U.S. patent application number 15/366791 was filed with the patent office on 2017-06-22 for human antibodies and antibody-drug conjugates against cd74.
The applicant listed for this patent is GENMAB A/S. Invention is credited to Willem Karel BLEEKER, Riemke van DIJKHUIZEN, Steen LISBY, Marije OVERDIJK, Paul PARREN, Patrick VAN BERKEL, Sandra VERPLOEGEN.
Application Number | 20170173151 15/366791 |
Document ID | / |
Family ID | 45560912 |
Filed Date | 2017-06-22 |
United States Patent
Application |
20170173151 |
Kind Code |
A1 |
VERPLOEGEN; Sandra ; et
al. |
June 22, 2017 |
HUMAN ANTIBODIES AND ANTIBODY-DRUG CONJUGATES AGAINST CD74
Abstract
Isolated human monoclonal antibodies which bind to human CD74
and related antibody-drug conjugates are disclosed. Pharmaceutical
compositions comprising the antibodies or antibody-drug conjugates,
and therapeutic and diagnostic methods for using the antibodies
and/or antibody-drug conjugates, are also disclosed.
Inventors: |
VERPLOEGEN; Sandra;
(Utrecht, NL) ; OVERDIJK; Marije; (Utrecht,
NL) ; DIJKHUIZEN; Riemke van; (Zeist, NL) ;
BLEEKER; Willem Karel; (Amsterdam, NL) ; VAN BERKEL;
Patrick; (Utrecht, NL) ; PARREN; Paul;
(Utrecht, NL) ; LISBY; Steen; (Frederiksberg,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENMAB A/S |
Copenhagen K |
|
DK |
|
|
Family ID: |
45560912 |
Appl. No.: |
15/366791 |
Filed: |
December 1, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13982959 |
Oct 7, 2013 |
9540433 |
|
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PCT/EP2012/051679 |
Feb 1, 2012 |
|
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15366791 |
|
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61438383 |
Feb 1, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/73 20130101;
A61K 39/39566 20130101; C07K 2317/92 20130101; A61K 35/76 20130101;
A61K 39/39533 20130101; A61P 37/06 20180101; A61K 47/6849 20170801;
A61K 39/39541 20130101; C07K 16/18 20130101; A61P 43/00 20180101;
A61K 31/7088 20130101; A61K 38/162 20130101; A61K 35/17 20130101;
A61K 38/19 20130101; C07K 2317/33 20130101; A61K 45/06 20130101;
A61P 35/02 20180101; C07K 2317/77 20130101; A61K 51/1027 20130101;
A61K 2121/00 20130101; C07K 2317/21 20130101; A61K 2039/505
20130101; C07K 16/2833 20130101; A61P 35/00 20180101 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 2011 |
DK |
PA201100064 |
Claims
1-56. (canceled)
57. A method of cancer prophylaxis comprising administering to a
subject in need thereof an antibody which binds to the same epitope
on variants 1 and 2 of human CD74 as at least one antibody selected
from: (a) an antibody comprising a VH region comprising the
sequence of SEQ ID NO:19 and a VL region comprising the sequence of
SEQ ID NO:26 [011]; (b) an antibody comprising a VH region
comprising the sequence of SEQ ID NO:7 and a VL region comprising
the sequence of SEQ ID NO:23 [005]; (c) an antibody comprising a VH
region comprising the sequence of SEQ ID NO:11 and a VL region
comprising the sequence of SEQ ID NO:26 [006]; and (d) an antibody
comprising a VH region comprising the sequence of SEQ ID NO:15 and
a VL region comprising the sequence of SEQ ID NO:26 [008].
58. The method of claim 57, wherein the method reduces the risk for
developing cancer.
59. The method of claim 57, wherein the method reduces the risk for
recurrence of a cancer.
60. The method of claim 57, wherein the antibody is administered in
association with surgical removal of a CD74-expressing primary
tumor.
61. The method of claim 57, wherein the antibody is administered in
association with radiotherapy of a CD74-expressing primary
tumor.
62. The method of claim 57, wherein the antibody (a) binds to the
extracellular domain of CD74 variant 1 with an EC.sub.50 of less
than about 500 ng/mL; (b) binds to the extracellular domain of CD74
variant 2 with an EC.sub.50 of less than about 400 ng/mL; or (c)
both of (a) and (b), when determined by enzyme-linked immunosorbant
assay.
63. The method of claim 57, wherein the antibody binds to
cynomolgous CD74.
64. The method of claim 57, wherein the antibody is internalized
after binding to CD74 expressed on the surface of a cell.
65. The method of claim 57, wherein the antibody has an EC.sub.50
of less than about 60 ng/mL in inducing killing of Raji cells in an
anti-kappa ETA' assay.
66. The method of claim 57, wherein the antibody has an off-rate at
0.degree. C. of 0.02 to 1.0 min.sup.-1.
67. The method of claim 57, wherein the antibody comprises a
V.sub.L region comprising the CDR1, 2 and 3 sequences of SEQ ID
NO:24, AAS and SEQ ID NO:25, and a) a V.sub.H region comprising the
CDR1, 2 and 3 sequences of SEQ ID NO: 20, 21 and 22 (011); b) a
V.sub.H region comprising the CDR1, 2 and 3 sequences of SEQ ID
NOS: 8, 9 and 10 (005); c) a V.sub.H region comprising the CDR1, 2
and 3 sequences of SEQ ID NO: 12, 13 and 14 (006); d) a V.sub.H
region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 16, 17
and 18 (008); or e) a variant of any of said antibodies, which
variant preferably has at most one, two or three amino acid
modifications in the V.sub.H and/or V.sub.L region, more preferably
amino acid substitutions, such as conservative amino acid
substitutions in said sequences.
68. The method of claim 57, wherein the antibody comprises: (a) a
V.sub.H region comprising the sequence of SEQ ID NO: 19 and a
V.sub.L region comprising the sequence of SEQ ID NO: 26 [011]; (b)
a V.sub.H region comprising the sequence of SEQ ID NO: 7 and a
V.sub.L region comprising the sequence of SEQ ID NO: 26 [005/011]
(c) a V.sub.H region comprising the sequence of SEQ ID NO: 7 and a
V.sub.L region comprising the sequence of SEQ ID NO: 23 [005]; (d)
a V.sub.H region comprising the sequence of SEQ ID NO: 11 and a
V.sub.L region comprising the sequence of SEQ ID NO: 26 [006]; or
(d) a V.sub.H region comprising the sequence of SEQ ID NO: 15 and a
V.sub.L region comprising the sequence of SEQ ID NO: 26 [008].
69. The method of claim 68, wherein the antibody is a human
monoclonal antibody.
70. The method of claim 69, wherein the antibody has an isotype
selected from IgG1 and IgG4.
71. The method of claim 57, wherein the antibody is conjugated to a
therapeutic moiety.
72. The method of claim 71, wherein the antibody is conjugated to
the therapeutic moiety via a linker attached to sulphydryl residues
in the antibody, obtained by at least partial reduction of the
antibody.
73. The method of claim 71, wherein the therapeutic moiety is a
cytotoxic moiety, a radioisotope, a chemotherapeutic agent, a lytic
peptide or a cytokine.
74. The method of claim 73, wherein the therapeutic moiety is a
cytotoxic moiety.
75. The method of claim 74, wherein the cytotoxic moiety is
selected from the group consisting of taxol; cytochalasin B;
gramicidin D; ethidium bromide; emetine; mitomycin; etoposide;
tenoposide; vincristine; vinblastine; colchicin; doxorubicin;
daunorubicin; dihydroxy anthracin dione; maytansine or an analog or
derivative thereof; an auristatin or a functional peptide analog or
derivative thereof dolastatin 10 or 15 or an analogue thereof;
irinotecan or an analogue thereof; mitoxantrone; mithramycin;
actinomycin D; 1-dehydrotestosterone; a glucocorticoid; procaine;
tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an
analog or derivative thereof; an antimetabolite such as
methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine,
fludarabin, 5 fluorouracil, decarbazine, hydroxyurea, asparaginase,
gemcitabine, or cladribine; an alkylating agent such as
mechlorethamine, thioepa, chlorambucil, melphalan, carmustine
(BSNU), lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine,
mitomycin C; a platinum derivative such as cisplatin or
carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065),
or an analog or derivative thereof; an antibiotic such as
dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin,
mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin
(AMC)); pyrrolo[2,1-c][1,4]-benzodiazepines (PDB); diphtheria toxin
and related molecules such as diphtheria A chain and active
fragments thereof and hybrid molecules, ricin toxin such as ricin A
or a deglycosylated ricin A chain toxin, cholera toxin, a
Shiga-like toxin such as SLT I, SLT II, SLT IIV, LT toxin, C3
toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean
Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin,
saporin, modeccin, gelanin, abrin A chain, modeccin A chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
Phytolacca americana proteins such as PAPI, PAPII, and PAP-S,
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, and enomycin toxins; ribonuclease (RNase); DNase I,
Staphylococcal enterotoxin A; pokeweed antiviral protein;
diphtherin toxin; and Pseudomonas endotoxin.
76. The method of claim 74, wherein the antibody is conjugated to a
cytotoxic moiety selected from the group consisting of an
anthracycline, a pyrrolo[2,1-c][1,4]-benzodiazepine, maytansine,
calicheamicin, duocarmycin, rachelmycin (CC-1065), dolastatin 10 or
15, irinotecan, or from an analog, derivative, or prodrug of any
thereof.
77. The method of claim 74, wherein the cytotoxic moiety is an
auristatin or a functional peptide analog or derivate thereof.
78. The method of claim 73, wherein the antibody is conjugated to a
cytokine selected from the group consisting of IL-2, IL-4, IL-6,
IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27,
IL-28a, IL-28b, IL-29, KGF, IFN.alpha., IFN.beta., IFN.gamma.,
GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and
TNF.alpha..
79. The method of claim 57, wherein the antibody is a multispecific
antibody and comprises at least one second antigen-binding region
having a different binding specificity.
80. The method of claim 79, wherein the antibody is a bispecific
antibody.
81. The method of claim 80, wherein the second antigen-binding
region has binding specificity for an antigen on a human effector
cell.
82. The method of claim 80, wherein, in the bispecific antibody the
first antigen-binding region is linked to a first Fc-region having
an amino acid substitution at a position selected from the group
consisting of 366, 368, 370, 399, 405, 407 and 409, and the second
antigen-binding region is linked to a second Fc-region having an
amino acid substitution at a position selected from the group
consisting of 366, 368, 370, 399, 405, 407 and 409, and the first
and second Fc-regions are not substituted in the same
positions.
83. The method of claim 57, wherein the cancer is selected from the
group consisting of breast cancer, colorectal cancer,
endometrial/cervical cancer, gastric cancer, head and neck cancer,
lung cancer, malignant glioma, malignant melanoma, ovarian cancer,
pancreatic cancer, prostate cancer, renal cancer, liver cancer,
thymus cancer, malignant fibrous histiosarcoma, acoustic
schwannoma, pituitary adenoma, and an adenoma.
84. The method of claim 57, wherein the cancer is selected from the
group consisting of malignant lymphoma, B cell chronic lymphocytic
leukemia (B-CLL), chronic myeloid leukemia (CML) in blast phase,
non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), monocytiod B
cell lymphoma (MBCL), hairy-cell leukemia (HCL), and T cell
lymphoma.
85. The method of claim 84, wherein the cancer is NHL.
86. The method of claim 84, wherein the cancer is MM.
87. The method of claim 83, wherein the cancer is ovarian
cancer.
88. The method of claim 83, wherein the cancer is breast
cancer.
89. The method of claim 83, wherein the cancer is pancreatic
cancer.
90. The method of claim 83, wherein the cancer is selected from
prostate cancer, gastric cancer, and colorectal cancer.
91. The method of claim 57, comprising administering the antibody
in combination with at least one further therapeutic agent.
92. The method of claim 91, wherein the at least one further
therapeutic agent is selected from a second antibody or ADC; a
chemotherapeutic agent; an inhibitor of angiogenesis,
neovascularization, and/or other vascularization; an anti-cancer
immunogen; a cytokine or chemokine; a cell cycle control or
apoptosis regulator; a hormonal regulating agent; an anti-anergic
agent; a tumor suppressor gene-containing nucleic acid or vector;
an anti-cancer nucleic acid; a virus or viral proteins; immune
system cells; a differentiation inducing agent; a CD74
up-regulating agent; and an anti-inflammatory, immunosuppressive
and/or immunomodulatory agent; or a combination of any thereof.
93. The method of claim 92, wherein at least one therapeutic agent
is selected from a CD20-specific antibody, a CD138-specific
antibody, a CD38-specific antibody, an anti-VEGF-A antibody,
melphalanan, lenalidomide, bortezomib, fluorouracil, gemticabine,
irinotecan, cisplatin, or a derivative or analog thereof.
94. A method for detecting the presence of CD74 antigen, or a cell
expressing CD74, in a sample comprising contacting the sample with
a CD74 specific antibody under conditions that allow for binding of
the CD74 specific antibody to CD74 in the sample; and analyzing
whether a complex has been formed, wherein the antibody binds to
the same epitope on variants 1 and 2 of human CD74 as at least one
antibody selected from: (a) an antibody comprising a VH region
comprising the sequence of SEQ ID NO:19 and a VL region comprising
the sequence of SEQ ID NO:26 [011]; (b) an antibody comprising a VH
region comprising the sequence of SEQ ID NO:7 and a VL region
comprising the sequence of SEQ ID NO:23 [005]; (c) an antibody
comprising a VH region comprising the sequence of SEQ ID NO:11 and
a VL region comprising the sequence of SEQ ID NO:26 [006]; and (d)
an antibody comprising a VH region comprising the sequence of SEQ
ID NO:15 and a VL region comprising the sequence of SEQ ID NO:26
[008].
95. The method of claim 94, wherein the sample is a biological
sample.
96. The method of claim 95, wherein the biological sample has been
taken from a patient, and the biological sample is known or
suspected of containing CD74 antigen and/or cells expressing
CD74.
97. The method of claim 96, wherein the patient suffers from a
disease in which cells expressing CD74 are indicative of disease or
involved in the pathogenesis.
98. The method of claim 97, wherein the disease is cancer.
99. The method of claim 94, wherein the analyzing step comprises
one or more of ELISA, RIA, FACS assays, plasmon resonance assays,
chromatographic assays, tissue immunohistochemistry, Western blot
and immunoprecipitation.
100. The method of claim 99, wherein the antibody is labeled with a
detectable substance or is detected by a second antibody labeled
with a detectable substance.
101. The method of claim 100, wherein the detectable substance is
selected from an enzyme, a prosthetic group, a fluorescent
material, a luminescent material, and a radioactive material.
102. The method of claim 94, wherein the antibody (a) binds to the
extracellular domain of CD74 variant 1 with an EC.sub.50 of less
than about 500 ng/mL; (b) binds to the extracellular domain of CD74
variant 2 with an EC.sub.50 of less than about 400 ng/mL; or (c)
both of (a) and (b), when determined by enzyme-linked immunosorbant
assay.
103. The method of claim 94, wherein the antibody binds to
cynomolgous CD74.
104. The method of claim 94, wherein the antibody is internalized
after binding to CD74 expressed on the surface of a cell.
105. The method of claim 94, wherein the antibody has an EC.sub.50
of less than about 60 ng/mL in inducing killing of Raji cells in an
anti-kappa ETA' assay.
106. The method of claim 94, wherein the antibody has an off-rate
at 0.degree. C. of 0.02 to 1.0 min.sup.-1.
