patient
has been previously administered an antibody or antigen-binding
fragment thereof capable of binding CD34 conjugated to a cytotoxin
in an amount sufficient to deplete a population of CD34+ cells in
the patient.
[0021] In an additional aspect, the invention features a method,
for example, of treating a human patient in need of a hematopoietic
stem cell transplant, including: administering to the patient an
antibody or antigen-binding fragment thereof capable of binding
CD34 conjugated to a cytotoxin in an amount sufficient to deplete a
population of CD34+ cells in the patient, and subsequently
administering to the patient a transplant including hematopoietic
stem cells.
[0022] In another aspect, the invention features a method of
depleting a population of CD90+ cells in a human patient by
administering an effective amount of an antibody or antigen-binding
fragment thereof capable of binding CD90 conjugated to a
cytotoxin.
[0023] In another aspect, the invention features a method of
depleting a population of CD90+ cells in a human patient in need of
a hematopoietic stem cell transplant by administering, prior to the
patient receiving a transplant including hematopoietic stem cells,
an effective amount of an antibody or antigen-binding fragment
thereof capable of binding CD90 conjugated to a cytotoxin.
[0024] In another aspect, the invention provides a method, for
example, of treating a human patient in need of a hematopoietic
stem cell transplant, including administering to a human patient a
transplant including hematopoietic stem cells, wherein the patient
has been previously administered an antibody or antigen-binding
fragment thereof capable of binding CD90 conjugated to a cytotoxin
in an amount sufficient to deplete a population of CD90+ cells in
the patient.
[0025] In an additional aspect, the invention features a method,
for example, of treating a human patient in need of a hematopoietic
stem cell transplant, including: administering to the patient an
antibody or antigen-binding fragment thereof capable of binding
CD90 conjugated to a cytotoxin in an amount sufficient to deplete a
population of CD90+ cells in the patient, and subsequently
administering to the patient a transplant including hematopoietic
stem cells.
[0026] In another aspect, the invention features a method of
depleting a population of CD110+ cells in a human patient by
administering an effective amount of an antibody or antigen-binding
fragment thereof capable of binding CD110 conjugated to a
cytotoxin.
[0027] In another aspect, the invention features a method of
depleting a population of CD110+ cells in a human patient in need
of a hematopoietic stem cell transplant by administering, prior to
the patient receiving a transplant including hematopoietic stem
cells, an effective amount of an antibody or antigen-binding
fragment thereof capable of binding CD110 conjugated to a
cytotoxin.
[0028] In another aspect, the invention provides a method, for
example, of treating a human patient in need of a hematopoietic
stem cell transplant, including administering to the patient a
transplant including hematopoietic stem cells, wherein the patient
has been previously administered an antibody or antigen-binding
fragment thereof capable of binding CD110 conjugated to a cytotoxin
in an amount sufficient to deplete a population of CD110+ cells in
the patient.
[0029] In an additional aspect, the invention features a method,
for example, of treating a human patient in need of a hematopoietic
stem cell transplant, including: administering to the patient an
antibody or antigen-binding fragment thereof capable of binding
CD110 conjugated to a cytotoxin in an amount sufficient to deplete
a population of CD110+ cells in the patient, and subsequently
administering to the patient a transplant including hematopoietic
stem cells.
[0030] In some embodiments of any of the foregoing aspects, the
cytotoxin conjugated to the antibody, antigen-binding fragment
thereof, or ligand is pseudomonas exotoxin A, deBouganin,
diphtheria toxin, an amatoxin, such as .alpha.-amanitin, saporin,
maytansine, a maytansinoid, an auristatin, an anthracycline, a
calicheamicin, irinotecan, SN-38, a duocarmycin, a
pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an
indolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a
variant thereof.
[0031] In another aspect, the invention provides a method of
depleting a population of CD45+ cells in a human patient by
administering an effective amount of an antibody or antigen-binding
fragment thereof capable of binding CD45 conjugated to a cytotoxin.
The cytotoxin may be, for example, pseudomonas exotoxin A,
deBouganin, diphtheria toxin, an amatoxin, such as
.alpha.-amanitin, saporin, maytansine, a maytansinoid, an
auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a
duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine
dimer, an indolinobenzodiazepine, or an indolinobenzodiazepine
dimer, or a variant thereof.
[0032] In another aspect, the invention provides a method of
depleting a population of CD45+ cells in a human patient in need of
a hematopoietic stem cell transplant by administering, prior to the
patient receiving a transplant including hematopoietic stem cells,
an effective amount of an antibody or antigen-binding fragment
thereof capable of binding CD45 conjugated to a cytotoxin. The
cytotoxin may be, for example, pseudomonas exotoxin A, deBouganin,
diphtheria toxin, an amatoxin, such as .alpha.-amanitin, saporin,
maytansine, a maytansinoid, an auristatin, an anthracycline, a
calicheamicin, irinotecan, SN-38, a duocarmycin, a
pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an
indolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a
variant thereof.
[0033] In another aspect, the invention features a method, for
example, of treating a human patient in need of a hematopoietic
stem cell transplant, including administering to a human patient a
transplant including hematopoietic stem cells, wherein the patient
has been previously administered an antibody or antigen-binding
fragment thereof capable of binding CD45 conjugated to a cytotoxin
in an amount sufficient to deplete a population of CD45+ cells in
the patient. The cytotoxin may be, for example, pseudomonas
exotoxin A, deBouganin, diphtheria toxin, an amatoxin, such as
.alpha.-amanitin, saporin, maytansine, a maytansinoid, an
auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a
duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine
dimer, an indolinobenzodiazepine, or an indolinobenzodiazepine
dimer, or a variant thereof
[0034] In an additional aspect, the invention features a method,
for example, of treating a human patient in need of a hematopoietic
stem cell transplant, including: administering to a human patient
an antibody or antigen-binding fragment thereof capable of binding
CD45 conjugated to a cytotoxin, such as pseudomonas exotoxin A,
deBouganin, diphtheria toxin, an amatoxin, such as
.alpha.-amanitin, saporin, maytansine, a maytansinoid, an
auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a
duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine
dimer, an indolinobenzodiazepine, or an indolinobenzodiazepine
dimer, or a variant thereof, in an amount sufficient to deplete a
population of CD45+ cells in the patient, and subsequently
administering to the patient a transplant including hematopoietic
stem cells.
[0035] In some embodiments of the preceding four aspects, the CD45
is CD45RO.
[0036] In another aspect, the invention provides a method of
depleting a population of CD45RO+ cells in a human patient by
administering an effective amount of an antibody or antigen-binding
fragment thereof capable of binding CD45RO.
[0037] In another aspect, the invention provides a method of
depleting a population of CD45RO+ cells in a human patient in need
of a hematopoietic stem cell transplant by administering, prior to
the patient receiving a transplant containing hematopoietic stem
cells, an effective amount of an antibody or antigen-binding
fragment thereof capable of binding CD45RO.
[0038] In another aspect, the invention features a method, for
example, of treating a human patient in need of a hematopoietic
stem cell transplant, including administering to a human patient a
transplant including hematopoietic stem cells, wherein the patient
has been previously administered an antibody or antigen-binding
fragment thereof capable of binding CD45RO in an amount sufficient
to deplete a population of CD45RO+ cells in the patient.
[0039] In an additional aspect, the invention features a method,
for example, of treating a human patient in need of a hematopoietic
stem cell transplant, including: administering to the patient an
antibody or antigen-binding fragment thereof capable of binding
CD45RO in an amount sufficient to deplete a population of CD45RO+
cells in the patient, and subsequently administering to the patient
a transplant including hematopoietic stem cells.
[0040] In some embodiments of any of the preceding four aspects of
the invention, the antibody or antigen-binding fragment thereof is
conjugated to a cytotoxin.
[0041] In another aspect, the invention provides a method of
depleting a population of CD45+ cells in a human patient by
administering an effective amount of a ligand or fragment thereof
capable of binding CD45.
[0042] In another aspect, the invention provides a method of
depleting a population of CD45+ cells in a human patient in need of
a hematopoietic stem cell transplant by administering, prior to the
patient receiving a transplant including hematopoietic stem cells,
an effective amount of a ligand or fragment thereof capable of
binding CD45.
[0043] In another aspect, the invention features a method, for
example, of treating a human patient in need of a hematopoietic
stem cell transplant, including administering to a human patient a
transplant including hematopoietic stem cells, wherein the patient
has been previously administered a ligand or fragment thereof
capable of binding CD45 in an amount sufficient to deplete a
population of CD45+ cells in the patient.
[0044] In an additional aspect, the invention features a method,
for example, of treating a human patient in need of a hematopoietic
stem cell transplant, including: administering to a human patient a
ligand or fragment thereof capable of binding CD45 in an amount
sufficient to deplete a population of CD45+ cells in the patient,
and subsequently administering to the patient a transplant
including hematopoietic stem cells.
[0045] In another aspect, the invention provides a method of
depleting a population of CD34+ cells in a human patient by
administering an effective amount of a ligand or fragment thereof
capable of binding CD34.
[0046] In another aspect, the invention provides a method of
depleting a population of CD34+ cells in a human patient in need of
a hematopoietic stem cell transplant by administering, prior to the
patient receiving a transplant including hematopoietic stem cells,
an effective amount of a ligand or fragment thereof capable of
binding CD34.
[0047] In another aspect, the invention features a method, for
example, of treating a human patient in need of a hematopoietic
stem cell transplant, including administering to a human patient a
transplant including hematopoietic stem cells, wherein the patient
has been previously administered a ligand or fragment thereof
capable of binding CD34 in an amount sufficient to deplete a
population of CD34+ cells in the patient.
[0048] In an additional aspect, the invention features a method,
for example, of treating a human patient in need of a hematopoietic
stem cell transplant, including: administering to a human patient a
ligand or fragment thereof capable of binding CD34 in an amount
sufficient to deplete a population of CD34+ cells in the patient,
and subsequently administering to the patient a transplant
including hematopoietic stem cells.
[0049] In another aspect, the invention provides a method of
depleting a population of CD90+ cells in a human patient by
administering an effective amount of a ligand or fragment thereof
capable of binding CD90.
[0050] In another aspect, the invention provides a method of
depleting a population of CD90+ cells in a human patient in need of
a hematopoietic stem cell transplant by administering, prior to the
patient receiving a transplant including hematopoietic stem cells,
an effective amount of a ligand or fragment thereof capable of
binding CD90.
[0051] In another aspect, the invention features a method, for
example, of treating a human patient in need of a hematopoietic
stem cell transplant, including administering to a human patient a
transplant including hematopoietic stem cells, wherein the patient
has been previously administered a ligand or fragment thereof
capable of binding CD90 in an amount sufficient to deplete a
population of CD90+ cells in the patient.
[0052] In an additional aspect, the invention features a method,
for example, of treating a human patient in need of a hematopoietic
stem cell transplant, including: administering to a human patient a
ligand or fragment thereof capable of binding CD90 in an amount
sufficient to deplete a population of CD90+ cells in the patient,
and subsequently administering to the patient a transplant
including hematopoietic stem cells.
[0053] In another aspect, the invention provides a method of
depleting a population of CD110+ cells in a human patient by
administering an effective amount of a ligand or fragment thereof
capable of binding CD110.
[0054] In another aspect, the invention provides a method of
depleting a population of CD110+ cells in a human patient in need
of a hematopoietic stem cell transplant by administering, prior to
the patient receiving a transplant including hematopoietic stem
cells, an effective amount of a ligand or fragment thereof capable
of binding CD110.
[0055] In another aspect, the invention features a method, for
example, of treating a human patient in need of a hematopoietic
stem cell transplant, including administering to a human patient a
transplant including hematopoietic stem cells, wherein the patient
has been previously administered a ligand or fragment thereof
capable of binding CD110 in an amount sufficient to deplete a
population of CD110+ cells in the patient.
[0056] In an additional aspect, the invention features a method,
for example, of treating a human patient in need of a hematopoietic
stem cell transplant, including: administering to a human patient a
ligand or fragment thereof capable of binding CD110 in an amount
sufficient to deplete a population of CD110+ cells in the patient,
and subsequently administering to the patient a transplant
including hematopoietic stem cells.
[0057] In some embodiments of the foregoing aspects, the ligand or
fragment thereof that binds one or more of CD45 (e.g., CD45RO),
CD34, CD90, and CD110 is covalently bound to an Fc domain, such as
a dimeric Fc domain isolated from a human antibody (for example,
isolated from an IgG1, IgG2, IgG3, or
[0058] IgG4 isotype human antibody). In some embodiments, the Fc
domain is a monomeric Fc domain containing a single polypeptide
strand. In some embodiments, the N-terminus of the ligand or
fragment thereof is bound to the Fc domain. In some embodiments,
the C-terminus of the ligand or fragment thereof is bound to the Fc
domain. The Fc domain may be conjugated to one or more copies of
the ligand or fragment thereof. For instance, conjugates that may
be used with the methods described herein include dimeric Fc
domains in which each polypeptide strand of the Fc domain is
conjugated to the ligand or fragment thereof. The Fc domain may in
turn be conjugated to a cytotoxin, such as a cytotoxin described
herein (for example, pseudomonas exotoxin A, deBouganin, diphtheria
toxin, an amatoxin, such as .alpha.-amanitin, saporin, maytansine,
a maytansinoid, an auristatin, an anthracycline, a calicheamicin,
irinotecan, SN-38, a duocarmycin, a pyrrolobenzodiazepine, a
pyrrolobenzodiazepine dimer, an indolinobenzodiazepine, and an
indolinobenzodiazepine dimer, or a variant thereof).
[0059] In some embodiments of any of the foregoing aspects, the
ligand or fragment thereof is covalently bound to a cytotoxin, such
as a cytotoxin described herein (for example, pseudomonas exotoxin
A, deBouganin, diphtheria toxin, an amatoxin, such as
.alpha.-amanitin, saporin, maytansine, a maytansinoid, an
auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a
duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine
dimer, an indolinobenzodiazepine, and an indolinobenzodiazepine
dimer, or a variant thereof). In some embodiments, the N-terminus
of the ligand or fragment thereof is bound to the cytotoxin. In
some embodiments, the C-terminus of the ligand or fragment thereof
is bound to the cytotoxin. The cytotoxin may in turn be conjugated
to an Fc domain.
[0060] In some embodiments of any of the foregoing aspects, the
ligand or fragment thereof is covalently bound to the cytotoxin at
one site on the ligand or fragment thereof (for example, the N- or
C-terminus of the ligand or fragment thereof) and is covalently
bound to an Fc domain at another site on the ligand or fragment
thereof (for example, the opposite terminus of the ligand or
fragment thereof).
[0061] In some embodiments of any of the foregoing aspects, the Fc
domain is a human IgG1 isotype Fc domain. In some embodiments, the
Fc domain is a human IgG2 isotype Fc domain. In some embodiments,
the Fc domain is a human IgG3 isotype Fc domain. In some
embodiments, the Fc domain is a human IgG4 isotype Fc domain.
[0062] In some embodiments of any of the above aspects, the
cytotoxin is an amatoxin or derivative thereof, such as
.alpha.-amanitin, .beta.-amanitin, .gamma.-amanitin,
.epsilon.-amanitin, amanin, amaninamide, amanullin, amanullinic
acid, and proamanullin. In some embodiments of any of the above
aspects, the cytotoxin is an amatoxin, and the antibody,
antigen-binding fragment thereof, or ligand conjugated to the
cytotoxin is represented by the formula Ab-Am, wherein Ab is the
antibody, antigen-binding fragment thereof, or ligand, and Am is an
amatoxin. In some embodiments, Am is represented by formula (I)
##STR00001##
[0063] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0064] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0065] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0066] R.sub.3 is H, R.sub.C, or R.sub.D;
[0067] R.sub.4 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0068] R.sub.5 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0069] R.sub.6 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0070] R.sub.7 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0071] R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or
NR.sub.CR.sub.D;
[0072] R.sub.9 is H, OH, OR.sub.C, or OR.sub.D;
[0073] X is --S--, --S(O)--, or --SO.sub.2--;
[0074] R.sub.C is -L-Z;
[0075] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl;
[0076] L is a linker, such as optionally substituted alkylene
(e.g., C.sub.1-C.sub.6 alkylene), optionally substituted
heteroalkylene (C.sub.1-C.sub.6 heteroalkylene), optionally
substituted alkenylene (e.g., C.sub.2-C.sub.6 alkenylene),
optionally substituted heteroalkenylene (e.g., C.sub.2-C.sub.6
heteroalkenylene), optionally substituted alkynylene (e.g.,
C.sub.2-C.sub.6 alkynylene), optionally substituted
heteroalkynylene (e.g., C.sub.2-C.sub.6 heteroalkynylene),
optionally substituted cycloalkylene, optionally substituted
heterocycloalkylene, optionally substituted arylene, or optionally
substituted heteroarylene; and
[0077] Z is a chemical moiety formed from a coupling reaction
between a reactive substituent present on L and a reactive
substituent present within an antibody, antigen-binding fragment
thereof, or ligand that binds CD45 (such as CD45RO), CD135, CD34,
CD90, and/or CD110.
[0078] In some embodiments, Am contains exactly one R.sub.C
substituent.
[0079] In some embodiments, Am is represented by formula (IA)
##STR00002##
[0080] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0081] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0082] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0083] R.sub.3 is H, R.sub.C, or R.sub.D;
[0084] R.sub.4 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0085] R.sub.5 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0086] R.sub.6 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0087] R.sub.7 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0088] R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or
NR.sub.CR.sub.D;
[0089] R.sub.9 is H, OH, OR.sub.C, or OR.sub.D;
[0090] X is --S--, --S(O)--, or --SO.sub.2--;
[0091] R.sub.C is -L-Z;
[0092] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted heteroaryl;
L is a linker, such as optionally substituted alkylene (e.g.,
C.sub.1-C.sub.6 alkylene), optionally substituted heteroalkylene
(C.sub.1-C.sub.6 heteroalkylene), optionally substituted alkenylene
(e.g., C.sub.2-C.sub.6 alkenylene), optionally substituted
heteroalkenylene (e.g., C.sub.2-C.sub.6 heteroalkenylene),
optionally substituted alkynylene (e.g., C.sub.2-C.sub.6
alkynylene), optionally substituted heteroalkynylene (e.g.,
C.sub.2-C.sub.6 heteroalkynylene), optionally substituted
cycloalkylene, optionally substituted heterocycloalkylene,
optionally substituted arylene, or optionally substituted
heteroarylene;
[0093] Z is a chemical moiety formed from a coupling reaction
between a reactive substituent present on L and a reactive
substituent present within an antibody, antigen-binding fragment
thereof, or ligand that binds CD45 (such as CD45RO), CD135, CD34,
CD90, and/or CD110; and
[0094] wherein Am contains exactly one R.sub.C substituent.
[0095] In some embodiments, Am is represented by formula (IB)
##STR00003##
[0096] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0097] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0098] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0099] R.sub.3 is H, R.sub.C, or R.sub.D;
[0100] R.sub.4 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0101] R.sub.5 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0102] R.sub.6 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0103] R.sub.7 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0104] R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or
NR.sub.CR.sub.D;
[0105] R.sub.9 is H, OH, OR.sub.C, or OR.sub.D;
[0106] X is --S--, --S(O)--, or --SO.sub.2--;
[0107] R.sub.C is -L-Z;
[0108] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl;
[0109] L is a linker, such as optionally substituted alkylene
(e.g., C.sub.1-C.sub.6 alkylene), optionally substituted
heteroalkylene (C.sub.1-C.sub.6 heteroalkylene), optionally
substituted alkenylene (e.g., C.sub.2-C.sub.6 alkenylene),
optionally substituted heteroalkenylene (e.g., C.sub.2-C.sub.6
heteroalkenylene), optionally substituted alkynylene (e.g.,
C.sub.2-C.sub.6 alkynylene), optionally substituted
heteroalkynylene (e.g., C.sub.2-C.sub.6 heteroalkynylene),
optionally substituted cycloalkylene, optionally substituted
heterocycloalkylene, optionally substituted arylene, or optionally
substituted heteroarylene;
[0110] Z is a chemical moiety formed from a coupling reaction
between a reactive substituent present on L and a reactive
substituent present within an antibody, antigen-binding fragment
thereof, or ligand that binds CD45 (such as CD45RO), CD135, CD34,
CD90, and/or CD110; and
[0111] wherein Am contains exactly one R.sub.C substituent.
[0112] In some embodiments, R.sub.A and R.sub.B, together with the
oxygen atoms to which they are bound, combine to form:
##STR00004##
[0113] wherein Y is selected from O, S, NR.sub.E, and
CR.sub.ER.sub.E', and
[0114] R.sub.E and R.sub.E' are each independently optionally
substituted C.sub.1-C.sub.6 alkylene-R.sub.C, optionally
substituted C.sub.1-C.sub.6 heteroalkylene-R.sub.C, optionally
substituted C.sub.2-C.sub.6 alkenylene-R.sub.C, optionally
substituted C.sub.2-C.sub.6 heteroalkenylene-R.sub.C, optionally
substituted C.sub.2-C.sub.6 alkynylene-R.sub.C, optionally
substituted C.sub.2-C.sub.6 heteroalkynylene-R.sub.C, optionally
substituted cycloalkylene-R.sub.C, optionally substituted
heterocycloalkylene-R.sub.C, optionally substituted
arylene-R.sub.C, or optionally substituted
heteroarylene-R.sub.C.
[0115] In some embodiments, Am is represented by formula (IA) or
formula (IB),
[0116] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0117] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0118] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form:
##STR00005##
[0119] R.sub.3 is H or R.sub.C;
[0120] R.sub.4 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0121] R.sub.5 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0122] R.sub.6 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0123] R.sub.7 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0124] R.sub.8 is OH, NH.sub.2, OR.sub.C, or NHR.sub.C;
[0125] R.sub.9 is H or OH; and
[0126] wherein R.sub.C and R.sub.D are each as defined above.
[0127] In some embodiments, Am is represented by formula (IA) or
formula (IB),
[0128] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0129] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0130] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form:
##STR00006##
[0131] R.sub.3 is H or R.sub.C;
[0132] R.sub.4 and R.sub.5 are each independently H, OH, OR.sub.C,
R.sub.C, or OR.sub.D;
[0133] R.sub.6 and R.sub.7 are each H;
[0134] R.sub.8 is OH, NH.sub.2, OR.sub.C, or NHR.sub.C;
[0135] R.sub.9 is H or OH; and
[0136] wherein R.sub.C is as defined above.
[0137] In some embodiments, Am is represented by formula (IA) or
formula (IB),
[0138] wherein R.sub.1 is H, OH, or OR.sub.A;
[0139] R.sub.2 is H, OH, or OR.sub.B;
[0140] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form:
##STR00007##
[0141] R.sub.3, R.sub.4, R.sub.6, and R.sub.7 are each H;
[0142] R.sub.5 is OR.sub.C;
[0143] R.sub.8 is OH or NH.sub.2;
[0144] R.sub.9 is H or OH; and
[0145] wherein R.sub.C is as defined above.
[0146] In some embodiments, Am is represented by formula (IA) or
formula (IB),
[0147] wherein R.sub.1 and R.sub.2 are each independently H or
OH;
[0148] R.sub.3 is R.sub.C;
[0149] R.sub.4, R.sub.6, and R.sub.7 are each H;
[0150] R.sub.5 is H, OH, or OC.sub.1-C.sub.6 alkyl;
[0151] R.sub.8 is OH or NH.sub.2;
[0152] R.sub.9 is H or OH; and
[0153] wherein R.sub.C is as defined above.
[0154] In some embodiments, Am is represented by formula (IA) or
formula (IB),
[0155] wherein R.sub.1 and R.sub.2 are each independently H or
OH;
[0156] R.sub.3, R.sub.6, and R.sub.7 are each H;
[0157] R.sub.4 and R.sub.5 are each independently H, OH, OR.sub.C,
or R.sub.C;
[0158] R.sub.8 is OH or NH.sub.2;
[0159] R.sub.9 is H or OH; and
[0160] wherein R.sub.C is as defined above.
[0161] In some embodiments, Am is represented by formula (IA) or
formula (IB),
[0162] wherein R.sub.1 and R.sub.2 are each independently H or
OH;
[0163] R.sub.3, R.sub.6, and R.sub.7 are each H;
[0164] R.sub.4 and R.sub.5 are each independently H or OH;
[0165] R.sub.8 is OH, NH.sub.2, OR.sub.C, or NHR.sub.C;
[0166] R.sub.9 is H or OH; and
[0167] wherein R.sub.C is as defined above.
[0168] In some embodiments, Am is represented by formula (II)
##STR00008##
wherein X is S, SO, or SO.sub.2; R.sub.1 is H or a linker
covalently bound to the antibody or antigen-binding fragment
thereof; and R.sub.2 is H or a linker covalently bound to the
antibody or antigen-binding fragment thereof; wherein when R.sub.1
is H, R.sub.2 is the linker, and when R.sub.2 is H, R.sub.1 is the
linker.
[0169] In some embodiments of any of the above aspects, the
cytotoxin is a maytansinoid selected from the group consisting of
DM1 and DM4. In some embodiments, the cytotoxin is an auristatin
selected from the group consisting of monomethyl auristatin E and
monomethyl auristatin F. In some embodiments, the cytotoxin is an
anthracycline selected from the group consisting of daunorubicin,
doxorubicin, epirubicin, and idarubicin.
[0170] In some embodiments of any of the above aspects, the
antibody or antigen-binding fragment thereof is selected from the
group consisting of a monoclonal antibody or antigen-binding
fragment thereof, a polyclonal antibody or antigen-binding fragment
thereof, a humanized antibody or antigen-binding fragment thereof,
a bispecific antibody or antigen-binding fragment thereof, a
dual-variable immunoglobulin domain, a single-chain Fv molecule
(scFv), a diabody, a triabody, a nanobody, an antibody-like protein
scaffold, a Fv fragment, a Fab fragment, a F(ab').sub.2 molecule,
and a tandem di-scFv. In some embodiments, the antibody has an
isotype selected from the group consisting of IgG, IgA, IgM, IgD,
and IgE.
[0171] In some embodiments of any of the above aspects, the
antibody, antigen-binding fragment thereof, or ligand is
internalized by a hematopoietic cell, such as a hematopoietic stem
cell, cancer cell, or autoimmune cell following administration to
the patient. For instance, the antibody, antigen-binding fragment
thereof, or ligand may be internalized by hematopoietic stem cells,
cancer cells, or autoimmune cells by receptor mediated endocytosis
(e.g., upon binding to cell-surface CD45, CD135, CD34, CD90, or
CD110). In some embodiments, a cytotoxin covalently bound to the
antibody or antigen-binding fragment thereof may be released
intracellularly by chemical cleavage (for instance, by enzymatic or
non-specific cleavage of a linker described herein). The cytotoxin
may then access its intracellular target (such as the mitotic
spindle apparatus, nuclear DNA, ribosomal RNA, or topoisomerases,
among others) so as to promote the death of an endogenous
hematopoietic cell, such as an endogenous hematopoietic stem cell
prior to transplantation therapy, an endogenous cancer cell, or an
endogenous autoimmune cell, among others.
[0172] In some embodiments, of any of the above aspects, the
antibody, antigen-binding fragment thereof, or ligand is capable of
promoting necrosis of a hematopoietic stem cell. In some
embodiments, the antibody or antigen-binding fragment thereof may
promote the death of an endogenous hematopoietic stem cell prior to
transplantation therapy by recruiting one or more complement
proteins, natural killer (NK) cells, macrophages, neutrophils,
and/or eosinophils to the hematopoietic stem cell upon
administration to the patient.
[0173] In some embodiments, the transplant containing hematopoietic
stem cells is administered to the patient after the concentration
of the antibody or antigen-binding fragment thereof has
substantially cleared from the blood of the patient.
[0174] In some embodiments, the hematopoietic stem cells or progeny
thereof maintain hematopoietic stem cell functional potential after
two or more days (for example, from about 2 to about 5 days, from
about 2 to about 7 days, from about 2 to about 20 days, from about
2 to about 30 days, such as 2 days, 3 days, 4 days, 5 days, 6 days,
7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14
days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21
days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28
days, 29 days, 30 days, or more) following transplantation of the
hematopoietic stem cells into the patient.
[0175] In some embodiments, the hematopoietic stem cells or progeny
thereof are capable of localizing to hematopoietic tissue, such as
the bone marrow, and/or reestablishing hematopoiesis following
transplantation of the hematopoietic stem cells into the
patient.
[0176] In some embodiments, upon transplantation into the patient,
the hematopoietic stem cells give rise to recovery of a population
of cells selected from the group consisting of megakaryocytes,
thrombocytes, platelets, erythrocytes, mast cells, myeoblasts,
basophils, neutrophils, eosinophils, microglia, granulocytes,
monocytes, osteoclasts, antigen-presenting cells, macrophages,
dendritic cells, natural killer cells, T-lymphocytes, and
B-lymphocytes.
[0177] In some embodiments of any of the above aspects, the method
is used to treat one or more disorders, such as by depleting a
population of hematopoietic stem cells in a patient prior to
hematopoietic stem cell transplant therapy so as to provide a niche
to which the transplanted hematopoietic stem cells may home.