107. The method of claim 94, wherein the antibody comprises a
V.sub.L region comprising the CDR1, 2 and 3 sequences of SEQ ID
NO:24, AAS and SEQ ID NO:25, and a) a V.sub.H region comprising the
CDR1, 2 and 3 sequences of SEQ ID NO: 20, 21 and 22 (011); b) a
V.sub.H region comprising the CDR1, 2 and 3 sequences of SEQ ID
NOS: 8, 9 and 10 (005); c) a V.sub.H region comprising the CDR1, 2
and 3 sequences of SEQ ID NO: 12, 13 and 14 (006); d) a V.sub.H
region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 16, 17
and 18 (008); or e) a variant of any of said antibodies, which
variant preferably has at most one, two or three amino acid
modifications in the V.sub.H and/or V.sub.L region, more preferably
amino acid substitutions, such as conservative amino acid
substitutions in said sequences.
108. The method of claim 94, wherein the antibody comprises: (a) a
V.sub.H region comprising the sequence of SEQ ID NO: 19 and a
V.sub.L region comprising the sequence of SEQ ID NO: 26 [011]; (b)
a V.sub.H region comprising the sequence of SEQ ID NO: 7 and a
V.sub.L region comprising the sequence of SEQ ID NO: 26 [005/011]
(c) a V.sub.H region comprising the sequence of SEQ ID NO: 7 and a
V.sub.L region comprising the sequence of SEQ ID NO: 23 [005]; (d)
a V.sub.H region comprising the sequence of SEQ ID NO: 11 and a
V.sub.L region comprising the sequence of SEQ ID NO: 26 [006]; or
(d) a V.sub.H region comprising the sequence of SEQ ID NO: 15 and a
V.sub.L region comprising the sequence of SEQ ID NO: 26 [008].
109. The method of claim 108, wherein the antibody is a human
monoclonal antibody.
110. The method of claim 109, wherein the antibody has an isotype
selected from IgG1 and IgG4.
111. The method of claim 98, wherein the cancer is selected from
the group consisting of breast cancer, colorectal cancer,
endometrial/cervical cancer, gastric cancer, head and neck cancer,
lung cancer, malignant glioma, malignant melanoma, ovarian cancer,
pancreatic cancer, prostate cancer, renal cancer, liver cancer,
thymus cancer, malignant fibrous histiosarcoma, acoustic
schwannoma, pituitary adenoma, and an adenoma.
112. The method of claim 98, wherein the cancer is selected from
the group consisting of malignant lymphoma, B cell chronic
lymphocytic leukemia (B-CLL), chronic myeloid leukemia (CML) in
blast phase, non-Hodgkin's lymphoma (NHL), multiple myeloma (MM),
monocytiod B cell lymphoma (MBCL), hairy-cell leukemia (HCL), and T
cell lymphoma.
113. The method of claim 112, wherein the cancer is NHL.
114. The method of claim 112, wherein the cancer is MM.
115. The method of claim 112, wherein the cancer is ovarian
cancer.
116. The method of claim 107, wherein the cancer is breast
cancer.
117. The method of claim 107, wherein the cancer is pancreatic
cancer.
118. The method of claim 107, wherein the cancer is selected from
prostate cancer, gastric cancer, and colorectal cancer.
119. A method for the in vivo imaging of a CD74-expressing tissue,
comprising administering an antibody which binds to the same
epitope on variants 1 and 2 of human CD74 as at least one antibody
selected from (a) an antibody comprising a VH region comprising the
sequence of SEQ ID NO:19 and a VL region comprising the sequence of
SEQ ID NO:26 [011]; (b) an antibody comprising a VH region
comprising the sequence of SEQ ID NO:7 and a VL region comprising
the sequence of SEQ ID NO:23 [005]; (c) an antibody comprising a VH
region comprising the sequence of SEQ ID NO:11 and a VL region
comprising the sequence of SEQ ID NO:26 [006]; and (d) an antibody
comprising a VH region comprising the sequence of SEQ ID NO:15 and
a VL region comprising the sequence of SEQ ID NO:26 [008], wherein
the antibody is conjugated to at least one of a radioisotope, a
radio-opaque agent, a dye, a contrast agent, a fluorescent compound
or molecule and a paramagnetic ion.
120. The method of claim 115, wherein the CD74-expressing tissue is
a tumor.
121. A kit for detecting the presence of CD74 antigen or a cell
expressing CD74, in a sample, the kit comprising: a CD74 specific
antibody, and instructions for use of the kit, wherein the antibody
binds to the same epitope on variants 1 and 2 of human CD74 as at
least one antibody selected from: (a) an antibody comprising a VH
region comprising the sequence of SEQ ID NO:19 and a VL region
comprising the sequence of SEQ ID NO:26 [011]; (b) an antibody
comprising a VH region comprising the sequence of SEQ ID NO:7 and a
VL region comprising the sequence of SEQ ID NO:23 [005]; (c) an
antibody comprising a VH region comprising the sequence of SEQ ID
NO:11 and a VL region comprising the sequence of SEQ ID NO:26
[006]; and (d) an antibody comprising a VH region comprising the
sequence of SEQ ID NO:15 and a VL region comprising the sequence of
SEQ ID NO:26 [008].
122. The kit of claim 121, further comprising one or more reagents
for detecting binding of the CD74-specific antibody to CD74.
123. The kit of claim 122, wherein the reagents comprise one or
more of a fluorescent tag, an enzymatic tag, a secondary antibody
and reagents for enzymatic reactions, wherein the enzymatic
reactions produce a product that may be visualized.
124. The kit of claim 121, wherein the antibody is labeled with a
detectable substance.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/982,959 (now U.S. Pat. No. 9,540,433),
filed Oct. 7, 2013, which is a 35 U.S.C. 371 national stage filing
of International Application No. PCT/EP2012/051679, filed Feb. 1,
2012, which claims priority to U.S. Provisional Application No.
61/438,383, filed Feb. 1, 2011 and Danish Patent Application No. PA
2011 00064, filed Feb. 1, 2011. The entire contents of the
aforementioned applications are hereby incorporated herein by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 1, 2016, is named GMI_138USCN_Sequence_Listing.txt and is
21,802 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to CD74-specific antibodies
and antibody-drug conjugates (ADCs) thereof, pharmaceutical
compositions of such antibodies or ADCs, and their use in
therapeutic applications.
BACKGROUND OF THE INVENTION
[0004] Human leukocyte antigen (HLA) class II histocompatibility
antigen gamma chain, also called HLA-DR antigen-associated
invariant chain, Ia antigen-associated invariant chain, Ii and
CD74, is a transmembrane protein with a short cytoplasmic tail. The
primary function of CD74 is to regulate peptide loading onto the
major histocompatibility complex (MHC) class II heterodimers in
intracellular compartments.
[0005] Only a small portion of the total cell CD74 content is
expressed on the cell surface. Cell surface CD74 is very rapidly
internalized both with and without CD74 antibodies bound (Roche P A
et al., PNAS 1993; 90: 8581-8585; Hansen H J et al., Biochem J
1996; 320: 293-300; Ong G L et al., Immunology 1999; 98:296-302).
The steady-state level of cell surface CD74 is therefore rather
low, varying in monocytes from a few hundred to a few thousand
molecules per cell.
[0006] The exact function of cell surface-expressed CD74 is not
known, but studies have documented CD74 as a membrane receptor for
the pro-inflammatory cytokine macrophage migration inhibitory
factor (MIF). MIF binding to CD74 activates downstream signaling
through the MAPK and Akt pathways and promotes cell proliferation
and survival. This interaction is likely regulated also by the
presence of CD44, CXCR2 or CXCR4 as co-receptors.
[0007] Upregulation of CD74 expression has been observed in many
types of cancer, as well as in certain infections and inflammatory
conditions. Various formats of a humanized CD74-specific monoclonal
antibody, hLL1, have been proposed for treatment of CD74-positive
tumors (Chang C H et al., Blood 2005; 106:4308-4314; Sapra P et
al., Clin Can Res 2005; 11:5257-5264; Stein R et al., Blood 2004;
104:3705-11; Govindan S V et al. J Nucl Med 2000; 41:2089-2097;
Hertlein E et al., Blood 2010; 116: 2554-2558; Stein R et al., Clin
Cancer Res 2009; 15: 2808-2817; Sharkey R M et al., J Nucl Med
2009; 50: 444-453; Lundberg B B et al., Drug Deliv 2007; 14:
171-175; Griffiths G L et al., Int J Cancer 1999; 81: 985-992;
Griffiths G L et al., Cancer Res 2003; 9: 6567-6571; Ochakovskaya R
et al., Clin Cancer Res 2001; 7: 1505-1510; Shih L et al., Cancer
Immunol Immunother; Burton J D et al., Clin Cancer Res 2004; 10:
6606-6611; Lundberg B B et al., J Control Release 2004; 94:
155-161).
[0008] Although much progress has been made, there remains a need
for improved methods of treating serious diseases, e.g. improved
treatment of cancer, based on therapeutic antibodies and ADCs.
SUMMARY OF THE INVENTION
[0009] It is an object of the present invention to provide novel
highly specific and effective monoclonal CD74-specific antibodies
and ADCs of such CD74-specific antibodies. The antibodies or ADCs
of the invention exhibit CD74 binding characteristics or other
effects on CD74-expressing cells that differ from antibodies
described in the art. Particularly, the antibodies are
characterized by rapid internalization upon binding to CD74
antigen, making them suitable for therapeutic applications in the
form of ADCs and for other applications where rapid internalization
is an advantage. The novel ADCs are characterized by a high
efficiency in killing CD74-expressing tumor cells.
[0010] The antibodies and corresponding ADCs can be provided in a
variety of formats, including, but not limited to, antibody
fragment and bispecific antibody formats. In preferred embodiments,
the antibodies are human.
[0011] It is also an object of the present invention to provide
ADCs based on such CD74-specific antibodies for medical use,
providing an efficient and selective way of causing cell death of
tumor cells.
[0012] These and other aspects of the invention are described in
further detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGS. 1A-1F: Amino acid sequences of recombinant CD74
proteins used in the Examples. CD74v1 and -v2, CD74del2-36v1 and
-v2, and HisCD74v1 and -v2 correspond to SEQ ID NOS: 1-6,
respectively.
[0014] FIGS. 2A and 2B: Alignment of variable heavy (VH) and
variable light (VL) chain sequences of the antibodies of the
present invention. The SEQ ID NO of each VH/VL sequence is listed
within parentheses to the right of the sequence.
Complementarity-determining regions (CDRs) according to IMGT
nomenclature are highlighted as follows: sequences in italics
represent CDR1, underlined sequences represent CDR2, and bold
sequences represent CDR3.
[0015] FIGS. 3A and 3B: Binding of CD74-specific antibodies to
recombinant protein representing the extracellular domain of the
variant 1 and 2 isoforms (CD74v1 and CD74v2), determined by ELISA.
All human antibodies were produced by transiently co-transfecting
HEK-293F cells with relevant heavy and light chain expression
vectors.
[0016] FIG. 4: Binding of CD74-specific antibodies to cellular CD74
on Raji cells, determined by FACS. All human antibodies were
produced by transiently co-transfecting HEK-293F cells with
relevant heavy and light chain expression vectors.
[0017] FIG. 5: Cross-reactivity of CD74-specific antibodies with
cynomolgus CD74. Human tonsil (upper panel) and cynomolgus lymph
nodes (lower panel) were stained with CD74-specific antibodies. *:
germinal center; Mf: macrophages; #: Mantle zone B cells.
[0018] FIG. 6: Dose-dependent induction of cell killing by
anti-kappa-ETA'-pre-incubated CD74-specific antibodies. A
representative experiment is shown. Data shown are mean percentages
viability of duplicate wells of cells treated with
anti-kappa-ETA'-pre-incubated CD74 HuMab antibodies. Percentage
viability was calculated as described in Example 14.
[0019] FIGS. 7A and 7B: Binding of CD74 HuMab antibodies 005 and
006 (FIG. 7A) and 011 (FIG. 7B) and the corresponding ADCs to
recombinant protein of the CD74v1 extracellular domain, as
determined by ELISA. One representative experiment is shown.
[0020] FIGS. 8A and 8B: Binding of CD74 HuMab antibodies 005 and
006 (FIG. 8A) and 011 (FIG. 8B) and the corresponding ADCs to
surface-expressed CD74, determined by FACS analysis on Daudi cells.
Data shown are mean fluorescence intensities (MFI), calculated from
three independent experiments.
[0021] FIGS. 9A-9D: Dose-dependent induction of cell killing by
CD74-specific ADCs. One representative experiment is shown for each
of the following cell lines: Daudi (FIG. 9A), Raji (FIG. 9B), M4A4
(FIG. 9C) and NCI-H747 (FIG. 9D) cells. Data shown are percentages
survival of duplicate wells of cells treated with CD74-specific
ADCs.
[0022] FIG. 10: In vivo efficacy of CD74-specific ADCs in
therapeutic treatment of Daudi-luc xenografts in SCID mice. Mice
with established Daudi-luc tumors were treated with CD74-specific
ADCs. Data shown are mean bioluminescence imaging (BLI)
signals.+-.S.E.M. per group (n=7 mice per group).
[0023] FIG. 11: In vivo efficacy of CD74-specific ADCs in
therapeutic treatment of Raji-luc xenografts in SCID mice. Mice
with established Raji-luc tumors were treated with CD74-specific
ADCs. Data shown are mean BLI signals.+-.S.E.M. per group (n=7 mice
per group).
[0024] FIG. 12: In vivo efficacy of CD74-specific ADCs in
therapeutic treatment of Raji xenografts in SCID mice. Mice with
established s.c. Raji tumors were treated with CD74-specific ADCs.
Data shown are mean tumor volumes.+-.S.E.M. per group (n=6 mice per
group).
[0025] FIG. 13: In vivo efficacy of anti CD74 ADCs in therapeutic
treatment of M4A4 xenografts in SCID mice. Mice with established
M4A4 tumors were treated with anti CD74 ADCs. Data shown are mean
tumor volumes.+-.S.E.M. per group (n=7 mice per group).
[0026] FIG. 14: Determination of off-rates of CD74-specific HuMab
antibodies. One representative experiment is shown. Data shown are
mean fluorescence intensities (MFI) of triplicate wells of cells
incubated with Alexa Fluor 488.RTM. Dye-labeled CD74 HuMab
antibodies, followed by incubation with unlabeled CD74 HuMab
antibodies for the indicated time intervals.
[0027] FIGS. 15A-15D: Time-dependent internalization and
accumulation of anti-CD74 HuMab antibodies. One representative
experiment is shown for each cell line. Data shown are mean
fluorescence intensities (MFI) of duplicate wells of cells
incubated with Alexa-488-labeled anti-CD74 HuMab antibodies. Daudi
cells were incubated with Alexa-488-labeled anti-CD74 HuMab
antibodies at 4.degree. C. (FIG. 15A) or 37.degree. C. (FIG. 15B).
Raji cells (FIG. 15C) and M4A4 cells (FIG. 15D) were incubated at
37.degree. C.
[0028] FIG. 16: In vivo efficacy of anti-CD74 HuMab antibodies in
prophylactic treatment of Daudi luc xenografts in SCID mice. Mice
were treated with anti CD74 HuMab antibodies within one hour after
intravenous inoculation of Daudi luc tumors. Data shown are mean
BLI signals.+-.S.E.M. per group (n=7 mice per group).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0029] The terms "CD74" and "CD74 antigen" are used interchangeably
herein. Unless specified otherwise, the terms include any variants,
isoforms and species homologs of human CD74 which are naturally
expressed by cells or are expressed on cells transfected with the
CD74 gene. At least four human isoforms are known to exist; p43,
p41, p35 and pp33 (Borghese F et al., Expert Opin Ther Targets
2011; 15(3): 237-251). These result from alternative transcript
splicing and two translation start sites. p43 (also known as CD74
isoform 1, isoform a, or "long"; see UniProt entry P04233-1 and
NCBI Reference Sequence NP 001020330) contains 296 amino acids,
with residues 73-296 forming the extracellular portion. Protein
constructs of CD74 having the extracellular part of isoform 1 are
herein referred to as "variant 1" or "CD74v1." p35 (also known as
CD74 isoform 2, isoform b or "short"; see Uniprot entry P04233-2
and NCBI Reference Sequence NP 004346) lacks residues 209-272 from
the extracellular part due to alternative splicing. Protein
constructs of CD74 having the extracellular part of isoform 2 are
herein referred to as "variant 2" or "CD74v2." p41 and p33 arise
from an alternative translation start site (48 bp downstream; 16
amino acids shorter protein) leading to variants lacking the
endoplasmic reticulum (ER) retention signal that is present within
these 16 amino acids, but having an identical extracellular part as
p43 and p35, respectively. The sequence of another isoform (known
as isoform 3 and isoform c), in which residues 148-160 are replaced
and residues 161-296 are lacking, is provided in NP 001020329. The
sequences of cynomolgus CD74 homologs are provided in, e.g., NCBI
Reference Sequence: XP_001099491.2 and NCBI Reference Sequence:
XP_002804624.1.
[0030] The term "immunoglobulin" refers to a class of structurally
related glycoproteins consisting of two pairs of polypeptide
chains, one pair of light (L) low molecular weight chains and one
pair of heavy (H) chains, all four inter-connected by disulfide
bonds. The structure of immunoglobulins has been well
characterized. See for instance Fundamental Immunology Ch. 7 (Paul,
W., ed., 2.sup.nd ed. Raven Press, N.Y. (1989)). Briefly, each
heavy chain typically is comprised of a heavy chain variable region
(abbreviated herein as V.sub.H or VH) and a heavy chain constant
region (C.sub.H or CH). The heavy chain constant region typically
is comprised of three domains, C.sub.H1, C.sub.H2, and C.sub.H3.
Each light chain typically is comprised of a light chain variable
region (abbreviated herein as V.sub.L or VL) and a light chain
constant region. The light chain constant region typically is
comprised of one domain, C.sub.L or CL. Typically, the numbering of
amino acid residues in the constant region is performed according
to the EU-index as described in Kabat et al., Sequences of Proteins
of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). The VH and VL regions
may be further subdivided into regions of hypervariability (or
hypervariable regions which may be hypervariable in sequence and/or
form of structurally defined loops), also termed complementarity
determining regions (CDRs), interspersed with regions that are more
conserved, termed framework regions (FRs). Each V.sub.H and V.sub.L
is typically composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol.
Biol. 196, 901 917 (1987)).
[0031] The term "antibody" or "Ab" in the context of the present
invention refers to an immunoglobulin molecule, a fragment of an
immunoglobulin molecule, or a derivative of either thereof, which
has the ability to specifically bind to an antigen under typical
physiological conditions with a half life of significant periods of
time, such as at least about 30 minutes, at least about 45 minutes,
at least about one hour, at least about two hours, at least about
four hours, at least about eight hours, at least about 12 hours,
about 24 hours or more, about 48 hours or more, about three, four,
five, six, seven or more days, etc., or any other relevant
functionally-defined period (such as a time sufficient to induce,
promote, enhance, and/or modulate a physiological response
associated with antibody binding to the antigen and/or time
sufficient for the antibody to recruit an effector activity). The
variable regions of the heavy and light chains of the
immunoglobulin molecule contain a binding domain that interacts
with an antigen. The constant regions of the antibodies (Abs) may
mediate the binding of the immunoglobulin to host tissues or
factors, including various cells of the immune system (such as
effector cells) and components of the complement system such as
C1q, the first component in the classical pathway of complement
activation. An antibody may also be multispecific, having
specificities for two or more different epitopes, typically
non-overlapping. Examples of multispecific antibodies include
bispecific antibodies, diabodies, and similar antibody molecules.
As indicated above, the term antibody herein, unless otherwise
stated or clearly contradicted by context, includes fragments of an
antibody that retain the ability to specifically bind to the
antigen. It has been shown that the antigen-binding function of an
antibody may be performed by fragments of a full-length antibody,
e.g., Fab and F(ab').sub.2 fragments. It also should be understood
that the term antibody, unless specified otherwise, also includes
polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like
polypeptides such as chimeric antibodies and humanized antibodies.
An antibody as generated can possess any isotype.
[0032] The terms "human antibody", "human Ab" or "HuMab", as used
herein, is intended to include antibodies having variable and
constant regions derived from human germline immunoglobulin
sequences. The human antibodies of the invention may include amino
acid residues not encoded by human germline immunoglobulin
sequences (e.g., mutations introduced by random or site-specific
mutagenesis in vitro or by somatic mutation in vivo). However, the
term "human antibody", as used herein, is not intended to include
antibodies in which CDR sequences derived from the germline of
another mammalian species, such as a mouse, have been grafted onto
human framework sequences.
[0033] As used herein, a human antibody is "derived from" a
particular germline sequence if the antibody is obtained from a
system using human immunoglobulin sequences, for instance by
immunizing a transgenic mouse carrying human immunoglobulin genes
or by screening a human immunoglobulin gene library, and wherein
the selected human antibody is at least 90%, such as at least 95%,
for instance at least 96%, such as at least 97%, for instance at
least 98%, or such as at least 99% identical in amino acid sequence
to the amino acid sequence encoded by the germline immunoglobulin
gene. Typically, outside the heavy chain CDR3, a human antibody
derived from a particular human germline sequence will display no
more than 20 amino acid differences, e.g. no more than 10 amino
acid differences, such as no more than 9, 8, 7, 6 or 5, for
instance no more than 4, 3, 2, or 1 amino acid difference from the
amino acid sequence encoded by the germline immunoglobulin
gene.
[0034] The terms "monoclonal antibody", "monoclonal Ab",
"monoclonal antibody composition", "mAb", or the like, as used
herein refer to a preparation of antibody molecules of single
molecular composition. A monoclonal antibody composition displays a
single binding specificity and affinity for a particular epitope.
Accordingly, the term "human monoclonal antibody" refers to
antibodies displaying a single binding specificity which have
variable and constant regions derived from human germline
immunoglobulin sequences. The human monoclonal antibodies may be
produced by a hybridoma which includes a B cell obtained from a
transgenic or transchromosomal non-human animal, such as a
transgenic mouse, having a genome comprising a human heavy chain
transgene and a light chain transgene, fused to an immortalized
cell.
[0035] As used herein, "isotype" refers to the immunoglobulin class
(for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that
is encoded by heavy chain constant region genes.
[0036] The term "full-length antibody" when used herein, refers to
an antibody which contains all heavy and light chain constant and
variable domains that are normally found in an antibody of that
isotype.
[0037] When used herein, unless contradicted by context, the term
"Fab-arm" or "arm" refers to one heavy chain-light chain pair.
[0038] When used herein, unless contradicted by context, the term
"Fc region" refers to an antibody region comprising at least one
hinge region, a C.sub.H2 domain, and a C.sub.H3 domain.
[0039] An "antibody deficient in effector function" or an "effector
function-deficient antibody" refers to an antibody which has a
significantly reduced or no ability to activate one or more
effector mechanisms, such as complement activation or Fc receptor
binding. Thus, effector-function deficient antibodies have
significantly reduced or no ability to mediate antibody-dependent
cell-mediated cytotoxicity (ADCC) and/or complement-dependent
cytotoxicity (CDC). An example of such an antibody is an antibody
of IgG4 isotype or a hinge-stabilized form thereof. Another example
is the introduction of mutations in Fc region which can strongly
reduce the interaction with complement proteins and Fc receptors.
See, for example, Bolt S et al., Eur J Immunol 1993, 23:403-411;
Oganesyan, Acta Crys. 2008, D64, 700-704; and Shields et al., JBC
2001, 276: 6591-6604.
[0040] As used herein, the term "effector cell" refers to an immune
cell which is involved in the effector phase of an immune response,
as opposed to the cognitive and activation phases of an immune
response. Exemplary immune cells include a cell of a myeloid or
lymphoid origin, for instance lymphocytes (such as B cells and T
cells including cytolytic T cells (CTLs)), killer cells, natural
killer cells, macrophages, monocytes, mast cells and granulocytes,
such as neutrophils, eosinophils and basophils. Some effector cells
express specific Fc receptors (FcRs) and carry out specific immune
functions. In some embodiments, an effector cell is capable of
inducing ADCC, such as a natural killer cell. For example,
monocytes, macrophages, which express FcRs, are involved in
specific killing of target cells and presenting antigens to other
components of the immune system. In some embodiments, an effector
cell may phagocytose a target antigen or target cell. The
expression of a particular FcR on an effector cell may be regulated
by humoral factors such as cytokines. An effector cell can
phagocytose a target antigen or phagocytose or lyse a target
cell.
[0041] In the context of the present invention, an "ADC" refers to
an antibody-drug conjugate, in the context of the present invention
typically referring to a CD74-specific antibody, which is coupled
to another moiety as described in the present application.
[0042] A "CD74 antibody", "anti-CD74 antibody", "CD74 Ab",
"CD74-specific antibody" or "anti-CD74 Ab" is an antibody as
described above, which binds specifically to the antigen CD74.
[0043] In a preferred embodiment, the antibody of the invention is
isolated. An "isolated Ab," as used herein, is intended to refer to
an antibody which is substantially free of other antibodies having
different antigenic specificities (for instance an isolated
antibody that specifically binds to CD74 is substantially free of
antibodies that specifically bind antigens other than CD74). An
isolated antibody that specifically binds to an epitope, isoform or
variant of human CD74 may, however, have cross-reactivity to other
related antigens, for instance from other species (such as CD74
species homologs). Moreover, an isolated antibody may be
substantially free of other cellular material and/or chemicals. In
one embodiment of the present invention, two or more "isolated"
monoclonal antibodies having different antigen-binding
specificities are combined in a well-defined composition.
[0044] When used herein in the context of two or more antibodies,
the term "competes with" or "cross-competes with" indicates that
the two or more antibodies compete for binding to CD74, e.g., to
CD74 variants 1, 2 or both. For example, the constructs described
in Example 1 can be used in such an assay. In one exemplary type of
assay, CD74 is coated on a plate and allowed to bind the first
antibody, after which the second, labeled antibody is added. If the
presence of the first antibody reduces binding of the second
antibody, the antibodies compete. The term "competes with" when
used herein is also intended to cover combinations of antibodies
where one antibody reduces binding of another antibody, but where
no competition is observed when the antibodies are added in the
reverse order.
[0045] The term "epitope" means a protein determinant capable of
specific binding to an antibody. Epitopes usually consist of
surface groupings of molecules such as amino acids or sugar side
chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics.
Conformational and non-conformational epitopes are distinguished in
that the binding to the former but not the latter is lost in the
presence of denaturing solvents. The epitope may comprise amino
acid residues which are directly involved in the binding, and other
amino acid residues, which are not directly involved in the
binding, such as amino acid residues which are effectively blocked
or covered by the specifically antigen binding peptide (in other
words, the amino acid residue is within the footprint of the
specifically antigen binding peptide).
[0046] As used herein, the term "binding" in the context of the
binding of an antibody to a predetermined antigen or epitope
typically is a binding with an affinity corresponding to a K.sub.D
of about 10.sup.-7 M or less, such as about 10.sup.-8 M or less,
such as about 10.sup.-9 M or less, about 10.sup.-10 M or less, or
about 10.sup.-11 M or even less when determined by for instance
surface plasmon resonance (SPR) technology in a BIAcore 3000
instrument using a soluble form of the antigen as the ligand and
the antibody as the analyte. Typically, an antibody binds to the
predetermined antigen with an affinity corresponding to a K.sub.D
that is at least ten-fold lower, such as at least 100-fold lower,
for instance at least 1,000-fold lower, such as at least
10,000-fold lower, for instance at least 100,000-fold lower than
its K.sub.D for binding to a non-specific antigen (e.g., BSA,
casein), which is not identical or closely related to the
predetermined antigen. When the K.sub.D of the antibody is very low
(that is, the antibody has a high affinity), then the K.sub.D with
which it binds the antigen is typically at least 10,000-fold lower
than its K.sub.D for a non-specific antigen.
[0047] The term "k.sub.d" (sec.sup.-1), as used herein, refers to
the dissociation rate constant of a particular Ab-antigen
interaction. Said value is also referred to as the k.sub.off
value.
[0048] The term "k.sub.a" (M.sup.-1.times.sec.sup.-1), as used
herein, refers to the association rate constant of a particular
Ab-antigen interaction.
[0049] The term "K.sub.D" (M), as used herein, refers to the
dissociation equilibrium constant of a particular Ab-antigen
interaction.
[0050] The term "K.sub.A" (M.sup.-1), as used herein, refers to the
association equilibrium constant of a particular Ab-antigen
interaction and is obtained by dividing the k.sub.a by the
k.sub.d.
[0051] As used herein, "internalization", when used in the context
of a CD74 antibody includes any mechanism by which the antibody is
internalized from the cell-surface into a CD74-expressing cell. The
internalization of an antibody can be evaluated in an indirect
assay measuring the effect of an internalized Ab-toxin conjugate or
a toxin specifically bound to an antibody by pre-incubation (such
as, e.g., the anti-kappa-ETA' assay of Example 14).
[0052] As used herein, the term "inhibits growth" (e.g. referring
to cells, such as tumor cells) is intended to include any
measurable decrease in the cell growth when contacted with a CD74
antibody or ADC as compared to the growth of the same cells not in
contact with a CD74 antibody or ADC, e.g., the inhibition of growth
of a cell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 99%, or 100%. Such a decrease in cell growth can
occur by a variety of mechanisms, e.g. internalization,
antibody-dependent cellular phagocytosis (ADCP), antibody-dependent
cell-mediated cytotoxicity (ADCC), complement-dependent
cytotoxicity (CDC), drug-mediated cell-kill and/or apoptosis.
[0053] The present invention also provides antibodies comprising
functional variants of the V.sub.L region, V.sub.H region, or one
or more CDRs of the antibodies of the examples. A functional
variant of a V.sub.L, V.sub.H, or CDR used in the context of a CD74
antibody still allows the antibody to retain at least a substantial
proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of
the affinity/avidity and/or the specificity/selectivity of the
parent antibody and in some cases such a CD74 antibody may be
associated with greater affinity, selectivity and/or specificity
than the parent Ab.
[0054] Such functional variants typically retain significant
sequence identity to the parent Ab. The percent identity between
two sequences is a function of the number of identical positions
shared by the sequences (i.e., % homology=# of identical
positions/total # of positions.times.100), taking into account the
number of gaps, and the length of each gap, which need to be
introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences may be accomplished using a mathematical
algorithm, as described in the non-limiting examples below.
[0055] The percent identity between two nucleotide sequences may be
determined using the GAP program in the GCG software package
(available at http://www.gcg.com), using a NWSgapdna.CMP matrix and
a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,
3, 4, 5, or 6. The percent identity between two nucleotide or amino
acid sequences may also be determined using the algorithm described
by E. Meyers and W. Miller (Comput. Appl. Biosci 4, 11-17 (1988)),
which has been incorporated into the ALIGN program (version 2.0),
using a PAM120 weight residue table, a gap length penalty of 12 and
a gap penalty of 4. In addition, the percent identity between two
amino acid sequences may be determined using the Needleman and
Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. 48, 444-453
(1970)), which has been incorporated into the GAP program in the
GCG software package (available at http://www.gcg.com), using
either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6.