Following transplantation, the hematopoietic stem cells may
establish productive hematopoiesis, so as to replenish a deficient
cell type in the patient or a cell type that is being actively
killed or has been killed, for instance, by chemotherapeutic
methods. For instance, the patient may be one that is suffering
from a stem cell disorder. In some embodiments, the patient is
suffering from a hemoglobinopathy disorder, such as sickle cell
anemia, thalassemia, Fanconi anemia, aplastic anemia, and
Wiskott-Aldrich syndrome. The patient may be suffering from an
immunodeficiency disorder, such as a congenital immunodeficiency
disorder or an acquired immunodeficiency disorder (e.g., human
immunodeficiency virus or acquired immune deficiency syndrome). In
some embodiments, the patient is suffering from a metabolic
disorder, such as glycogen storage diseases, mucopolysaccharidoses,
Gaucher's Disease, Hurlers Disease, sphingolipidoses, and
metachromatic leukodystrophy. In some embodiments, the patient is
suffering from a disorder selected from the group consisting of
adenosine deaminase deficiency and severe combined
immunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashi
disease, hereditary lymphohistiocytosis, osteopetrosis,
osteogenesis imperfecta, storage diseases, thalassemia major,
systemic sclerosis, systemic lupus erythematosus, and juvenile
rheumatoid arthritis. In some embodiments, the patient is suffering
from an autoimmune disease, such as scleroderma, multiple
sclerosis, ulcerative colitis, Chron's disease, ant Type 1
diabetes. In some embodiments, the patient is suffering from cancer
or myeloproliferative disease, such as a hematological cancer. In
some embodiments, the patient is suffering from acute myeloid
leukemia, acute lymphoid leukemia, chronic myeloid leukemia,
chronic lymohoid leukemia, multiple meloma, diffuse large B-cell
lymphoma, or non-Hodgkin's lymphoma. In some embodiments, the
patient is suffering from a myelodysplastic disease, such as
myelodysplastic syndrome.
[0178] In some embodiments of any of the above aspects, the method
is used to directly treat a cancer, such as a cancer characterized
by CD45+, CD135+, CD34+, CD90+, or CD110+ cells (e.g., a leukemia
characterized by CD45+, CD135+, CD34+, CD90+, or CD110+ cells), by
administration of an antibody, antigen-binding fragment thereof, or
ligand that depletes a population of CD45+, CD135+, CD34+, CD90+,
or CD110+ cancer cells in the patient and/or by administration of
an antibody, antigen-binding fragment thereof, or ligand so as to
deplete a population of endogenous hematopoietic stem cells prior
to hematopoietic stem cell transplantation. In the latter case, the
transplantation may in turn re-constitute, for example, a
population of cells depleted during the process of eradicating
cancer cells. The cancer may be a hematological cancer, such as
acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid
leukemia, chronic lymohoid leukemia, multiple meloma, diffuse large
B-cell lymphoma, or non-Hodgkin's lymphoma.
[0179] In some embodiments of any of the above aspects, the method
is used to treat an autoimmune disease, such as by administration
of an antibody, antigen-binding fragment thereof, or ligand so as
to deplete a population of CD45+, CD135+, CD34+, CD90+, or CD110+
autoimmune cells and/or by administration of an antibody,
antigen-binding fragment thereof, or ligand so as to deplete a
population of endogenous hematopoietic stem cells prior to
hematopoietic stem cell transplantation. In the latter case, the
transplantation may in turn re-constitute, for example, a
population of cells depleted during the process of eradicating
autoimmune cells. The autoimmune disease may be, for example,
scleroderma, multiple sclerosis (MS), human systemic lupus (SLE),
rheumatoid arthritis (RA), inflammatory bowel disease (IBD),
treating psoriasis, Type 1 diabetes mellitus (Type 1 diabetes),
acute disseminated encephalomyelitis (ADEM), Addison's disease,
alopecia universalis, ankylosing spondylitisis, antiphospholipid
antibody syndrome (APS), aplastic anemia, autoimmune hemolytic
anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED),
autoimmune lymphoproliferative syndrome (ALPS), autoimmune
oophoritis, Balo disease, Behcet's disease, bullous pemphigoid,
cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction
syndrome (CFIDS), chronic inflammatory demyelinating
polyneuropathy, Crohn's disease, cicatrical pemphigoid, coeliac
sprue-dermatitis herpetiformis, cold agglutinin disease, CREST
syndrome, Degos disease, discoid lupus, dysautonomia,
endometriosis, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, Goodpasture's syndrome, Grave's
disease, Guillain-Barre syndrome (GBS), Hashimoto's thyroiditis,
Hidradenitis suppurativa, idiopathic and/or acute thrombocytopenic
purpura, idiopathic pulmonary fibrosis, IgA neuropathy,
interstitial cystitis, juvenile arthritis, Kawasaki's disease,
lichen planus, Lyme disease, Meniere disease, mixed connective
tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus
myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis,
pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis
and dermatomyositis, primary biliary cirrhosis, polyarteritis
nodosa, polyglandular syndromes, polymyalgia rheumatica, primary
agammaglobulinemia, Raynaud phenomenon, Reiter's syndrome,
rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome,
stiff person syndrome, Takayasu's arteritis, temporal arteritis
(also known as "giant cell arteritis"), ulcerative colitis,
uveitis, vasculitis, vitiligo, vulvodynia ("vulvar vestibulitis"),
and Wegener's granulomatosis.
[0180] Thus, in some embodiments of any of the above aspects, the
invention features a method of treating a hemoglobinopathy
disorder, such as sickle cell anemia, thalassemia, Fanconi anemia,
aplastic anemia, and Wiskott-Aldrich syndrome. In some embodiments,
the invention features a method of treating an immunodeficiency
disorder, such as a congenital immunodeficiency disorder or an
acquired immunodeficiency disorder (e.g., human immunodeficiency
virus or acquired immune deficiency syndrome). In some embodiments,
the invention features a method of treating a metabolic disorder,
such as glycogen storage diseases, mucopolysaccharidoses, Gaucher's
Disease, Hurlers Disease, sphingolipidoses, and metachromatic
leukodystrophy. In some embodiments, the invention features a
method of treating a disorder selected from the group consisting of
adenosine deaminase deficiency and severe combined
immunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashi
disease, hereditary lymphohistiocytosis, osteopetrosis,
osteogenesis imperfecta, storage diseases, thalassemia major,
systemic sclerosis, systemic lupus erythematosus, and juvenile
rheumatoid arthritis In some embodiments, the invention features a
method of treating an autoimmune disease, such as scleroderma,
multiple sclerosis, ulcerative colitis, Chron's disease, ant Type 1
diabetes. In some embodiments, the invention features a method of
treating a cancer or myeloproliferative disease, such as a
hematological cancer. In some embodiments, the invention features a
method of treating acute myeloid leukemia, acute lymphoid leukemia,
chronic myeloid leukemia, chronic lymohoid leukemia, multiple
meloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.
In some embodiments, the patient is suffering from a myelodyplastic
disease, such as myelodysplastic syndrome. In these embodiments,
the method may include the steps of administering an antibody,
antigen-binding fragment thereof, or ligand that binds CD45, CD135,
CD34, CD90, or CD110 and/or a hematopoietic stem cell transplant
according to the method of any of the above-described aspects and
embodiments of the invention.
[0181] Similarly, in some embodiments of any of the above aspects,
the invention provides a method of treating cancer directly, such
as a cancer characterized by CD45+, CD135+, CD34+, CD90+, or CD110+
cells (e.g., a leukemia characterized by CD45+, CD135+, CD34+,
CD90+, or CD110+ cells). In these embodiments, the method includes
administering an antibody, antigen-binding fragment thereof, or
ligand that binds CD45, CD135, CD34, CD90, or CD110. The cancer may
be a hematological cancer, such as acute myeloid leukemia, acute
lymphoid leukemia, chronic myeloid leukemia, chronic lymohoid
leukemia, multiple meloma, diffuse large B-cell lymphoma, or
non-Hodgkin's lymphoma.
[0182] Additionally, in some embodiments of any of the above
aspects, the invention provides a method of treating an autoimmune
disease, such as multiple sclerosis (MS), human systemic lupus
(SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD),
treating psoriasis, Type 1 diabetes mellitus (Type 1 diabetes)
acute disseminated encephalomyelitis (ADEM), Addison's disease,
alopecia universalis, ankylosing spondylitisis, antiphospholipid
antibody syndrome (APS), aplastic anemia, autoimmune hemolytic
anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED),
autoimmune lymphoproliferative syndrome (ALPS), autoimmune
oophoritis, Balo disease, Behcet's disease, bullous pemphigoid,
cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction
syndrome (CFIDS), chronic inflammatory demyelinating
polyneuropathy, Crohn's disease, cicatrical pemphigoid, coeliac
sprue-dermatitis herpetiformis, cold agglutinin disease, CREST
syndrome, Degos disease, discoid lupus, dysautonomia,
endometriosis, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, Goodpasture's syndrome, Grave's
disease, Guillain-Barre syndrome (GBS), Hashimoto's thyroiditis,
Hidradenitis suppurativa, idiopathic and/or acute thrombocytopenic
purpura, idiopathic pulmonary fibrosis, IgA neuropathy,
interstitial cystitis, juvenile arthritis, Kawasaki's disease,
lichen planus, Lyme disease, Meniere disease, mixed connective
tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus
myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis,
pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis
and dermatomyositis, primary biliary cirrhosis, polyarteritis
nodosa, polyglandular syndromes, polymyalgia rheumatica, primary
agammaglobulinemia, Raynaud phenomenon, Reiter's syndrome,
rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome,
stiff person syndrome, Takayasu's arteritis, temporal arteritis
(also known as "giant cell arteritis"), ulcerative colitis,
uveitis, vasculitis, vitiligo, vulvodynia ("vulvar vestibulitis"),
and Wegener's granulomatosis. In these embodiments, the method
includes administering an antibody, antigen-binding fragment
thereof, or ligand that binds CD45, CD135, CD34, CD90, or
CD110.
[0183] In another aspect, the invention features a method of
depleting a population of CD45+ cells by contacting the population
with an effective amount of a conjugate represented by the formula
Ab-Am, wherein Ab is an antibody or antigen-binding fragment
thereof that binds CD45 and Am is an amatoxin. Am may be
represented by formula (IA)
##STR00009##
[0184] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0185] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0186] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0187] R.sub.3 is H, R.sub.C, or R.sub.D;
[0188] R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each
independently H, OH, OR.sub.C, OR.sub.D, R.sub.C, or R.sub.D;
[0189] R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or
NR.sub.CR.sub.D;
[0190] R.sub.9 is H, OH, OR.sub.C, or OR.sub.D;
[0191] X is --S--, --S(O)--, or --SO.sub.2--;
[0192] R.sub.C is -L-Z;
[0193] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl;
[0194] L is a linker, such as optionally substituted alkylene
(e.g., C.sub.1-C.sub.6 alkylene), optionally substituted
heteroalkylene (C.sub.1-C.sub.6 heteroalkylene), optionally
substituted alkenylene (e.g., C.sub.2-C.sub.6 alkenylene),
optionally substituted heteroalkenylene (e.g., C.sub.2-C.sub.6
heteroalkenylene), optionally substituted alkynylene (e.g.,
C.sub.2-C.sub.6 alkynylene), optionally substituted
heteroalkynylene (e.g., C.sub.2-C.sub.6 heteroalkynylene),
optionally substituted cycloalkylene, optionally substituted
heterocycloalkylene, optionally substituted arylene, or optionally
substituted heteroarylene; and
[0195] Z is a chemical moiety formed from a coupling reaction
between a reactive substituent present on L and a reactive
substituent present within the antibody or antigen-binding fragment
thereof,
[0196] wherein Am contains exactly one R.sub.C substituent.
[0197] In some embodiments, Am is represented by formula (IB)
##STR00010##
[0198] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0199] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0200] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0201] R.sub.3 is H, R.sub.C, or R.sub.D;
[0202] R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each
independently H, OH, OR.sub.C, OR.sub.D, R.sub.C, or R.sub.D;
[0203] R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or
NR.sub.CR.sub.D;
[0204] R.sub.9 is H, OH, OR.sub.C, or OR.sub.D;
[0205] X is --S--, --S(O)--, or --SO.sub.2--;
[0206] R.sub.C is -L-Z;
[0207] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl;
[0208] L is a linker, such as optionally substituted alkylene
(e.g., C.sub.1-C.sub.6 alkylene), optionally substituted
heteroalkylene (C.sub.1-C.sub.6 heteroalkylene), optionally
substituted alkenylene (e.g., C.sub.2-C.sub.6 alkenylene),
optionally substituted heteroalkenylene (e.g., C.sub.2-C.sub.6
heteroalkenylene), optionally substituted alkynylene (e.g.,
C.sub.2-C.sub.6 alkynylene), optionally substituted
heteroalkynylene (e.g., C.sub.2-C.sub.6 heteroalkynylene),
optionally substituted cycloalkylene, optionally substituted
heterocycloalkylene, optionally substituted arylene, or optionally
substituted heteroarylene; and
[0209] Z is a chemical moiety formed from a coupling reaction
between a reactive substituent present on L and a reactive
substituent present within the antibody or antigen-binding fragment
thereof,
[0210] wherein Am contains exactly one R.sub.C substituent.
[0211] In another aspect, the invention features a conjugate
represented by the formula Ab-Am, wherein Ab is an antibody or
antigen-binding fragment thereof that binds CD45 and Am is an
amatoxin. In some embodiments, Am is represented by formula (IA) or
formula (IB), above.
[0212] In some embodiments of the preceding two aspects, the
antibody or antigen-binding fragment thereof is conjugated to the
amatoxin by way of a cysteine residue in the Fc domain of the
antibody or antigen-binding fragment thereof. In some embodiments,
the cysteine residue is introduced by way of a mutation in the Fc
domain of the antibody or antigen-binding fragment thereof. For
instance, the cysteine residue may be selected from the group
consisting of Cys118, Cys239, and Cys265.
[0213] In some embodiments of these aspects, the cysteine residue
is naturally occurring in the Fc domain of the antibody or
antigen-binding fragment thereof. For instance, the Fc domain may
be an IgG Fc domain, such as a human IgG1 Fc domain, and the
cysteine residue may be selected from the group consisting of
Cys261, Csy321, Cys367, and Cys425.
[0214] In some embodiments of these aspects, R.sub.1 is H, OH, or
OR.sub.A;
[0215] R.sub.2 is H, OH, or OR.sub.B;
[0216] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form:
##STR00011##
[0217] R.sub.3, R.sub.4, R.sub.6, and R.sub.7 are each H;
[0218] R.sub.5 is OR.sub.C;
[0219] R.sub.8 is OH or NH.sub.2; and
[0220] R.sub.9 is H or OH.
[0221] In some embodiments, R.sub.1 and R.sub.2 are each
independently H or OH;
[0222] R.sub.3 is R.sub.C;
[0223] R.sub.4, R.sub.6, and R.sub.7 are each H;
[0224] R.sub.5 is H, OH, or OC.sub.1-C.sub.6 alkyl;
[0225] R.sub.8 is OH or NH.sub.2; and
[0226] R.sub.9 is H or OH.
[0227] In some embodiments, R.sub.1 and R.sub.2 are each
independently H or OH;
[0228] R.sub.3, R.sub.6, and R.sub.7 are each H;
[0229] R.sub.4 is OR.sub.C, or R.sub.C;
[0230] R.sub.5 is H, OH, or OC.sub.1-C.sub.6 alkyl;
[0231] R.sub.8 is OH or NH.sub.2; and
[0232] R.sub.9 is H or OH.
[0233] In some embodiments, R.sub.1 and R.sub.2 are each
independently H or OH;
[0234] R.sub.3, R.sub.6, and R.sub.7 are each H;
[0235] R.sub.4 and R.sub.5 are each independently H or OH;
[0236] R.sub.8 is OR.sub.C or NHR.sub.C; and
[0237] R.sub.9 is H or OH.
[0238] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof is internalized by a CD45+
cell.
[0239] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof binds CD45 with a K.sub.d of less
than 1 .mu.M, less than 750 nM, less than 500 nM, less than 250 nM,
less than 200 nM, less than 150 nM, less than 100 nM, less than 75
nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1
nM, less than 10 pM, less than 1 pM, or less than 0.1 pM. In some
embodiments, the K.sub.d is from about 0.1 pM to about 1 .mu.M. In
some embodiments, the K.sub.d is about 357 pM, the k.sub.on is
about 2.56.times.10.sup.5 M.sup.-1s.sup.-1, and/or the k.sub.off is
about 9.14.times.10.sup.-5 s.sup.-1.
[0240] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof binds CD45 with a k.sub.on of from
about 9.times.10.sup.-2 M.sup.-1 s.sup.-1 to about 1.times.10.sup.2
M.sup.-1s.sup.-1.
[0241] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof competitively inhibits the binding
of CD45 to a second antibody or antigen binding fragment thereof,
wherein the second antibody or antigen-binding fragment thereof has
the following complementarity determining regions (CDRs):
TABLE-US-00003 a. a CDR-H1 having the amino acid sequence SYAMS;
(SEQ ID NO: 16) b. a CDR-H2 having the amino acid sequence
AISGSGGSTFYADSVRG; (SEQ ID NO: 17) c. a CDR-H3 having the amino
acid sequence EVMGPIFFDY; (SEQ ID NO: 18) d. a CDR-L1 having the
amino acid sequence RASQSIISSALA; (SEQ ID NO: 19) e. a CDR-L2
having the amino acid sequence GASSRAT; (SEQ ID NO: 20) and f. a
CDR-L3 having the amino acid sequence QQYGSTPLT. (SEQ ID NO:
21)
[0242] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof is rat YTH24.5 or a humanized
variant thereof.
[0243] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof competitively inhibits the binding
of CD45 to a second antibody or antigen binding fragment thereof,
wherein the second antibody or antigen-binding fragment thereof is
rat YTH24.5 or a humanized variant thereof.
[0244] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof is selected from the group
consisting of a monoclonal antibody or antigen-binding fragment
thereof, a polyclonal antibody or antigen-binding fragment thereof,
a humanized antibody or antigen-binding fragment thereof, a
bispecific antibody or antigen-binding fragment thereof, a
dual-variable immunoglobulin domain, a single-chain Fv molecule
(scFv), a diabody, a triabody, a nanobody, an antibody-like protein
scaffold, a Fv fragment, a Fab fragment, a F(ab').sub.2 molecule,
and a tandem di-scFV.
[0245] In another aspect, the invention features a method of
depleting a population of CD135+ cells by contacting the population
with an effective amount of a conjugate represented by the formula
Ab-Am, wherein Ab is an antibody or antigen-binding fragment
thereof that binds CD135 and Am is an amatoxin. Am may be
represented by formula (IA)
##STR00012##
[0246] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0247] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0248] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0249] R.sub.3 is H, R.sub.C, or R.sub.D;
[0250] R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each
independently H, OH, OR.sub.C, OR.sub.D, R.sub.C, or R.sub.D;
[0251] R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or
NR.sub.CR.sub.D;
[0252] R.sub.9 is H, OH, OR.sub.C, or OR.sub.D;
[0253] X is --S--, --S(O)--, or --SO.sub.2--;
[0254] R.sub.C is -L-Z;
[0255] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl;
[0256] L is a linker, such as optionally substituted alkylene
(e.g., C.sub.1-C.sub.6 alkylene), optionally substituted
heteroalkylene (C.sub.1-C.sub.6 heteroalkylene), optionally
substituted alkenylene (e.g., C.sub.2-C.sub.6 alkenylene),
optionally substituted heteroalkenylene (e.g., C.sub.2-C.sub.6
heteroalkenylene), optionally substituted alkynylene (e.g.,
C.sub.2-C.sub.6 alkynylene), optionally substituted
heteroalkynylene (e.g., C.sub.2-C.sub.6 heteroalkynylene),
optionally substituted cycloalkylene, optionally substituted
heterocycloalkylene, optionally substituted arylene, or optionally
substituted heteroarylene; and
[0257] Z is a chemical moiety formed from a coupling reaction
between a reactive substituent present on L and a reactive
substituent present within the antibody or antigen-binding fragment
thereof,
[0258] wherein Am contains exactly one R.sub.C substituent.
[0259] In some embodiments, Am is represented by formula (IB)
##STR00013##
[0260] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0261] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0262] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0263] R.sub.3 is H, R.sub.C, or R.sub.D;
[0264] R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each
independently H, OH, OR.sub.C, OR.sub.D, R.sub.C, or R.sub.D;
[0265] R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or
NR.sub.CR.sub.D;
[0266] R.sub.9 is H, OH, OR.sub.C, or OR.sub.D;
[0267] X is --S--, --S(O)--, or --SO.sub.2--;
[0268] R.sub.C is -L-Z;
[0269] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl;
[0270] L is a linker, such as optionally substituted alkylene
(e.g., C.sub.1-C.sub.6 alkylene), optionally substituted
heteroalkylene (C.sub.1-C.sub.6 heteroalkylene), optionally
substituted alkenylene (e.g., C.sub.2-C.sub.6 alkenylene),
optionally substituted heteroalkenylene (e.g., C.sub.2-C.sub.6
heteroalkenylene), optionally substituted alkynylene (e.g.,
C.sub.2-C.sub.6 alkynylene), optionally substituted
heteroalkynylene (e.g., C.sub.2-C.sub.6 heteroalkynylene),
optionally substituted cycloalkylene, optionally substituted
heterocycloalkylene, optionally substituted arylene, or optionally
substituted heteroarylene; and
[0271] Z is a chemical moiety formed from a coupling reaction
between a reactive substituent present on L and a reactive
substituent present within the antibody or antigen-binding fragment
thereof,
[0272] wherein Am contains exactly one R.sub.C substituent.
[0273] In another aspect, the invention features a conjugate
represented by the formula Ab-Am, wherein Ab is an antibody or
antigen-binding fragment thereof that binds CD135 and Am is an
amatoxin. In some embodiments, Am is represented by formula (IA) or
formula (IB), above.
[0274] In some embodiments of the preceding two aspects, the
antibody or antigen-binding fragment thereof is conjugated to the
amatoxin by way of a cysteine residue in the Fc domain of the
antibody or antigen-binding fragment thereof. In some embodiments,
the cysteine residue is introduced by way of a mutation in the Fc
domain of the antibody or antigen-binding fragment thereof. For
instance, the cysteine residue may be selected from the group
consisting of Cys118, Cys239, and Cys265.
[0275] In some embodiments of these aspects, the cysteine residue
is naturally occurring in the Fc domain of the antibody or
antigen-binding fragment thereof. For instance, the Fc domain may
be an IgG Fc domain, such as a human IgG1 Fc domain, and the
cysteine residue may be selected from the group consisting of
Cys261, Csy321, Cys367, and Cys425.
[0276] In some embodiments of these aspects, R.sub.1 is H, OH, or
OR.sub.A;
[0277] R.sub.2 is H, OH, or OR.sub.B;
[0278] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form:
##STR00014##
[0279] R.sub.3, R.sub.4, R.sub.6, and R.sub.7 are each H;
[0280] R.sub.5 is OR.sub.C;
[0281] R.sub.8 is OH or NH.sub.2; and
[0282] R.sub.9 is H or OH.
[0283] In some embodiments, R.sub.1 and R.sub.2 are each
independently H or OH;
[0284] R.sub.3 is R.sub.C;
[0285] R.sub.4, R.sub.6, and R.sub.7 are each H;
[0286] R.sub.5 is H, OH, or OC.sub.1-C.sub.6 alkyl;
[0287] R.sub.8 is OH or NH.sub.2; and
[0288] R.sub.9 is H or OH.
[0289] In some embodiments, R.sub.1 and R.sub.2 are each
independently H or OH;
[0290] R.sub.3, R.sub.6, and R.sub.7 are each H;
[0291] R.sub.4 is OR.sub.C, or R.sub.C;
[0292] R.sub.5 is H, OH, or OC.sub.1-C.sub.6 alkyl;
[0293] R.sub.8 is OH or NH.sub.2; and
[0294] R.sub.9 is H or OH.
[0295] In some embodiments, R.sub.1 and R.sub.2 are each
independently H or OH;
[0296] R.sub.3, R.sub.6, and R.sub.7 are each H;
[0297] R.sub.4 and R.sub.5 are each independently H or OH;
[0298] R.sub.8 is OR.sub.C or NHR.sub.C; and
[0299] R.sub.9 is H or OH.
[0300] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof is internalized by a CD135+
cell.
[0301] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof binds CD135 with a K.sub.d of less
than 1 .mu.M, less than 750 nM, less than 500 nM, less than 250 nM,
less than 200 nM, less than 150 nM, less than 100 nM, less than 75
nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1
nM, less than 10 pM, less than 1 pM, or less than 0.1 pM, In some
embodiments, the K.sub.d is from about 0.1 pM to about 1 .mu.M.
[0302] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof binds CD135 with a k.sub.on of
from about 9.times.10.sup.-2 M.sup.-1 s.sup.-1 to about
1.times.10.sup.2 M.sup.-1s.sup.-1.
[0303] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof has the following CDRs:
TABLE-US-00004 a. a CDR-H1 having the amino acid sequence SYYMH;
(SEQ ID NO: 1) b. a CDR-H2 having the amino acid sequence
IINPSGGSTSYAQKFQG; (SEQ ID NO: 2) c. a CDR-H3 having the amino acid
sequence GVGAHDAFDI (SEQ ID NO: 3) or VVAAAVADY; (SEQ ID NO: 4) d.
a CDR-L1 having the amino acid sequence RSSQSLLHSNGNNYLD (SEQ ID
NO: 5) or RSSQSLLHSNGYNYLD; (SEQ ID NO: 6) e. a CDR-L2 having the
amino acid sequence LGSNRAS; (SEQ ID NO: 7) and f. a CDR-L3 having
the amino acid sequence MQGTHPAIS (SEQ ID NO: 8) or MQSLQTPFT. (SEQ
ID NO: 9)
[0304] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof has the following CDRs:
TABLE-US-00005 a. a CDR-H1 having the amino acid sequence SYAIS;
(SEQ ID NO: 10) b. a CDR-H2 having the amino acid sequence
GIIPIFGTANYAQKFQG; (SEQ ID NO: 11) c. a CDR-H3 having the amino
acid sequence FALFGFREQAFDI; (SEQ ID NO: 12) d. a CDR-L1 having the
amino acid sequence RASQSISSYLN; (SEQ ID NO: 13) e. a CDR-L2 having
the amino acid sequence AASSLQS; (SEQ ID NO: 14) and f. a CDR-L3
having the amino acid sequence QQSYSTPFT. (SEQ ID NO: 15)
[0305] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof competitively inhibits the binding
of CD135 to a second antibody or antigen binding fragment thereof,
wherein the second antibody or antigen-binding fragment thereof has
the following CDRs:
TABLE-US-00006 a. a CDR-H1 having the amino acid sequence SYYMH;
(SEQ ID NO: 1) b. a CDR-H2 having the amino acid sequence
IINPSGGSTSYAQKFQG; (SEQ ID NO: 2) c. a CDR-H3 having the amino acid
sequence GVGAHDAFDI (SEQ ID NO: 3) or VVAAAVADY; (SEQ ID NO: 4) d.
a CDR-L1 having the amino acid sequence RSSQSLLHSNGNNYLD (SEQ ID
NO: 5) or RSSQSLLHSNGYNYLD; (SEQ ID NO: 6) e. a CDR-L2 having the
amino acid sequence LGSNRAS; (SEQ ID NO: 7) and f. a CDR-L3 having
the amino acid sequence MQGTHPAIS (SEQ ID NO: 8) or MQSLQTPFT. (SEQ
ID NO: 9)
[0306] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof competitively inhibits the binding
of CD135 to a second antibody or antigen binding fragment thereof,
wherein the second antibody or antigen-binding fragment thereof has
the following CDRs:
TABLE-US-00007 (SEQ ID NO: 10) a. a CDR-H1 having the amino acid
sequence SYAIS; (SEQ ID NO: 11) b. a CDR-H2 having the amino acid
sequence GIIPIFGTANYAQKFQG; (SEQ ID NO: 12) c. a CDR-H3 having the
amino acid sequence FALFGFREQAFDI; (SEQ ID NO: 13) d. a CDR-L1
having the amino acid sequence RASQSISSYLN; (SEQ ID NO: 14) e. a
CDR-L2 having the amino acid sequence AASSLQS; and (SEQ ID NO: 15)
f. a CDR-L3 having the amino acid sequence QQSYSTPFT.
[0307] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof is selected from the group
consisting of a monoclonal antibody or antigen-binding fragment
thereof, a polyclonal antibody or antigen-binding fragment thereof,
a humanized antibody or antigen-binding fragment thereof, a
bispecific antibody or antigen-binding fragment thereof, a
dual-variable immunoglobulin domain, a single-chain Fv molecule
(scFv), a diabody, a triabody, a nanobody, an antibody-like protein
scaffold, a Fv fragment, a Fab fragment, a F(ab').sub.2 molecule,
and a tandem di-scFV.
[0308] In another aspect, the invention features a conjugate
represented by the formula Ab-Cy, wherein Ab is an antibody or
antigen-binding fragment thereof that binds CD45 (e.g., CD45RO) and
Cy is a cytotoxin. In some embodiments of this aspect, the
cytotoxin is pseudomonas exotoxin A, deBouganin, diphtheria toxin,
saporin, maytansine, a maytansinoid, an auristatin, an
anthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, a
pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an
indolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a
variant of any of the foregoing cytotoxins.
[0309] In another aspect, the invention features a conjugate
represented by the formula Ab-Cy, wherein Ab is an antibody or
antigen-binding fragment thereof that binds CD135 and Cy is a
cytotoxin. In some embodiments of this aspect, the cytotoxin is
pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin,
maytansine, a maytansinoid, an auristatin, an anthracycline, a
calicheamicin, irinotecan, SN-38, a duocarmycin, a
pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an
indolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a
variant of any of the foregoing cytotoxins.
[0310] In another aspect, the invention features a conjugate
represented by the formula Ab-Cy, wherein Ab is an antibody or
antigen-binding fragment thereof that binds CD34 and Cy is a
cytotoxin. In some embodiments of this aspect, the cytotoxin is
pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin,
maytansine, a maytansinoid, an auristatin, an anthracycline, a
calicheamicin, irinotecan, SN-38, a duocarmycin, a
pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an
indolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a
variant of any of the foregoing cytotoxins.
[0311] In another aspect, the invention features a conjugate
represented by the formula Ab-Cy, wherein Ab is an antibody or
antigen-binding fragment thereof that binds CD90 and Cy is a
cytotoxin. In some embodiments of this aspect, the cytotoxin is
pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin,
maytansine, a maytansinoid, an auristatin, an anthracycline, a
calicheamicin, irinotecan, SN-38, a duocarmycin, a
pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an
indolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a
variant of any of the foregoing cytotoxins.
[0312] In another aspect, the invention features a conjugate
represented by the formula Ab-Cy, wherein Ab is an antibody or
antigen-binding fragment thereof that binds CD110 and Cy is a
cytotoxin. In some embodiments of this aspect, the cytotoxin is
pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin,
maytansine, a maytansinoid, an auristatin, an anthracycline, a
calicheamicin, irinotecan, SN-38, a duocarmycin, a
pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an
indolinobenzodiazepine, or an indolinobenzodiazepine dimer, or a
variant of any of the foregoing cytotoxins.
[0313] In some embodiments, of the foregoing five aspects, the
antibody or antigen-binding fragment thereof is internalized by a
hematopoietic cell, such as a hematopoietic stem cell (e.g., a
CD45+(such as a CD45RO+), CD135+, CD34+, CD90+, and/or CD110+
cell).
[0314] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof binds CD45 (e.g., CD45RO), CD135,
CD34, CD90, or CD110 with a K.sub.d of less than 1 .mu.M, less than
750 nM, less than 500 nM, less than 250 nM, less than 200 nM, less
than 150 nM, less than 100 nM, less than 75 nM, less than 50 nM,
less than 10 nM, less than 1 nM, less than 0.1 nM, less than 10 pM,
less than 1 pM, or less than 0.1 pM. In some embodiments, the
K.sub.d is from about 0.1 pM to about 1 .mu.M.
[0315] In some embodiments of this aspect, the antibody or
antigen-binding fragment thereof binds CD45 (e.g., CD45RO), CD135,
CD34, CD90, or CD110 with a k.sub.on of from about
9.times.10.sup.-2 M.sup.-1 s.sup.-1 to about 1.times.10.sup.2
M.sup.-1s.sup.-1.
[0316] In some embodiments of these aspects, the antibody or
antigen-binding fragment thereof is selected from the group
consisting of a monoclonal antibody or antigen-binding fragment
thereof, a polyclonal antibody or antigen-binding fragment thereof,
a humanized antibody or antigen-binding fragment thereof, a
bispecific antibody or antigen-binding fragment thereof, a
dual-variable immunoglobulin domain, a single-chain Fv molecule
(scFv), a diabody, a triabody, a nanobody, an antibody-like protein
scaffold, a Fv fragment, a Fab fragment, a F(ab').sub.2 molecule,
and a tandem di-scFv. In some embodiments, the antibody has an
isotype selected from the group consisting of IgG, IgA, IgM, IgD,
and IgE.
BRIEF DESCRIPTION OF THE FIGURES
[0317] FIG. 1 is a graph demonstrating the effect of various
concentrations of anti-CD45 monoclonal antibodies Ab1, Ab2, and
Ab3, or isotype-matched negative controls, each bound to saporin by
way of saporin-conjugated Fab fragments, on the viability of Reh
cells in vitro. Cell viability was assessed using the
CellTiter-Glo.TM. assay kit as described in Example 8, below.
[0318] FIG. 2 is a graph demonstrating the effect of various
concentrations of anti-CD45 monoclonal antibody or isotype-matched
negative control, each bound to saporin by way of
saporin-conjugated Fab fragments, on the viability of human CD34+
cells in vitro. Cell viability was assessed using the
CellTiter-Glo.TM. assay kit as described in Example 8, below.
DETAILED DESCRIPTION
[0319] The invention provides methods of treating a variety of
disorders, such as diseases of a cell type in the hematopoietic
lineage, cancers, autoimmune diseases, metabolic disorders, and
stem cell disorders, among others. The compositions and methods
described herein may (i) directly deplete a population of cells
that give rise to a pathology, such as a population of cancer cells
(e.g., leukemia cells) and autoimmune cells (e.g., autoreactive
T-cells), and/or (ii) deplete a population of endogenous
hematopoietic stem cells so as to promote the engraftment of
transplanted hematopoietic stem cells by providing a niche to which
the transplanted cells may home. The foregoing activities can be
achieved by administration of an antibody, antigen-binding fragment
thereof, or ligand capable of binding an antigen expressed by an
endogenous disease-causing cell or a hematopoietic stem cell. In
the case of direct treatment of a disease, this administration can
cause a reduction in the quantity of the cells that give rise to
the pathology of interest. In the case of preparing a patient for
hematopoietic stem cell transplant therapy, this administration can
cause the selective depletion of a population of endogenous
hematopoietic stem cells, thereby creating a vacancy in the
hematopoietic tissue, such as the bone marrow, that can
subsequently be filled by transplanted, exogenous hematopoietic
stem cells. The invention is based in part on the discovery that
antibodies, antigen-binding fragments thereof, and ligands capable
of binding CD45, CD135, CD34, CD90, or CD110 can be administered to
a patient to effect both of the above activities. Antibodies,
antigen-binding fragments thereof, and ligands that bind CD45,
CD135, CD34, CD90, or CD110 can be administered to a patient
suffering from a cancer or autoimmune disease to directly deplete a
population of cancerous cells or autoimmune cells, and can also be
administered to a patient in need of hematopoietic stem cell
transplant therapy in order to promote the survival and engraftment
potential of transplanted hematopoietic stem cells.
[0320] Engraftment of hematopoietic stem cell transplants due to
the administration of anti-CD45, anti-CD135, anti-CD34, anti-CD90,
or anti-CD110 antibodies, antigen-binding fragments thereof, or
ligands can manifest in a variety of empirical measurements. For
instance, engraftment of transplanted hematopoietic stem cells can
be evaluated by assessing the quantity of competitive repopulating
units (CRU) present within the bone marrow of a patient following
administration of an antibody or antigen-binding fragment thereof
capable of binding CD45, CD135, CD34, CD90, or CD110 and subsequent
administration of a hematopoietic stem cell transplant.
Additionally, one can observe engraftment of a hematopoietic stem
cell transplant by incorporating a reporter gene, such as an enzyme
that catalyzes a chemical reaction yielding a fluorescent,
chromophoric, or luminescent product, into a vector with which the
donor hematopoietic stem cells have been transfected and
subsequently monitoring the corresponding signal in a tissue into
which the hematopoietic stem cells have homed, such as the bone
marrow. One can also observe hematopoietic stem cell engraftment by
evaluation of the quantity and survival of hematopoietic stem and
progenitor cells, for instance, as determined by fluorescence
activated cell sorting (FACS) analysis methods known in the art.
Engraftment can also be determined by measuring white blood cell
counts in peripheral blood during a post-transplant period, and/or
by measuring recovery of marrow cells by donor cells in a bone
marrow aspirate sample.
[0321] The sections that follow provide a description of
antibodies, antigen-binding fragments thereof, and ligands that can
be administered to a patient, such as a patient suffering from a
cancer or autoimmune disease, or a patient in need of hematopoietic
stem cell transplant therapy in order to promote engraftment of
hematopoietic stem cell grafts, as well as methods of administering
such therapeutics to a patient (e.g., prior to hematopoietic stem
cell transplantation).
Definitions
[0322] As used herein, the term "about" refers to a value that is
within 10% above or below the value being described. For example,
the term "about 5 nM" indicates a range of from 4.5 nM to 5.5
nM.
[0323] As used herein, the term "amatoxin" refers to a member of
the amatoxin family of peptides produced by Amanita phalloides
mushrooms, or a variant or derivative thereof, such as a variant or
derivative thereof capable of inhibiting RNA polymerase II
activity. Amatoxins useful in conjunction with the compositions and
methods described herein include compounds according to formula
(III), below, such as .alpha.-amanitin, .beta.-amanitin,
.gamma.-amanitin, .epsilon.-amanitin, amanin, amaninamide,
amanullin, amanullinic acid, and proamanullin. Formula (III) is as
follows:
##STR00015##
[0324] wherein R.sub.1 is H, OH, or OR.sub.A;
[0325] R.sub.2 is H, OH, or OR.sub.B;
[0326] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0327] R.sub.3 is H or R.sub.D;
[0328] R.sub.4 is H, OH, OR.sub.D, or R.sub.D;
[0329] R.sub.5 is H, OH, OR.sub.D, or R.sub.D;
[0330] R.sub.6 is H, OH, OR.sub.D, or R.sub.D;
[0331] R.sub.7 is H, OH, OR.sub.D, or R.sub.D;
[0332] R.sub.8 is OH, NH.sub.2, or OR.sub.D;
[0333] R.sub.9 is H, OH, or OR.sub.D;
[0334] X is --S--, --S(O)--, or --SO.sub.2--; and
[0335] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl.
[0336] For instance, amatoxins useful in conjunction with the
compositions and methods described herein include compounds
according to formula (IIIA), below:
##STR00016##
[0337] wherein R.sub.1 is H, OH, or OR.sub.A;
[0338] R.sub.2 is H, OH, or OR.sub.B;
[0339] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0340] R.sub.3 is H or R.sub.D;
[0341] R.sub.4 is H, OH, OR.sub.D, or R.sub.D;
[0342] R.sub.5 is H, OH, OR.sub.D, or R.sub.D;
[0343] R.sub.6 is H, OH, OR.sub.D, or R.sub.D;
[0344] R.sub.7 is H, OH, OR.sub.D, or R.sub.D;
[0345] R.sub.8 is OH, NH.sub.2, or OR.sub.D;
[0346] R.sub.9 is H, OH, or OR.sub.D;
[0347] X is --S--, --S(O)--, or --SO.sub.2--; and
[0348] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl.
[0349] Amatoxins useful in conjunction with the compositions and
methods described herein also include compounds according to
formula (IIIB), below:
##STR00017##
[0350] wherein R.sub.1 is H, OH, or OR.sub.A;
[0351] R.sub.2 is H, OH, or OR.sub.B;
[0352] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0353] R.sub.3 is H or R.sub.D;
[0354] R.sub.4 is H, OH, OR.sub.D, or R.sub.D;
[0355] R.sub.5 is H, OH, OR.sub.D, or R.sub.D;
[0356] R.sub.6 is H, OH, OR.sub.D, or R.sub.D;
[0357] R.sub.7 is H, OH, OR.sub.D, or R.sub.D;
[0358] R.sub.8 is OH, NH.sub.2, or OR.sub.D;
[0359] R.sub.9 is H, OH, or OR.sub.D;
[0360] X is --S--, --S(O)--, or --SO.sub.2--; and
[0361] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl.
[0362] As described herein, amatoxins may be conjugated to an
antibody, antigen-binding fragment thereof, or ligand, for
instance, by way of a linker moiety. Exemplary methods of amatoxin
conjugation and linkers useful for such processes are described in
the section entitled "Linkers for chemical conjugation," as well as
in Table 2, below. Exemplary linker-containing amatoxins useful for
conjugation to an antibody, antigen-binding fragment, or ligand in
accordance with the compositions and methods described herein are
shown in structural formulas (I), (IA), (IB) and (II), recited
herein.
[0363] As used herein, the term "antibody" refers to an
immunoglobulin molecule that specifically binds to, or is
immunologically reactive with, a particular antigen, and includes
polyclonal, monoclonal, genetically engineered, and otherwise
modified forms of antibodies, including but not limited to chimeric
antibodies, humanized antibodies, heteroconjugate antibodies (e.g.,
bi- tri- and quad-specific antibodies, diabodies, triabodies, and
tetrabodies), and antigen binding fragments of antibodies,
including, for example, Fab', F(ab').sub.2, Fab, Fv, rIgG, and scFv
fragments. Unless otherwise indicated, the term "monoclonal
antibody" (mAb) is meant to include both intact molecules, as well
as antibody fragments (including, for example, Fab and F(ab').sub.2
fragments) that are capable of specifically binding to a target
protein. As used herein, the Fab and F(ab').sub.2 fragments refer
to antibody fragments that lack the Fc fragment of an intact
antibody. Examples of these antibody fragments are described
herein.
[0364] The term "antigen-binding fragment," as used herein, refers
to one or more fragments of an antibody that retain the ability to
specifically bind to a target antigen. The antigen-binding function
of an antibody can be performed by fragments of a full-length
antibody. The antibody fragments can be, for example, a Fab,
F(ab').sub.2, scFv, diabody, a triabody, an affibody, a nanobody,
an aptamer, or a domain antibody. Examples of binding fragments
encompassed of the term "antigen-binding fragment" of an antibody
include, but are not limited to: (i) a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, C.sub.L, and C.sub.H1
domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
containing two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the V.sub.H and
C.sub.H1 domains; (iv) a Fv fragment consisting of the V.sub.L and
V.sub.H domains of a single arm of an antibody, (v) a dAb including
V.sub.H and V.sub.L domains; (vi) a dAb fragment that consists of a
V.sub.H domain (see, e.g., Ward et al., Nature 341:544-546, 1989);
(vii) a dAb which consists of a V.sub.H or a V.sub.L domain; (viii)
an isolated complementarity determining region (CDR); and (ix) a
combination of two or more (e.g., two, three, four, five, or six)
isolated CDRs which may optionally be joined by a synthetic linker.
Furthermore, although the two domains of the Fv fragment, V.sub.L
and V.sub.H, are coded for by separate genes, they can be joined,
using recombinant methods, by a linker that enables them to be made
as a single protein chain in which the V.sub.L and V.sub.H regions
pair to form monovalent molecules (known as single chain Fv (scFv);
see, for example, Bird et al., Science 242:423-426, 1988 and Huston
et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These
antibody fragments can be obtained using conventional techniques
known to those of skill in the art, and the fragments can be
screened for utility in the same manner as intact antibodies.
Antigen-binding fragments can be produced by recombinant DNA
techniques, enzymatic or chemical cleavage of intact
immunoglobulins, or, in certain cases, by chemical peptide
synthesis procedures known in the art.
[0365] As used herein, the term "anti-CD45 antibody" refers to a
protein or peptide-containing molecule that includes at least a
portion of an immunoglobulin molecule, such as but not limited to
at least one complementarity determining region (CDR) of a heavy or
light chain or a ligand binding portion thereof, a heavy chain or
light chain variable region, a heavy chain or light chain constant
region, a framework region, or any portion thereof, that is capable
of specifically binding to CD45 (for example, CD45RO). Anti-CD45
antibodies also include antibody-like protein scaffolds, such as
the tenth fibronectin type III domain (.sup.10Fn3), which contains
BC, DE, and FG structural loops similar in structure and solvent
accessibility to antibody CDRs. The tertiary structure of the
.sup.10Fn3 domain resembles that of the variable region of the IgG
heavy chain, and one of skill in the art can graft, for example,
the CDRs of an anti-CD45 monoclonal antibody onto the fibronectin
scaffold by replacing residues of the BC, DE, and FG loops of
.sup.10Fn3 with residues from the CDRH-1, CDRH-2, or CDRH-3 regions
of an anti-CD45 monoclonal antibody.
[0366] As used herein, the term "anti-CD135 antibody" refers to a
protein or peptide-containing molecule that includes at least a
portion of an immunoglobulin molecule, such as but not limited to
at least one complementarity determining region (CDR) of a heavy or
light chain or a ligand binding portion thereof, a heavy chain or
light chain variable region, a heavy chain or light chain constant
region, a framework region, or any portion thereof, that is capable
of specifically binding to CD135. Anti-CD135 antibodies also
include antibody-like protein scaffolds, such as the tenth
fibronectin type III domain (.sup.10Fn3), which contains BC, DE,
and FG structural loops similar in structure and solvent
accessibility to antibody CDRs. The tertiary structure of the
.sup.10Fn3 domain resembles that of the variable region of the IgG
heavy chain, and one of skill in the art can graft, for example,
the CDRs of an anti-CD135 monoclonal antibody onto the fibronectin
scaffold by replacing residues of the BC, DE, and FG loops of
.sup.10Fn3 with residues from the CDRH-1, CDRH-2, or CDRH-3 regions
of an anti-CD135 monoclonal antibody.
[0367] As used herein, the term "anti-CD34 antibody" refers to a
protein or peptide-containing molecule that includes at least a
portion of an immunoglobulin molecule, such as but not limited to
at least one complementarity determining region (CDR) of a heavy or
light chain or a ligand binding portion thereof, a heavy chain or
light chain variable region, a heavy chain or light chain constant
region, a framework region, or any portion thereof, that is capable
of specifically binding to CD34. Anti-CD34 antibodies also include
antibody-like protein scaffolds, such as the tenth fibronectin type
III domain (.sup.10Fn3), which contains BC, DE, and FG structural
loops similar in structure and solvent accessibility to antibody
CDRs. The tertiary structure of the .sup.10Fn3 domain resembles
that of the variable region of the IgG heavy chain, and one of
skill in the art can graft, for example, the CDRs of an anti-CD34
monoclonal antibody onto the fibronectin scaffold by replacing
residues of the BC, DE, and FG loops of .sup.10Fn3 with residues
from the CDRH-1, CDRH-2, or CDRH-3 regions of an anti-CD34
monoclonal antibody.
[0368] As used herein, the term "anti-CD90 antibody" refers to a
protein or peptide-containing molecule that includes at least a
portion of an immunoglobulin molecule, such as but not limited to
at least one complementarity determining region (CDR) of a heavy or
light chain or a ligand binding portion thereof, a heavy chain or
light chain variable region, a heavy chain or light chain constant
region, a framework region, or any portion thereof, that is capable
of specifically binding to CD90. Anti-CD90 antibodies also include
antibody-like protein scaffolds, such as the tenth fibronectin type
III domain (.sup.10Fn3), which contains BC, DE, and FG structural
loops similar in structure and solvent accessibility to antibody
CDRs. The tertiary structure of the .sup.10Fn3 domain resembles
that of the variable region of the IgG heavy chain, and one of
skill in the art can graft, for example, the CDRs of an anti-CD90
monoclonal antibody onto the fibronectin scaffold by replacing
residues of the BC, DE, and FG loops of .sup.10Fn3 with residues
from the CDRH-1, CDRH-2, or CDRH-3 regions of an anti-CD90
monoclonal antibody.
[0369] As used herein, the term "anti-CD110 antibody" refers to a
protein or peptide-containing molecule that includes at least a
portion of an immunoglobulin molecule, such as but not limited to
at least one complementarity determining region (CDR) of a heavy or
light chain or a ligand binding portion thereof, a heavy chain or
light chain variable region, a heavy chain or light chain constant
region, a framework region, or any portion thereof, that is capable
of specifically binding to CD110. Anti-CD110 antibodies also
include antibody-like protein scaffolds, such as the tenth
fibronectin type III domain (.sup.10Fn3), which contains BC, DE,
and FG structural loops similar in structure and solvent
accessibility to antibody CDRs. The tertiary structure of the
.sup.10Fn3 domain resembles that of the variable region of the IgG
heavy chain, and one of skill in the art can graft, for example,
the CDRs of an anti-CD110 monoclonal antibody onto the fibronectin
scaffold by replacing residues of the BC, DE, and FG loops of
.sup.10Fn3 with residues from the CDRH-1, CDRH-2, or CDRH-3 regions
of an anti-CD110 monoclonal antibody.
[0370] As used herein, the term "bispecific antibody" refers to,
for example, a monoclonal, often a human or humanized antibody that
is capable of binding at least two different antigens. For
instance, one of the binding specificities can be directed towards
a hematopoietic stem cell surface antigen, such as CD45 (e.g.,
CD45RO), CD135, CD34, CD90, or CD110, the other can be for a
different hematopoietic stem cell surface antigen or another cell
surface protein, such as a receptor or receptor subunit involved in
a signal transduction pathway that potentiates cell growth, among
others.
[0371] As used herein, the term "complementarity determining
region" (CDR) refers to a hypervariable region found both in the
light chain and the heavy chain variable domains of an antibody.
The more highly conserved portions of variable domains are referred
to as framework regions (FRs). The amino acid positions that
delineate a hypervariable region of an antibody can vary, depending
on the context and the various definitions known in the art. Some
positions within a variable domain may be viewed as hybrid
hypervariable positions in that these positions can be deemed to be
within a hypervariable region under one set of criteria while being
deemed to be outside a hypervariable region under a different set
of criteria. One or more of these positions can also be found in
extended hypervariable regions. The antibodies described herein may
contain modifications in these hybrid hypervariable positions. The
variable domains of native heavy and light chains each contain four
framework regions that primarily adopt a .beta.-sheet
configuration, connected by three CDRs, which form loops that
connect, and in some cases form part of, the .beta.-sheet
structure. The CDRs in each chain are held together in close
proximity by the framework regions in the order
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other
antibody chains, contribute to the formation of the target binding
site of antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, National Institute of Health, Bethesda,
Md., 1987). As used herein, numbering of immunoglobulin amino acid
residues is performed according to the immunoglobulin amino acid
residue numbering system of Kabat et al., unless otherwise
indicated.
[0372] As used herein, the terms "condition" and "conditioning"
refer to processes by which a patient is prepared for receipt of a
transplant containing hematopoietic stem cells. Such procedures
promote the engraftment of a hematopoietic stem cell transplant
(for instance, as inferred from a sustained increase in the
quantity of viable hematopoietic stem cells within a blood sample
isolated from a patient following a conditioning procedure and
subsequent hematopoietic stem cell transplantation. According to
the methods described herein, a patient may be conditioned for
hematopoietic stem cell transplant therapy by administration to the
patient of an antibody or antigen-binding fragment thereof capable
of binding an antigen expressed by hematopoietic stem cells, such
as CD45 (e.g., CD45RO), CD135, CD34, CD90, or CD110. As described
herein, the antibody may be covalently conjugated to a cytotoxin so
as to form a drug-antibody conjugate. Administration of an
antibody, antigen-binding fragment thereof, or drug-antibody
conjugate capable of binding one or more of the foregoing antigens
to a patient in need of hematopoietic stem cell transplant therapy
can promote the engraftment of a hematopoietic stem cell graft, for
example, by selectively depleting endogenous hematopoietic stem
cells, thereby creating a vacancy filled by an exogenous
hematopoietic stem cell transplant.
[0373] As used herein, the term "conjugate" refers to a compound
formed by the chemical bonding of a reactive functional group of
one molecule, such as an antibody or antigen-binding fragment
thereof, with an appropriately reactive functional group of another
molecule, such as a cytotoxin described herein. Conjugates may
include a linker between the two molecules bound to one another.
Examples of linkers that can be used for the formation of a
conjugate include peptide-containing linkers, such as those that
contain naturally occurring or non-naturally occurring amino acids,
such as D-amino acids. Linkers can be prepared using a variety of
strategies described herein and known in the art. Depending on the
reactive components therein, a linker may be cleaved, for example,
by enzymatic hydrolysis, photolysis, hydrolysis under acidic
conditions, hydrolysis under basic conditions, oxidation, disulfide
reduction, nucleophilic cleavage, or organometallic cleavage (see,
for example, Leriche et al., Bioorg. Med. Chem., 20:571-582,
2012).
[0374] As used herein, the term "coupling reaction" refers to a
chemical reaction in which two or more substituents suitable for
reaction with one another react so as to form a chemical moiety
that joins (e.g., covalently) the molecular fragments bound to each
substituent. Coupling reactions include those in which a reactive
substituent bound to a fragment that is a cytotoxin, such as a
cytotoxin known in the art or described herein, reacts with a
suitably reactive substituent bound to a fragment that is an
antibody, antigen-binding fragment thereof, or ligand, such as an
antibody, antigen-binding fragment thereof, or ligand specific for
CD45 (such as CD45RO), CD135, CD34, CD90, and/or CD110 known in the
art or described herein. Examples of suitably reactive substituents
include a nucleophile/electrophile pair (e.g., a thiol/haloalkyl
pair, an amine/carbonyl pair, or a thiol/.alpha.,.beta.-unsaturated
carbonyl pair, among others), a diene/dienophile pair (e.g., an
azide/alkyne pair, among others), and the like. Coupling reactions
include, without limitation, thiol alkylation, hydroxyl alkylation,
amine alkylation, amine condensation, amidation, esterification,
disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder
cycloaddition, [3+2] Huisgen cycloaddition, among others),
nucleophilic aromatic substitution, electrophilic aromatic
substitution, and other reactive modalities known in the art or
described herein.
[0375] As used herein, "CRU (competitive repopulating unit)" refers
to a unit of measure of long-term engrafting stem cells, which can
be detected after in-vivo transplantation.
[0376] As used herein, the term "donor" refers to a human or animal
from which one or more cells are isolated prior to administration
of the cells, or progeny thereof, into a recipient. The one or more
cells may be, for example, a population of hematopoietic stem
cells.
[0377] As used herein, the term "diabody" refers to a bivalent
antibody containing two polypeptide chains, in which each
polypeptide chain includes V.sub.H and V.sub.L domains joined by a
linker that is too short (e.g., a linker composed of five amino
acids) to allow for intramolecular association of V.sub.H and
V.sub.L domains on the same peptide chain. This configuration
forces each domain to pair with a complementary domain on another
polypeptide chain so as to form a homodimeric structure.
Accordingly, the term "triabody" refers to trivalent antibodies
containing three peptide chains, each of which contains one V.sub.H
domain and one V.sub.L domain joined by a linker that is
exceedingly short (e.g., a linker composed of 1-2 amino acids) to
permit intramolecular association of V.sub.H and V.sub.L domains
within the same peptide chain. In order to fold into their native
structures, peptides configured in this way typically trimerize so
as to position the V.sub.H and V.sub.L domains of neighboring
peptide chains spatially proximal to one another (see, for example,
Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993).
[0378] As used herein, a "dual variable domain immunoglobulin"
("DVD-Ig") refers to an antibody that combines the target-binding
variable domains of two monoclonal antibodies via linkers to create
a tetravalent, dual-targeting single agent (see, for example, Gu et
al., Meth. Enzymol., 502:25-41, 2012).
[0379] As used herein, the term "endogenous" describes a substance,
such as a molecule, cell, tissue, or organ (e.g., a hematopoietic
stem cell or a cell of hematopoietic lineage, such as a
megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell,
myeoblast, basophil, neutrophil, eosinophil, microglial cell,
granulocyte, monocyte, osteoclast, antigen-presenting cell,
macrophage, dendritic cell, natural killer cell, T-lymphocyte, or
B-lymphocyte) that is found naturally in a particular organism,
such as a human patient.
[0380] As used herein, the term "engraftment potential" is used to
refer to the ability of hematopoietic stem and progenitor cells to
repopulate a tissue, whether such cells are naturally circulating
or are provided by transplantation. The term encompasses all events
surrounding or leading up to engraftment, such as tissue homing of
cells and colonization of cells within the tissue of interest. The
engraftment efficiency or rate of engraftment can be evaluated or
quantified using any clinically acceptable parameter as known to
those of skill in the art and can include, for example, assessment
of competitive repopulating units (CRU); incorporation or
expression of a marker in tissue(s) into which stem cells have
homed, colonized, or become engrafted; or by evaluation of the
progress of a subject through disease progression, survival of
hematopoietic stem and progenitor cells, or survival of a
recipient. Engraftment can also be determined by measuring white
blood cell counts in peripheral blood during a post-transplant
period. Engraftment can also be assessed by measuring recovery of
marrow cells by donor cells in a bone marrow aspirate sample.
[0381] As used herein, the term "exogenous" describes a substance,
such as a molecule, cell, tissue, or organ (e.g., a hematopoietic
stem cell or a cell of hematopoietic lineage, such as a
megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell,
myeoblast, basophil, neutrophil, eosinophil, microglial cell,
granulocyte, monocyte, osteoclast, antigen-presenting cell,
macrophage, dendritic cell, natural killer cell, T-lymphocyte, or
B-lymphocyte) that is not found naturally in a particular organism,
such as a human patient. Exogenous substances include those that
are provided from an external source to an organism or to cultured
matter extracted therefrom.