[0056] The sequence of CDR variants may differ from the sequence of
the CDR of the parent antibody sequences through mostly
conservative substitutions; for instance at least about 35%, about
50% or more, about 60% or more, about 70% or more, about 75% or
more, about 80% or more, about 85% or more, about 90% or more,
(e.g., about 65-95%, such as about 92%, 93% or 94%) of the
substitutions in the variant are conservative amino acid residue
replacements.
[0057] The sequences of CDR variants may differ from the sequence
of the CDRs of the parent antibody sequences through mostly
conservative substitutions; for instance at least 10, such as at
least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the
variant are conservative amino acid residue replacements.
[0058] The term "stabilized IgG4 antibody" refers to an IgG4
antibody which has been modified to reduce half-molecule exchange
(see, e.g., international patent application publication
WO2008145142 or van der Neut Kolfschoten M et al. (2007) Science
14; 317(5844) and references therein.
[0059] In the context of the present invention, conservative
substitutions may be defined by substitutions within the classes of
amino acids reflected in one or more of the following three
tables:
Amino Acid Residue Classes for Conservative Substitutions
TABLE-US-00001 [0060] Acidic Residues Asp (D) and Glu (E) Basic
Residues Lys (K), Arg (R), and His (H) Hydrophilic Uncharged
Residues Ser (S), Thr (T), Asn (N), and Gln (Q) Aliphatic Uncharged
Residues Gly (G), Ala (A), Val (V), Leu (L), and Ile (I) Non-polar
Uncharged Residues Cys (C), Met (M), and Pro (P) Aromatic Residues
Phe (F), Tyr (Y), and Trp (W)
Alternative Conservative Amino Acid Residue Substitution
Classes
TABLE-US-00002 [0061] 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W
Alternative Physical and Functional Classifications of Amino Acid
Residues
TABLE-US-00003 [0062] Alcohol group-containing residues S and T
Aliphatic residues I, L, V, and M Cycloalkenyl-associated residues
F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T,
V, W, and Y Negatively charged residues D and E Polar residues C,
D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and
R Small residues A, C, D, G, N, P, S, T, and V Very small residues
A, G, and S Residues involved in turn A, C, D, E, G, H, K, N, Q, R,
S, P, formation and T Flexible residues Q, T, K, S, G, P, D, E, and
R
[0063] More conservative substitutions groupings include:
valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,
alanine-valine, and asparagine-glutamine.
[0064] Additional groups of amino acids may also be formulated
using the principles described in e.g. Creighton (1984) Proteins:
Structure and Molecular Properties (2d Ed. 1993), W.H. Freeman and
Company.
[0065] Conservation in terms of hydropathic/hydrophilic properties
and residue weight/size also is substantially retained in a variant
CDR as compared to a CDR of an antibody of the examples (e.g., the
weight class, hydropathic score, or both, of the sequences are at
least about 50%, at least about 60%, at least about 70%, at least
about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 95%, or more (e.g., about 65-99%) retained).
For example, conservative residue substitutions may also or
alternatively be based on the replacement of strong or weak
weight-based conservation groups, which are known in the art.
[0066] The retention of similar residues may also or alternatively
be measured by a similarity score, as determined by use of a BLAST
program (e.g., BLAST 2.2.8 available through the NCBI using
standard settings BLOSUM62, Open Gap=11 and Extended Gap=1).
Suitable variants typically exhibit at least about 45%, such as at
least about 55%, at least about 65%, at least about 75%, at least
about 85%, at least about 90%, at least about 95%, or more (e.g.,
about 70-99%) similarity to the parent peptide.
[0067] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (for
instance bacterial vectors having a bacterial origin of replication
and episomal mammalian vectors). Other vectors (such as
non-episomal mammalian vectors) may be integrated into the genome
of a host cell upon introduction into the host cell, and thereby
are replicated along with the host genome. Moreover, certain
vectors are capable of directing the expression of genes to which
they are operatively linked. Such vectors are referred to herein as
"recombinant expression vectors" (or simply, "expression vectors").
In general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" may be used interchangeably
as the plasmid is the most commonly used form of vector. However,
the present invention is intended to include such other forms of
expression vectors, such as viral vectors (such as
replication-defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0068] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which an
expression vector has been introduced. It should be understood that
such terms are intended to refer not only to the particular subject
cell, but also to the progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
Recombinant host cells include, for example, transfectomas, such as
CHO cells, HEK-293 cells, PER.C6, NSO cells, and lymphocytic cells,
and prokaryotic cells such as E. coli.
[0069] The term "transfectoma", as used herein, includes
recombinant eukaryotic host cells expressing the Ab, such as CHO
cells, PER.C6, NSO cells, HEK-293 cells, plant cells, or fungi,
including yeast cells.
[0070] The term "transgenic non-human animal" refers to a non-human
animal having a genome comprising one or more human heavy and/or
light chain transgenes or transchromosomes (either integrated or
non-integrated into the animal's natural genomic DNA) and which is
capable of expressing fully human Abs. For example, a transgenic
mouse can have a human light chain transgene and either a human
heavy chain transgene or human heavy chain transchromosome, such
that the mouse produces human CD74 antibodies when immunized with
CD74 antigen and/or cells expressing CD74. The human heavy chain
transgene may be integrated into the chromosomal DNA of the mouse,
as is the case for transgenic mice, for instance HuMAb.RTM. mice,
such as HCo7 or HCo12 mice, or the human heavy chain transgene may
be maintained extrachromosomally, as is the case for
transchromosomal KM mice as described in WO02/43478. Such
transgenic and transchromosomal mice (collectively referred to
herein as "transgenic mice") are capable of producing multiple
isotypes of human monoclonal antibodies to a given antigen (such as
IgG, IgA, IgM, IgD and/or IgE) by undergoing V-D-J recombination
and isotype switching. Transgenic, non-human animal can also be
used for production of antibodies against a specific antigen by
introducing genes encoding such specific Ab, for example by
operatively linking the genes to a gene which is expressed in the
milk of the animal.
[0071] "Treatment" refers to the administration of an effective
amount of a therapeutically active compound of the present
invention with the purpose of easing, ameliorating, arresting or
eradicating (curing) symptoms or disease states.
[0072] An "effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve a desired
therapeutic result. A therapeutically effective amount of a CD74
antibody may vary according to factors such as the disease state,
age, sex, and weight of the individual, and the ability of the CD74
antibody to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the antibody or antibody portion are
outweighed by the therapeutically beneficial effects.
[0073] An "anti-idiotypic" (Id) antibody is an antibody which
recognizes unique determinants generally associated with the
antigen-binding site of an Ab.
Further Aspects and Embodiments of the Invention
[0074] The invention provides an isolated antibody, such as a human
monoclonal antibody, which binds to human CD74 isoforms 1 and 2.
The antibody may additionally bind to other CD74 isoforms or
species homologs, such as the cynomolgus homolog. In particular,
the antibody of the invention efficiently internalizes after
binding to CD74 expressed on the surface of a cell, which is
advantageous for therapeutic applications of an ADC approach. As
shown in Examples 19 to 22, ADCs of CD74 antibodies and linker-drug
combinations vcMMAE or mcMMAF effectively reduced the size of
tumors in several in vivo tumor models. CD74 ADCs were surprisingly
effective despite the low surface expression of the CD74 target on
the tumor cells (Example 22). Further, CD74 antibodies were shown
effective in preventing the outgrowth of tumors in an in vivo model
of tumor prophylaxis (Example 25).
[0075] The antibody can be further characterized by one or more
functional properties such that it binds to one or more human CD74
variants with high affinity, inhibits MIF binding to CD74, or any
combination of the foregoing properties.
[0076] In one aspect, the antibody of the invention binds with high
affinity to human CD74 variants 1 and/or 2 or to human cells
naturally expressing CD74. For example, in one embodiment, the
antibody (a) binds to the extracellular domain of CD74 variant 1
with an EC.sub.50 (apparent affinity) of less than about 500 ng/mL,
less than about 400 ng/mL, less than about 350 ng/mL, or less than
about 330 ng/mL; b) binds to the extracellular domain of CD74
variant 2 with an EC.sub.50 of less than about 400 ng/mL, less than
about 300 ng/mL, less than about 250 ng/mL, or less than about 220
ng/mL; or (c) both of (a) and (b), when determined as described in
Example 11. Also, or alternatively, the antibody may bind to CD74
on Raji cells with an EC.sub.50 of less than about 400 ng/mL, less
than about 300 ng/mL, less than about 250 ng/mL, or less than about
200 ng/mL, when determined as described in Example 12. Also, or
alternatively, the antibody may bind to CD74 variants 1, 2 or both
with a K.sub.D of about 10.sup.-8 M or less, such as about
10.sup.-9 M or less, or even about 10.sup.-10 M or less.
[0077] In one aspect, the antibody is internalized after binding to
CD74 expressed on the surface of a cell. This can be determined
according to the assay described in Example 24 using fluorescently
labeled antibodies, according to the assay described in Example 14,
using an ADC approach reflecting antibody internalization, or using
a method described in Ong G L et al., Immunology 1999; 98:296-302;
Hansen H J et al., Biochem J 1996; 320: 293-300; Koch N G et al., J
Immunol 1991; 147: 2643-2651; Roche P A et al., PNAS 1993; 90:
8581-8585). The cell may be from a B cell line, such as Raji cells,
or from another type of tumor cell line induced to express high
levels of CD74 by treatment with IFN.gamma. (for example HT-29
colon cancer cells or SK-MEL-37 melanoma cells). In one embodiment,
the cell is a Raji cell. In another embodiment, the antibody has an
EC.sub.50 of less than about 60 ng/mL, less than about 40 ng/mL,
less than about 30 ng/mL, or about 25 ng/mL or less in inducing
killing of Raji cells in an anti-kappa ETA' assay, when determined
as described in Example 14. Alternatively, the antibody has an
EC.sub.50 between about 25 to about 60 ng/L about 25 to 40 ng/mL,
or about 25 to about 30 ng/mL in such an assay.
[0078] In one aspect, an antibody of the invention has an EC.sub.50
of less than 30 ng/mL, or an EC.sub.50 of about 25 ng/mL or less,
when determined as described in Example 14.
[0079] In one aspect, the antibody is characterized by its off-rate
from CD74 antigen, optionally expressed on the surface of a cell.
The off-rate can be determined, for example, using a cellular assay
such as the one in Example 23, typically using fluorescently (or
otherwise) labeled antibodies and determining the off-rate at
0.degree. C. The cell may be from a B cell line, such as, e.g.,
Daudi or Raji cells, or from another suitable type of tumor cell
line (e.g., M4A4 cells or NCI-H747 cells). In one embodiment, the
cell is a Daudi cell. In one embodiment, the antibody has an
off-rate in the range of 0.02 to 1.0 min.sup.-1, such as about 0.03
to about 0.30 min.sup.-1, such as 0.04 to 0.10 or 0.15 to 0.30
min.sup.-1. In one embodiment, the antibody has an off-rate of
about 0.07 min.sup.-1. In one embodiment, the antibody has an
off-rate of about 0.20 or 0.24 min.sup.-1.
[0080] The antibody of the invention may also, or alternatively, be
characterized by cross-competing with, or binding to the same
epitope as, a reference antibody to human CD74 variant 1, variant
2, or both of variants 1 and 2.
[0081] An assay testing for competitive binding of the antibody
with a reference antibody can utilize, e.g., the extracellular
domain of a CD74 variant (e.g., the constructs described in Example
1), CD74-expressing cells and/or cell-membranes prepared from
CD74-expressing cells. In an exemplary assay, CD74-expressing cells
are pre-incubated with the test antibody at different
concentrations, ranging from 1 to 100 .mu.g/mL, subsequently
incubated with a fluorophore-labeled reference antibody at a
concentration of 10 .mu.g/mL. Binding of the reference antibody is
determined using FACS analysis.
[0082] In one aspect, the antibody competes for binding to variants
1 and 2 of human CD74 with at least one reference antibody selected
from
[0083] (a) an antibody comprising a VH region comprising the
sequence of SEQ ID NO:7 and a VL region comprising the sequence of
SEQ ID NO:23 [005];
[0084] (b) an antibody comprising a VH region comprising the
sequence of SEQ ID NO:11 and a VL region comprising the sequence of
SEQ ID NO:26 [006];
[0085] (c) an antibody comprising a VH region comprising the
sequence of SEQ ID NO:15 and a VL region comprising the sequence of
SEQ ID NO:26 [008]; and
[0086] (d) an antibody comprising a VH region comprising the
sequence of SEQ ID NO:19 and a VL region comprising the sequence of
SEQ ID NO:26 [011].
[0087] In separate and specific embodiments, the antibody competes
with the antibody of (a) and (b), (a) and (c), (a) and (d), (b) and
(c), (b) and (d), (c) and (d), at least three of (a) to (d), or all
of (a), (b), (c) and (d).
[0088] In one embodiment, the antibody binds to the same epitope on
human CD74 as at least one of the reference antibodies defined in
(a), (b), (c), and (d). This can be determined using known
techniques for epitope determination, such as, e.g., testing for
antibody binding to CD74 variants with differing point-mutations,
or phage display techniques (see, e.g., Binder et al., Cancer Res
2007; 67:3518-3523; Carter J M et al., Curr Protocols Immunol 2004;
Ch 9: Unit 9.4; Hjelm B et al., N Biotechnol 2010; 27: 129-137;
Rockberg J et al., Curr Protocols Immunol 2010; Ch 9: Unit 9.9;
Benjamin D C et al., Methods 1996; 9: 508-515).
[0089] An antibody or immunoglobulin of the invention may also or
alternatively be characterized by comprising specific V.sub.H,
V.sub.L, or CDR sequences, or specific combinations thereof.
[0090] In one aspect, the antibody or immunoglobulin comprises the
V.sub.H CDR3 region of any one of HuMab-CD74-005, -006, -008, and
-011. The invention thus provides for an antibody or immunoglobulin
comprising a V.sub.H CDR3 comprising or consisting of a sequence
selected from SEQ ID NOS:10, 14, 18, and 22. In one embodiment, the
antibody or immunoglobulin comprises SEQ ID NO:22.
[0091] In one aspect, the antibody or immunoglobulin comprises a
V.sub.L region comprising the CDR1, 2 and 3 sequences of SEQ ID
NO:24, AAS and SEQ ID NO:26 and [0092] a) a V.sub.H region
comprising the CDR1, 2 and 3 sequences of SEQ ID NOS:8, 9 and 10
(005); [0093] b) a V.sub.H region comprising the CDR1, 2 and 3
sequences of SEQ ID NO:12, 13 and 14 (006); [0094] c) a V.sub.H
region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:16, 17
and 18 (008); [0095] d) a V.sub.H region comprising the CDR1, 2 and
3 sequences of SEQ ID NO:20, 21 and 22 (011), or [0096] e) a
variant of any of said antibodies or immunoglobulins, wherein said
variant preferably has at most 1, 2 or 3 amino acid modifications,
more preferably amino acid substitutions, such as conservative
amino acid substitutions in any of said sequences. In one aspect,
the antibody comprises a V.sub.H region comprising the CDR1, 2 and
3 sequences of SEQ ID NO:20, 21 and 22 and a V.sub.L region
comprising the CDR1, 2 and 3 sequences of SEQ ID NO:24, AAS and 25,
with at most 3 amino acid modifications as compared to the original
sequences. In one embodiment, the antibody comprises a V.sub.H
region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 20, 21
and 22 and a V.sub.L region comprising the CDR1, 2 and 3 sequences
of SEQ ID NO:24, AAS and SEQ ID NO:25, with at most 1 amino acid
modification. In a particular embodiment of (e), the variant
comprises an amino acid-substitution of residue 7 of the V.sub.H
CDR2 of (d), such as a conservative amino acid substitution.