[0382] As used herein, the term "framework region" or "FW region"
includes amino acid residues that are adjacent to the CDRs of an
antibody or antigen-binding fragment thereof. FW region residues
may be present in, for example, human antibodies, humanized
antibodies, monoclonal antibodies, antibody fragments, Fab
fragments, single chain antibody fragments, scFv fragments,
antibody domains, and bispecific antibodies, among others.
[0383] As used herein, the term "hematopoietic stem cells" ("HSCs")
refers to immature blood cells having the capacity to self-renew
and to differentiate into mature blood cells containing diverse
lineages including but not limited to granulocytes (e.g.,
promyelocytes, neutrophils, eosinophils, basophils), erythrocytes
(e.g., reticulocytes, erythrocytes), thrombocytes (e.g.,
megakaryoblasts, platelet producing megakaryocytes, platelets),
monocytes (e.g., monocytes, macrophages), dendritic cells,
microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells
and T-cells). Such cells may include CD34+ cells. CD34+ cells are
immature cells that express the CD34 cell surface marker. In
humans, CD34+ cells are believed to include a subpopulation of
cells with the stem cell properties defined above, whereas in mice,
HSCs are CD34-. In addition, HSCs also refer to long term
repopulating HSCs (LT-HSC) and short term repopulating HSCs
(ST-HSC). LT-HSCs and ST-HSCs are differentiated, based on
functional potential and on cell surface marker expression. For
example, human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and
lin- (negative for mature lineage markers including CD2, CD3, CD4,
CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A). In mice, bone
marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamfl/CD150+,
CD48-, and lin- (negative for mature lineage markers including
Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra), whereas ST-HSCs
are CD34+, SCA-1+, C-kit+, CD135-, Slamfl/CD150+, and lin-
(negative for mature lineage markers including Ter119, CD11b, Gr1,
CD3, CD4, CD8, B220, IL7ra). In addition, ST-HSCs are less
quiescent and more proliferative than LT-HSCs under homeostatic
conditions. However, LT-HSC have greater self renewal potential
(i.e., they survive throughout adulthood, and can be serially
transplanted through successive recipients), whereas ST-HSCs have
limited self renewal (i.e., they survive for only a limited period
of time, and do not possess serial transplantation potential). Any
of these HSCs can be used in the methods described herein. ST-HSCs
are particularly useful because they are highly proliferative and
thus, can more quickly give rise to differentiated progeny.
[0384] As used herein, the term "hematopoietic stem cell functional
potential" refers to the functional properties of hematopoietic
stem cells which include 1) multi-potency (which refers to the
ability to differentiate into multiple different blood lineages
including, but not limited to, granulocytes (e.g., promyelocytes,
neutrophils, eosinophils, basophils), erythrocytes (e.g.,
reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts,
platelet producing megakaryocytes, platelets), monocytes (e.g.,
monocytes, macrophages), dendritic cells, microglia, osteoclasts,
and lymphocytes (e.g., NK cells, B-cells and T-cells), 2)
self-renewal (which refers to the ability of hematopoietic stem
cells to give rise to daughter cells that have equivalent potential
as the mother cell, and further that this ability can repeatedly
occur throughout the lifetime of an individual without exhaustion),
and 3) the ability of hematopoietic stem cells or progeny thereof
to be reintroduced into a transplant recipient whereupon they home
to the hematopoietic stem cell niche and re-establish productive
and sustained hematopoiesis.
[0385] As used herein, the term "human antibody" refers to an
antibody in which substantially every part of the protein (for
example, all CDRs, framework regions, C.sub.L, C.sub.H domains
(e.g., C.sub.H1, C.sub.H2, C.sub.H3), hinge, and V.sub.L and
V.sub.H domains) is substantially non-immunogenic in humans, with
only minor sequence changes or variations. A human antibody can be
produced in a human cell (for example, by recombinant expression)
or by a non-human animal or a prokaryotic or eukaryotic cell that
is capable of expressing functionally rearranged human
immunoglobulin (such as heavy chain and/or light chain) genes. When
a human antibody is a single chain antibody, it can include a
linker peptide that is not found in native human antibodies. For
example, an Fv can contain a linker peptide, such as two to about
eight glycine or other amino acid residues, which connects the
variable region of the heavy chain and the variable region of the
light chain. Such linker peptides are considered to be of human
origin. Human antibodies can be made by a variety of methods known
in the art including phage display methods using antibody libraries
derived from human immunoglobulin sequences. Human antibodies can
also be produced using transgenic mice that are incapable of
expressing functional endogenous immunoglobulins, but which can
express human immunoglobulin genes (see, for example, PCT
Publication Nos. WO 1998/24893; WO 1992/01047; WO 1996/34096; WO
1996/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425;
5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771;
and 5,939,598).
[0386] As used herein, the term "humanized" antibody refers to a
non-human antibody that contains minimal sequences derived from
non-human immunoglobulin. In general, a humanized antibody contains
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin. All or
substantially all of the FW regions may also be those of a human
immunoglobulin sequence. The humanized antibody can also contain at
least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin consensus sequence.
Methods of antibody humanization are known in the art and have been
described, for example, in Riechmann et al., Nature 332:323-7,
1988; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762;
and 6,180,370.
[0387] As used herein, patients that are "in need of" a
hematopoietic stem cell transplant include patients that exhibit a
defect or deficiency in one or more blood cell types, as well as
patients having a stem cell disorder, autoimmune disease, cancer,
or other pathology described herein. Hematopoietic stem cells
generally exhibit 1) multi-potency, and can thus differentiate into
multiple different blood lineages including, but not limited to,
granulocytes (e.g., promyelocytes, neutrophils, eosinophils,
basophils), erythrocytes (e.g., reticulocytes, erythrocytes),
thrombocytes (e.g., megakaryoblasts, platelet producing
megakaryocytes, platelets), monocytes (e.g., monocytes,
macrophages), dendritic cells, microglia, osteoclasts, and
lymphocytes (e.g., NK cells, B-cells and T-cells), 2) self-renewal,
and can thus give rise to daughter cells that have equivalent
potential as the mother cell, and 3) the ability to be reintroduced
into a transplant recipient whereupon they home to the
hematopoietic stem cell niche and re-establish productive and
sustained hematopoiesis. Hematopoietic stem cells can thus be
administered to a patient defective or deficient in one or more
cell types of the hematopoietic lineage in order to re-constitute
the defective or deficient population of cells in vivo. For
example, the patient may be suffering from cancer, and the
deficiency may be caused by administration of a chemotherapeutic
agent or other medicament that depletes, either selectively or
non-specifically, the cancerous cell population. Additionally or
alternatively, the patient may be suffering from a hemoglobinopathy
(e.g., a non-malignant hemoglobinopathy), such as sickle cell
anemia, thalassemia, Fanconi anemia, aplastic anemia, and
Wiskott-Aldrich syndrome. The subject may be one that is suffering
from adenosine deaminase severe combined immunodeficiency (ADA
SCID), HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan
anemia, and Schwachman-Diamond syndrome. The subject may have or be
affected by an inherited blood disorder (e.g., sickle cell anemia)
or an autoimmune disorder. Additionally or alternatively, the
subject may have or be affected by a malignancy, such as
neuroblastoma or a hematologic cancer. For instance, the subject
may have a leukemia, lymphoma, or myeloma. In some embodiments, the
subject has acute myeloid leukemia, acute lymphoid leukemia,
chronic myeloid leukemia, chronic lymphoid leukemia, multiple
myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.
In some embodiments, the subject has myelodysplastic syndrome. In
some embodiments, the subject has an autoimmune disease, such as
scleroderma, multiple sclerosis, ulcerative colitis, Crohn's
disease, Type 1 diabetes, or another autoimmune pathology described
herein. In some embodiments, the subject is in need of chimeric
antigen receptor T-cell (CART) therapy. In some embodiments, the
subject has or is otherwise affected by a metabolic storage
disorder. The subject may suffer or otherwise be affected by a
metabolic disorder selected from the group consisting of glycogen
storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers
Disease, sphingolipidoses, metachromatic leukodystrophy, or any
other diseases or disorders which may benefit from the treatments
and therapies disclosed herein and including, without limitation,
severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyper
immunoglobulin M (IgM) syndrome, Chediak-Higashi disease,
hereditary lymphohistiocytosis, osteopetrosis, osteogenesis
imperfecta, storage diseases, thalassemia major, sickle cell
disease, systemic sclerosis, systemic lupus erythematosus, multiple
sclerosis, juvenile rheumatoid arthritis and those diseases, or
disorders described in "Bone Marrow Transplantation for
Non-Malignant Disease," ASH Education Book, 1:319-338 (2000), the
disclosure of which is incorporated herein by reference in its
entirety as it pertains to pathologies that may be treated by
administration of hematopoietic stem cell transplant therapy.
Additionally or alternatively, a patient "in need of" a
hematopoietic stem cell transplant may one that is or is not
suffering from one of the foregoing pathologies, but nonetheless
exhibits a reduced level (e.g., as compared to that of an otherwise
healthy subject) of one or more endogenous cell types within the
hematopoietic lineage, such as megakaryocytes, thrombocytes,
platelets, erythrocytes, mast cells, myeoblasts, basophils,
neutrophils, eosinophils, microglia, granulocytes, monocytes,
osteoclasts, antigen-presenting cells, macrophages, dendritic
cells, natural killer cells, T-lymphocytes, and B-lymphocytes. One
of skill in the art can readily determine whether one's level of
one or more of the foregoing cell types, or other blood cell type,
is reduced with respect to an otherwise healthy subject, for
instance, by way of flow cytometry and fluorescence activated cell
sorting (FACS) methods, among other procedures, known in the
art.
[0388] As used herein, the term "monoclonal antibody" refers to an
antibody that is derived from a single clone, including any
eukaryotic, prokaryotic, or phage clone, and not the method by
which it is produced.
[0389] As used herein, the term "recipient" refers to a patient
that receives a transplant, such as a transplant containing a
population of hematopoietic stem cells. The transplanted cells
administered to a recipient may be, e.g., autologous, syngeneic, or
allogeneic cells.
[0390] As used herein, the term "sample" refers to a specimen
(e.g., blood, blood component (e.g., serum or plasma), urine,
saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g.,
placental or dermal), pancreatic fluid, chorionic villus sample,
and cells) taken from a subject.
[0391] As used herein, the term "scFv" refers to a single chain Fv
antibody in which the variable domains of the heavy chain and the
light chain from an antibody have been joined to form one chain.
scFv fragments contain a single polypeptide chain that includes the
variable region of an antibody light chain (V.sub.L) (e.g., CDR-L1,
CDR-L2, and/or CDR-L3) and the variable region of an antibody heavy
chain (V.sub.H) (e.g., CDR-H1, CDR-H2, and/or CDR-H3) separated by
a linker. The linker that joins the V.sub.L and V.sub.H regions of
a scFv fragment can be a peptide linker composed of proteinogenic
amino acids. Alternative linkers can be used to so as to increase
the resistance of the scFv fragment to proteolytic degradation (for
example, linkers containing D-amino acids), in order to enhance the
solubility of the scFv fragment (for example, hydrophilic linkers
such as polyethylene glycol-containing linkers or polypeptides
containing repeating glycine and serine residues), to improve the
biophysical stability of the molecule (for example, a linker
containing cysteine residues that form intramolecular or
intermolecular disulfide bonds), or to attenuate the immunogenicity
of the scFv fragment (for example, linkers containing glycosylation
sites). It will also be understood by one of ordinary skill in the
art that the variable regions of the scFv molecules described
herein can be modified such that they vary in amino acid sequence
from the antibody molecule from which they were derived. For
example, nucleotide or amino acid substitutions leading to
conservative substitutions or changes at amino acid residues can be
made (e.g., in CDR and/or framework residues) so as to preserve or
enhance the ability of the scFv to bind to the antigen recognized
by the corresponding antibody.
[0392] As used herein, the terms "subject" and "patient" refer to
an organism, such as a human, that receives treatment for a
particular disease or condition as described herein. For instance,
a patient, such as a human patient, may receive treatment prior to
hematopoietic stem cell transplant therapy in order to promote the
engraftment of exogenous hematopoietic stem cells.
[0393] As used herein, the phrase "substantially cleared from the
blood" refers to a point in time following administration of a
therapeutic agent (such as an anti-CD45, anti-CD135, anti-CD34,
anti-CD90, or anti-CD110 antibody, antigen-binding fragment
thereof, or ligand) to a patient when the concentration of the
therapeutic agent in a blood sample isolated from the patient is
such that the therapeutic agent is not detectable by conventional
means (for instance, such that the therapeutic agent is not
detectable above the noise threshold of the device or assay used to
detect the therapeutic agent). A variety of techniques known in the
art can be used to detect antibodies, antibody fragments, and
protein ligands, such as ELISA-based detection assays known in the
art or described herein. Additional assays that can be used to
detect antibodies, antibody fragments, and protein ligands include
immunoprecipitation techniques and immunoblot assays, among others
known in the art.
[0394] As used herein, the phrase "stem cell disorder" broadly
refers to any disease, disorder, or condition that may be treated
or cured by conditioning a subjects target tissues, and/or by
ablating an endogenous stem cell population in a target tissue
(e.g., ablating an endogenous hematopoietic stem or progenitor cell
population from a subject's bone marrow tissue) and/or by
engrafting or transplanting stem cells in a subjects target
tissues. For example, Type I diabetes has been shown to be cured by
hematopoietic stem cell transplant and may benefit from
conditioning in accordance with the compositions and methods
described herein. Additional disorders that can be treated using
the compositions and methods described herein include, without
limitation, sickle cell anemia, thalassemias, Fanconi anemia,
aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV/AIDS,
metachromatic leukodystrophy, Diamond-Blackfan anemia, and
Schwachman-Diamond syndrome. Additional diseases that may be
treated using the patient conditioning and/or hematopoietic stem
cell transplant methods described herein influde inherited blood
disorders (e.g., sickle cell anemia) and autoimmune disorders, such
as scleroderma, multiple sclerosis, ulcerative colitis, and
Chrohn's disease. Additional diseases that may be treated using the
conditioning and/or transplantation methods described herein
include a malignancy, such as a neuroblastoma or a hematologic
cancers, such as leukemia, lymphoma, and myeloma. For instance, the
cancer may be acute myeloid leukemia, acute lymphoid leukemia,
chronic myeloid leukemia, chronic lymphoid leukemia, multiple
myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.
Additional diseases treatable using the conditioning and/or
transplantation methods described herein include myelodysplastic
syndrome. In some embodiments, the subject has or is otherwise
affected by a metabolic storage disorder. For example, the subject
may suffer or otherwise be affected by a metabolic disorder
selected from the group consisting of glycogen storage diseases,
mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease,
sphingolipidoses, metachromatic leukodystrophy, or any other
diseases or disorders which may benefit from the treatments and
therapies disclosed herein and including, without limitation,
severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyper
immunoglobulin M (IgM) syndrome, Chediak-Higashi disease,
hereditary lymphohistiocytosis, osteopetrosis, osteogenesis
imperfecta, storage diseases, thalassemia major, sickle cell
disease, systemic sclerosis, systemic lupus erythematosus, multiple
sclerosis, juvenile rheumatoid arthritis and those diseases, or
disorders described in "Bone Marrow Transplantation for
Non-Malignant Disease," ASH Education Book, 1:319-338 (2000), the
disclosure of which is incorporated herein by reference in its
entirety as it pertains to pathologies that may be treated by
administration of hematopoietic stem cell transplant therapy.
[0395] As used herein, the terms "treat" or "treatment" refer to
therapeutic treatment, in which the object is to prevent or slow
down (lessen) an undesired physiological change or disorder or to
promote a beneficial phenotype in the patient being treated.
Beneficial or desired clinical results include, but are not limited
to, promoting the engraftment of exogenous hematopoietic cells in a
patient following antibody conditioning therapy as described herein
and subsequent hematopoietic stem cell transplant therapy.
Additional beneficial results include an increase in the cell count
or relative concentration of hematopoietic stem cells in a patient
in need of a hematopoietic stem cell transplant following
conditioning therapy and subsequent administration of an exogenous
hematopoietic stem cell graft to the patient. Beneficial results of
therapy described herein may also include an increase in the cell
count or relative concentration of one or more cells of
hematopoietic lineage, such as a megakaryocyte, thrombocyte,
platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil,
eosinophil, microglial cell, granulocyte, monocyte, osteoclast,
antigen-presenting cell, macrophage, dendritic cell, natural killer
cell, T-lymphocyte, or B-lymphocyte, following conditioning therapy
and subsequent hematopoietic stem cell transplant therapy.
Additional beneficial results may include the reduction in quantity
of a disease-causing cell population, such as a population of
cancer cells (e.g., CD45+, CD135+, CD34+, CD90+, or CD110+ leukemic
cells) or autoimmune cells (e.g., CD45+, CD135+, CD34+, CD90+, or
CD110+ autoimmune lymphocytes, such as a CD45+, CD135+, CD34+,
CD90+, or CD110+ T-cell that expresses a T-cell receptor that
cross-reacts with a self antigen).
[0396] As used herein, the terms "variant" and "derivative" are
used interchangeably and refer to naturally-occurring, synthetic,
and semi-synthetic analogues of a compound, peptide, protein, or
other substance described herein. A variant or derivative of a
compound, peptide, protein, or other substance described herein may
retain or improve upon the biological activity of the original
material.
[0397] As used herein, the term "vector" includes a nucleic acid
vector, such as a plasmid, a DNA vector, a plasmid, a RNA vector,
virus, or other suitable replicon. Expression vectors described
herein may contain a polynucleotide sequence as well as, for
example, additional sequence elements used for the expression of
proteins and/or the integration of these polynucleotide sequences
into the genome of a mammalian cell. Certain vectors that can be
used for the expression of antibodies and antibody fragments of the
invention include plasmids that contain regulatory sequences, such
as promoter and enhancer regions, which direct gene transcription.
Other useful vectors for expression of antibodies and antibody
fragments contain polynucleotide sequences that enhance the rate of
translation of these genes or improve the stability or nuclear
export of the mRNA that results from gene transcription. These
sequence elements may include, for example, 5' and 3' untranslated
regions and a polyadenylation signal site in order to direct
efficient transcription of the gene carried on the expression
vector. The expression vectors described herein may also contain a
polynucleotide encoding a marker for selection of cells that
contain such a vector. Examples of a suitable marker include genes
that encode resistance to antibiotics, such as ampicillin,
chloramphenicol, kanamycin, and nourseothricin.
[0398] As used herein, the term "alkyl" refers to a straight- or
branched-chain alkyl group having, for example, from 1 to 20 carbon
atoms in the chain. Examples of alkyl groups include methyl, ethyl,
n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,
pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like.
[0399] As used herein, the term "alkylene" refers to a straight- or
branched-chain divalent alkyl group. The divalent positions may be
on the same or different atoms within the alkyl chain. Examples of
alkylene include methylene, ethylene, propylene, isopropylene, and
the like.
[0400] As used herein, the term "heteroalkyl" refers to a straight
or branched-chain alkyl group having, for example, from 1 to 20
carbon atoms in the chain, and further containing one or more
heteroatoms (e.g., oxygen, nitrogen, or sulfur, among others) in
the chain.
[0401] As used herein, the term "heteroalkylene" refers to a
straight- or branched-chain divalent heteroalkyl group. The
divalent positions may be on the same or different atoms within the
heteroalkyl chain. The divalent positions may be one or more
heteroatoms.
[0402] As used herein, the term "alkenyl" refers to a straight- or
branched-chain alkenyl group having, for example, from 2 to 20
carbon atoms in the chain. Examples of alkenyl groups include
vinyl, propenyl, isopropenyl, butenyl, tert-butylenyl, hexenyl, and
the like.
[0403] As used herein, the term "alkenylene" refers to a straight-
or branched-chain divalent alkenyl group. The divalent positions
may be on the same or different atoms within the alkenyl chain.
Examples of alkenylene include ethenylene, propenylene,
isopropenylene, butenylene, and the like.
[0404] As used herein, the term "heteroalkenyl" refers to a
straight- or branched-chain alkenyl group having, for example, from
2 to 20 carbon atoms in the chain, and further containing one or
more heteroatoms (e.g., oxygen, nitrogen, or sulfur, among others)
in the chain.
[0405] As used herein, the term "heteroalkenylene" refers to a
straight- or branched-chain divalent heteroalkenyl group. The
divalent positions may be on the same or different atoms within the
heteroalkenyl chain. The divalent positions may be one or more
heteroatoms.
[0406] As used herein, the term "alkynyl" refers to a straight- or
branched-chain alkynyl group having, for example, from 2 to 20
carbon atoms in the chain. Examples of alkynyl groups include
propargyl, butynyl, pentynyl, hexynyl, and the like.
[0407] As used herein, the term "alkynylene" refers to a straight-
or branched-chain divalent alkynyl group. The divalent positions
may be on the same or different atoms within the alkynyl chain.
[0408] As used herein, the term "heteroalkynyl" refers to a
straight- or branched-chain alkynyl group having, for example, from
2 to 20 carbon atoms in the chain, and further containing one or
more heteroatoms (e.g., oxygen, nitrogen, or sulfur, among others)
in the chain.
[0409] As used herein, the term "heteroalkynylene" refers to a
straight- or branched-chain divalent heteroalkynyl group. The
divalent positions may be on the same or different atoms within the
heteroalkynyl chain. The divalent positions may be one or more
heteroatoms.
[0410] As used herein, the term "cycloalkyl" refers to a
monocyclic, or fused, bridged, or spiro polycyclic ring structure
that is saturated and has, for example, from 3 to 12 carbon ring
atoms. Examples of cycloalkyl groups include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,
bicyclo[3.1.0]hexane, and the like.
[0411] As used herein, the term "cycloalkylene" refers to a
divalent cycloalkyl group. The divalent positions may be on the
same or different atoms within the ring structure. Examples of
cycloalkylene include cyclopropylene, cyclobutylene,
cyclopentylene, cyclohexylene, and the like.
[0412] As used herein, the term "heterocyloalkyl" refers to a
monocyclic, or fused, bridged, or spiro polycyclic ring structure
that is saturated and has, for example, from 3 to 12 ring atoms per
ring structure selected from carbon atoms and heteroatoms selected
from, e.g., nitrogen, oxygen, and sulfur, among others. The ring
structure may contain, for example, one or more oxo groups on
carbon, nitrogen, or sulfur ring members.
[0413] As used herein, the term "heterocycloalkylene" refers to a
divalent heterocyclolalkyl group. The divalent positions may be on
the same or different atoms within the ring structure. The divalent
positions may be one or more heteroatoms.
[0414] As used herein, the term "aryl" refers to a monocyclic or
multicyclic aromatic ring system containing, for example, from 6 to
19 carbon atoms. Aryl groups include, but are not limited to,
phenyl, fluorenyl, naphthyl, and the like.
[0415] As used herein, the term "arylene" refers to a divalent aryl
group. The divalent positions may be on the same or different
atoms.
[0416] As used herein, the term "heteroaryl" refers to a monocyclic
heteroaromatic, or a bicyclic or a tricyclic fused-ring
heteroaromatic group. Heteroaryl groups include pyridyl, pyrrolyl,
furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl,
isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl,
1,2,3-oxadiazolyl, 1,2,4-oxadia-zolyl, 1,2,5-oxadiazolyl,
1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl,
[2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl,
benzotriazolyl, isobenzothienyl, indolyl, isoindolyl, 3H-indolyl,
benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl,
benzoxazolyl, quinolizinyl, quinazolinyl, pthalazinyl,
quinoxalinyl, cinnolinyl, napthyridinyl, pyrido[3,4-b]pyridyl,
pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl,
tetrazolyl, 5,6,7,8-tetrahydroquinolyl,
5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl,
xanthenyl, benzoquinolyl, and the like.
[0417] As used herein, the term "heteroarylene" refers to a
divalent heteroaryl group. The divalent positions may be on the
same or different atoms. The divalent positions may be one or more
heteroatoms.
[0418] Unless otherwise constrained by the definition of the
individual substituent, the foregoing chemical moieties, such as
"alkyl", "alkylene", "heteroalkyl", "heteroalkylene", "alkenyl",
"alkenylene", "heteroalkenyl", "heteroalkenylene", "alkynyl",
"alkynylene", "heteroalkynyl", "heteroalkynylene", "cycloalkyl",
"cycloalkylene", "heterocyclolalkyl", heterocycloalkylene", "aryl,"
"arylene", "heteroaryl", and "heteroarylene" groups can optionally
be substituted with, for example, from 1 to 5 substituents selected
from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, alkyl aryl, alkyl heteroaryl, alkyl cycloalkyl,
alkyl heterocycloalkyl, amino, ammonium, acyl, acyloxy, acylamino,
aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl,
sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxy,
trihalomethyl, cyano, hydroxy, mercapto, nitro, and the like. The
substitution may include situations in which neighboring
substituents have undergone ring closure, such as ring closure of
vicinal functional substituents, to form, for instance, lactams,
lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals,
aminals, and hemiaminals, formed by ring closure, for example, to
furnish a protecting group.
Antibodies that Recognize Hematopoietic Cell Antigens
[0419] The present invention is based in part on the discovery that
antibodies, antigen-binding fragments thereof, and ligands capable
of binding CD45, CD135, CD34, CD90, or CD110 can be used as
therapeutic agents to (i) directly treat cancers and autoimmune
diseases characterized by cells that express one or more of these
antigens and (ii) promote the engraftment of transplanted
hematopoietic stem cells in a patient in need of transplant
therapy. These therapeutic activities can be caused, for instance,
by the binding of antibodies, antigen-binding fragments thereof,
and/or ligands to one or more of the foregoing antigens expressed
on the surface of a cell, such as a cancer cell, autoimmune cell,
or hematopoietic stem cell and subsequently inducing cell death.
The depletion of endogenous hematopoietic stem cells can provide a
niche toward which transplanted hematopoietic stem cells can home,
and subsequently establish productive hematopoiesis. In this way,
transplanted hematopoietic stem cells may successfully engraft in a
patient, such as human patient suffering from a stem cell disorder
described herein. The following sections describe each of these
antigens as well as antibodies and antigen-binding fragments
thereof capable of binding these targets.
Anti-CD45 Antibodies
[0420] Antibodies and antigen-binding fragments capable of binding
human CD45 (mRNA NCBI Reference Sequence: NM_080921.3, Protein NCBI
Reference Sequence: NP_563578.2), including those capable of
binding the isoform CD45RO, can be used in conjunction with the
compositions and methods disclosed herein, such as to promote
engraftment of hematopoietic stem cell grafts in a patient in need
of hematopoietic stem cell transplant therapy. Multiple isoforms of
CD45 arise from the alternative splicing of 34 exons in the primary
transcript. Splicing of exons 4, 5, 6, and potentially 7 give rise
to multiple CD45 variations. Selective exon expression is observed
in the CD45 isoforms described in Table 1, below.
TABLE-US-00008 TABLE 1 Exon expression in various CD45 isoforms
CD45 Isoform Exon Expression Pattern CD45RA Expresses exon 4 only
CD45RB Expresses exon 5 only CD45RC Expresses exon 6 only CD45RO
Does not express exons 4-6
[0421] Alternative splicing can result in individual exons or
combinations of exons expressed in various isoforms of the CD45
protein (for example, CD45RA, CD45RAB, CD45RABC). In contrast,
CD45RO lacks expression of exons 4-6 and is generated from a
combination of exons 1-3 and 7-34. There is evidence that exon 7
can also be excluded from the protein, resulting in splicing
together of exons 1-3 and 8-34. This protein, designated E3-8, has
been detected at the mRNA level but has not been currently
identified by flow cytometry.
[0422] CD45RO is currently the only known CD45 isoform expressed on
hematopoietic stem cells. CD45RA and CD45RABC have not been
detected or are excluded from the phenotype of hematopoietic stem
cells. There is evidence from studies conducted in mice that CD45RB
is expressed on fetal hematopoietic stem cells, but it is not
present on adult bone marrow hematopoietic stem cells. Notably,
CD45RC has a high rate of polymorphism in exon 6 found within Asian
populations (a polymorphism at exon 6 in CD45RC is found in
approximately 25% of the Japanese population). This polymorphism
leads to high expression of CD45RO and decreased levels of CD45RA,
CD45RB, and CD45RC. Additionally, CD45RA variants (such as CD45RAB
and CD45RAC) exhibit a polymorphism in exon 4 that has been
associated with autoimmune disease.
[0423] The presence of CD45RO on hematopoietic stem cells and its
comparatively limited expression on other immune cells (such as T
and B lymphocyte subsets and various myeloid cells) renders CD45RO
a particularly well-suited target for conditioning therapy for
patients in need of a hematopoietic stem cell transplant. As CD45RO
only lacks expression of exons 4, 5, and 6, its use as an immunogen
enables the screening of pan CD45 Abs and CD45RO-specific
antibodies.
[0424] Anti-CD45 antibodies that can be used in conjunction with
the patient conditioning methods described herein include, for
example, the anti-CD45 antibody clone HI30, which is commercially
available from BIOLEGEND.RTM. (San Diego, Calif.), as well as
humanized variants thereof. Humanization of antibodies can be
performed by replacing framework residues and constant region
residues of a non-human antibody with those of a germline human
antibody according to procedures known in the art (as described,
for instance, in Example 7, below). Additional anti-CD45 antibodies
that can be used in conjunction with the methods described herein
include the anti-CD45 antibodies ab10558, EP322Y, MEM-28, ab10559,
0.N.125, F10-89-4, Hle-1, 2611, YTH24.5, PD7/26/16, F10-89-4, 1B7,
ab154885, B-A11, phosphor S1007, ab170444, EP350, Y321, GA90, D3/9,
X1 6/99, and LT45, which are commercially available from ABCAM.RTM.
(Cambridge, Mass.), as well as humanized variants thereof. Further
anti-CD45 antibodies that may be used in conjunction with the
patient conditioning procedures described herein include anti-CD45
antibody HPA000440, which is commercially available from
SIGMA-ALDRICH.RTM. (St. Louis, Mo.), and humanized variants
thereof. Additional anti-CD45 antibodies that can be used in
conjunction with the patient conditioning methods described herein
include murine monoclonal antibody BC8, which is described, for
instance, in Matthews et al., Blood 78:1864-1874, 1991, the
disclosure of which is incorporated herein by reference as it
pertains to anti-CD45 antibodies, as well as humanized variants
thereof. Further anti-CD45 antibodies that can be used in
conjunction with the methods described herein include monoclonal
antibody YAML568, which is described, for instance, in Glatting et
al., J. Nucl. Med. 8:1335-1341, 2006, the disclosure of which is
incorporated herein by reference as it pertains to anti-CD45
antibodies, as well as humanized variants thereof. Additional
anti-CD45 antibodies that can be used in conjunction with the
patient conditioning procedures described herein include monoclonal
antibodies YTH54.12 and YTH25.4, which are described, for instance,
in Brenner et al., Ann. N.Y. Acad. Sci. 996:80-88, 2003, the
disclosure of which is incorporated herein by reference as it
pertains to anti-CD45 antibodies, as well as humanized variants
thereof. Additional anti-CD45 antibodies for use with the patient
conditioning methods described herein include UCHL1, 2H4, SN130,
MD4.3, MBI, and MT2, which are described, for instance, in Brown et
al., Immunology 64:331-336, 1998, the disclosure of which is
incorporated herein by reference as it pertains to anti-CD45
antibodies, as well as humanized variants thereof. Additional
anti-CD45 antibodies that can be used in conjunction with the
methods described herein include those produced and released from
American Type Culture Collection (ATCC) Accession Nos. RA3-6132,
RA3-2C2, and TIB122, as well as monoclonal antibodies C363.16A, and
13/2, which are described, for instance, in Johnson et al., J. Exp.
Med. 169:1179-1184, 1989, the disclosure of which is incorporated
herein by reference as it pertains to anti-CD45 antibodies, as well
as humanized variants thereof. Further anti-CD45 antibodies that
can be used in conjunction with the patient conditioning methods
described herein include the monoclonal antibodies AHN-12.1,
AHN-12, AHN-12.2, AHN-12.3, AHN-12.4, HLe-1, and KC56(T200), which
are described, for instance, in Harvath et al., J. Immunol.
146:949-957, 1991, the disclosure of which is incorporated herein
by reference as it pertains to anti-CD45 antibodies, as well as
humanized variants thereof.
[0425] Additional anti-CD45 antibodies that can be used in
conjunction with the patient conditioning methods described herein
include those described, for example, in U.S. Pat. No. 7,265,212
(which describes, e.g., anti-CD45 antibodies 39E11, 16C9, and 1G10,
among other clones); U.S. Pat. No. 7,160,987 (which describe, e.g.,
anti-CD45 antibodies produced and released by ATCC Accession No.
HB-11873, such as monoclonal antibody 6G3); and U.S. Pat. No.
6,099,838 (which describes, e.g., anti-CD45 antibody MT3, as well
as antibodies produced and released by ATCC Accession Nos. HB220
(also designated MB23G2) and HB223), as well as US 2004/0096901 and
US 2008/0003224 (which describes, e.g., anti-CD45 antibodies
produced and released by ATCC Accession No. PTA-7339, such as
monoclonal antibody 17.1), the disclosures of each of which are
incorporated herein by reference as they pertain to anti-CD45
antibodies.
[0426] Further anti-CD45 antibodies that can be used in conjunction
with the patient conditioning methods described herein include
antibodies produced and released from ATCC Accession Nos. MB4B4,
MB23G2, 14.8, GAP 8.3, 74-9-3, I/24.D6, 9.4, 4B2, M1/9.3.4.HL.2, as
well as humanized and/or affinity-matured variants thereof.
Affinity maturation can be performed, for instance, using in vitro
display techniques described herein or known in the art, such as
phage display, as described in Example 6, below.
[0427] Additional anti-CD45 antibodies that can be used in
conjunction with the patient conditioning methods described herein
include anti-CD45 antibody T29/33, which is described, for
instance, in Morikawa et al., Int. J. Hematol. 54:495-504, 1991,
the disclosure of which is incorporated herein by reference as it
pertains to anti-CD45 antibodies.
[0428] The disclosures of each of the foregoing publications are
incorporated herein by reference as they pertain to anti-CD45
antibodies. Antibodies and antigen-binding fragments that may be
used in conjunction with the compositions and methods described
herein include the above-described antibodies and antigen-binding
fragments thereof, as well as humanized variants of those non-human
antibodies and antigen-binding fragments described above and
antibodies or antigen-binding fragments that bind the same epitope
as those described above, as assessed, for instance, by way of a
competitive CD45 binding assay.
Anti-CD135 Antibodies
[0429] The present invention is based in part on the discovery that
antibodies and antigen-binding fragments thereof capable of binding
CD135 (also referred to as Flk2 and Flt3) can be used as
therapeutic agents to treat cancers and autoimmune diseases
directly, as well as to promote the engraftment of transplanted
hematopoietic stem cells in a patient in need of transplant
therapy. Additionally, it has been discovered that ligands that
bind CD135, such as human Flt3 ligand, can be used to treat cancers
and autoimmune diseases directly, as well as to promote the
engraftment of transplanted hematopoietic stem cells in a patient
in need of transplant therapy. These ligands, such as human Flt3
ligand, can be covalently bound to an effector domain, such as an
Fc domain, for instance, in order to promote antibody-dependent
cell-mediated cytotoxicity (ADCC).
[0430] Hematopoietic stem cells have been shown to express CD135,
as this antigen is a receptor tyrosine kinase that has been
reported to promote the up-regulation of Mcl-1, which in turn
modulates cell survival throughout hematopoiesis (see, e.g.,
Kikushige et al., J. Immunol. 180:7358-7367, 2008, the disclosure
of which is incorporated herein by reference as it pertains to the
expression of CD135 by hematopoietic stem cells). Antibodies,
antigen-binding fragments thereof, and ligands capable of binding
this cell-surface antigen can be identified using techniques known
in the art and described herein, such as by immunization,
computational modeling techniques, and in vitro selection methods,
such as the phage display and cell-based display platforms
described below.
[0431] Anti-CD135 antibodies that can be used in conjunction with
the patient conditioning methods disclosed herein include those
that have one or more, or all, of the following CDRs:
TABLE-US-00009 (SEQ ID NO: 1) a. a CDR-H1 having the amino acid
sequence SYYMH; (SEQ ID NO: 2) b. a CDR-H2 having the amino acid
sequence IINPSGGSTSYAQKFQG; (SEQ ID NO: 3) c. a CDR-H3 having the
amino acid sequence GVGAHDAFDI or (SEQ ID NO: 4) VVAAAVADY; (SEQ ID
NO: 5) d. a CDR-L1 having the amino acid sequence RSSQSLLHSNGNNYLD
or (SEQ ID NO: 6) RSSQSLLHSNGYNYLD; (SEQ ID NO: 7) e. a CDR-L2
having the amino acid sequence LGSNRAS; and (SEQ ID NO: 8) f. a
CDR-L3 having the amino acid sequence MQGTHPAIS or (SEQ ID NO: 9)
MQSLQTPFT.
[0432] Additional Anti-CD135 antibodies that can be used in
conjunction with the patient conditioning methods disclosed herein
include those that have one or more, or all, of the following
CDRs:
TABLE-US-00010 (SEQ ID NO: 10) a. a CDR-H1 having the amino acid
sequence SYAIS; (SEQ ID NO: 11) b. a CDR-H2 having the amino acid
sequence GIIPIFGTANYAQKFQG; (SEQ ID NO: 12) c. a CDR-H3 having the
amino acid sequence FALFGFREQAFDI; (SEQ ID NO: 13) d. a CDR-L1
having the amino acid sequence RASQSISSYLN; (SEQ ID NO: 14) e. a
CDR-L2 having the amino acid sequence AASSLQS; and (SEQ ID NO: 15)
f. a CDR-L3 having the amino acid sequence QQSYSTPFT.
[0433] The foregoing antibodies are described, e.g., in U.S. Pat.
No. 8,071,099, the disclosure of which is incorporated herein by
reference as it pertains to anti-CD135 antibodies and
antigen-binding fragments thereof. The antibodies and fragments
thereof disclosed in U.S. Pat. No. 8,071,099, such as IMC-EB10 and
IMC-NC7, can be used in conjunction with the methods disclosed
herein.
[0434] In addition to the above, anti-CD135 antibodies that can be
used in conjunction with the patient conditioning methods described
herein include those that have one or more, or all, of the
following CDRs:
TABLE-US-00011 (SEQ ID NO: 22) a. a CDR-H1 having the amino acid
sequence SYWMH; (SEQ ID NO: 23) b. a CDR-H2 having the amino acid
sequence EIDPSDSYKDYNQKFK; (SEQ ID NO: 24) c. a CDR-H3 having the
amino acid sequence AITTTPFDF; (SEQ ID NO: 25) d. a CDR-L1 having
the amino acid sequence RASQSISNNLH; (SEQ ID NO: 26) e. a CDR-L2
having the amino acid sequence YASQSIS; and (SEQ ID NO: 27) f. a
CDR-L3 having the amino acid sequence QQSNTWPYT.
[0435] Additional Anti-CD135 antibodies that can be used in
conjunction with the patient conditioning methods disclosed herein
include those that have one or more, or all, of the following
CDRs:
TABLE-US-00012 (SEQ ID NO: 28) a. a CDR-H1 having the amino acid
sequence NYGLH; (SEQ ID NO: 29) b. a CDR-H2 having the amino acid
sequence VIWSGGSTDYNAAFIS; (SEQ ID NO: 30) c. a CDR-H3 having the
amino acid sequence KGGIYYANHYYAMDY; (SEQ ID NO: 31) d. a CDR-L1
having the amino acid sequence KSSQSLLNSGNQKNYM; (SEQ ID NO: 32) e.
a CDR-L2 having the amino acid sequence GASTRES; and (SEQ ID NO:
33) f. a CDR-L3 having the amino acid sequence QNDHSYPLT.
[0436] The foregoing antibodies are described, e.g., in U.S. Pat.
No. 9,023,996, the disclosure of which is incorporated herein by
reference as it pertains to anti-CD135 antibodies and
antigen-binding fragments thereof. The antibodies and fragments
thereof disclosed in U.S. Pat. No. 9,023,996, such as clones 4G8
and BV10, can be used in conjunction with the methods disclosed
herein.
[0437] Other anti-CD135 antibodies that can be used in conjunction
with the patient conditioning methods described herein include, for
instance, antibodies produced and released by American Type Culture
Collection (ATCC) Accession No. ATCC HB 11,557, which is described,
for example, in U.S. Pat. No. 5,635,388, the disclosure of which is
incorporated herein by reference as it pertains to anti-CD135
antibodies. Additional anti-CD135 antibodies that can be used in
conjunction with the patient conditioning methods described herein
include, for instance, antibodies produced and released by
hybridoma cells that are deposited at the International Depositary
Authority DSMZ-Deutsche Sammlung von Mikroorganismen and
Zellkulturen GmbH Mascheroder Weg 1 b, D-38124 Braunschweig,
Germany, as of Dec. 19, 1995 under No. DSM ACC2249 in accordance
with the Budapest Treaty, and designated 4G8B4B 12, which are
described, for example, in U.S. Pat. No. 6,156,882, the disclosure
of which is incorporated herein by reference as it pertains to
anti-CD135 antibodies. Additional anti-CD135 antibodies that can be
used in conjunction with the patient conditioning methods described
herein include, for instance, antibodies produced and released by
hybridoma cells that were deposited on Dec. 19, 1995 under No. DSM
ACC2248 at the German Collection of Microorganisms and Cell
Cultures Ltd. in accordance with the Budapest Treaty, and
designated BV10A4H2, which are described, for example, in U.S. Pat.
No. 5,777,084, the disclosure of which is incorporated herein by
reference as it pertains to anti-CD135 antibodies. Additional
anti-CD135 antibodies that can be used in conjunction with the
patient-conditioning methods described herein include, for
instance, antibodies produced and released by ATCC Accession No.
FTA-4089, which is described, for example, in U.S. Pat. No.
7,183,385, the disclosure of which is incorporated herein by
reference as it pertains to anti-CD135 antibodies. Additional
anti-CD135 antibodies are described in U.S. Pat. No. 5,548,065
(including, for instance, anti-CD135 antibodies, antigen-binding
fragments thereof, and ligands produced and released by ATCC
Accession Nos. CRL 10907, CRL 10935, CRL 10936, and CRL 11005) and
U.S. Pat. No. 9,109,227, as well as in US 2009/0054358, the
disclosures of each of which are incorporated herein by reference
as they pertain to anti-CD135 antibodies. Other anti-CD135
antibodies for use in conjunction with the methods described herein
include antibodies produced by the rabbit hybridoma cell line
C24D9, and humanized variants thereof, as described, for example,
in US 2010/0093008, the disclosure of which is incorporated herein
by reference as it pertains to anti-CD135 antibodies.
[0438] In addition to the above, antibodies and antigen-binding
fragments thereof capable of binding CD135 include those described
in WO 1995/007348, the disclosure of which is incorporated herein
by reference as it pertains to anti-CD135 antibodies. For instance,
humanized variants of rat antibody 19A, produced and released by
ATCC Accession No. HB 11442, humanized variants of antibody 23H,
produced and released by ATCC Accession No. HB 11443, humanized
variants of antibody 2A13, produced and released by ATCC Accession
No. 11444, humanized variants of murine antibody 6J11, produced and
released by ATCC Accession No. HB 11445, and humanized variants of
rat antibody 7 IE, as described in WO 1995/007348, can be used in
conjunction with the methods described herein to condition a
patient prior to hematopoietic stem cell transplant therapy.
[0439] In addition to antibodies and antigen-binding fragments,
CD135 ligands, such as human Flt3 ligand, can be administered to a
patient according to the methods described herein, such as to treat
a cancer it autoimmune disorder or to condition a patient prior to
hematopoietic stem cell transplant therapy. For instance, CD135
ligands, such as human Flt3 ligand, can be conjugated to a
cytotoxin (e.g., according to the methods described below or known
in the art) or another effector molecule, such as an Fc domain.
CD135 ligands for use with the methods described herein include,
for example, human Flt3 ligand-IgG1 Fc conjugates, human Flt3
ligand-IgG2 Fc conjugates, human Flt3 ligand-IgG3 Fc conjugates,
and human Flt3 ligand-IgG4 Fc conjugates, such as those produced by
ADIPOGEN.RTM. (San Diego, Calif.), product number AG-40B-0119.
[0440] The disclosures of each of the foregoing publications are
incorporated herein by reference as they pertain to anti-CD135
antibodies. Antibodies and antigen-binding fragments that may be
used in conjunction with the compositions and methods described
herein include the above-described antibodies and antigen-binding
fragments thereof, as well as humanized variants of those non-human
antibodies and antigen-binding fragments described above and
antibodies or antigen-binding fragments that bind the same epitope
as those described above, as assessed, for instance, by way of a
competitive CD135 binding assay.
Anti-CD34 Antibodies
[0441] Antibodies and antigen-binding fragments capable of binding
human CD34 (mRNA NCBI Reference Sequence: NM_001025109.1, Protein
NCBI Reference Sequence: NP_001020280.1) can be used in conjunction
with the compositions and methods described herein. An
understanding of the polymorphisms affecting the coding region or
extracellular domain of CD34 in a significant percentage of the
population has not yet been established. There are two isoforms of
CD34 that differ in the length of their cytoplasmic tail (long and
short). Recently, the long isoform was used to generate a stable
cell line expressing CD34 that could be used as an immunogen (see,
e.g., Adv. Pharm. Bull. 5:69-75, 2015). CD34, such as the long
isoform of CD34, can be used as an immunogen in order to identify
antibodies and antigen-binding fragments thereof capable of binding
CD34 and suitable, for example, for the treatment of cancers and
autoimmune diseases, as well as for use as a conditioning agent
prior to hematopoietic stem cell transplant therapy.
[0442] CD34 antibodies that can be used in conjunction with the
methods described herein include, without limitation, antibodies
produced and released from ATCC Accession No. AC133.1 and HB 12346,
as described, for example, in U.S. Pat. No. 5,843,633.
[0443] The disclosure of the foregoing publication is incorporated
herein by reference as it pertains to anti-CD34 antibodies.
Antibodies and antigen-binding fragments that may be used in
conjunction with the compositions and methods described herein
include the above-described antibodies and antigen-binding
fragments thereof, as well as humanized variants of those non-human
antibodies and antigen-binding fragments described above and
antibodies or antigen-binding fragments that bind the same epitope
as those described above, as assessed, for instance, by way of a
competitive CD34 binding assay.
Anti-CD90 Antibodies
[0444] Antibodies and antigen-binding fragments thereof capable of
binding human CD90 can be used in conjunction with the compositions
and methods described herein. Hematopoietic stem cells have been
shown to express CD90, as Lin-CD34+CD38-CD90+CD45RA- fractions of
human cord blood are enriched have been reported to contain
hematopoietic stem cells (see, e.g., Majeti et al., Cell Stem Cell
1:635-645, 2007, the disclosure of which is incorporated herein by
reference as it pertains to the expression of CD90 by hematopoietic
stem cells). Antibodies and antigen-binding fragments capable of
binding this cell-surface antigen can be raised using techniques
known in the art and described herein, such as by immunization and
in vitro selection techniques including phage display and
cell-based display platforms described below.
[0445] Anti-CD90 antibodies that can be used in conjunction with
the methods described herein include, without limitation, EPR3132,
EPR3133, AF-9, 5E10, F15-42-1, 7E1B11, ab189367, aTHy-1A1,
ab106934, and ab110477, which are commercially available from
ABCAM.RTM. (Cambridge, Mass.), as well as humanized variants
thereof.
[0446] Antibodies and antigen-binding fragments that may be used in
conjunction with the compositions and methods described herein
include the above-described antibodies and antigen-binding
fragments thereof, as well as humanized variants of those non-human
antibodies and antigen-binding fragments described above and
antibodies or antigen-binding fragments that bind the same epitope
as those described above, as assessed, for instance, by way of a
competitive CD90 binding assay.
Anti-CD110 Antibodies
[0447] Antibodies and antigen-binding fragments thereof capable of
binding human CD110 (also referred to as c-mpl and thrombopoietic
receptor (TPO)) can be used in conjunction with the compositions
and methods described herein. Hematopoietic stem cells have been
shown to express CD110, and the expression of CD110 has been
correlated with hematopoietic stem cell functional potential and
decreases as hematopoietic stem cells differentiate and progress
through the hematopoietic lineage (see, e.g., Ninos et al., J.
Transl. Med. 4:9, 2006, the disclosure of which is incorporated
herein by reference as it pertains to the expression of CD110 by
hematopoietic stem cells). Antibodies and antigen-binding fragments
capable of binding this cell-surface antigen can be raised using
techniques known in the art and described herein, such as by
immunization and in vitro selection techniques including phage
display and cell-based display platforms described below.
[0448] Anti-CD110 antibodies that can be used in conjunction with
the methods described herein, such as the cancer treatment methods,
autoimmune disease treatment methods, and patient conditioning
methods described herein include clones BAH-1, 1.78.1, and 1.6.1,
as described, for example, in De Gobbi et al., Epigenetics
Chromatin 4:9, 2011; Erickson-Miller et al., BMC Cancer 12:405,
2012; and Petit Cocault et al., Exp. Hematol. 44:297-302, 2016, the
disclosures of each of which are incorporated herein by reference
as they pertain to anti-CD110 antibodies.
[0449] The disclosures of each of the foregoing publications are
incorporated herein by reference as they pertain to anti-CD110
antibodies. Antibodies and antigen-binding fragments that may be
used in conjunction with the compositions and methods described
herein include the above-described antibodies and antigen-binding
fragments thereof, as well as humanized variants of those non-human
antibodies and antigen-binding fragments described above and
antibodies or antigen-binding fragments that bind the same epitope
as those described above, as assessed, for instance, by way of a
competitive CD110 binding assay.
Additional Antibodies and Antigen-Binding Fragments Thereof
[0450] Antibodies and ligands for use in conjunction with the
methods described herein include variants of those antibodies
described above, such as antibody fragments that contain or lack an
Fc domain, as well as humanized variants of non-human antibodies
described herein and antibody-like protein scaffolds (e.g.,
.sup.10Fn3 domains) containing one or more, or all, of the CDRs or
equivalent regions thereof of an antibody, antibody fragment, or
ligand described herein. Exemplary antigen-binding fragments of the
foregoing antibodies include a dual-variable immunoglobulin domain,
a single-chain Fv molecule (scFv), a diabody, a triabody, a
nanobody, an antibody-like protein scaffold, a Fv fragment, a Fab
fragment, a F(ab').sub.2 molecule, and a tandem di-scFv, among
others.
Methods of Combination Therapy
[0451] Using the methods described herein, a physician of skill in
the art may treat a patient for cancer or an autoimmune disease or
may condition a patient prior to hematopoietic stem cell transplant
therapy by administering one or more antibodies, antigen-binding
fragments thereof, or ligands described herein. For instance, a
physician may treat a cancer or autoimmune disease or condition a
patient for hematopoietic stem cell transplant therapy by
administering an antibody, antigen-binding fragment thereof, or
ligand that recognizes and binds CD45 (e.g., CD45RO) alone or in
combination with an antibody, antigen-binding fragment thereof, or
ligand that recognizes and binds CD135, an antibody,
antigen-binding fragment thereof, or ligand that recognizes and
binds CD34, an antibody, antigen-binding fragment thereof, or
ligand that recognizes and binds CD90, and/or an antibody,
antigen-binding fragment thereof, or ligand that recognizes and
binds CD110. Dosing regimens for combination therapy may include
sequential administration of two or more of the above antibodies,
antigen-binding fragments thereof, or ligands, such that a first
antibody, antigen-binding fragment thereof, or ligand is
administered to a patient at a designated time point, and a second
antibody, antigen-binding fragment thereof, or ligand is
subsequently administered to a patient at a later time point, for
example, so as to optimize the pharmacokinetic profile of the
combined therapeutic dosage. Two or more antibodies,
antigen-binding fragments thereof, or ligands that each bind one or
more of the hematopoietic stem cell antigens described herein can
be administered to a patient (e.g., a human patient) concurrently,
may be admixed with one another in a single pharmaceutical
composition, or may be administered at different times (e.g.,
within 1-24 hours of one another, such as within 1 hour, 2 hours, 3
hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10
hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours,
17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23
hours, 24 hours, or more, of one another).
[0452] Antibodies, antigen-binding fragments thereof, and ligands
can be administered to a patient so as to recognize and bind
multiple hematopoietic stem cell antigens using a single antibody.
This can be achieved, for example, using bi-specific and
multi-specific antibodies. For instance, bi-specific antibodies
containing an antigen-binding fragment that selectively binds CD45
(e.g., CD45RO) and another antigen-binding fragment that
selectively binds CD135 can be administered to a patient in need of
hematopoietic stem cell transplant therapy in order to deplete
CD45+ CD135+ cells, thereby creating a vacancy in hematopoietic
tissue (e.g., the bone marrow) that can be filled by an exogenous
hematopoietic stem cell transplant. Similarly, bi-specific
antibodies containing an antigen-binding fragment that selectively
binds CD45 (e.g., CD45RO) and CD34 can be administered to a patient
in order to deplete CD34+ CD45+ cells prior to hematopoietic stem
cell transplant therapy. Bi-specific antibodies containing an
antigen-binding fragment that selectively binds CD45 (e.g., CD45RO)
and CD90 can be similarly administered to a patient, such as a
patient suffering from cancer or an autoimmune disease, or in order
to deplete CD45+CD90+ cells prior to hematopoietic stem cell
transplant therapy. Additionally, bi-specific antibodies containing
an antigen-binding fragment that selectively binds CD45 (e.g.,
CD45RO) and CD110 can be administered to a patient, for instance,
in order to deplete CD45+ CD110+ cells prior to hematopoietic stem
cell transplantation.
[0453] In some embodiments, bi-specific antibodies containing an
antigen-binding fragment that selectively binds CD135 and another
antigen-binding fragment that selectively binds CD34 can be
administered to a patient, such as a patient suffering from cancer,
an autoimmune disease, or in need of hematopoietic stem cell
transplant therapy in order to deplete CD34+ CD135+ cells, thereby
creating a vacancy in hematopoietic tissue (e.g., the bone marrow)
that can be filled by an exogenous hematopoietic stem cell
transplant. Similarly, bi-specific antibodies containing an
antigen-binding fragment that selectively binds CD135 and CD90 can
be administered to a patient in order to deplete CD90+ CD135+ cells
prior to hematopoietic stem cell transplant therapy. Bi-specific
antibodies containing an antigen-binding fragment that selectively
binds CD135 and CD110 can be similarly administered to a patient,
for instance, in order to deplete CD110+ CD135+ cells prior to
hematopoietic stem cell transplant therapy.
[0454] In some embodiments, bi-specific antibodies containing an
antigen-binding fragment that selectively binds CD34 and another
antigen-binding fragment that selectively binds CD90 can be
administered to a patient suffering from cancer, an autoimmune
disease, or in need of hematopoietic stem cell transplant therapy
in order to deplete CD34+ CD90+ cells, thereby creating a vacancy
in hematopoietic tissue (e.g., the bone marrow) that can be filled
by an exogenous hematopoietic stem cell transplant. Similarly,
bi-specific antibodies containing an antigen-binding fragment that
selectively binds CD34 and CD110 can be administered to a patient,
for instance, in order to deplete CD34+ CD110+ cells prior to
hematopoietic stem cell transplant therapy.
[0455] In some embodiments, bi-specific antibodies containing an
antigen-binding fragment that selectively binds CD90 and another
antigen-binding fragment that selectively binds CD110 can be
administered to a patient suffering from cancer, an autoimmune
disease, or in need of hematopoietic stem cell transplant therapy
in order to deplete CD90+ CD110+ cells, thereby creating a vacancy
in hematopoietic tissue (e.g., the bone marrow) that can be filled
by an exogenous hematopoietic stem cell transplant.
Methods of Identifying Antibodies and Ligands
[0456] Methods for high throughput screening of antibody, antibody
fragment, and ligand libraries for molecules capable of binding
CD45 (e.g., CD45RO), CD135, CD34, CD90, and CD110 can be used to
identify and affinity mature antibodies useful for treating
cancers, autoimmune diseases, and conditioning a patient (e.g., a
human patient) in need of hematopoietic stem cell therapy as
described herein. Such methods include in vitro display techniques
known in the art, such as phage display, bacterial display, yeast
display, mammalian cell display, ribosome display, mRNA display,
and cDNA display, among others. The use of phage display to isolate
ligands that bind biologically relevant molecules has been
reviewed, for example, in Felici et al., Biotechnol. Annual Rev.
1:149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct.
26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4:1-20,
1998, the disclosures of each of which are incorporated herein by
reference as they pertain to in vitro display techniques.
Randomized combinatorial peptide libraries have been constructed to
select for polypeptides that bind cell surface antigens as
described in Kay, Perspect. Drug Discovery Des. 2:251-268, 1995 and
Kay et al., Mol. Divers. 1:139-140, 1996, the disclosures of each
of which are incorporated herein by reference as they pertain to
the discovery of antigen-binding molecules. Proteins, such as
multimeric proteins, have been successfully phage-displayed as
functional molecules (see, for example, EP 0349578; EP 4527839; and
EP 0589877, as well as Chiswell and McCafferty, Trends Biotechnol.
10:80-84 1992, the disclosures of each of which are incorporated
herein by reference as they pertain to the use of in vitro display
techniques for the discovery of antigen-binding molecules). In
addition, functional antibody fragments, such as Fab and scFv
fragments, have been expressed in in vitro display formats (see,
for example, McCafferty et al., Nature 348:552-554, 1990; Barbas et
al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et
al., Nature 352:624-628, 1991, the disclosures of each of which are
incorporated herein by reference as they pertain to in vitro
display platforms for the discovery of antigen-binding molecules).