[0097] In one aspect, the antibody or immunoglobulin comprises a
V.sub.H having [0098] a) at least 80% identity, such as at least
90%, at least 95%, or at least 98% or 100% identity to a V.sub.H
region sequence selected from the group consisting of SEQ ID NO:7,
11, 15 and 19, or [0099] b) at most 20, such as 15, or 10, or 5, 4,
3, 2 or 1 amino acid modifications, more preferably amino acid
substitutions, such as conservative amino acid substitutions as
compared to a V.sub.H region sequence selected from the group
consisting of SEQ ID NO:7, 11, 15 and 19.
[0100] In one aspect, the antibody or immunoglobulin comprises a
V.sub.L having [0101] a) at least 80% identity, such as at least
90%, at least 95%, or at least 98% or 100% identity to a V.sub.L
region sequence selected from the group consisting of: SEQ ID NO:23
and 26 or [0102] b) at most 20, such as 15, or 10, or 5, 4, 3, 2 or
1 amino acid modifications, more preferably amino acid
substitutions, such as conservative amino acid substitutions as
compared to a V.sub.L region sequence selected from the group
consisting of: SEQ ID NO:23 and 26.
[0103] In one embodiment of (b), the V.sub.L region comprises an
amino acid substitution in the position corresponding to residue 36
in SEQ ID NO:23 and 26. In SEQ ID NOS: 23 and 26, the amino acid at
this position is F and Y, respectively.
[0104] In separate and specific aspects, the antibody or
immunoglobulin comprises a V.sub.H and a V.sub.L region selected
from any one of the following combinations: [0105] a) a V.sub.H
region comprising the sequence of SEQ ID NO:7 and a V.sub.L region
comprising the sequence of SEQ ID NO:23 (005); [0106] b) a V.sub.H
region comprising the sequence of SEQ ID NO:11 and a V.sub.L region
comprising the sequence of SEQ ID NO:26 (006); [0107] c) a V.sub.H
region comprising the sequence of SEQ ID NO:15 and a V.sub.L region
comprising the sequence of SEQ ID NO:26 (008), [0108] d) a V.sub.H
region comprising the sequence of SEQ ID NO:19 and a V.sub.L region
comprising the sequence of SEQ ID NO:26 (011), and [0109] e) a
V.sub.H region comprising the sequence of SEQ ID NO:7 and a V.sub.L
region comprising the sequence of SEQ ID NO:26 (005/011); and
[0110] f) a variant of any of said antibodies or immunoglobulins,
wherein said variant preferably has at most 1, 2 or 3 amino acid
modifications, more preferably amino acid substitutions, such as
conservative amino acid substitutions in any of said VH and/or VL
region sequences.
[0111] In one aspect, the invention provides an antibody or
immunoglobulin comprising a V.sub.L region comprising the sequence
of SEQ ID NO: 26. In one embodiment, the antibody or immunoglobulin
comprises the V.sub.H CDR3 of SEQ ID NO: 22. In another embodiment,
the antibody comprises the V.sub.H CDR1, 2 and 3 sequences of SEQ
ID NOS: 20, 21 and 22, respectively.
The antibody of the invention can be characterized by one or more
of the functional or structural features of the aspects described
above, or by any combination of selected functional and structural
features. For example, in one embodiment, the antibody or
immunoglobulin of the invention is characterized by any one of the
following characteristics: [0112] a) an EC.sub.50 of less than 30
ng/mL, or an EC.sub.50 of about 25 ng/mL or less in an
anti-kappa-ETA' assay, when determined as described in Example 14;
[0113] b) competing with, or binding the same epitope as, an
antibody having the V.sub.H and V.sub.L sequences of SEQ ID NOS:19
and 26, respectively; [0114] c) an off-rate in the range of 0.03 to
0.30 min.sup.-1, when determined according to Example 23; [0115] d)
a V.sub.H CDR3 comprising SEQ ID NO:22; [0116] e) a combination of
(a) and (b); [0117] f) a combination of (a) and (c); [0118] g) a
combination of (a) and (d) [0119] h) a combination of (b) and (c);
[0120] i) a combination of (b) and (d); [0121] j) a combination of
(c) and (d); or [0122] k) a combination of (a), (b), (c) and (d).
The antibodies of the invention are preferably monoclonal.
Monoclonal antibodies of the present invention may e.g. be produced
by the hybridoma method first described by Kohler et al. (Nature
256, 495 (1975)), or may be produced by recombinant DNA methods.
Monoclonal antibodies may also be isolated from phage antibody
libraries using the techniques described in, for example, Clackson
et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol.
222, 581-597 (1991). Monoclonal antibodies may be obtained from any
suitable source. Thus, for example, monoclonal antibodies may be
obtained from hybridomas prepared from murine splenic and lymph
node B cells obtained from mice immunized with an antigen of
interest, for instance in the form of cells expressing an antigen
of interest on the surface, or a nucleic acid encoding an antigen
of interest. Monoclonal antibodies may also be obtained from
hybridomas derived from antibody-expressing cells of immunized
humans or non-human mammals such as rats, dogs, primates, etc.
[0123] In one embodiment, the antibody of the invention is a human
antibody. Human monoclonal antibodies directed against CD74 may be
generated using transgenic or transchromosomal mice carrying parts
of the human immune system rather than the mouse system. Such
transgenic and transchromosomic mice include mice referred to
herein as HuMAb mice and KM mice, respectively, and are
collectively referred to herein as "transgenic mice".
[0124] The HuMAb mouse contains a human immunoglobulin gene
minilocus that encodes unrearranged human heavy (.mu. and .gamma.)
and .kappa. light chain immunoglobulin sequences, together with
targeted mutations that inactivate the endogenous .mu. and .kappa.
chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)).
Accordingly, the mice exhibit reduced expression of mouse IgM or
.kappa. and, in response to immunization, the introduced human
heavy and light chain transgenes undergo class switching and
somatic mutation to generate high affinity human IgG,.kappa.
monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed
in Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101
(1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13
65-93 (1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci
764 536-546 (1995)). The preparation of HuMAb mice is described in
detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295
(1992), Chen, J. et al., International Immunology 5, 647-656
(1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor,
L. et al., International Immunology 6, 579-591 (1994), Fishwild, D.
et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat.
No. 5,545,806, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126,
U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,789,650, U.S. Pat. No.
5,877,397, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,814,318, U.S.
Pat. No. 5,874,299, U.S. Pat. No. 5,770,429, U.S. Pat. No.
5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO
92/03918 and WO 01/09187.
[0125] The HCo7, HCo12, HCo17 and HCo20 mice have a JKD disruption
in their endogenous light chain (kappa; .kappa.) genes (as
described in Chen et al., EMBO J. 12, 821-830 (1993)), a CMD
disruption in their endogenous heavy chain genes (as described in
Example 1 of WO 01/14424), and a KCo5 human kappa light chain
transgene (as described in Fishwild et al., Nature Biotechnology
14, 845-851 (1996)). Additionally, the Hco7 mice have a HCo7 human
heavy chain transgene (as described in U.S. Pat. No. 5,770,429),
the HCo12 mice have a HCo12 human heavy chain transgene (as
described in Example 2 of WO 01/14424), the HCo17 mice have a HCo17
human heavy chain transgene (as described in Example 2 of WO
01/09187) and the HCo20 mice have a HCo20 human heavy chain
transgene. The resulting mice express human immunoglobulin heavy
and kappa light chain transgenes in a background homozygous for
disruption of the endogenous mouse heavy and kappa light chain
loci.
[0126] In the KM mouse strain, the endogenous mouse kappa light
chain gene has been homozygously disrupted as described in Chen et
al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy
chain gene has been homozygously disrupted as described in Example
1 of WO 01/09187. This mouse strain carries a human kappa light
chain transgene, KCo5, as described in Fishwild et al., Nature
Biotechnology 14, 845-851 (1996). This mouse strain also carries a
human heavy chain transchromosome composed of chromosome 14
fragment hCF (SC20) as described in WO 02/43478. HCo12-BALB/C mice
can be generated by crossing HCo12 to KCo5[J/K]-Balb/C as described
in WO 097006.
[0127] Splenocytes and lymph node cells from these transgenic mice
may be used to generate hybridomas that secrete human monoclonal
antibodies according to well known techniques.
[0128] Human monoclonal or polyclonal antibodies of the present
invention, or antibodies of the present invention originating from
other species may also be generated transgenically through the
generation of another non-human mammal or plant that is transgenic
for the immunoglobulin heavy and light chain sequences of interest
and production of the antibody in a recoverable form therefrom. In
connection with the transgenic production in mammals, antibodies
may be produced in, and recovered from, the milk of goats, cows, or
other mammals. See for instance U.S. Pat. No. 5,827,690, U.S. Pat.
No. 5,756,687, U.S. Pat. No. 5,750,172 and U.S. Pat. No.
5,741,957.
[0129] Further, human antibodies of the present invention or
antibodies of the present invention from other species may be
generated through display-type technologies, including, without
limitation, phage display, retroviral display, ribosomal display,
and other techniques, using techniques well known in the art and
the resulting molecules may be subjected to additional maturation,
such as affinity maturation, as such techniques are well known in
the art (see for instance Hoogenboom et al., J. Mol. Biol. 227, 381
(1991) (phage display), Vaughan et al., Nature Biotech 14, 309
(1996) (phage display), Hanes and Plucthau, PNAS USA 94, 4937-4942
(1997) (ribosomal display), Parmley and Smith, Gene 73, 305-318
(1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et
al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. Acids
Research 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews
130, 43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84
(1992), and U.S. Pat. No. 5,733,743). If display technologies are
utilized to produce antibodies that are not human, such antibodies
may be humanized.
[0130] The antibody of the invention may be of any isotype. The
choice of isotype typically will be guided by the desired effector
functions, such as ADCC induction. Exemplary isotypes are IgG1,
IgG2, IgG3, and IgG4. Either of the human light chain constant
regions, kappa or lambda, may be used. If desired, the class of a
CD74-specific antibody of the present invention may be switched by
known methods. For example, an antibody of the present invention
that was originally IgM may be class switched to an IgG antibody of
the present invention. Further, class switching techniques may be
used to convert one IgG subclass to another, for instance from IgG1
to IgG2. Thus, the effector function of the antibodies of the
present invention may be changed by isotype switching to, e.g., an
IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various
therapeutic uses. In one embodiment an antibody of the present
invention is an IgG1 antibody, for instance an IgG1,.kappa..
[0131] In one embodiment, the antibody of the invention is a
full-length antibody.
[0132] In one embodiment, the full-length antibody is an IgG1
antibody, such as an IgG1,.kappa. antibody.
[0133] In another embodiment, the full-length antibody is an IgG4
antibody.
[0134] In a particular embodiment, the CD74-specific IgG4 antibody
is a stabilized IgG4 antibody. Examples of suitable stabilized IgG4
antibodies are antibodies wherein arginine at position 409 in a
heavy chain constant region of human IgG4, which is indicated in
the EU index as in Kabat et al. supra, is substituted with lysine,
threonine, methionine, or leucine, preferably lysine (described in
WO2006033386) and/or wherein the hinge region comprises a
Cys-Pro-Pro-Cys sequence. Other suitable stabilized IgG4 antibodies
are disclosed in WO2008145142, which is hereby incorporated by
reference in its entirety.
[0135] In one embodiment, the stabilized IgG4 CD74-specific
antibody is an IgG4 antibody comprising a heavy chain and a light
chain, wherein said heavy chain comprises a human IgG4 constant
region having a residue selected from the group consisting of: Lys,
Ala, Thr, Met and Leu at the position corresponding to 409 and/or a
residue selected from the group consisting of: Ala, Val, Gly, Ile
and Leu at the position corresponding to 405, and wherein said
antibody optionally comprises one or more further substitutions,
deletions and/or insertions, but does not comprise a
Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said
antibody comprises a Lys or Ala residue at the position
corresponding to 409 or the C.sub.H3 region of the antibody has
been replaced by the C.sub.H3 region of human IgG1, of human IgG2
or of human IgG3.
[0136] In another embodiment, the stabilized IgG4 CD74-specific
antibody is an IgG4 antibody comprising a heavy chain and a light
chain, wherein said heavy chain comprises a human IgG4 constant
region having a residue selected from the group consisting of: Lys,
Ala, Thr, Met and Leu at the position corresponding to 409 and/or a
residue selected from the group consisting of: Ala, Val, Gly, Ile
and Leu at the position corresponding to 405, and wherein said
antibody optionally comprises one or more further substitutions,
deletions and/or insertions and wherein said antibody comprises a
Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said
antibody comprises a Lys or Ala residue at the position
corresponding to 409 or the C.sub.H3 region of the antibody has
been replaced by the C.sub.H3 region of human IgG1, of human IgG2
or of human IgG3.
[0137] In another embodiment, the CD74-specific antibody is an
antibody of a non-IgG4 type, e.g. IgG1, IgG2 or IgG3 which has been
mutated such that the ability to mediate effector functions, such
as ADCC, has been reduced or even eliminated. Such mutations have
e.g. been described in Dall'Acqua W F et al., J Immunol.
177(2):1129-1138 (2006) and Hezareh M, J Virol. 75(24):12161-12168
(2001).
[0138] In one embodiment, the respective isotypes and/or sequences
of the two heavy chain constant (Fc) regions are the same. In
another embodiment, the respective isotypes and/or sequences of the
two heavy-chain constant (Fc) regions of a single CD74-specific
antibody are different. This is particularly applicable to
multispecific, such as bispecific, CD74-specific antibodies, which
are described in further detail below.
[0139] In another aspect, the antibody is an antigen-binding
fragment. Antibody fragments can be obtained by conventional
techniques, such as by fragmentation of full-length antibodies or
by expression of nucleic acids encoding antibody fragments in
recombinant cells (see, for instance Evans et al., J. Immunol.
Meth. 184, 123-38 (1995)). The fragments can then be tested or
screened for their properties in the same manner as described
herein for full-length antibodies. The following describe exemplary
formats for CD74-specific antigen-binding fragments of the
invention:
[0140] F(ab').sub.2 fragments, which are bivalent fragments
comprising two Fab fragments linked by a disulfide bridge at the
hinge region. These can be generated by, e.g., treating a
full-length antibody with pepsin.
[0141] Fab' or Fab fragments, which are monovalent fragments
consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains.
Fab fragments can be obtained, e.g., by treating an IgG antibody
with papain. Fab' fragments can be obtained, e.g., by reducing the
disulfide bridges of a F(ab').sub.2 fragment using a reducing agent
such as dithiothreitol.
[0142] Monovalent antibodies or "antibody half-molecules", which
exist in aqueous solutions as a heterodimer of a single light and
single heavy chain, described in WO2007059782 (Genmab A/S).
[0143] Fd fragments, which consist essentially of the V.sub.H and
C.sub.H1 domains.
[0144] Fv fragments, which consist essentially of the V.sub.L and
V.sub.H domains of a single arm of an antibody and single-chain
antibodies thereof. Single-chain antibodies (also known as single
chain Fv (scFv) antibodies) are constructs where the V.sub.L and
V.sub.H domains of an Fv fragment are joined, using recombinant
methods, by a synthetic linker that enables them to be expressed as
a single protein chain in which the V.sub.L and V.sub.H regions
pair to form monovalent molecules (see for instance Bird et al.,
Science 242, 423-426 (1988) and Huston et al., PNAS USA 85,
5879-5883 (1988)).