These techniques, among others, can be used to identify and improve
the affinity of antibodies that bind hematopoietic stem cell
antigens (such as CD45 (e.g., CD45RO), CD135, CD34, CD90, and
CD110) that can in turn be used to deplete endogenous hematopoietic
stem cells in a patient (e.g., a human patient) in need of
hematopoietic stem cell transplant therapy.
[0457] In addition to in vitro display techniques, computational
modeling techniques can be used to design and identify antibodies,
antibody fragments, and ligands in silico that bind CD45 (e.g.,
CD45RO), CD135, CD34, CD90, or CD110. For example, using
computational modeling techniques, one of skill in the art can
screen libraries of antibodies, antibody fragments, and ligands in
silico for molecules capable of binding specific epitopes on CD45
(e.g., CD45RO), CD135, CD34, CD90, and CD110, such as extracellular
epitopes of these antigens. The antibodies, antigen-binding
fragments thereof, and ligands identified by these computational
techniques can be used in conjunction with the therapeutic methods
described herein, such as the cancer and autoimmune disease
treatment methods described herein and the patient conditioning
procedures described herein.
[0458] Additional techniques can be used to identify antibodies,
antigen-binding fragments thereof, and ligands that bind CD45
(e.g., CD45RO), CD135, CD34, CD90, and/or CD110 on the surface of a
cell (e.g., a cancer cell, autoimmune cell, or a hematopoietic stem
cell) and that are internalized by the cell, for instance, by
receptor-mediated endocytosis. For example, the in vitro display
techniques described above can be adapted to screen for antibodies,
antigen-binding fragments thereof, and ligands that bind CD45
(e.g., CD45RO), CD135, CD34, CD90, and/or CD110 on the surface of a
cancer cell, autoimmune cell, or hematopoietic stem cell and that
are subsequently internalized. Phage display represents one such
technique that can be used in conjunction with this screening
paradigm. To identify antibodies, fragments thereof, and ligands
that bind one or more of the above targets and are subsequently
internalized by cancer cells, autoimmune cells, or hematopoietic
stem cells, one of skill in the art can adapt the phage display
techniques described, for example, in Williams et al., Leukemia
19:1432-1438, 2005, the disclosure of which is incorporated herein
by reference in its entirety. For example, using mutagenesis
methods known in the art, recombinant phage libraries can be
produced that encode antibodies, antibody fragments, such as scFv
fragments, Fab fragments, diabodies, triabodies, and .sup.10Fn3
domains, among others, or ligands that contain randomized amino
acid cassettes (e.g., in one or more, or all, of the CDRs or
equivalent regions thereof or an antibody or antibody fragment).
The framework regions, hinge, Fc domain, and other regions of the
antibodies or antibody fragments may be designed such that they are
non-immunogenic in humans, for instance, by virtue of having human
germline antibody sequences or sequences that exhibit only minor
variations relative to human germline antibodies.
[0459] Using phage display techniques described herein or known in
the art, phage libraries containing randomized antibodies, antibody
fragments, or ligands covalently bound to the phage particles can
be incubated with CD45 (e.g., CD45RO), CD135, CD34, CD90, and/or
CD110 antigen, for instance, by first incubating the phage library
with blocking agents (such as, for instance, milk protein, bovine
serum albumin, and/or IgG so as to remove phage encoding
antibodies, fragments thereof, or ligands that exhibit non-specific
protein binding and phage that encode antibodies or fragments
thereof that bind Fc domains, and then incubating the phage library
with a population of hematopoietic stem cells. The phage library
can be incubated with the target cells, such as cancer cells,
autoimmune cells, or hematopoietic stem cells for a time sufficient
to allow CD45-specific, CD135-specific, CD34-specific,
CD90-specific, and/or CD110-specific antibodies, antigen-binding
fragments thereof, or ligands to bind cell-surface antigen and to
subsequently be internalized by the cancer cells, autoimmune cells,
or hematopoietic stem cells (e.g., from 30 minutes to 6 hours at
4.degree. C., such as 1 hour at 4.degree. C.). Phage containing
antibodies, fragments thereof, or ligands that do not exhibit
sufficient affinity for one or more of these antigens so as to
permit binding to, and internalization by, cancer cells, autoimmune
cells, or hematopoietic stem cells can subsequently be removed by
washing the cells, for instance, with cold (4.degree. C.) 0.1 M
glycine buffer at pH 2.8. Phage bound to antibodies, fragments
thereof, or ligands that have been internalized by the cancer
cells, autoimmune cells, or hematopoietic stem cells can be
identified, for instance, by lysing the cells and recovering
internalized phage from the cell culture medium. The phage can then
be amplified in bacterial cells, for example, by incubating
bacterial cells with recovered phage in 2.times.YT medium using
methods known in the art. Phage recovered from this medium can then
be characterized, for instance, by determining the nucleic acid
sequence of the gene(s) encoding the antibodies, fragments thereof,
or ligands inserted within the phage genome. The encoded
antibodies, fragments thereof, or ligands can subsequently be
prepared de novo by chemical synthesis (for instance, of antibody
fragments, such as scFv fragments, or ligands) or by recombinant
expression (for instance, of full-length antibodies).
[0460] The internalizing capacity of the prepared antibodies,
fragments thereof, or ligands can be assessed, for instance, using
radionuclide internalization assays known in the art. For example,
antibodies, fragments thereof, or ligands identified using in vitro
display techniques described herein or known in the art can be
functionalized by incorporation of a radioactive isotope, such as
.sup.18F, .sup.75Br, .sup.77Br, .sup.122I, .sup.123I, .sup.124I,
.sup.125I, .sup.129I, .sup.131I, .sup.211At, .sup.67Ga, .sup.111In,
.sup.99Tc, .sup.169Yb, .sup.186Re, .sup.64Cu, .sup.67Cu,
.sup.177Lu, .sup.77As, .sup.72As, .sup.86Y, .sup.90Y, .sup.89Zr,
.sup.212Bi, .sup.213Bi, or .sup.225Ac. For instance, radioactive
halogens, such as .sup.18F, .sup.75Br, .sup.77Br, .sup.122I,
.sup.123I, .sup.124I, .sup.125I, .sup.129I, .sup.131I, .sup.211At,
can be incorporated into antibodies, fragments thereof, or ligands
using beads, such as polystyrene beads, containing electrophilic
halogen reagents (e.g., Iodination Beads, Thermo Fisher Scientific,
Inc., Cambridge, Mass.). Radiolabeled antibodies, fragments
thereof, or ligands can be incubated with cancer cells, autoimmune
cells, or hematopoietic stem cells for a time sufficient to permit
internalization (e.g., from 30 minutes to 6 hours at 4.degree. C.,
such as 1 hour at 4.degree. C.). The cells can then be washed to
remove non-internalized antibodies, fragments thereof, or ligands
(e.g., using cold (4.degree. C.) 0.1 M glycine buffer at pH 2.8).
Internalized antibodies, fragments thereof, or ligands can be
identified by detecting the emitted radiation (e.g.,
.gamma.-radiation) of the resulting cancer cells, autoimmune cells,
or hematopoietic stem cells in comparison with the emitted
radiation (e.g., .gamma.-radiation) of the recovered wash
buffer.
Drug-Antibody Conjugates and Drug-Ligand Conjugates
Cytotoxins
[0461] Antibodies, antigen-binding fragments thereof, and ligands
described herein (e.g., antibodies, antigen-binding fragments, and
ligands that recognize and bind CD45 (such as CD45RO), CD135, CD34,
CD90, and/or CD110) can be conjugated to a cytotoxin, such as
pseudomonas exotoxin A, deBouganin, diphtheria toxin, an amatoxin,
such as .alpha.-amanitin, saporin, maytansine, a maytansinoid, an
auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a
duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine
dimer, an indolinobenzodiazepine, and an indolinobenzodiazepine
dimer, or a variant thereof, or another cytotoxic compound
described herein or known in the art, for example, in order to
treat a cancer or autoimmune disease described herein or to promote
the depletion of endogenous hematopoietic stem cells upon
administration to a patient (e.g., a human patient) in need of
hematopoietic stem cell transplant therapy. In some embodiments,
the cytotoxic molecule is conjugated to an internalizing antibody,
antigen-binding fragment thereof, or ligand, such that following
the cellular uptake of the antibody, antigen-binding fragment, or
ligand, the cytotoxin may access its intracellular target and
mediate endogenous hematopoietic cell death. Cytotoxins suitable
for use with the compositions and methods described herein include
DNA-intercalating agents, (e.g., anthracyclines), agents capable of
disrupting the mitotic spindle apparatus (e.g., vinca alkaloids,
maytansine, maytansinoids, and derivatives thereof), RNA polymerase
inhibitors (e.g., an amatoxin, such as .alpha.-amanitin and
derivatives thereof), agents capable of disrupting protein
biosynthesis (e.g., agents that exhibit rRNA N-glycosidase
activity, such as saporin and ricin A-chain), among others known in
the art.
[0462] In some embodiments, the cytotoxin is an amatoxin or
derivative thereof, such as .alpha.-amanitin, .beta.-amanitin,
.gamma.-amanitin, .epsilon.-amanitin, amanin, amaninamide,
amanullin, amanullinic acid, and proamanullin. For instance, the
antibodies, antigen-binding fragments, and ligands described herein
may be bound to an amatoxin so as to form a conjugate represented
by the formula Ab-Am, wherein Ab is the antibody, antigen-binding
fragment thereof, or ligand, and Am is an amatoxin. In some
embodiments, Am is represented by formula (I)
##STR00018##
[0463] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0464] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0465] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0466] R.sub.3 is H, R.sub.C, or R.sub.D;
[0467] R.sub.4 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0468] R.sub.5 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0469] R.sub.6 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0470] R.sub.7 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0471] R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or
NR.sub.CR.sub.D;
[0472] R.sub.9 is H, OH, OR.sub.C, or OR.sub.D;
[0473] X is --S--, --S(O)--, or --SO.sub.2--;
[0474] R.sub.C is -L-Z;
[0475] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl;
[0476] L is a linker, such as optionally substituted alkylene
(e.g., C.sub.1-C.sub.6 alkylene), optionally substituted
heteroalkylene (C.sub.1-C.sub.6 heteroalkylene), optionally
substituted alkenylene (e.g., C.sub.2-C.sub.6 alkenylene),
optionally substituted heteroalkenylene (e.g., C.sub.2-C.sub.6
heteroalkenylene), optionally substituted alkynylene (e.g.,
C.sub.2-C.sub.6 alkynylene), optionally substituted
heteroalkynylene (e.g., C.sub.2-C.sub.6 heteroalkynylene),
optionally substituted cycloalkylene, optionally substituted
heterocycloalkylene, optionally substituted arylene, or optionally
substituted heteroarylene; and
[0477] Z is a chemical moiety formed from a coupling reaction
between a reactive substituent present on L and a reactive
substituent present within an antibody, antigen-binding fragment
thereof, or ligand that binds CD45 (such as CD45RO), CD135, CD34,
CD90, and/or CD110.
[0478] In some embodiments, Am contains exactly one R.sub.C
substituent.
[0479] In some embodiments, Am is represented by formula (IA)
##STR00019##
[0480] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0481] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0482] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0483] R.sub.3 is H, R.sub.C, or R.sub.D;
[0484] R.sub.4 is H, OH, OR.sub.C, OR.sub.B, R.sub.C, or
R.sub.D;
[0485] R.sub.5 is H, OH, OR.sub.C, OR.sub.B, R.sub.C, or
R.sub.D;
[0486] R.sub.6 is H, OH, OR.sub.C, OR.sub.B, R.sub.C, or
R.sub.D;
[0487] R.sub.7 is H, OH, OR.sub.C, OR.sub.B, R.sub.C, or
R.sub.D;
[0488] R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.B, NHR.sub.C, or
NR.sub.CR.sub.D;
[0489] R.sub.9 is H, OH, OR.sub.C, or OR.sub.D;
[0490] X is --S--, --S(O)--, or --SO.sub.2--;
[0491] R.sub.C is -L-Z;
[0492] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl;
[0493] L is a linker, such as optionally substituted alkylene
(e.g., C.sub.1-C.sub.6 alkylene), optionally substituted
heteroalkylene (C.sub.1-C.sub.6 heteroalkylene), optionally
substituted alkenylene (e.g., C.sub.2-C.sub.6 alkenylene),
optionally substituted heteroalkenylene (e.g., C.sub.2-C.sub.6
heteroalkenylene), optionally substituted alkynylene (e.g.,
C.sub.2-C.sub.6 alkynylene), optionally substituted
heteroalkynylene (e.g., C.sub.2-C.sub.6 heteroalkynylene),
optionally substituted cycloalkylene, optionally substituted
heterocycloalkylene, optionally substituted arylene, or optionally
substituted heteroarylene;
[0494] Z is a chemical moiety formed from a coupling reaction
between a reactive substituent present on L and a reactive
substituent present within an antibody, antigen-binding fragment
thereof, or ligand that binds CD45 (such as CD45RO), CD135, CD34,
CD90, and/or CD110; and
[0495] wherein Am contains exactly one R.sub.C substituent.
[0496] In some embodiments, Am is represented by formula (IB)
##STR00020##
[0497] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0498] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0499] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form an optionally substituted
5-membered heterocyclolalkyl group;
[0500] R.sub.3 is H, R.sub.C, or R.sub.D;
[0501] R.sub.4 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0502] R.sub.5 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0503] R.sub.6 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0504] R.sub.7 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0505] R.sub.8 is OH, NH.sub.2, OR.sub.C, OR.sub.D, NHR.sub.C, or
NR.sub.CR.sub.D;
[0506] R.sub.9 is H, OH, OR.sub.C, or OR.sub.D;
[0507] X is --S--, --S(O)--, or --SO.sub.2--;
[0508] R.sub.C is -L-Z;
[0509] R.sub.D is optionally substituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), optionally substituted heteroalkyl (e.g.,
C.sub.1-C.sub.6 heteroalkyl), optionally substituted alkenyl (e.g.,
C.sub.2-C.sub.6 alkenyl), optionally substituted heteroalkenyl
(e.g., C.sub.2-C.sub.6 heteroalkenyl), optionally substituted
alkynyl (e.g., C.sub.2-C.sub.6 alkynyl), optionally substituted
heteroalkynyl (e.g., C.sub.2-C.sub.6 heteroalkynyl), optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, or optionally substituted
heteroaryl;
[0510] L is a linker, such as optionally substituted alkylene
(e.g., C.sub.1-C.sub.6 alkylene), optionally substituted
heteroalkylene (C.sub.1-C.sub.6 heteroalkylene), optionally
substituted alkenylene (e.g., C.sub.2-C.sub.6 alkenylene),
optionally substituted heteroalkenylene (e.g., C.sub.2-C.sub.6
heteroalkenylene), optionally substituted alkynylene (e.g.,
C.sub.2-C.sub.6 alkynylene), optionally substituted
heteroalkynylene (e.g., C.sub.2-C.sub.6 heteroalkynylene),
optionally substituted cycloalkylene, optionally substituted
heterocycloalkylene, optionally substituted arylene, or optionally
substituted heteroarylene;
[0511] Z is a chemical moiety formed from a coupling reaction
between a reactive substituent present on L and a reactive
substituent present within an antibody, antigen-binding fragment
thereof, or ligand that binds CD45 (such as CD45RO), CD135, CD34,
CD90, and/or CD110; and
[0512] wherein Am contains exactly one R.sub.C substituent.
[0513] In some embodiments, R.sub.A and R.sub.B, together with the
oxygen atoms to which they are bound, combine to form:
##STR00021##
[0514] wherein Y is selected from O, S, NR.sub.E, and
CR.sub.ER.sub.E', and
[0515] R.sub.E and R.sub.E' are each independently optionally
substituted C.sub.1-C.sub.6 alkylene-R.sub.C, optionally
substituted C.sub.1-C.sub.6 heteroalkylene-R.sub.C, optionally
substituted C.sub.2-C.sub.6 alkenylene-R.sub.C, optionally
substituted C.sub.2-C.sub.6 heteroalkenylene-R.sub.C, optionally
substituted C.sub.2-C.sub.6 alkynylene-R.sub.C, optionally
substituted C.sub.2-C.sub.6 heteroalkynylene-R.sub.C, optionally
substituted cycloalkylene-R.sub.C, optionally substituted
heterocycloalkylene-R.sub.C, optionally substituted
arylene-R.sub.C, or optionally substituted
heteroarylene-R.sub.C.
[0516] In some embodiments, Am is represented by formula (IA) or
formula (IB),
[0517] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0518] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0519] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form:
##STR00022##
[0520] R.sub.3 is H or R.sub.C;
[0521] R.sub.4 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0522] R.sub.5 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0523] R.sub.6 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0524] R.sub.7 is H, OH, OR.sub.C, OR.sub.D, R.sub.C, or
R.sub.D;
[0525] R.sub.8 is OH, NH.sub.2, OR.sub.C, or NHR.sub.C;
[0526] R.sub.9 is H or OH; and
[0527] wherein R.sub.C and R.sub.D are each as defined above.
[0528] In some embodiments, Am is represented by formula (IA) or
formula (IB),
[0529] wherein R.sub.1 is H, OH, OR.sub.A, or OR.sub.C;
[0530] R.sub.2 is H, OH, OR.sub.B, or OR.sub.C;
[0531] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form:
##STR00023##
[0532] R.sub.3 is H or R.sub.C;
[0533] R.sub.4 and R.sub.5 are each independently H, OH, OR.sub.C,
R.sub.C, or OR.sub.D;
[0534] R.sub.6 and R.sub.7 are each H;
[0535] R.sub.8 is OH, NH.sub.2, OR.sub.C, or NHR.sub.C;
[0536] R.sub.9 is H or OH; and
[0537] wherein R.sub.C is as defined above.
[0538] In some embodiments, Am is represented by formula (IA) or
formula (IB),
[0539] wherein R.sub.1 is H, OH, or OR.sub.A;
[0540] R.sub.2 is H, OH, or OR.sub.B;
[0541] R.sub.A and R.sub.B, together with the oxygen atoms to which
they are bound, combine to form:
##STR00024##
[0542] R.sub.3, R.sub.4, R.sub.6, and R.sub.7 are each H;
[0543] R.sub.5 is OR.sub.C;
[0544] R.sub.8 is OH or NH.sub.2;
[0545] R.sub.9 is H or OH; and
[0546] wherein R.sub.C is as defined above. Such amatoxin
conjugates are described, for example, in US Patent Application
Publication No. 2016/0002298, the disclosure of which is
incorporated herein by reference in its entirety.
[0547] In some embodiments, Am is represented by formula (IA) or
formula (IB),
[0548] wherein R.sub.1 and R.sub.2 are each independently H or
OH;
[0549] R.sub.3 is R.sub.C;
[0550] R.sub.4, R.sub.6, and R.sub.7 are each H;
[0551] R.sub.5 is H, OH, or OC.sub.1-C.sub.6 alkyl;
[0552] R.sub.8 is OH or NH.sub.2;
[0553] R.sub.9 is H or OH; and
[0554] wherein R.sub.C is as defined above. Such amatoxin
conjugates are described, for example, in US Patent Application
Publication No. 2014/0294865, the disclosure of which is
incorporated herein by reference in its entirety.
[0555] In some embodiments, Am is represented by formula (IA) or
formula (IB),
[0556] wherein R.sub.1 and R.sub.2 are each independently H or
OH;
[0557] R.sub.3, R.sub.6, and R.sub.7 are each H;
[0558] R.sub.4 and R.sub.5 are each independently H, OH, OR.sub.C,
or R.sub.C;
[0559] R.sub.8 is OH or NH.sub.2;
[0560] R.sub.9 is H or OH; and
[0561] wherein R.sub.C is as defined above. Such amatoxin
conjugates are described, for example, in US Patent Application
Publication No. 2015/0218220, the disclosure of which is
incorporated herein by reference in its entirety.
[0562] In some embodiments, Am is represented by formula (IA) or
formula (IB),
[0563] wherein R.sub.1 and R.sub.2 are each independently H or
OH;
[0564] R.sub.3, R.sub.6, and R.sub.7 are each H;
[0565] R.sub.4 and R.sub.5 are each independently H or OH;
[0566] R.sub.8 is OH, NH.sub.2, OR.sub.C, or NHR.sub.C;
[0567] R.sub.9 is H or OH; and
[0568] wherein R.sub.C is as defined above. Such amatoxin
conjugates are described, for example, in U.S. Pat. Nos. 9,233,173
and 9,399,681, as well as in US 2016/0089450, the disclosures of
each of which are incorporated herein by reference in their
entirety.
[0569] Additional amatoxins that may be used for conjugation to an
antibody, antigen-binding fragment thereof, or ligand in accordance
with the compositions and methods described herein are described,
for example, in WO 2016/142049; WO 2016/071856; and WO 2017/046658,
the disclosures of each of which are incorporated herein by
reference in their entirety.
[0570] In some embodiments, Am is represented by formula (II),
##STR00025##
[0571] wherein X is S, SO, or SO.sub.2;
[0572] R.sub.1 is H or a linker covalently bound to the antibody or
antigen-binding fragment thereof;
[0573] R.sub.2 is H or a linker covalently bound to the antibody or
antigen-binding fragment thereof; and
[0574] wherein when R.sub.1 is H, R.sub.2 is the linker, and when
R.sub.2 is H, R.sub.1 is the linker.
[0575] Antibodies, antigen-binding fragments, and ligands for use
with the compositions and methods described herein can be
conjugated to an amatoxin, such as .alpha.-amanitin or a variant
thereof, using conjugation techniques known in the art or described
herein. For instance, antibodies, antigen-binding fragments
thereof, and ligands that recognize and bind CD45 (such as CD45RO),
CD135, CD34, CD90, and/or CD110 can be conjugated to an amatoxin,
such as .alpha.-amanitin or a variant thereof, as described in US
2015/0218220, the disclosure of which is incorporated herein by
reference as it pertains, for example, to amatoxins, such as
.alpha.-amanitin and variants thereof, as well as covalent linkers
that can be used for covalent conjugation.
[0576] Exemplary antibody-drug and ligand-drug conjugates useful in
conjunction with the methods described herein may be formed by the
reaction of an antibody, antigen-binding fragment thereof, or
ligand with an amatoxin that is conjugated to a linker containing a
substituent suitable for reaction with a reactive residue on the
antibody, antigen-binding fragment thereof, or ligand. Amatoxins
that are conjugated to a linker containing a substituent suitable
for reaction with a reactive residue on the antibody,
antigen-binding fragment thereof, or ligand described herein
include, without limitation,
7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin;
7'C-(4-(6-(maleimido)hexanamido)piperidin-1-yl)-amatoxin;
7'C-(4-(6-(6-(maleimido)hexanamido)hexanoyl)piperazin-1-yl)-amatoxin;
7'C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)piperazin-1-yl)-amatoxin-
;
7'C-(4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazi-
n-1-yl)-amatoxin;
7'C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;
7'C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)-am-
atoxin;
7'C-(4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piper-
idin-1-yl)-amatoxin;
7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethy-
l)piperidin-1-yl)-amatoxin;
7'C-(4-(2-(3-carboxypropanamido)ethyl)piperidin-1-yl)-amatoxin;
7'C-(4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)-amatoxin;
7'C-(4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)-am-
atoxin;
7'C-(4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)-amatoxin;
7'C-(4-(2-(maleimido)acetyl)piperazin-1-yl)-amatoxin;
7'C-(4-(3-(maleimido)propanoyl)piperazin-1-yl)-amatoxin;
7'C-(4-(4-(maleimido)butanoyl)piperazin-1-yl)-amatoxin;
7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethy-
l)piperidin-1-yl)-amatoxin;
7'C-(3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;
7'C-(3-((6-(6-(maleimido)hexanamido)hexanamido)methyl)pyrrolidin-1-yl)-am-
atoxin;
7'C-(3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)pyrrol-
idin-1-yl)-amatoxin;
7'C-(3-((6-((4-(maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl-
)pyrrolidin-1-yl)-amatoxin;
7'C-(4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amat-
oxin;
7'C-(4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)-
-amatoxin;
7'C-(4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)-amato-
xin;
7'C-(4-(6-(2-(aminooxy)acetamido)hexanoyl)piperazin-1-yl)-amatoxin;
7'C-((4-(6-(maleimido)hexanamido)piperidin-1-yl)methyl)-amatoxin;
7'C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin-
; 7'C-((4-(6-(maleimido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;
(R)-7'C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amat-
oxin;
(S)-7'C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-
-amatoxin;
7'C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperid-
in-1-yl)methyl)-amatoxin;
7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-
-yl)methyl)-amatoxin;
7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)eth-
yl)piperidin-1-yl)methyl)-amatoxin;
7'C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxin-
;
7'C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)m-
ethyl)-amatoxin;
7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-
-yl)methyl)-amatoxin;
7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)eth-
yl)piperazin-1-yl)methyl)-amatoxin;
7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-S-methyl)pyrrolidin-1-yl-
)methyl)-amatoxin;
7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-R-methyl)pyrrolidin-1-yl-
)methyl)-amatoxin;
7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidi-
n-1-yl)methyl)-amatoxin;
7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-R-methyl)pyrrolidi-
n-1-yl)methyl)-amatoxin;
7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methy-
l)pyrrolidin-1-yl)methyl)-amatoxin;
7'C-((4-(2-(3-carboxypropanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;
7'C-((4-(6-(6-(maleimido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amato-
xin;
7'C-((4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)pipe-
razin-1-yl)methyl)-amatoxin;
7'C-((4-(2-(maleimido)acetyl)piperazin-1-yl)methyl)-amatoxin;
7'C-((4-(3-(maleimido)propanoyl)piperazin-1-yl)methyl)-amatoxin;
7'C-((4-(4-(maleimido)butanoyl)piperazin-1-yl)methyl)-amatoxin;
7'C-((4-(2-(2-(maleimido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;
7'C-((4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxin-
;
7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)et-
hyl)piperidin-1-yl)methyl)-amatoxin;
7'C-((3-((6-(maleimido)hexanamido)methyl)azetidin-1-yl)methyl)-amatoxin;
7'C-((3-(2-(6-(maleimido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxin;
7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-y-
l)methyl)-amatoxin;
7'C-((3-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)azetidin-1y-
l)methyl)-amatoxin;
7'C-((3-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)eth-
yl)azetidin-1-yl)methyl)-amatoxin;
7'C-(((2-(6-(maleimido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-am-
atoxin;
7'C-(((4-(6-(maleimido)-N-methylhexanamido)butyl(methyl)amino)meth-
yl)-amatoxin;
7'C-((2-(2-(6-(maleimido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxin;
7'C-((2-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)eth-
yl)aziridin-1-yl)methyl)-amatoxin;
7'C-((4-(6-(6-(2-(aminooxy)acetamido)hexanamido)hexanoyl)piperazin-1-yl)m-
ethyl)-amatoxin;
7'C-((4-(1-(aminooxy)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-oyl)pip-
erazin-1-yl)methyl)-amatoxin;
7'C-((4-(2-(2-(aminooxy)acetamido)acetyl)piperazin-1-yl)methyl)-amatoxin;
7'C-((4-(3-(2-(aminooxy)acetamido)propanoyl)piperazin-1-yl)methyl)-amatox-
in;
7'C-((4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)methyl)-amat-
oxin;
7'C-((4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl-
)methyl)-amatoxin;
7'C-((4-(2-(2-(2-(aminooxy)acetamido)acetamido)ethyl)piperidin-1-yl)methy-
l)-amatoxin;
7'C-((4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)meth-
yl)-amatoxin;
7'C-((4-(20-(aminooxy)-4,19-dioxo-6,9,12,15-tetraoxa-3,18-diazaicosyl)pip-
eridin-1-yl)methyl)-amatoxin;
7'C-(((2-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)ethyl)(methyl)amin-
o)methyl)-amatoxin;
7'C-(((4-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)butyl)(methyl)amin-
o)methyl)-amatoxin;
7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methy-
l)pyrrolidin-1-yl)-S-methyl)-amatoxin;
7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)-R-me-
thyl)pyrrolidin-1-yl)methyl)-amatoxin;
7'C-((4-(2-(2-bromoacetamido)ethyl)piperazin-1-yl)methyl)-amatoxin;
7'C-((4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;
7'C-((4-(2-(3-(pyridine-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)m-
ethyl)-amatoxin; 6'O-(6-(6-(maleimido)hexanamido)hexyl)-amatoxin;
6'O-(5-(4-((maleimido)methyl)cyclohexanecarboxamido)pentyl)-amatoxin;
6'O-(2-((6-(maleimido)hexyl)oxy)-2-oxoethyl)-amatoxin;
6'O-((6-(maleimido)hexyl)carbamoyl)-amatoxin;
6'O-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexyl)carbamoyl)-ama-
toxin; 6'O-(6-(2-bromoacetamido)hexyl)-amatoxin;
7'C-(4-(6-(azido)hexanamido)piperidin-1-yl)-amatoxin;
7'C-(4-(hex-5-ynoylamino)piperidin-1-yl)-amatoxin;
7'C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;
7'C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)-am-
atoxin;
6'O-(6-(6-(11,12-didehydro-5,6-dihydro-dibenz[b,f]azocin-5-yl)-6-o-
xohexanamido)hexyl)-amatoxin;
6'O-(6-(hex-5-ynoylamino)hexyl)-amatoxin;
6'O-(6-(2-(aminooxy)acetylamido)hexyl)-amatoxin;
6'O-((6-aminooxy)hexyl)-amatoxin; and
6'O-(6-(2-iodoacetamido)hexyl)-amatoxin. The foregoing linkers,
among others useful in conjunction with the compositions and
methods described herein, are described, for example, in US Patent
Application Publication No. 2015/0218220, the disclosure of which
is incorporated herein by reference in its entirety.