[0145] Domain antibodies (also called dAb fragments), which
consists essentially of a V.sub.H domain (see, e.g., Ward et al.,
Nature 341, 544-546 (1989); Holt et al; Trends Biotechnol. 2003
November; 21(11):484-90).
[0146] Other exemplary formats include camelids or nanobodies (see,
e.g., Revets et al; Expert Opin Biol Ther. 2005 January;
5(1):111-24).
Multispecific Antibody Formats
[0147] In another embodiment, the invention provides a
multispecific antibody comprising a first antigen binding site from
a CD74-specific antibody molecule described herein above and at
least one second antigen binding site.
[0148] In a particular embodiment, the second antigen-binding site
is used for recruiting a killing mechanism such as, for example, by
binding an antigen on a human effector cell or by binding a
cytotoxic agent or a second therapeutic agent. Exemplary effector
cells include a T cell such as, for example, a cytolytic T cell
(CTL)), a natural killer (NK) cell, a macrophage, a monocyte, a
mast cell, and a granulocyte, such as, for example, a neutrophil,
an eosinophil and a basophil. Exemplary effector cell-antigens
include, but are not limited to, CD1, CD3, CD4, CD8, CD16, CD25,
CD28, CD32, CD40, CD64, CD89, Fc.epsilon.RI and HLA-DR. Suitable
cytotoxic agents and second therapeutic agents are exemplified
below, and include toxins (such as radiolabeled peptides),
chemotherapeutic agents and prodrugs.
[0149] In another particular embodiment, the second antigen-binding
site binds to an antigen on a human B cell, such as, e.g., CD19,
CD20, CD21, CD22, CD23, CD46, CD80, CD138 and HLA-DR.
[0150] In another particular embodiment, the second antigen-binding
site binds a tissue-specific antigen, promoting localization of the
bispecific antibody to a specific tissue.
[0151] In another particular embodiment, the second antigen-binding
site binds to an antigen located on the same type of cell as the
CD74-expressing cell, typically a tumor-associated antigen (TAA),
but has a binding specificity different from that of the first
antigen-binding site. Such multi- or bispecific antibodies can
enhance the specificity of the tumor cell binding and/or engage
multiple effector pathways. Exemplary TAAs include carcinoembryonic
antigen (CEA), prostate specific antigen (PSA), RAGE (renal
antigen), .alpha.-fetoprotein, CAMEL (CTL-recognized antigen on
melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D;
Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin
antigens (e.g., MUC1, mucin-CA125, etc.), ganglioside antigens,
tyrosinase, gp75, c-Met, C-myc, Marti, MelanA, MUM-1, MUM-2, MUM-3,
HLA-B7, Ep-CAM or a cancer-associated integrin, such as
.alpha.5.beta.3 integrin. Alternatively, the second antigen-binding
site binds to a different epitope of CD74. The second
antigen-binding site may alternatively bind an angiogenic factor or
other cancer-associated growth factor, such as a vascular
endothelial growth factor, a fibroblast growth factor, epidermal
growth factor, angiogenin or a receptor of any of these,
particularly receptors associated with cancer progression.
[0152] In another particular embodiment, the second antigen-binding
site is from a second CD74-specific antibody, such as a
CD74-specific antibody of the invention.
[0153] Exemplary formats for the multispecific antibody molecules
of the invention include, but are not limited to (i) two antibodies
cross-linked by chemical heteroconjugation, one with a specificity
to CD74 and another with a specificity to a second antigen; (ii) a
single antibody that comprises two different antigen-binding
regions; (iii) a single-chain antibody that comprises two different
antigen-binding regions, e.g., two scFvs linked in tandem by an
extra peptide linker; (iv) a dual-variable-domain antibody
(DVD-Ig), where each light chain and heavy chain contains two
variable domains in tandem through a short peptide linkage (Wu et
al., Generation and Characterization of a Dual Variable Domain
Immunoglobulin (DVD-Ig.TM.) Molecule, In: Antibody Engineering,
Springer Berlin Heidelberg (2010)); (v) a chemically-linked
bispecific (Fab').sub.2 fragment; (vi) a Tandab, which is a fusion
of two single chain diabodies resulting in a tetravalent bispecific
antibody that has two binding sites for each of the target
antigens; (vii) a flexibody, which is a combination of scFvs with a
diabody resulting in a multivalent molecule; (viii) a so called
"dock and lock" molecule, based on the "dimerization and docking
domain" in Protein Kinase A, which, when applied to Fabs, can yield
a trivalent bispecific binding protein consisting of two identical
Fab fragments linked to a different Fab fragment; (ix) a so-called
Scorpion molecule, comprising, e.g., two scFvs fused to both
termini of a human Fab-arm; and (x) a diabody.
[0154] Another exemplary format for bispecific antibodies is
IgG-like molecules with complementary CH3 domains to force
heterodimerization. Such molecules can be prepared using known
technologies, such as, e.g., those known as Triomab/Quadroma (Trion
Pharma/Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb
(Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech),
Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono),
Biclonic (Merus) and DuoBody (Genmab A/S) technologies.
[0155] In one embodiment, the bispecific antibody is obtained or
obtainable via a controlled Fab-arm exchange, typically using
DuoBody technology. In vitro methods for producing bispecific
antibodies by controlled Fab-arm exchange have been described in WO
2008119353 and WO 2011131746 (both by Genmab A/S). In one exemplary
method, described in WO 2008119353, a bispecific antibody is formed
by "Fab-arm" or "half-molecule" exchange (swapping of a heavy chain
and attached light chain) between two monospecific antibodies, both
comprising IgG4-like CH3 regions, upon incubation under reducing
conditions. The resulting product is a bispecific antibody having
two Fab arms which may comprise different sequences. In another
exemplary method, described in WO 2011131746, bispecific antibodies
of the present invention are prepared by a method comprising the
following steps, wherein at least one of the first and second
antibodies is a CD74 antibody of the present invention: [0156] a)
providing a first antibody comprising an Fc region of an
immunoglobulin, said Fc region comprising a first CH3 region;
[0157] b) providing a second antibody comprising an Fc region of an
immunoglobulin, said Fc region comprising a second CH3 region;
wherein the sequences of said first and second CH3 regions are
different and are such that the heterodimeric interaction between
said first and second CH3 regions is stronger than each of the
homodimeric interactions of said first and second CH3 regions;
[0158] c) incubating said first antibody together with said second
antibody under reducing conditions; and [0159] d) obtaining said
bispecific antibody, wherein the first antibody is a CD74 antibody
of the present invention and the second antibody has a different
binding specificity, or vice versa.
[0160] The reducing conditions may, for example, be provided by
adding a reducing agent, e.g. selected from 2-mercaptoethylamine,
dithiothreitol and tris(2-carboxyethyl)phosphine. Step d) may
further comprise restoring the conditions to become non-reducing or
less reducing, for example by removal of a reducing agent, e.g. by
desalting.
[0161] Preferably, the sequences of the first and second CH3
regions are different, comprising only a few, fairly conservative,
asymmetrical mutations, such that the heterodimeric interaction
between said first and second CH3 regions is stronger than each of
the homodimeric interactions of said first and second CH3 regions.
More details on these interactions and how they can be achieved are
provided in WO 2011131746, which is hereby incorporated by
reference in its entirety. The following are exemplary embodiments
of combinations of such assymetrical mutations, optionally wherein
one or both Fc-regions are of the IgG1 isotype.
[0162] In one embodiment, the first Fc region has an amino acid
substitution at a position selected from the group consisting of:
366, 368, 370, 399, 405, 407 and 409, and the second Fc region has
an amino acid substitution at a position selected from the group
consisting of: 366, 368, 370, 399, 405, 407 and 409, and wherein
the first and second Fc regions are not substituted in the same
positions.
[0163] In one embodiment, the first Fc region has an amino acid
substitution at position 405, and said second Fc region has an
amino acid substitution at a position selected from the group
consisting of: 366, 368, 370, 399, 407 and 409, optionally 409.
[0164] In one embodiment, the first Fc region has an amino acid
substitution at position 409, and said second Fc region has an
amino acid substitution at a position selected from the group
consisting of: 366, 368, 370, 399, 405, and 407, optionally 405 or
368.
[0165] In a particular embodiment, both the first and second Fc
regions are of the IgG1 isotype, with the first Fc region having a
Leu at position 405, and the second Fc region having an Arg at
position 409.
Conjugates
[0166] The present invention provides a CD74-specific antibody
conjugated to a therapeutic moiety, i.e. a drug. The therapeutic
moiety can be, e.g., a cytotoxin, a chemotherapeutic agent, a
cytokine, an immunosuppressant, an immune stimulator, a lytic
peptide, or a radioisotope. Such conjugates are referred to herein
as an "antibody-drug conjugates" or "ADCs".
[0167] Accordingly, in one aspect, the antibody according to any
above-described aspect or embodiment is conjugated to a therapeutic
moiety. Exemplary therapeutic moieties include a cytotoxic moiety,
a radioisotope, a cytokine, and a lytic peptide.
[0168] In one embodiment, the antibody is capable of inducing
cytotoxicity in a Raji cell by internalization of the antibody
conjugated to or associated with a therapeutic moiety in the Raji
cell, e.g., as described in Example 14 or a similar type of assay.
In one embodiment, the antibody induces cytotoxicity by
internalization as described in Example 14, with an EC.sub.50 value
between about 25 ng/mL and about 60 ng/ml, such as between 25 ng/mL
and 30 ng/mL, or an EC.sub.50 value less than 60 ng/mL, such as
less than 40 ng/mL, or less than 30 ng/mL for inducing killing of
Raji cells in an anti-kappa ETA' assay. In another embodiment, an
ADC according to the present invention induces cytotoxicity with an
EC.sub.50 value less than 10 ng/mL, such as less than 5 ng/mL, less
than 1 ng/mL, less than 0.5 ng/mL or less than 0.1 ng/mL in
inducing killing of Raji cells or other CD74-expressing cells.
[0169] In one embodiment, the antibody is conjugated to a cytotoxic
moiety. The cytotoxic moiety may, for example, be selected from the
group consisting of taxol; cytochalasin B; gramicidin D; ethidium
bromide; emetine; mitomycin; etoposide; tenoposide; vincristine;
vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxy
anthracin dione; a tubulin-inhibitor such as maytansine or an
analog or derivative thereof; an antimitotic agent such as
monomethyl auristatin E or F or an analog or derivative thereof;
dolastatin 10 or 15 or an analogue thereof; irinotecan or an
analogue thereof; mitoxantrone; mithramycin; actinomycin D;
1-dehydrotestosterone; a glucocorticoid; procaine; tetracaine;
lidocaine; propranolol; puromycin; calicheamicin or an analog or
derivative thereof; an antimetabolite such as methotrexate, 6
mercaptopurine, 6 thioguanine, cytarabine, fludarabin, 5
fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine,
or cladribine; an alkylating agent such as mechlorethamine,
thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine
(CCNU), cyclophosphamide, busulfan, dibromomannitol,
streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; a
platinum derivative such as cisplatin or carboplatin; duocarmycin
A, duocarmycin SA, rachelmycin (CC-1065), or an analog or
derivative thereof; an antibiotic such as dactinomycin, bleomycin,
daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin,
mitoxantrone, plicamycin, anthramycin (AMC));
pyrrolo[2,1-c][1,4]-benzodiazepines (PDB); diphtheria toxin and
related molecules such as diphtheria A chain and active fragments
thereof and hybrid molecules, ricin toxin such as ricin A or a
deglycosylated ricin A chain toxin, cholera toxin, a Shiga-like
toxin such as SLT I, SLT II, SLT IIV, LT toxin, C3 toxin, Shiga
toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease
inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin,
gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolacca americana proteins
such as PAPI, PAPII, and PAP-S, momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, and enomycin toxins;
ribonuclease (RNase); DNase I, Staphylococcal enterotoxin A;
pokeweed antiviral protein; diphtherin toxin; and Pseudomonas
endotoxin.
[0170] In one embodiment, the antibody is conjugated to an
auristatin or a peptide analog, derivative or prodrug thereof.
Auristatins have been shown to interfere with microtubule dynamics,
GTP hydrolysis and nuclear and cellular division (Woyke et al
(2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and
have anti-cancer (U.S. Pat. No. 5,663,149) and anti-fungal activity
(Pettit et al., (1998) Antimicrob. Agents and Chemother.
42:2961-2965. For example, auristatin E can be reacted with
para-acetyl benzoic acid or benzoylvaleric acid to produce AEB and
AEVB, respectively. Other typical auristatin derivatives include
AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl
auristatin E). Suitable auristatins and auristatin analogs,
derivatives and prodrugs, as well as suitable linkers for
conjugation of auristatins to Abs, are described in, e.g., U.S.
Pat. Nos. 5,635,483, 5,780,588 and 6,214,345 and in International
patent application publications WO02088172, WO2004010957,
WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860,
WO207011968 and WO205082023.
[0171] In one embodiment, the antibody is conjugated to
pyrrolo[2,1-c][1,4]-benzodiazepine (PDB) or an analog, derivative
or prodrug thereof. Suitable PDBs and PDB derivatives, and related
technologies are described in, e.g., Hartley J. A. et al., Cancer
Res 2010; 70(17): 6849-6858; Antonow D. et al., Cancer 3 2008;
14(3):154-169; Howard P. W. et al., Bioorg Med Chem Lett 2009; 19:
6463-6466 and Sagnou et al., Bioorg Med Chem Lett 2000; 10(18):
2083-2086.
[0172] In one embodiment, the antibody is conjugated to a cytotoxic
moiety selected from the group consisting of an anthracycline,
maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065),
dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E,
monomethyl auristatin F, a PDB, or an analog, derivative, or
prodrug of any thereof.
[0173] In a particular embodiment, the antibody is conjugated to an
anthracycline or an analog, derivative or prodrug thereof. In
another particular embodiment, the antibody is conjugated to
maytansine or an analog, derivative or prodrug thereof. In another
particular embodiment, the antibody is conjugated to calicheamicin
or an analog, derivative or prodrug thereof. In another particular
embodiment, the antibody is conjugated to duocarmycin or an analog,
derivative or prodrug thereof. In another particular embodiment,
the antibody is conjugated to rachelmycin (CC-1065) or an analog,
derivative or prodrug thereof. In another particular embodiment,
the antibody is conjugated to dolastatin 10 or an analog,
derivative or prodrug thereof. In another particular embodiment,
the antibody is conjugated to dolastatin 15 or an analog,
derivative or prodrug thereof. In another particular embodiment,
the antibody is conjugated to monomethyl auristatin E or an analog,
derivative or prodrug thereof. In another particular embodiment,
the antibody is conjugated to monomethyl auristatin F or an analog,
derivative or prodrug thereof. In another particular embodiment,
the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine or
an analog, derivative or prodrug thereof. In another particular
embodiment, the antibody is conjugated to irinotecan or an analog,
derivative or prodrug thereof.
[0174] In one embodiment, a CD74-specific antibody of the invention
is conjugated to a nucleic acid or nucleic acid-associated
molecule. In one such embodiment, the conjugated nucleic acid is a
cytotoxic ribonuclease (RNase) or deoxy-ribonuclease (e.g., DNase
I), an antisense nucleic acid, an inhibitory RNA molecule (e.g., a
siRNA molecule) or an immunostimulatory nucleic acid (e.g., an
immunostimulatory CpG motif-containing DNA molecule). In another
embodiment, a CD74-specific antibody of the invention is conjugated
to an aptamer or a ribozyme.
[0175] In one embodiment, a CD74-specific antibody of the invention
is conjugated, e.g., as a fusion protein, to a lytic peptide such
as CLIP, Magainin 2, mellitin, Cecropin and P18.
[0176] In one embodiment, the antibody is conjugated to a cytokine,
such as, e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15,
IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFN.alpha.,
IFN.beta., IFN.gamma., GM-CSF, CD40L, Flt3 ligand, stem cell
factor, ancestim, and TNF.alpha..
[0177] In one embodiment, the antibody is conjugated to a
radioisotope or to a radioisotope-containing chelate. For example,
the antibody can be conjugated to a chelator linker, e.g. DOTA,
DTPA or tiuxetan, which allows for the antibody to be complexed
with a radioisotope. The antibody may also or alternatively
comprise or be conjugated to one or more radiolabeled amino acids
or other radiolabeled molecules. A radiolabeled CD74-specific
antibody may be used for both diagnostic and therapeutic purposes.
Non-limiting examples of radioisotopes include .sup.3H, .sup.14C,
.sup.15N, .sup.35S, .sup.90Y, .sup.99Tc, .sup.125I, .sup.111In,
.sup.131I, .sup.186Re, .sup.213Bi, .sup.225Ac and .sup.227Th. For
therapeutic purposes, a radioisotope emitting beta- or
alpha-particle radiation can be used, e.g., .sup.131I, .sup.90Y,
.sup.211At, .sup.212Bi, .sup.67Cu, .sup.186Re, .sup.188Re, and
.sup.212Pb.
[0178] A therapeutic agent that may be administered in combination
with a CD74-specific antibody of the present invention as described
elsewhere herein, such as, e.g., a chemotherapeutic agent,
anti-cancer cytokine or chemokine, is also a candidate for a
therapeutic moiety useful for conjugation to an antibody of the
present invention.
[0179] A CD74-specific antibody of the present invention may also
be chemically modified by covalent conjugation to a polymer to, for
instance, increase its circulating half-life. Exemplary polymers,
and methods to attach them to polypeptides, are illustrated in for
instance U.S. Pat. No. 4,766,106, U.S. Pat. No. 4,179,337, U.S.
Pat. No. 4,495,285 and U.S. Pat. No. 4,609,546. Additional polymers
include polyoxyethylated polyols and polyethylene glycol (PEG)
(e.g., a PEG with a molecular weight of between about 1,000 and
about 40,000, such as between about 2,000 and about 20,000).
[0180] A therapeutic or other agent may be conjugated either
directly or indirectly to a CD74-specific antibody of the present
invention, according to methods known in the art. One example of
indirect conjugation of a second agent is via a spacer moiety to
cysteine or lysine residues in the antibody. The therapeutic or
other moiety may also or alternatively be conjugated to an
N-(amino-) terminal or C-(carboxy-) terminal residue of a
CD74-specific antibody polypeptide or fragment thereof (e.g., a
CD74-specific antibody H or L chain) (see, e.g., Antibody
Engineering Handbook, edited by Osamu Kanemitsu, published by
Chijin Shokan (1994)). Conjugated antibody derivatives may also be
generated by conjugation at internal residues or sugars, where
appropriate. Exemplary methods are also described in, e.g., Hunter
et al., Nature 144, 945 (1962), David et al., Biochemistry 13, 1014
(1974), Pain et al., J. Immunol. Meth. 40, 219 (1981) and Nygren,
J. Histochem. and Cytochem. 30, 407 (1982).
[0181] In one embodiment, a CD74-specific antibody is conjugated to
a prodrug molecule via a spacer or linker that can be activated in
vivo to a therapeutic drug. For example, the prodrug moiety may be
attached to the antibody via a linker, through the N- or C-terminus
of the peptidic or non-peptidic drug moiety. After administration,
the spacers or linkers are cleaved by tumor cell-associated enzymes
or other tumor-specific conditions, by which the active drug is
formed. Examples of such prodrug technologies and linkers are
described in WO02083180, WO2004043493, WO2007018431, WO2007089149,
WO2009017394 and WO201062171 by Syntarga B V, et al. (all
incorporated herein by reference) Suitable antibody-prodrug
technology and duocarmycin analogs can also be found in U.S. Pat.
No. 6,989,452 (Medarex) (incorporated herein by reference).
Suitable prodrug technology for auristatins is described in
WO03026577 (Seatte Genetics) and other auristatin references
mentioned above.
[0182] In one embodiment, a CD74-specific antibody is conjugated to
a therapeutic moiety or prodrug via a linker sensitive to changes
in pH or reducing conditions. Suitable linker technologies are
known in the art, and include those described in, e.g., Ducry, L
and Stump, Bioconjugate Chem. 2010; 21:5-13; Senter P. D., Current
Opinion in Chemical Biology 2009; 13:235-244; and Carter, P. J. and
Senter, P. D., The Cancer Journal 2010; 14:154-169.
[0183] In some embodiments, the linker is cleavable under
intracellular conditions, such that the cleavage of the linker
releases the drug unit from the antibody in the intracellular
environment. In some embodiments, the linker is cleavable by a
cleavable agent that is present in the intracellular environment
(e. g. within a lysosome or endosome or caveolus). The linker can
be, e. g. a peptidyl linker that is cleaved by an intracellular
peptidase or protease enzyme, including but not limited to, a
lysosomal or endosomal protease. In some embodiments, the peptidyl
linker is at least two amino acids long or at least three amino
acids long. Cleaving agents can include cathepsins B and D and
plasmin, all of which are known to hydrolyze dipeptide drug
derivatives resulting in the release of active drug inside the
target cells (see e. g. Dubowchik and Walker, 1999, Pharm.
Therapeutics 83:67-123). In a specific embodiment, the peptidyl
linker cleavable by an intracellular protease is a Val-Cit
(valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine)
linker (see e.g. U.S. Pat. No. 6,214,345, which describes the
synthesis of doxorubicin with the Val-Cit linker and different
examples of Phe-Lys linkers). Examples of the structures of a
Val-Cit and a Phe-Lys linker include but are not limited to
MC-vc-PAB described below, MC-vc-GABA, MC-Phe-Lys-PAB or
MC-Phe-Lys-GABA, wherein MC or mc is an abbreviation for maleimido
caproyl, vc is an abbreviation for Val-Cit, PAB is an abbreviation
for p-aminobenzylcarbamate and GABA is an abbreviation for
.gamma.-aminobutyric acid. An advantage of using intracellular
proteolytic release of the therapeutic agent is that the agent is
typically attenuated when conjugated and the serum stabilities of
the conjugates are typically high.
[0184] In yet another embodiment, the linker unit is not cleavable
and the drug is released by antibody degradation (see, e.g., US
2005/0238649). Typically, such a linker is not substantially
sensitive to the extracellular environment. As used herein, "not
substantially sensitive to the extracellular environment" in the
context of a linker means that no more than 20%, typically no more
than about 15%, more typically no more than about 10%, and even
more typically no more than about 5%, no more than about 3%, or no
more than about 1% of the linkers, in a sample of antibody-drug
conjugate compound, are cleaved when the antibody-drug conjugate
compound presents in an extracellular environment (e.g. plasma).
Whether a linker is not substantially sensitive to the
extracellular environment can be determined for example by
incubating with plasma the antibody-drug conjugate for a
predetermined time period (e.g. 2, 4, 8, 16 or 24 hours) and then
quantitating the amount of free drug present in the plasma.
In a specific embodiment, the CD74-specific antibody is conjugated
to MMAE (formula I):
##STR00001##
wherein the wavy line indicates the covalent attachment site for
the linker.
[0185] In another specific embodiment, the CD74-specific antibody
is conjugated to MMAF (formula II):
##STR00002##
wherein the wavy line indicates the covalent attachment site for
the linker.
[0186] In a particular embodiment, the linker to MMAE or MMAF is
attached to sulfhydryl groups (free cysteine residues) of the
CD74-specific antibody, obtained by (partial) reduction of the
CD74-specific antibody.
[0187] In another particular embodiment, the linker-auristatin is
MC-vc-PAB-MMAF (also designated as vcMMAF) or MC-vc-PAB-MMAE (also
designated as vcMMAE (formula III and IV, respectively):
##STR00003##
wherein p denotes a number of from 1 to 8, S represents a free
cysteine thiol residue of the CD74-specific antibody, and Ab
designates the CD74-specific antibody. In one embodiment thereof,
the linker-auristatin is vcMMAE. The vcMMAE drug linker moiety and
conjugation methods are disclosed in WO2004010957, U.S. Pat. No.
7,659,241, U.S. Pat. No. 7,829,531, U.S. Pat. No. 7,851,437 and
U.S. Ser. No. 11/833,028 (Seattle Genetics, Inc.), (which are
incorporated herein by reference), and the vcMMAE drug linker
moiety can be bound to the CD74-specific antibodies at the
cysteines using a method similar to those disclosed in therein.
[0188] In another particular embodiment, the linker-conjugate is
mcMMAF (formula V):
##STR00004##
[0189] wherein p denotes a number of from 1 to 8, S represents a
free cysteine thiol residue of the CD74-specific antibody, and Ab
designates the CD74-specific antibody. The mcMMAF drug linker
moiety and conjugation methods are disclosed in U.S. Pat. No.
7,498,298, U.S. Ser. No. 11/833,954, and WO2005081711 (Seattle
Genetics, Inc.) (which are incorporated herein by reference), and
the mcMMAF drug linker moiety can be bound to the CD74-specific
antibodies at the cysteines using a method similar to those
disclosed in therein.
[0190] In one aspect, the invention provides a CD74-specific ADC
comprising an antibody binding to the same epitope as an antibody
selected from 005, 006, 008 and 011, and a drug which is auristatin
or an analog, derivative or prodrug thereof. In one embodiment, the
EC.sub.50 of the ADC in binding to the extracellular domain of
CD47v1 is lower than about 0.2 .mu.g/mL, such as lower than 0.1
.mu.g/mL, or lower than about 0.05 .mu.g/mL, optionally higher than
0.01 .mu.g/mL, such as higher than 0.02 .mu.g/mL, when determined
in an assay as described in Example 16. In one embodiment, the
CD74-specific ADC induces a cell kill higher than 70%, 80% or 90%
when measured for Raji, Daudi or M4A4 cells in an assay as
described in Example 18. In one embodiment, the CD74-specific ADC
has an IC.sub.50 of less than about 0.5 .mu.g/mL, less than about
0.3 .mu.g/mL, less than about 0.2 .mu.g/mL, or less than about 0.1
.mu.g/mL, and optionally higher than 0.005 .mu.g/mL or about 0.01
.mu.g/mL, in inducing killing of Raji, Daudi or M4A4 cells, when
determined in an assay as described in Example 18. In one
embodiment, the antibody comprises at least the VH CDR3, such as
the VH CDR1, 2 and 3, optionally the VH CDR1, 2 and 3 and VL CDR1,
2 and 3 of 005, described in Table 3. In one embodiment, the
antibody comprises at least the VH CDR3, such as the VH CDR1, 2 and
3, optionally the VH CDR1, 2 and 3 and VL CDR1, 2 and 3 of 006,
described in Table 3. In one embodiment, the antibody comprises at
least the VH CDR3, such as the VH CDR1, 2 and 3, optionally the VH
CDR1, 2 and 3 and VL CDR1, 2 and 3 of 011, described in Table 3. In
one embodiment, the drug is a monomethyl auristatin derivative,
optionally selected from MMAE and MMAF.
[0191] In one aspect, the invention provides a CD74-specific ADC
comprising an antibody comprising the CDR, VH and/or VL sequences
of an antibody selected from the group consisting of 005, 006 and
011, and a drug selected from MMAE and MMAF. In one embodiment, the
antibody is 005. In one embodiment, the antibody is 006. In one
embodiment, the antibody is 011. In one particular embodiment, the
antibody is 005 and the drug is MMAE, optionally vcMMAE. In one
particular embodiment, the antibody is 005 and the drug is MMAF,
optionally mcMMAF. In one particular embodiment, the antibody is
006 and the drug is MMAE, optionally vcMMAE. In one particular
embodiment, the antibody is 006 and the drug is MMAF, optionally
mcMMAF. In one particular embodiment, the antibody is 011 and the
drug is MMAE, optionally vcMMAE. In one particular embodiment, the
antibody is 011 and the drug is MMAF, optionally mcMMAF.
[0192] In specific and separate embodiments, the invention provides
the following CD74-specific ADCs: 011-vcMMAE, 006-vcMMAE,
005-vcMMAE, 011-mcMMAF, 006-mcMMAF and 005-mcMMAF.
[0193] The cytostatic drug loading is represented by p and is the
average number of cytostatic drug moieties per antibody in a
molecule (also designated as the drug to antibody ratio, DAR). The
cytostatic drug loading may range from 1 to 20 drug moieties per
antibody and may occur on amino acids with useful functional groups
such as, but not limited to, amino or sulfhydryl groups, as in
lysine or cysteine.
[0194] Depending on the way of conjugation, p may be limited by the
number of attachment sites on the antibody, for example where the
attachment is a cysteine thiol, i.e., a sulphydryl group.
Generally, antibodies do not contain many free and reactive
cysteine thiol groups, i.e., sulphydryl groups, which may be linked
to a drug moiety, as most cysteine thiol residues in antibodies
exist as disulfide bridges. Therefore, in certain embodiments, an
antibody may be reduced with a reducing agent such as
dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under
partially or fully reducing conditions, to generate reactive
sulphydryl groups. In certain embodiments, the drug loading for an
ADC of the invention ranges from 1 to about 8, such as about 2 to
5, such as about 3 to 5, such as about 4. A maximum of 8 free
sulphydryl groups can become available after (partial) reduction of
the antibody (there are 8 cysteines involved in inter-chain
disulfide bonding).
Expression Constructs
[0195] In further and separate aspects, the invention relates to
nucleic acids encoding a sequence of an antibody of the invention,
to expression vectors encoding the sequences of an antibody of the
invention, to host cells comprising such expression vectors, to
hybridomas which produce antibodies of the invention, and to
methods of producing an antibody of the invention by culturing such
host cells or hybridomas under appropriate conditions whereby the
antibody is produced and, optionally, retrieved.
[0196] In one embodiment, the invention provides an expression
vector comprising a nucleotide sequence encoding one or more amino
acid sequences selected from SEQ ID NOS: 7-26. In one embodiment,
the expression vector comprises one or more nucleotide sequences
encoding one or more of the amino acid sequences selected from the
group consisting of SEQ ID NOS: 7, 11, 15, 19, 23 and 26, or any
combination thereof. In another embodiment, the expression vector
comprises a nucleotide sequence encoding any one or more of the
V.sub.H CDR3 amino acid sequences of SEQ ID NOS: 10, 14, 18 or 22.
In another embodiment, the expression vector comprises a nucleotide
sequence encoding a V.sub.H amino acid sequence selected from SEQ
ID NOS: 7, 11, 15 and 19. In another embodiment, the expression
vector comprises a nucleotide sequence encoding a V.sub.L amino
acid sequence selected from SEQ ID NOS: 23 and 26. In another
embodiment, the expression vector further comprises a nucleotide
sequence encoding the constant region of a human antibody light
chain, of a human antibody heavy chain, or both.
[0197] In a particular embodiment, the expression vector of the
invention comprises a nucleotide sequence encoding variants of one
or more of the above amino acid sequences, said variants having at
most 25 amino acid modifications, such as at most 20, such as at
most 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9,
8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as
deletions or insertions, preferably substitutions, such as
conservative substitutions or at least 80% identity to any of said
sequences, such as at least 85% identity or 90% identity or 95%
identity, such as 96% identity or 97% identity or 98% identity or
99% identity to any of the afore-mentioned amino acid
sequences.