[0577] Additional cytotoxins that can be conjugated to antibodies,
antigen-binding fragments thereof, and ligands that recognize and
bind CD45 (such as CD45RO), CD135, CD34, CD90, and/or CD110 for use
in directly treating a cancer, autoimmune condition, or for
conditioning a patient (e.g., a human patient) in preparation for
hematopoietic stem cell transplant therapy include, without
limitation, 5-ethynyluracil, abiraterone, acylfulvene, adecypenol,
adozelesin, aldesleukin, altretamine, ambamustine, amidox,
amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide,
anastrozole, andrographolide, angiogenesis inhibitors, antarelix,
anti-dorsalizing morphogenetic protein-1, antiandrogen, prostatic
carcinoma, antiestrogen, antineoplaston, antisense
oligonucleotides, aphidicolin glycinate, apoptosis gene modulators,
apoptosis regulators, apurinic acid, asulacrine, atamestane,
atrimustine, axinastatin 1, axinastatin 2, axinastatin 3,
azasetron, azatoxin, azatyrosine, baccatin III derivatives,
balanol, batimastat, BCR/ABL antagonists, benzochlorins,
benzoylstaurosporine, beta lactam derivatives, beta-alethine,
betaclamycin B, betulinic acid, bFGF inhibitors, bicalutamide,
bisantrene, bisaziridinylspermine, bisnafide, bistratene A,
bizelesin, breflate, bleomycin A2, bleomycin B2, bropirimine,
budotitane, buthionine sulfoximine, calcipotriol, calphostin C,
camptothecin derivatives (e.g., 10-hydroxy-camptothecin),
capecitabine, carboxamide-amino-triazole, carboxyamidotriazole,
carzelesin, casein kinase inhibitors, castanospermine, cecropin B,
cetrorelix, chlorins, chloroquinoxaline sulfonamide, cicaprost,
cis-porphyrin, cladribine, clomifene and analogues thereof,
clotrimazole, collismycin A, collismycin B, combretastatin A4,
combretastatin analogues, conagenin, crambescidin 816, crisnatol,
cryptophycin 8, cryptophycin A derivatives, curacin A,
cyclopentanthraquinones, cycloplatam, cypemycin, cytarabine
ocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine,
dehydrodidemnin B, 2'deoxycoformycin (DCF), deslorelin,
dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didemnin B,
didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol,
dioxamycin, diphenyl spiromustine, discodermolide, docosanol,
dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA,
ebselen, ecomustine, edelfosine, edrecolomab, eflornithine,
elemene, emitefur, epothilones, epithilones, epristeride,
estramustine and analogues thereof, etoposide, etoposide
4'-phosphate (also referred to as etopofos), exemestane, fadrozole,
fazarabine, fenretinide, filgrastim, finasteride, flavopiridol,
flezelastine, fluasterone, fludarabine, fluorodaunorunicin
hydrochloride, forfenimex, formestane, fostriecin, fotemustine,
gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix,
gelatinase inhibitors, gemcitabine, glutathione inhibitors,
hepsulfam, homoharringtonine (HHT), hypericin, ibandronic acid,
idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridones,
imiquimod, immunostimulant peptides, iobenguane, iododoxorubicin,
ipomeanol, irinotecan, iroplact, irsogladine, isobengazole,
jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,
leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,
lipophilic platinum compounds, lissoclinamide 7, lobaplatin,
lometrexol, lonidamine, losoxantrone, loxoribine, lurtotecan,
lutetium texaphyrin, lysofylline, masoprocol, maspin, matrix
metalloproteinase inhibitors, menogaril, rnerbarone, meterelin,
methioninase, metoclopramide, MIF inhibitor, ifepristone,
miltefosine, mirimostim, mithracin, mitoguazone, mitolactol,
mitomycin and analogues thereof, mitonafide, mitoxantrone,
mofarotene, molgramostim, mycaperoxide B, myriaporone,
N-acetyldinaline, N-substituted benzamides, nafarelin, nagrestip,
napavin, naphterpin, nartograstim, nedaplatin, nemorubicin,
neridronic acid, nilutamide, nisamycin, nitrullyn, octreotide,
okicenone, onapristone, ondansetron, oracin, ormaplatin,
oxaliplatin, oxaunomycin, paclitaxel and analogues thereof,
palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol,
panomifene, parabactin, pazelliptine, pegaspargase, peldesine,
pentosan polysulfate sodium, pentostatin, pentrozole, perflubron,
perfosfamide, phenazinomycin, picibanil, pirarubicin, piritrexim,
podophyllotoxin, porfiromycin, purine nucleoside phosphorylase
inhibitors, raltitrexed, rhizoxin, rogletimide, rohitukine,
rubiginone B1, ruboxyl, safingol, saintopin, sarcophytol A,
sargramostim, sobuzoxane, sonermin, sparfosic acid, spicamycin D,
spiromustine, stipiamide, sulfinosine, tallimustine, tegafur,
temozolomide, teniposide, thaliblastine, thiocoraline,
tirapazamine, topotecan, topsentin, triciribine, trimetrexate,
veramine, vinorelbine, vinxaltine, vorozole, zeniplatin, and
zilascorb, among others.
Linkers for Chemical Conjugation
[0578] A variety of linkers can be used to conjugate antibodies,
antigen-binding fragments, and ligands described herein (e.g.,
antibodies, antigen-binding fragments thereof, and ligands that
recognize and bind CD45 (such as CD45RO), CD135, CD34, CD90, and/or
CD110) with a cytotoxic molecule. Linkers include those that may be
cleaved, for instance, by enzymatic hydrolysis, photolysis,
hydrolysis under acidic conditions, hydrolysis under basic
conditions, oxidation, disulfide reduction, nucleophilic cleavage,
or organometallic cleavage (see, for example, Leriche et al.,
Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is
incorporated herein by reference as it pertains to linkers suitable
for covalent conjugation). Examples of linkers useful for the
synthesis of drug-antibody conjugates and drug-ligand conjugates
include those that contain electrophiles, such as Michael acceptors
(e.g., maleimides), activated esters, electron-deficient carbonyl
compounds, and aldehydes, among others, suitable for reaction with
nucleophilic substituents present within antibodies or
antigen-binding fragments, such as amine and thiol moieties. For
instance, linkers suitable for the synthesis of drug-antibody
conjugates and drug-ligand conjugates include, without limitation,
succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate
(SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC,
m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS,
and succinimidyl iodoacetate, among others described, for instance,
Liu et al., 18:690-697, 1979, the disclosure of which is
incorporated herein by reference as it pertains to linkers for
chemical conjugation. Additional linkers include the non-cleavable
maleimidocaproyl linkers, which are particularly useful for the
conjugation of microtubule-disrupting agents such as auristatins,
are described by Doronina et al., Bioconjugate Chem. 17:14-24,
2006, the disclosure of which is incorporated herein by reference
as it pertains to linkers for chemical conjugation. Additional
linkers suitable for the synthesis of drug-antibody conjugates and
drug-ligand conjugates as described herein include those capable of
releasing a cytotoxin by a 1,6-elimination process, such as
p-aminobenzyl alcohol (PABC), 6-maleimidohexanoic acid,
pH-sensitive carbonates, and other reagents described in Jain et
al., Pharm. Res. 32:3526-3540, 2015, the disclosure of which is
incorporated herein by reference in its entirety.
[0579] Linkers that can be used to conjugate an antibody,
antigen-binding fragment thereof, or ligand to a cytotoxic agent
include those that are covalently bound to the cytotoxic agent on
one end of the linker and, on the other end of the linker, contain
a chemical moiety formed from a coupling reaction between a
reactive substituent present on the linker and a reactive
substituent present within the antibody, antigen-binding fragment
thereof, or ligand that binds CD45 (such as CD45RO), CD135, CD34,
CD90, and/or CD110. Reactive substituents that may be present
within an antibody, antigen-binding fragment thereof, or ligand
that binds CD45 (such as CD45RO), CD135, CD34, CD90, and/or CD110
include, without limitation, hydroxyl moieties of serine,
threonine, and tyrosine residues; amino moieties of lysine
residues; carboxyl moieties of aspartic acid and glutamic acid
residues; and thiol moieties of cysteine residues, as well as
propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl
(e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties
of non-naturally occurring amino acids. Linkers useful in
conjunction with the antibody-drug and ligand-drug conjugates
described herein include, without limitation, linkers containing
chemical moieties formed by coupling reactions as depicted in Table
2, below. Curved lines designate points of attachment to the
antibody, antigen-binding fragment, or ligand and the cytotoxic
molecule, respectively.
TABLE-US-00013 TABLE 2 Exemplary chemical moieties formed by
coupling reactions in the formation of antibody-drug and
ligand-drug conjugates Exemplary Coupling Reactions Chemical Moiety
Formed by Coupling Reactions [3 + 2] Cycloaddition ##STR00026## [3
+ 2] Cycloaddition ##STR00027## [3 + 2] Cycloaddition,
Esterification ##STR00028## [3 + 2] Cycloaddition, Esterification
##STR00029## [3 + 2] Cycloaddition, Esterification ##STR00030## [3
+ 2] Cycloaddition, Esterification ##STR00031## [3 + 2]
Cycloaddition, Esterification ##STR00032## [3 + 2] Cycloaddition,
Esterification ##STR00033## [3 + 2] Cycloaddition, Esterification
##STR00034## [3 + 2] Cycloaddition, Esterification ##STR00035## [3
+ 2] Cycloaddition, Esterification ##STR00036## [3 + 2]
Cycloaddition, Esterification ##STR00037## [3 + 2] Cycloaddition,
Esterification ##STR00038## [3 + 2] Cycloaddition, Etherification
##STR00039## [3 + 2] Cycloaddition ##STR00040## Michael addition
##STR00041## Michael addition ##STR00042## Imine condensation,
Amidation ##STR00043## Imine condensation ##STR00044## Disulfide
formation ##STR00045## Thiol alkylation ##STR00046## Condensation,
Michael addition ##STR00047##
Methods of Treatment
[0580] As described herein, hematopoietic stem cell transplant
therapy can be administered to a subject in need of treatment so as
to populate or re-populate one or more blood cell types.
Hematopoietic stem cells generally exhibit multi-potency, and can
thus differentiate into multiple different blood lineages
including, but not limited to, granulocytes (e.g., promyelocytes,
neutrophils, eosinophils, basophils), erythrocytes (e.g.,
reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts,
platelet producing megakaryocytes, platelets), monocytes (e.g.,
monocytes, macrophages), dendritic cells, microglia, osteoclasts,
and lymphocytes (e.g., NK cells, B-cells and T-cells).
Hematopoietic stem cells are additionally capable of self-renewal,
and can thus give rise to daughter cells that have equivalent
potential as the mother cell, and also feature the capacity to be
reintroduced into a transplant recipient whereupon they home to the
hematopoietic stem cell niche and re-establish productive and
sustained hematopoiesis.
[0581] Hematopoietic stem cells can thus be administered to a
patient defective or deficient in one or more cell types of the
hematopoietic lineage in order to re-constitute the defective or
deficient population of cells in vivo, thereby treating the
pathology associated with the defect or depletion in the endogenous
blood cell population. The compositions and methods described
herein can thus be used to treat a non-malignant hemoglobinopathy
(e.g., a hemoglobinopathy selected from the group consisting of
sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia,
and Wiskott-Aldrich syndrome). Additionally or alternatively, the
compositions and methods described herein can be used to treat an
immunodeficiency, such as a congenital immunodeficiency.
Additionally or alternatively, the compositions and methods
described herein can be used to treat an acquired immunodeficiency
(e.g., an acquired immunodeficiency selected from the group
consisting of HIV and AIDS). The compositions and methods described
herein can be used to treat a metabolic disorder (e.g., a metabolic
disorder selected from the group consisting of glycogen storage
diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers
Disease, sphingolipidoses, and metachromatic leukodystrophy).
[0582] Additionally or alternatively, the compositions and methods
described herein can be used to treat a malignancy or proliferative
disorder, such as a hematologic cancer, myeloproliferative disease.
In the case of cancer treatment, the compositions and methods
described herein may be administered to a patient so as to deplete
a population of endogenous hematopoietic stem cells prior to
hematopoietic stem cell transplantation therapy, in which case the
transplanted cells can home to a niche created by the endogenous
cell depletion step and establish productive hematopoiesis. This,
in turn, can re-constitute a population of cells depleted during
cancer cell eradication, such as during systemic chemotherapy.
Exemplary hematological cancers that can be treated using the
compositions and methods described heein include, without
limitation, acute myeloid leukemia, acute lymphoid leukemia,
chronic myeloid leukemia, chronic lymphoid leukemia, multiple
myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma,
as well as other cancerous conditions, including neuroblastoma.
[0583] Additional diseases that can be treated with the
compositions and methods described herein include, without
limitation, adenosine deaminase deficiency and severe combined
immunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashi
disease, hereditary lymphohistiocytosis, osteopetrosis,
osteogenesis imperfecta, storage diseases, thalassemia major,
systemic sclerosis, systemic lupus erythematosus, multiple
sclerosis, and juvenile rheumatoid arthritis.
[0584] The antibodies, antigen-binding fragments thereof, ligands,
and conjugates described herein may be used to induce solid organ
transplant tolerance. For instance, the compositions and methods
described herein may be used to deplete or ablate a population of
cells from a target tissue (e.g., to deplete hematopoietic stem
cells from the bone marrow stem cell niche). Following such
depletion of cells from the target tissues, a population of stem or
progenitor cells from an organ donor (e.g., hematopoietic stem
cells from the organ donor) may be administered to the transplant
recipient, and following the engraftment of such stem or progenitor
cells, a temporary or stable mixed chimerism may be achieved,
thereby enabling long-term transplant organ tolerance without the
need for further immunosuppressive agents. For example, the
compositions and methods described herein may be used to induce
transplant tolerance in a solid organ transplant recipient (e.g., a
kidney transplant, lung transplant, liver transplant, and heart
transplant, among others). The compositions and methods described
herein are well-suited for use in connection the induction of solid
organ transplant tolerance, for instance, because a low percentage
temporary or stable donor engraftment is sufficient to induce
long-term tolerance of the transplanted organ.
[0585] In addition, the compositions and methods described herein
can be used to treat cancers directly, such as cancers
characterized by cells that are CD45+, CD135+, CD34+, CD90+, or
CD110+. For instance, the compositions and methods described herein
can be used to treat leukemia, particularly in patients that
exhibit CD45+, CD135+, CD34+, CD90+, or CD110+ leukemic cells. By
depleting CD45+, CD135+, CD34+, CD90+, or CD110+ cancerous cells,
such as leukemic cells, the compositions and methods described
herein can be used to treat various cancers directly. Exemplary
cancers that may be treated in this fashion include hematological
cancers, such as acute myeloid leukemia, acute lymphoid leukemia,
chronic myeloid leukemia, chronic lymphoid leukemia, multiple
myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's
lymphoma,
[0586] In addition, the compositions and methods described herein
can be used to treat autoimmune disorders. For instance, an
antibody, antigen-binding fragment thereof, or ligand can be
administered to a subject, such as a human patient suffering from
an autoimmune disorder, so as to kill a CD45+, CD135+, CD34+,
CD90+, or CD110+ immune cell. The CD45+, CD135+, CD34+, CD90+, or
CD110+ immune cell may be an autoreactive lymphocyte, such as a
T-cell that expresses a T-cell receptor that specifically binds,
and mounts an immune response against, a self antigen. By depleting
self-reactive, CD45+, CD135+, CD34+, CD90+, or CD110+ cells, the
compositions and methods described herein can be used to treat
autoimmune pathologies, such as those described below. Additionally
or alternatively, the compositions and methods described herein can
be used to treat an autoimmune disease by depleting a population of
endogenous hematopoietic stem cells prior to hematopoietic stem
cell transplantation therapy, in which case the transplanted cells
can home to a niche created by the endogenous cell depletion step
and establish productive hematopoiesis. This, in turn, can
re-constitute a population of cells depleted during autoimmune cell
eradication.
[0587] Autoimmune diseases that can be treated using the
compositions and methods described herein include, without
limitation, psoriasis, psoriatic arthritis, Type 1 diabetes
mellitus (Type 1 diabetes), rheumatoid arthritis (RA), human
systemic lupus (SLE), multiple sclerosis (MS), inflammatory bowel
disease (IBD), lymphocytic colitis, acute disseminated
encephalomyelitis (ADEM), Addison's disease, alopecia universalis,
ankylosing spondylitisis, antiphospholipid antibody syndrome (APS),
aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis,
autoimmune inner ear disease (AIED), autoimmune lymphoproliferative
syndrome (ALPS), autoimmune oophoritis, Balo disease, Behcet's
disease, bullous pemphigoid, cardiomyopathy, Chagas' disease,
chronic fatigue immune dysfunction syndrome (CFIDS), chronic
inflammatory demyelinating polyneuropathy, Crohn's disease,
cicatrical pemphigoid, coeliac sprue-dermatitis herpetiformis, cold
agglutinin disease, CREST syndrome, Degos disease, discoid lupus,
dysautonomia, endometriosis, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, Goodpasture's syndrome, Grave's
disease, Guillain-Barre syndrome (GBS), Hashimoto's thyroiditis,
Hidradenitis suppurativa, idiopathic and/or acute thrombocytopenic
purpura, idiopathic pulmonary fibrosis, IgA neuropathy,
interstitial cystitis, juvenile arthritis, Kawasaki's disease,
lichen planus, Lyme disease, Meniere disease, mixed connective
tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus
myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis,
pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis
and dermatomyositis, primary biliary cirrhosis, polyarteritis
nodosa, polyglandular syndromes, polymyalgia rheumatica, primary
agammaglobulinemia, Raynaud phenomenon, Reiter's syndrome,
rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome,
stiff person syndrome, Takayasu's arteritis, temporal arteritis
(also known as "giant cell arteritis"), ulcerative colitis,
collagenous colitis, uveitis, vasculitis, vitiligo, vulvodynia
("vulvar vestibulitis"), and Wegener's granulomatosis.
Routes of Administration and Dosing
[0588] Antibodies, antigen-binding fragments thereof, and ligands
described herein can be administered to a patient (e.g., a human
patient suffering from cancer, an autoimmune disease, or in need of
hematopoietic stem cell transplant therapy) in a variety of dosage
forms. For instance, antibodies, antigen-binding fragments thereof,
and ligands described herein can be administered to a patient
suffering from cancer, an autoimmune disease, or in need of
hematopoietic stem cell transplant therapy in the form of an
aqueous solution, such as an aqueous solution containing one or
more pharmaceutically acceptable excipients. Pharmaceutically
acceptable excipients for use with the compositions and methods
described herein include viscosity-modifying agents. The aqueous
solution may be sterilized using techniques known in the art.
[0589] The antibodies, antigen-binding fragments, and ligands
described herein may be administered by a variety of routes, such
as orally, transdermally, subcutaneously, intranasally,
intravenously, intramuscularly, intraocularly, or parenterally. The
most suitable route for administration in any given case will
depend on the particular antibody, antigen-binding fragment, or
ligand administered, the patient, pharmaceutical formulation
methods, administration methods (e.g., administration time and
administration route), the patient's age, body weight, sex,
severity of the diseases being treated, the patient's diet, and the
patient's excretion rate.
[0590] The effective dose of an antibody, antigen-binding fragment
thereof, or ligand described herein can range, for example from
about 0.001 to about 100 mg/kg of body weight per single (e.g.,
bolus) administration, multiple administrations, or continuous
administration, or to achieve an optimal serum concentration (e.g.,
a serum concentration of 0.0001-5000 .mu.g/mL) of the antibody,
antigen-binding fragment thereof, or ligand. The dose may be
administered one or more times (e.g., 2-10 times) per day, week, or
month to a subject (e.g., a human) suffering from cancer, an
autoimmune disease, or undergoing conditioning therapy in
preparation for receipt of a hematopoietic stem cell transplant. In
the case of a conditioning procedure prior to hematopoietic stem
cell transplantation, the antibody, antigen-binding fragment
thereof, or ligand can be administered to the patient at a time
that optimally promotes engraftment of the exogenous hematopoietic
stem cells, for instance, from 1 hour to 1 week (e.g., 1 hour, 2
hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9
hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours,
16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22
hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days,
or 7 days) or more prior to administration of the exogenous
hematopoietic stem cell transplant.
EXAMPLES
[0591] The following examples are put forth so as to provide those
of ordinary skill in the art with a description of how the
compositions and methods described herein may be used, made, and
evaluated, and are intended to be purely exemplary of the invention
and are not intended to limit the scope of what the inventors
regard as their invention.
Example 1. Administration of an Anti-CD45 Antibody to a Human
Patient in Preparation for Hematopoietic Stem Cell Transplant
Therapy
[0592] According to the methods disclosed herein, a physician of
skill in the art can condition a patient, such as a human patient,
so as to promote the engraftment of exogenous hematopoietic stem
cell grafts prior to hematopoietic stem cell transplant therapy. To
this end, a physician of skill in the art can administer to the
human patient an antibody or antigen-binding fragment thereof
capable of binding an antigen expressed by hematopoietic stem
cells, such as an antibody or antigen-biding fragment thereof that
binds CD45 (for example, an antibody or antigen-binding fragment
thereof that binds CD45RO). The antibody may be covalently
conjugated to a toxin, such as a cytotoxic molecule described
herein or known in the art. For instance, an anti-CD45 antibody or
antigen-binding fragment thereof (such as an anti-CD45RO antibody
or antigen-binding fragment thereof) can be covalently conjugated
to a cytotoxin, such as pseudomonas exotoxin A, deBouganin,
diphtheria toxin, an amatoxin, such as .alpha.-amanitin, saporin,
maytansine, a maytansinoid, an auristatin, an anthracycline, a
calicheamicin, irinotecan, SN-38, a duocarmycin, a
pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an
indolinobenzodiazepine, an indolinobenzodiazepine dimer, or a
variant thereof. This conjugation can be performed using covalent
bond-forming techniques described herein or known in the art. The
antibody, antigen-binding fragment thereof, or drug-antibody
conjugate can subsequently be administered to the patient, for
example, by intravenous administration, prior to transplantation of
exogenous hematopoietic stem cells (such as autologous, syngeneic,
or allogeneic hematopoietic stem cells) to the patient.
[0593] The anti-CD45 (e.g., anti-CD45RO) antibody, antigen-binding
fragment thereof, or drug-antibody conjugate can be administered in
an amount sufficient to reduce the quantity of endogenous
hematopoietic stem cells, for example, by 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, or more prior to hematopoietic stem cell
transplant therapy. The reduction in hematopoietic stem cell count
can be monitored using conventional techniques known in the art,
such as by FACS analysis of cells expressing characteristic
hematopoietic stem cell surface antigens in a blood sample
withdrawn from the patient at varying intervals during conditioning
therapy. For instance, a physician of skill in the art can withdraw
a blood sample from the patient at various time points during
conditioning therapy and determine the extent of endogenous
hematopoietic stem cell reduction by conducting a FACS analysis to
elucidate the relative concentrations of hematopoietic stem cells
in the sample using antibodies that bind to hematopoietic stem cell
marker antigens. According to some embodiments, when the
concentration of hematopoietic stem cells has reached a minimum
value in response to conditioning therapy with an anti-CD45 (e.g.,
anti-CD45RO) antibody, antigen-binding fragment thereof, or
drug-antibody conjugate, the physician may conclude the
conditioning therapy, and may begin preparing the patient for
hematopoietic stem cell transplant therapy.
[0594] The anti-CD45 (e.g., anti-CD45RO) antibody, antigen-binding
fragment thereof, or drug-antibody conjugate can be administered to
the patient in an aqueous solution containing one or more
pharmaceutically acceptable excipients, such as a
viscosity-modifying agent. The aqueous solution may be sterilized
using techniques described herein or known in the art. The
antibody, antigen-binding fragment thereof, or drug-antibody
conjugate can be administered to the patient at a dosage of, for
example, from 0.001 mg/kg to 100 mg/kg prior to administration of a
hematopoietic stem cell graft to the patient. The antibody,
antigen-binding fragment thereof, or drug-antibody conjugate can be
administered to the patient at a time that optimally promotes
engraftment of the exogenous hematopoietic stem cells, for
instance, from 1 hour to 1 week (e.g., 1 hour, 2 hours, 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11
hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours,
18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24
hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) or more
prior to administration of the exogenous hematopoietic stem cell
transplant.
[0595] Following the conclusion of conditioning therapy, the
patient may then receive an infusion (e.g., an intravenous
infusion) of exogenous hematopoietic stem cells, such as from the
same physician that performed the conditioning therapy or from a
different physician. The physician may administer the patient an
infusion of autologous, syngeneic, or allogeneic hematopoietic stem
cells, for instance, at a dosage of from 1.times.10.sup.3 to
1.times.10.sup.9 hematopoietic stem cells/kg. The physician may
monitor the engraftment of the hematopoietic stem cell transplant,
for example, by withdrawing a blood sample from the patient and
determining the increase in concentration of hematopoietic stem
cells or cells of the hematopoietic lineage (such as
megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells,
myeoblasts, basophils, neutrophils, eosinophils, microglia,
granulocytes, monocytes, osteoclasts, antigen-presenting cells,
macrophages, dendritic cells, natural killer cells, T-lymphocytes,
and B-lymphocytes) following administration of the transplant. This
analysis may be conducted, for example, from 1 hour to 6 months, or
more, following hematopoietic stem cell transplant therapy (e.g., 1
hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8
hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours,
15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21
hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks,
7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks,
14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20
weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, or more). A finding
that the concentration of hematopoietic stem cells or cells of the
hematopoietic lineage has increased (e.g., by 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
200%, 500%, or more) following the transplant therapy relative to
the concentration of the corresponding cell type prior to
transplant therapy provides one indication that treatment with the
anti-CD45 (e.g., anti-CD45RO) antibody, antigen-binding fragment
thereof, or drug-antibody conjugate has successfully promoted
engraftment of the transplanted hematopoietic stem cell graft.
Example 2. Administration of an Anti-CD135 Antibody to a Human
Patient in Preparation for Hematopoietic Stem Cell Transplant
Therapy
[0596] Using the methods disclosed herein, a physician of skill in
the art can administer to a human patient in need of hematopoietic
stem cell transplant therapy an antibody or antigen-binding
fragment thereof capable of binding an antigen expressed by
hematopoietic stem cells, such as an antibody or antigen-biding
fragment thereof that binds CD135. In this fashion, a population of
endogenous hematopoietic stem cells can be depleted prior to
administration of an exogenous hematopoietic stem cell graft so as
to promote engraftment of the hematopoietic stem cell graft. The
antibody may be covalently conjugated to a toxin, such as a
cytotoxic molecule described herein or known in the art. For
instance, an anti-CD135 antibody or antigen-binding fragment
thereof can be covalently conjugated to a cytotoxin, such as
pseudomonas exotoxin A, deBouganin, diphtheria toxin, an amatoxin,
such as .alpha.-amanitin, saporin, maytansine, a maytansinoid, an
auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a
duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine
dimer, an indolinobenzodiazepine, an indolinobenzodiazepine dimer,
or a variant thereof. This conjugation can be performed using
covalent bond-forming techniques described herein or known in the
art. The antibody, antigen-binding fragment thereof, or
drug-antibody conjugate can subsequently be administered to the
patient, for example, by intravenous administration, prior to
transplantation of exogenous hematopoietic stem cells (such as
autologous, syngeneic, or allogeneic hematopoietic stem cells) to
the patient.
[0597] The anti-CD135 antibody, antigen-binding fragment thereof,
or drug-antibody conjugate can be administered in an amount
sufficient to reduce the quantity of endogenous hematopoietic stem
cells, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or more prior to hematopoietic stem cell transplant therapy.
The reduction in hematopoietic stem cell count can be monitored
using conventional techniques known in the art, such as by FACS
analysis of cells expressing characteristic hematopoietic stem cell
surface antigens in a blood sample withdrawn from the patient at
varying intervals during conditioning therapy. For instance, a
physician of skill in the art can withdraw a blood sample from the
patient at various time points during conditioning therapy and
determine the extent of endogenous hematopoietic stem cell
reduction by conducting a FACS analysis to elucidate the relative
concentrations of hematopoietic stem cells in the sample using
antibodies that bind to hematopoietic stem cell marker antigens.
According to some embodiments, when the concentration of
hematopoietic stem cells has reached a minimum value in response to
conditioning therapy with an anti-CD135 antibody, antigen-binding
fragment thereof, or drug-antibody conjugate, the physician may
conclude the conditioning therapy, and may begin preparing the
patient for hematopoietic stem cell transplant therapy.
[0598] The anti-CD135 antibody, antigen-binding fragment thereof,
or drug-antibody conjugate can be administered to the patient in an
aqueous solution containing one or more pharmaceutically acceptable
excipients, such as a viscosity-modifying agent. The aqueous
solution may be sterilized using techniques described herein or
known in the art. The antibody, antigen-binding fragment thereof,
or drug-antibody conjugate can be administered to the patient at a
dosage of, for example, from 0.001 mg/kg to 100 mg/kg prior to
administration of a hematopoietic stem cell graft to the patient.
The antibody, antigen-binding fragment thereof, or drug-antibody
conjugate can be administered to the patient at a time that
optimally promotes engraftment of the exogenous hematopoietic stem
cells, for instance, from 1 hour to 1 week (e.g., 1 hour, 2 hours,
3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10
hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours,
17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23
hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days)
or more prior to administration of the exogenous hematopoietic stem
cell transplant.
[0599] Following the conclusion of conditioning therapy, the
patient may then receive an infusion (e.g., an intravenous
infusion) of exogenous hematopoietic stem cells, such as from the
same physician that performed the conditioning therapy or from a
different physician. The physician may administer the patient an
infusion of autologous, syngeneic, or allogeneic hematopoietic stem
cells, for instance, at a dosage of from 1.times.10.sup.3 to
1.times.10.sup.9 hematopoietic stem cells/kg. The physician may
monitor the engraftment of the hematopoietic stem cell transplant,
for example, by withdrawing a blood sample from the patient and
determining the increase in concentration of hematopoietic stem
cells or cells of the hematopoietic lineage (such as
megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells,
myeoblasts, basophils, neutrophils, eosinophils, microglia,
granulocytes, monocytes, osteoclasts, antigen-presenting cells,
macrophages, dendritic cells, natural killer cells, T-lymphocytes,
and B-lymphocytes) following administration of the transplant. This
analysis may be conducted, for example, from 1 hour to 6 months, or
more, following hematopoietic stem cell transplant therapy (e.g., 1
hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8
hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours,
15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21
hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks,
7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks,
14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20
weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, or more). A finding
that the concentration of hematopoietic stem cells or cells of the
hematopoietic lineage has increased (e.g., by 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
200%, 500%, or more) following the transplant therapy relative to
the concentration of the corresponding cell type prior to
transplant therapy provides one indication that treatment with the
anti-CD135 antibody, antigen-binding fragment thereof, or
drug-antibody conjugate has successfully promoted engraftment of
the transplanted hematopoietic stem cell graft.
Example 3. Administration of an Anti-CD34 Antibody to a Human
Patient in Preparation for Hematopoietic Stem Cell Transplant
Therapy
[0600] Using the methods disclosed herein, a physician of skill in
the art can administer to a human patient in need of hematopoietic
stem cell transplant therapy an antibody or antigen-binding
fragment thereof capable of binding an antigen expressed by
hematopoietic stem cells, such as an antibody or antigen-biding
fragment thereof that binds CD34. In this way, a population of
endogenous hematopoietic stem cells can be depleted prior to
administration of an exogenous hematopoietic stem cell graft so as
to promote engraftment of the hematopoietic stem cell graft. The
antibody may be covalently conjugated to a toxin, such as a
cytotoxic molecule described herein or known in the art. For
instance, an anti-CD34 antibody or antigen-binding fragment thereof
can be covalently conjugated to a cytotoxin, such as pseudomonas
exotoxin A, deBouganin, diphtheria toxin, an amatoxin, such as
.alpha.-amanitin, saporin, maytansine, a maytansinoid, an
auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a
duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine
dimer, an indolinobenzodiazepine, an indolinobenzodiazepine dimer,
or a variant thereof. This conjugation can be performed using
covalent bond-forming techniques described herein or known in the
art. The antibody, antigen-binding fragment thereof, or
drug-antibody conjugate can subsequently be administered to the
patient, for example, by intravenous administration, prior to
transplantation of exogenous hematopoietic stem cells (such as
autologous, syngeneic, or allogeneic hematopoietic stem cells) to
the patient.
[0601] The anti-CD34 antibody, antigen-binding fragment thereof, or
drug-antibody conjugate can be administered in an amount sufficient
to reduce the quantity of endogenous hematopoietic stem cells, for
example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or
more prior to hematopoietic stem cell transplant therapy. The
reduction in hematopoietic stem cell count can be monitored using
conventional techniques known in the art, such as by FACS analysis
of cells expressing characteristic hematopoietic stem cell surface
antigens in a blood sample withdrawn from the patient at varying
intervals during conditioning therapy. For instance, a physician of
skill in the art can withdraw a blood sample from the patient at
various time points during conditioning therapy and determine the
extent of endogenous hematopoietic stem cell reduction by
conducting a FACS analysis to elucidate the relative concentrations
of hematopoietic stem cells in the sample using antibodies that
bind to hematopoietic stem cell marker antigens. According to some
embodiments, when the concentration of hematopoietic stem cells has
reached a minimum value in response to conditioning therapy with an
anti-CD34 antibody, antigen-binding fragment thereof, or
drug-antibody conjugate, the physician may conclude the
conditioning therapy, and may begin preparing the patient for
hematopoietic stem cell transplant therapy.
[0602] The anti-CD34 antibody, antigen-binding fragment thereof, or
drug-antibody conjugate can be administered to the patient in an
aqueous solution containing one or more pharmaceutically acceptable
excipients, such as a viscosity-modifying agent. The aqueous
solution may be sterilized using techniques described herein or
known in the art. The antibody, antigen-binding fragment thereof,
or drug-antibody conjugate can be administered to the patient at a
dosage of, for example, from 0.001 mg/kg to 100 mg/kg prior to
administration of a hematopoietic stem cell graft to the patient.
The antibody, antigen-binding fragment thereof, or drug-antibody
conjugate can be administered to the patient at a time that
optimally promotes engraftment of the exogenous hematopoietic stem
cells, for instance, from 1 hour to 1 week (e.g., 1 hour, 2 hours,
3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10
hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours,
17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23
hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days)
or more prior to administration of the exogenous hematopoietic stem
cell transplant.
[0603] Following the conclusion of conditioning therapy, the
patient may then receive an infusion (e.g., an intravenous
infusion) of exogenous hematopoietic stem cells, such as from the
same physician that performed the conditioning therapy or from a
different physician. The physician may administer the patient an
infusion of autologous, syngeneic, or allogeneic hematopoietic stem
cells, for instance, at a dosage of from 1.times.10.sup.3 to
1.times.10.sup.9 hematopoietic stem cells/kg. The physician may
monitor the engraftment of the hematopoietic stem cell transplant,
for example, by withdrawing a blood sample from the patient and
determining the increase in concentration of hematopoietic stem
cells or cells of the hematopoietic lineage (such as
megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells,
myeoblasts, basophils, neutrophils, eosinophils, microglia,
granulocytes, monocytes, osteoclasts, antigen-presenting cells,
macrophages, dendritic cells, natural killer cells, T-lymphocytes,
and B-lymphocytes) following administration of the transplant. This
analysis may be conducted, for example, from 1 hour to 6 months, or
more, following hematopoietic stem cell transplant therapy (e.g., 1
hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8
hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours,
15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21
hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks,
7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks,
14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20
weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, or more). A finding
that the concentration of hematopoietic stem cells or cells of the
hematopoietic lineage has increased (e.g., by 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
200%, 500%, or more) following the transplant therapy relative to
the concentration of the corresponding cell type prior to
transplant therapy provides one indication that treatment with the
anti-CD34 antibody, antigen-binding fragment thereof, or
drug-antibody conjugate has successfully promoted engraftment of
the transplanted hematopoietic stem cell graft.
Example 4. Administration of an Anti-CD90 Antibody to a Human
Patient in Preparation for Hematopoietic Stem Cell Transplant
Therapy
[0604] Using the methods disclosed herein, a physician of skill in
the art can administer to a human patient in need of hematopoietic
stem cell transplant therapy an antibody or antigen-binding
fragment thereof capable of binding an antigen expressed by
hematopoietic stem cells, such as an antibody or antigen-biding
fragment thereof that binds CD90. In this way, a population of
endogenous hematopoietic stem cells can be depleted prior to
administration of an exogenous hematopoietic stem cell graft so as
to promote engraftment of the hematopoietic stem cell graft. The
antibody may be covalently conjugated to a toxin, such as a
cytotoxic molecule described herein or known in the art. For
instance, an anti-CD90 antibody or antigen-binding fragment thereof
(such as an anti-GNNK+ CD90 antibody or antigen-binding fragment
thereof) can be covalently conjugated to a cytotoxin, such as
pseudomonas exotoxin A, deBouganin, diphtheria toxin, an amatoxin,
such as .alpha.-amanitin, saporin, maytansine, a maytansinoid, an
auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a
duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine
dimer, an indolinobenzodiazepine, an indolinobenzodiazepine dimer,
or a variant thereof. This conjugation can be performed using
covalent bond-forming techniques described herein or known in the
art. The antibody, antigen-binding fragment thereof, or
drug-antibody conjugate can subsequently be administered to the
patient, for example, by intravenous administration, prior to
transplantation of exogenous hematopoietic stem cells (such as
autologous, syngeneic, or allogeneic hematopoietic stem cells) to
the patient.
[0605] The anti-CD90 antibody, antigen-binding fragment thereof, or
drug-antibody conjugate can be administered in an amount sufficient
to reduce the quantity of endogenous hematopoietic stem cells, for
example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or
more prior to hematopoietic stem cell transplant therapy. The
reduction in hematopoietic stem cell count can be monitored using
conventional techniques known in the art, such as by FACS analysis
of cells expressing characteristic hematopoietic stem cell surface
antigens in a blood sample withdrawn from the patient at varying
intervals during conditioning therapy. For instance, a physician of
skill in the art can withdraw a blood sample from the patient at
various time points during conditioning therapy and determine the
extent of endogenous hematopoietic stem cell reduction by
conducting a FACS analysis to elucidate the relative concentrations
of hematopoietic stem cells in the sample using antibodies that
bind to hematopoietic stem cell marker antigens. According to some
embodiments, when the concentration of hematopoietic stem cells has
reached a minimum value in response to conditioning therapy with an
anti-CD90 antibody, antigen-binding fragment thereof, or
drug-antibody conjugate, the physician may conclude the
conditioning therapy, and may begin preparing the patient for
hematopoietic stem cell transplant therapy.
[0606] The anti-CD90 antibody, antigen-binding fragment thereof, or
drug-antibody conjugate can be administered to the patient in an
aqueous solution containing one or more pharmaceutically acceptable
excipients, such as a viscosity-modifying agent. The aqueous
solution may be sterilized using techniques described herein or
known in the art. The antibody, antigen-binding fragment thereof,
or drug-antibody conjugate can be administered to the patient at a
dosage of, for example, from 0.001 mg/kg to 100 mg/kg prior to
administration of a hematopoietic stem cell graft to the patient.
The antibody, antigen-binding fragment thereof, or drug-antibody
conjugate can be administered to the patient at a time that
optimally promotes engraftment of the exogenous hematopoietic stem
cells, for instance, from 1 hour to 1 week (e.g., 1 hour, 2 hours,
3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10
hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours,
17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23
hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days)
or more prior to administration of the exogenous hematopoietic stem
cell transplant.
[0607] Following the conclusion of conditioning therapy, the
patient may then receive an infusion (e.g., an intravenous
infusion) of exogenous hematopoietic stem cells, such as from the
same physician that performed the conditioning therapy or from a
different physician. The physician may administer the patient an
infusion of autologous, syngeneic, or allogeneic hematopoietic stem
cells, for instance, at a dosage of from 1.times.10.sup.3 to
1.times.10.sup.9 hematopoietic stem cells/kg. The physician may
monitor the engraftment of the hematopoietic stem cell transplant,
for example, by withdrawing a blood sample from the patient and
determining the increase in concentration of hematopoietic stem
cells or cells of the hematopoietic lineage (such as
megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells,
myeoblasts, basophils, neutrophils, eosinophils, microglia,
granulocytes, monocytes, osteoclasts, antigen-presenting cells,
macrophages, dendritic cells, natural killer cells, T-lymphocytes,
and B-lymphocytes) following administration of the transplant. This
analysis may be conducted, for example, from 1 hour to 6 months, or
more, following hematopoietic stem cell transplant therapy (e.g., 1
hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8
hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours,
15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21
hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks,
7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks,
14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20
weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, or more). A finding
that the concentration of hematopoietic stem cells or cells of the
hematopoietic lineage has increased (e.g., by 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
200%, 500%, or more) following the transplant therapy relative to
the concentration of the corresponding cell type prior to
transplant therapy provides one indication that treatment with the
anti-CD90 antibody, antigen-binding fragment thereof, or
drug-antibody conjugate has successfully promoted engraftment of
the transplanted hematopoietic stem cell graft.
Example 5. Administration of an Anti-CD110 Antibody to a Human
Patient in Preparation for Hematopoietic Stem Cell Transplant
Therapy
[0608] Using the methods disclosed herein, a physician of skill in
the art can administer to a human patient in need of hematopoietic
stem cell transplant therapy an antibody or antigen-binding
fragment thereof capable of binding an antigen expressed by
hematopoietic stem cells, such as an antibody or antigen-biding
fragment thereof that binds CD110. In this way, a population of
endogenous hematopoietic stem cells can be depleted prior to
administration of an exogenous hematopoietic stem cell graft so as
to promote engraftment of the hematopoietic stem cell graft. The
antibody may be covalently conjugated to a toxin, such as a
cytotoxic molecule described herein or known in the art. For
instance, an anti-CD110 antibody or antigen-binding fragment
thereof can be covalently conjugated to a cytotoxin, such as
pseudomonas exotoxin A, deBouganin, diphtheria toxin, an amatoxin,
such as .alpha.-amanitin, saporin, maytansine, a maytansinoid, an
auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a
duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine
dimer, an indolinobenzodiazepine, an indolinobenzodiazepine dimer,
or a variant thereof. This conjugation can be performed using
covalent bond-forming techniques described herein or known in the
art. The antibody, antigen-binding fragment thereof, or
drug-antibody conjugate can subsequently be administered to the
patient, for example, by intravenous administration, prior to
transplantation of exogenous hematopoietic stem cells (such as
autologous, syngeneic, or allogeneic hematopoietic stem cells) to
the patient.
[0609] The anti-CD110 antibody, antigen-binding fragment thereof,
or drug-antibody conjugate can be administered in an amount
sufficient to reduce the quantity of endogenous hematopoietic stem
cells, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or more prior to hematopoietic stem cell transplant therapy.
The reduction in hematopoietic stem cell count can be monitored
using conventional techniques known in the art, such as by FACS
analysis of cells expressing characteristic hematopoietic stem cell
surface antigens in a blood sample withdrawn from the patient at
varying intervals during conditioning therapy. For instance, a
physician of skill in the art can withdraw a blood sample from the
patient at various time points during conditioning therapy and
determine the extent of endogenous hematopoietic stem cell
reduction by conducting a FACS analysis to elucidate the relative
concentrations of hematopoietic stem cells in the sample using
antibodies that bind to hematopoietic stem cell marker antigens.
According to some embodiments, when the concentration of
hematopoietic stem cells has reached a minimum value in response to
conditioning therapy with an anti-CD135 antibody, antigen-binding
fragment thereof, or drug-antibody conjugate, the physician may
conclude the conditioning therapy, and may begin preparing the
patient for hematopoietic stem cell transplant therapy.
[0610] The anti-CD110 antibody, antigen-binding fragment thereof,
or drug-antibody conjugate can be administered to the patient in an
aqueous solution containing one or more pharmaceutically acceptable
excipients, such as a viscosity-modifying agent. The aqueous
solution may be sterilized using techniques described herein or
known in the art. The antibody, antigen-binding fragment thereof,
or drug-antibody conjugate can be administered to the patient at a
dosage of, for example, from 0.001 mg/kg to 100 mg/kg prior to
administration of a hematopoietic stem cell graft to the patient.
The antibody, antigen-binding fragment thereof, or drug-antibody
conjugate can be administered to the patient at a time that
optimally promotes engraftment of the exogenous hematopoietic stem
cells, for instance, from 1 hour to 1 week (e.g., 1 hour, 2 hours,
3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10
hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours,
17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23
hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days)
or more prior to administration of the exogenous hematopoietic stem
cell transplant.
[0611] Following the conclusion of conditioning therapy, the
patient may then receive an infusion (e.g., an intravenous
infusion) of exogenous hematopoietic stem cells, such as from the
same physician that performed the conditioning therapy or from a
different physician. The physician may administer the patient an
infusion of autologous, syngeneic, or allogeneic hematopoietic stem
cells, for instance, at a dosage of from 1.times.10.sup.3 to
1.times.10.sup.9 hematopoietic stem cells/kg. The physician may
monitor the engraftment of the hematopoietic stem cell transplant,
for example, by withdrawing a blood sample from the patient and
determining the increase in concentration of hematopoietic stem
cells or cells of the hematopoietic lineage (such as
megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells,
myeoblasts, basophils, neutrophils, eosinophils, microglia,
granulocytes, monocytes, osteoclasts, antigen-presenting cells,
macrophages, dendritic cells, natural killer cells, T-lymphocytes,
and B-lymphocytes) following administration of the transplant. This
analysis may be conducted, for example, from 1 hour to 6 months, or
more, following hematopoietic stem cell transplant therapy (e.g., 1
hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8
hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours,
15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21
hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks,
7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks,
14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20
weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, or more). A finding
that the concentration of hematopoietic stem cells or cells of the
hematopoietic lineage has increased (e.g., by 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
200%, 500%, or more) following the transplant therapy relative to
the concentration of the corresponding cell type prior to
transplant therapy provides one indication that treatment with the
anti-CD110 antibody, antigen-binding fragment thereof, or
drug-antibody conjugate has successfully promoted engraftment of
the transplanted hematopoietic stem cell graft.
Example 6. Generating Antibodies Capable of Binding Hematopoietic
Stem Cells by Phage Display
[0612] An exemplary method for in vitro evolution of anti-CD45
(e.g., anti-CD45RO), anti-CD135, anti-CD34, anti-CD90, or
anti-CD110 antibodies for use with the compositions and methods
described herein is phage display. Phage display libraries can be
created by making a designed series of mutations or variations
within a coding sequence for the CDRs of an antibody or the
analogous regions of an antibody-like scaffold (e.g., the BC, CD,
and DE loops of .sup.10Fn3 domains). The template antibody-encoding
sequence into which these mutations are introduced may be, for
example, a naive human germline sequence. These mutations can be
performed using standard mutagenesis techniques known in the art.
Each mutant sequence thus encodes an antibody corresponding to the
template save for one or more amino acid variations. Retroviral and
phage display vectors can be engineered using standard vector
construction techniques known in the art. P3 phage display vectors
along with compatible protein expression vectors can be used to
generate phage display vectors for antibody diversification.
[0613] The mutated DNA provides sequence diversity, and each
transformant phage displays one variant of the initial template
amino acid sequence encoded by the DNA, leading to a phage
population (library) displaying a vast number of different but
structurally related amino acid sequences. Due to the well-defined
structure of antibody hypervariable regions, the amino acid
variations introduced in a phage display screen are expected to
alter the binding properties of the binding peptide or domain
without significantly altering its overall molecular structure.
[0614] In a typical screen, a phage library may be contacted with
and allowed to bind one of the foregoing antigens or an epitope
thereof. To facilitate separation of binders and non-binders, it is
convenient to immobilize the target on a solid support. Phage
bearing a CD45-, CD135-, CD34-, CD90-, or CD110-binding moiety can
form a complex with the target on the solid support, whereas
non-binding phage remain in solution and can be washed away with
excess buffer. Bound phage can then liberated from the target by
changing the buffer to an extreme pH (pH 2 or pH 10), changing the
ionic strength of the buffer, adding denaturants, or other known
means.
[0615] The recovered phage can then be amplified through infection
of bacterial cells, and the screening process can be repeated with
the new pool that is now depleted in non-binding antibodies and
enriched for antibodies that bind CD45 (e.g., CD45RO), CD135, CD34,
CD90, or CD110. The recovery of even a few binding phage is
sufficient to amplify the phage for a subsequent iteration of
screening. After a few rounds of selection, the gene sequences
encoding the antibodies or antigen-binding fragments thereof
derived from selected phage clones in the binding pool are
determined by conventional methods, thus revealing the peptide
sequence that imparts binding affinity of the phage to the target.
During the panning process, the sequence diversity of the
population diminishes with each round of selection until desirable
peptide-binding antibodies remain. The sequences may converge on a
small number of related antibodies or antigen-binding fragments
thereof. An increase in the number of phage recovered at each round
of selection is an indication that convergence of the library has
occurred in a screen.
Example 7. Producing Humanized Antibodies that Bind a Hematopoietic
Stem Cell Antigen
[0616] Non-human antibodies that bind CD45 (e.g., CD45RO), CD135,
CD34, CD90, or CD110 can be humanized, for instance, according to
the following procedure. Consensus human antibody heavy chain and
light chain sequences are known in the art (see e.g., the "VBASE"
human germline sequence database; Kabat et al. Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242, 1991;
Tomlinson et al., J. Mol. Biol. 227:776-798, 1992; and Cox et al.
Eur. J. Immunol. 24:827-836, 1994, the disclosures of each of which
are incorporated herein by reference as they pertain to consensus
human antibody heavy chain and light chain sequences. Using
established procedures, one of skill in the art can identify the
variable domain framework residues and CDRs of a consensus antibody
sequence (e.g., by sequence alignment). One can substitute one or
more CDRs of the heavy chain and/or light chain variable domains of
consensus human antibody with one or more corresponding CDRs of a
non-human antibody that binds CD45 (e.g., CD45RO), CD135, CD34,
CD90, or CD110 as described herein in order to produce a humanized
antibody. This CDR exchange can be performed using gene editing
techniques described herein or known in the art.
[0617] One example of a variable domain of a consensus human
antibody contains the heavy chain variable domain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGSDTYYADSVKGR
FTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVWGQGTLVTVSS (SEQ ID NO:
34) and the light chain variable domain
DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGT
DFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 35),
identified in U.S. Pat. No. 6,054,297, the disclosure of which is
incorporated herein by reference as it pertains to human antibody
consensus sequences. The CDRs in the above sequences are shown in
bold.
[0618] To produce humanized antibodies, one can recombinantly
express a polynucleotide encoding the above consensus sequence in
which one or more variable region CDRs have been replaced with one
or more variable region CDR sequences of a non-human antibody that
binds CD45 (e.g., CD45RO), CD135, CD34, CD90, or CD110. As the
affinity of the antibody for the hematopoietic stem cell antigen is
determined primarily by the CDR sequences, the resulting humanized
antibody is expected to exhibit an affinity for the hematopoietic
stem cell antigen that is about the same as that of the non-human
antibody from which the humanized antibody was derived. Methods of
determining the affinity of an antibody for a target antigen
include, for instance, ELISA-based techniques described herein and
known in the art, as well as surface plasmon resonance,
fluorescence anisotropy, and isothermal titration calorimetry,
among others.
Example 8. Ability of Anti-CD45 Antibody-Drug Conjugates to Deplete
Populations of CD45+ Cells
[0619] To investigate the ability of anti-CD45 antibody-drug
conjugates to kill CD45+ Reh cells, a series of anti-CD45
monoclonal antibodies of various isotypes, Ab1, Ab2, and Ab3, were
bound to saporin-conjugated Fab fragments and were subsequently
incubated with CD45+ Reh cells for three days. As a negative
control, an isotype-matched antibody-saporin conjugate was
generated for each of Ab1, Ab2, and Ab3, and these were was
incubated with CD45+ Reh cells for the same three-day period. Cell
viability was then assessed using the CellTiter-Glo.TM. assay. As
shown in FIG. 1, the various antibody-saporin conjugates were
capable of killing CD45+ Reh cells with different potencies, and
each was capable of depleting the CD45+ Reh cell line in a
dose-dependent fashion.
[0620] To investigate the ability of anti-CD45 monoclonal
antibody-saporin conjugates to deplete human CD34+ hematopoietic
stem cells in vitro, a human IgG1 anti-CD45 monoclonal
antibody-saporin conjugate or isotype-matched control was incubated
with CD34+ cells at various concentrations. Cell viability was
subsequently assessed following a six-day incubation period using
flow cytometry. The results of this experiment are reported in FIG.
2. As shown therein, anti-CD45 antibody-saporin conjugates were
found to be capable of depleting human CD34+ hematopoietic stem
cells in a dose-dependent manner.
[0621] Taken together, these results demonstrate that anti-CD45
antibodies conjugated to saporin can be used to deplete a
population of hematopoietic stem cells in a subject, for instance,
in preparation for hematopoietic stem cell transplant therapy, so
as to provide a niche for to which the hematopoietic stem cells may
home.
Other Embodiments
[0622] All publications, patents, and patent applications mentioned
in this specification are incorporated herein by reference to the
same extent as if each independent publication or patent
application was specifically and individually indicated to be
incorporated by reference.
[0623] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the invention that come within known
or customary practice within the art to which the invention
pertains and may be applied to the essential features hereinbefore
set forth, and follows in the scope of the claims.
[0624] Other embodiments are within the claims.
Sequence CWU 1
1
3515PRTHomo sapiens 1Ser Tyr Tyr Met His1 5217PRTHomo sapiens 2Ile
Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln1 5 10
15Gly310PRTHomo sapiens 3Gly Val Gly Ala His Asp Ala Phe Asp Ile1 5
1049PRTHomo sapiens 4Val Val Ala Ala Ala Val Ala Asp Tyr1
5516PRTHomo sapiens 5Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly
Asn Asn Tyr Leu Asp1 5 10 15616PRTHomo sapiens 6Arg Ser Ser Gln Ser
Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp1 5 10 1577PRTHomo
sapiens 7Leu Gly Ser Asn Arg Ala Ser1 589PRTHomo sapiens 8Met Gln
Gly Thr His Pro Ala Ile Ser1 599PRTHomo sapiens 9Met Gln Ser Leu
Gln Thr Pro Phe Thr1 5105PRTHomo sapiens 10Ser Tyr Ala Ile Ser1
51117PRTHomo sapiens 11Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr
Ala Gln Lys Phe Gln1 5 10 15Gly1213PRTHomo sapiens 12Phe Ala Leu
Phe Gly Phe Arg Glu Gln Ala Phe Asp Ile1 5 101311PRTHomo sapiens
13Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn1 5 10147PRTHomo
sapiens 14Ala Ala Ser Ser Leu Gln Ser1 5159PRTHomo sapiens 15Gln
Gln Ser Tyr Ser Thr Pro Phe Thr1 5165PRTHomo sapiens 16Ser Tyr Ala
Met Ser1 51717PRTHomo sapiens 17Ala Ile Ser Gly Ser Gly Gly Ser Thr
Phe Tyr Ala Asp Ser Val Arg1 5 10 15Gly1810PRTHomo sapiens 18Glu
Val Met Gly Pro Ile Phe Phe Asp Tyr1 5 101912PRTHomo sapiens 19Arg
Ala Ser Gln Ser Ile Ile Ser Ser Ala Leu Ala1 5 10207PRTHomo sapiens
20Gly Ala Ser Ser Arg Ala Thr1 5219PRTHomo sapiens 21Gln Gln Tyr
Gly Ser Thr Pro Leu Thr1 5225PRTArtificial Sequencesynthetic
construct 22Ser Tyr Trp Met His1 52316PRTArtificial
Sequencesynthetic construct 23Glu Ile Asp Pro Ser Asp Ser Tyr Lys
Asp Tyr Asn Gln Lys Phe Lys1 5 10 15249PRTArtificial
Sequencesynthetic construct 24Ala Ile Thr Thr Thr Pro Phe Asp Phe1
52511PRTArtificial Sequencesynthetic construct 25Arg Ala Ser Gln
Ser Ile Ser Asn Asn Leu His1 5 10267PRTArtificial Sequencesynthetic
construct 26Tyr Ala Ser Gln Ser Ile Ser1 5279PRTArtificial
Sequencesynthetic construct 27Gln Gln Ser Asn Thr Trp Pro Tyr Thr1
5285PRTArtificial Sequencesynthetic construct 28Asn Tyr Gly Leu
His1 52916PRTArtificial Sequencesynthetic construct 29Val Ile Trp
Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile Ser1 5 10
153015PRTArtificial Sequencesynthetic construct 30Lys Gly Gly Ile
Tyr Tyr Ala Asn His Tyr Tyr Ala Met Asp Tyr1 5 10
153116PRTArtificial Sequencesynthetic construct 31Lys Ser Ser Gln
Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Met1 5 10
15327PRTArtificial Sequencesynthetic construct 32Gly Ala Ser Thr
Arg Glu Ser1 5339PRTArtificial Sequencesynthetic construct 33Gln
Asn Asp His Ser Tyr Pro Leu Thr1 534120PRTHomo sapiens 34Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Val Ile Ser Glu Asn Gly Ser Asp Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Arg Gly Gly Ala Val Ser Tyr Phe
Asp Val Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12035109PRTHomo sapiens 35Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Glu
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Leu Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105
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