[0198] An expression vector in the context of the present invention
may be any suitable vector, including chromosomal, non-chromosomal,
and synthetic nucleic acid vectors (a nucleic acid sequence
comprising a suitable set of expression control elements). Examples
of such vectors include derivatives of SV40, bacterial plasmids,
phage DNA, baculovirus, yeast plasmids, vectors derived from
combinations of plasmids and phage DNA, and viral nucleic acid (RNA
or DNA) vectors. In one embodiment, a CD74-specific
antibody-encoding nucleic acid is comprised in a naked DNA or RNA
vector, including, for example, a linear expression element (as
described in for instance Sykes and Johnston, Nat Biotech 17,
355-59 (1997)), a compacted nucleic acid vector (as described in
for instance U.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid
vector such as pBR322, pUC 19/18, or pUC 118/119, a "midge"
minimally-sized nucleic acid vector (as described in for instance
Schakowski et al., Mol Ther 3, 793-800 (2001)), or as a
precipitated nucleic acid vector construct, such as a
CaPO.sub.4.sup.--precipitated construct (as described in for
instance WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55
(1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson,
Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and
the usage thereof are well known in the art (see for instance U.S.
Pat. No. 5,589,466 and U.S. Pat. No. 5,973,972).
[0199] In one embodiment, the vector is suitable for expression of
the CD74-specific antibody in a bacterial cell. Examples of such
vectors include expression vectors such as BlueScript (Stratagene),
pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509
(1989)), pET vectors (Novagen, Madison Wis.) and the like.
[0200] An expression vector may also, or alternatively, be a vector
suitable for expression in a yeast system. Any vector suitable for
expression in a yeast system may be employed. Suitable vectors
include, for example, vectors comprising constitutive or inducible
promoters such as alpha factor, alcohol oxidase and PGH (reviewed
in: F. Ausubel et al., ed. Current Protocols in Molecular Biology,
Greene Publishing and Wiley InterScience New York (1987), and Grant
et al., Methods in Enzymol 153, 516-544 (1987)).
[0201] A nucleic acid and/or vector may also comprise a nucleic
acid sequence encoding a secretion/localization sequence, which can
target a polypeptide, such as a nascent polypeptide chain, to the
periplasmic space or into cell culture media. Such sequences are
known in the art, and include secretion leader or signal peptides,
organelle-targeting sequences (e. g., nuclear localization
sequences, ER retention signals, mitochondrial transit sequences,
chloroplast transit sequences), membrane localization/anchor
sequences (e. g., stop transfer sequences, GPI anchor sequences),
and the like.
[0202] In an expression vector of the invention, CD74-specific
antibody-encoding nucleic acids may comprise or be associated with
any suitable promoter, enhancer, and other expression-facilitating
elements. Examples of such elements include strong expression
promoters (e. g., human CMV IE promoter/enhancer as well as RSV,
SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A)
termination sequences, an origin of replication for plasmid product
in E. coli, an antibiotic resistance gene as selectable marker,
and/or a convenient cloning site (e.g., a polylinker). Nucleic
acids may also comprise an inducible promoter as opposed to a
constitutive promoter such as CMV IE (the skilled artisan will
recognize that such terms are actually descriptors of a degree of
gene expression under certain conditions).
[0203] In one embodiment, the CD74-specific antibody-encoding
expression vector is positioned in and/or delivered to the host
cell or host animal via a viral vector.
[0204] Such expression vectors may be used for recombinant
production of antibodies of the invention.
[0205] In one aspect, the invention provides a recombinant
eukaryotic or prokaryotic host cell which produces the antibody of
any aspect or embodiment described herein. Accordingly, the
invention provides a recombinant eukaryotic or prokaryotic host
cell, such as a transfectoma, which produces an antibody or
immunoglobulin of the invention as defined herein. Examples of host
cells include yeast, bacterial and mammalian cells, such as CHO or
HEK-293 cells. For example, in one embodiment, the present
invention provides a cell comprising a nucleic acid stably
integrated into the cellular genome that comprises a sequence
coding for expression of a CD74-specific antibody of the present
invention. In another embodiment, the present invention provides a
cell comprising a non-integrated nucleic acid, such as a plasmid,
cosmid, phagemid, or linear expression element, which comprises a
sequence coding for expression of a CD74-specific antibody of the
invention.
[0206] In a further aspect, the invention relates to a hybridoma
which produces an antibody of the invention as defined herein. In
an even further aspect, the invention relates to a transgenic
non-human animal or plant comprising nucleic acids encoding a human
heavy chain and a human light chain, wherein the animal or plant
produces an antibody of the invention. Generation of such
hybridomas and transgenic animals or plants has been described
above, and is further described in the Examples.
[0207] In a further aspect, the invention relates to a method for
producing a CD74-specific antibody of the invention, said method
comprising the steps of [0208] a) culturing a hybridoma or a host
cell of the invention as described herein above, and [0209] b)
retrieving and/or purifying the antibody of the invention from the
culture media and, optionally, [0210] c) preparing an ADC from the
CD74-specific antibody.
[0211] In a further aspect, the nucleotide sequence encoding a
sequence of an antibody of the invention further encodes a second
moiety, such as a therapeutic polypeptide. Exemplary therapeutic
polypeptides are described elsewhere herein. In one embodiment, the
invention relates to a method for producing a CD74-specific
antibody fusion protein, said method comprising the steps of [0212]
a) culturing a host cell comprising an expression vector comprising
such a nucleotide sequence, and [0213] b) retrieving and/or
purifying the CD74-specific antibody fusion protein from the
culture media.
Pharmaceutical Compositions
[0214] In one aspect, the invention provides a pharmaceutical
composition comprising an antibody or ADC as defined in any of the
above aspects and embodiments, and a pharmaceutically acceptable
carrier.
[0215] The pharmaceutical compositions may be formulated with
pharmaceutically acceptable carriers or diluents as well as any
other known adjuvants and excipients in accordance with
conventional techniques such as those disclosed in Remington: The
Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack
Publishing Co., Easton, Pa., 1995.
[0216] The pharmaceutically acceptable carriers or diluents as well
as any other known adjuvants and excipients should be suitable for
the antibody or antibody conjugate of the present invention and the
chosen mode of administration. Suitability for carriers and other
components of pharmaceutical compositions is determined based on
the lack of significant negative impact on the desired biological
properties of the chosen compound or pharmaceutical composition of
the present invention (e.g., less than a substantial impact (10% or
less relative inhibition, 5% or less relative inhibition, etc.) on
antigen binding).
[0217] A pharmaceutical composition of the present invention may
also include diluents, fillers, salts, buffers, detergents (e. g.,
a nonionic detergent, such as Tween-20 or Tween-80), stabilizers
(e.g., sugars or protein-free amino acids), preservatives, tissue
fixatives, solubilizers, and/or other materials suitable for
inclusion in a pharmaceutical composition.
[0218] The actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the amide thereof, the route of administration, the
time of administration, the rate of excretion of the particular
compound being employed, the duration of the treatment, other
drugs, compounds and/or materials used in combination with the
particular compositions employed, the age, sex, weight, condition,
general health and prior medical history of the patient being
treated, and like factors well known in the medical arts.
[0219] The pharmaceutical composition may be administered by any
suitable route and mode. Suitable routes of administering a
compound of the present invention in vivo and in vitro are well
known in the art and may be selected by those of ordinary skill in
the art.
[0220] In one embodiment, a pharmaceutical composition of the
present invention is administered parenterally.
[0221] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
include epidermal, intravenous, intramuscular, intra-arterial,
intrathecal, intracapsular, intra-orbital, intracardiac,
intradermal, intraperitoneal, intratendinous, transtracheal,
subcutaneous, subcuticular, intra-articular, subcapsular,
subarachnoid, intraspinal, intracranial, intrathoracic, epidural
and intrasternal injection and infusion.
[0222] In one embodiment that pharmaceutical composition is
administered by intravenous or subcutaneous injection or
infusion.
[0223] Pharmaceutically acceptable carriers include any and all
suitable solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonicity agents, antioxidants and
absorption-delaying agents, and the like that are physiologically
compatible with a compound of the present invention.
[0224] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the present
invention include water, saline, phosphate-buffered saline,
ethanol, dextrose, polyols (such as glycerol, propylene glycol,
polyethylene glycol, and the like), and suitable mixtures thereof,
vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed
oil, and sesame oil, carboxymethyl cellulose colloidal solutions,
tragacanth gum and injectable organic esters, such as ethyl oleate,
and/or various buffers. Other carriers are well known in the
pharmaceutical arts.
[0225] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the present invention is
contemplated.
[0226] Proper fluidity may be maintained, for example, by the use
of coating materials, such as lecithin, by the maintenance of the
required particle size in the case of dispersions, and by the use
of surfactants.
[0227] Pharmaceutical compositions of the present invention may
also comprise pharmaceutically acceptable antioxidants for instance
(1) water-soluble antioxidants, such as ascorbic acid, cysteine
hydrochloride, sodium bisulfate, sodium metabisulfite, sodium
sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal-chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0228] Pharmaceutical compositions of the present invention may
also comprise isotonicity agents, such as sugars, polyalcohols,
such as mannitol, sorbitol, glycerol or sodium chloride in the
compositions.
[0229] The pharmaceutical compositions of the present invention may
also contain one or more adjuvants appropriate for the chosen route
of administration such as preservatives, wetting agents,
emulsifying agents, dispersing agents, preservatives or buffers,
which may enhance the shelf life or effectiveness of the
pharmaceutical composition. The compounds of the present invention
may be prepared with carriers that will protect the compound
against rapid release, such as a controlled release formulation,
including implants, transdermal patches, and micro-encapsulated
delivery systems. Such carriers may include gelatin, glyceryl
monostearate, glyceryl distearate, biodegradable, biocompatible
polymers such as ethylene vinyl acetate, polyanhydrides,
polyglycolic acid, collagen, poly-ortho-esters, and polylactic acid
alone or with a wax, or other materials well known in the art.
Methods for the preparation of such formulations are generally
known to those skilled in the art. See e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978.
[0230] In one embodiment, the compounds of the present invention
may be formulated to ensure proper distribution in vivo.
Pharmaceutically acceptable carriers for parenteral administration
include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. The use of such media and agents for
pharmaceutically active substances is known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the pharmaceutical compositions of
the present invention is contemplated. Other active or therapeutic
compounds may also be incorporated into the compositions.
[0231] Pharmaceutical compositions for injection must typically be
sterile and stable under the conditions of manufacture and storage.
The composition may be formulated as a solution, micro-emulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier may be an aqueous or a non-aqueous
solvent or dispersion medium containing for instance water,
ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable
oils, such as olive oil, and injectable organic esters, such as
ethyl oleate. The proper fluidity may be maintained, for example,
by the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols such as
glycerol, mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
may be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
Sterile injectable solutions may be prepared by incorporating the
active compound in the required amount in an appropriate solvent
with one or a combination of ingredients e.g. as enumerated above,
as required, followed by sterilization microfiltration. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients e.g. from those enumerated above. In the
case of sterile powders for the preparation of sterile injectable
solutions, examples of methods of preparation are vacuum-drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0232] Sterile injectable solutions may be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions,
examples of methods of preparation are vacuum-drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0233] The pharmaceutical composition of the present invention may
contain one antibody or ADC of the present invention, a combination
of an antibody or ADC according to the invention with another
therapeutic compound, or a combination of compounds of the present
invention.
Therapeutic Applications
[0234] In another aspect, the invention relates to the antibody or
ADC of the invention, as defined in any aspect or embodiment
herein, for use as a medicament.
[0235] The CD74-specific antibodies of the present invention can be
used in the treatment or prevention of disorders involving cells
expressing CD74. For example, the antibodies may be administered to
cells in culture, e.g., in vitro or ex vivo, or to human subjects,
e.g., in vivo, to treat or prevent disorders involving
CD74-expressing cells. As used herein, the term "subject" is
typically a human who responds to the CD74-specific antibody or
ADC. Subjects may for instance include human patients having
disorders that may be corrected or ameliorated by modulating CD74
function or by killing of the cell, directly or indirectly.
[0236] In one embodiment, the invention provides a method for
modulating CD74-associated signaling in a CD74-expressing cell by
contacting the cell with a CD74-specific antibody. A CD74-specific
antibody of the invention may, for example, interfere with
MIF-binding to CD74, which is a non-limiting example of how an
antibody of the invention can modulate CD74-associated
signaling.
[0237] In one embodiment, the invention provides a method for
killing a CD74-expressing cell by contacting the cell with a
CD74-specific antibody of the invention. Without being limited to
theory, antibody-mediated crosslinking or clustering (e.g., due to
the Fc-region of CD74-bound antibodies binding to FcR-expressing
cells) of CD74 molecules on the surface of a cell can lead to
apoptosis of the cell.
[0238] In one embodiment, the invention provides a method for
killing a CD74-expressing cell by contacting the cell with a
CD74-specific antibody of the invention in the presence of effector
cells capable of inducing an Fc-mediated effector cell response
such as a CDC, ADCC or ADCP response. In this embodiment, the
antibody is typically full-length and of an isotype leading to a
CDC or ADCC response, such as, e.g., an IgG1,.kappa. isotype.
[0239] The CD74-specific antibodies of the invention are
characterized by efficient internalization upon binding to CD74,
making them suitable for an ADC approach using an ADC as described
in any aspect or embodiment described herein.
[0240] Accordingly, in one embodiment, the invention provides a
method for killing a CD74-expressing cell by contacting the cell
with an ADC of the invention which requires internalization and
trafficking to lysosomes for specific (i.e. cleavable linker) or
non-specific (non-cleavable linker) proteolytic cleavage of the
antibody-linker-drug complex. In another embodiment, the invention
provides for a method of killing a CD74-expressing cell by
contacting the cell with an ADC of the invention wherein the
CD74-specific antibody is linked to a therapeutic moiety via a
linker allowing for release of the drug once the ADC is
internalized, e.g., by a change in pH or reducing conditions.
Suitable linker technology is known in the art, as described
above.
[0241] In another aspect, the present invention provides methods
for treating or preventing a disorder involving cells expressing
CD74 in a subject, which method comprises administration of a
therapeutically effective amount of a CD74-specific antibody or ADC
of the present invention to a subject in need thereof. The method
typically involves administering to a subject a CD74-specific
antibody or ADC in an amount effective to treat or prevent the
disorder.
[0242] In a particular aspect, a CD74-specific antibody or ADC is
administered prophylactically in order to reduce the risk of
developing cancer, delay the onset of an event in cancer
progression or reduce the risk of recurrence when a cancer is in
remission and/or a primary tumor has been surgically removed. In
the latter case, the CD74-specific antibody could, for example, be
administered in association with (i.e., before, during, or after)
the surgery. Prophylactic administration may also be useful in
patients wherein it is difficult to locate a tumor that is believed
to be present due to other biological factors.
[0243] Cells over-expressing CD74, such as cancer cells, are
particularly good targets for the CD74-specific antibodies or ADCs
of the invention, since more antibodies or ADCs may be bound per
cell. Thus, in one aspect, the disorder involving cells expressing
CD74 is cancer, i.e., a tumorigenic disorder, such as a disorder
characterized by the presence of tumor cells expressing CD74
including, for example, disorders where the cells are from a solid
tumor or hematological tumor. CD74 expression has been described
in, e.g., breast cancer (Koretz K et al., Int J Cancer 1989; 44:
816-822), colorectal cancer (Cuthbert R J et al., Eur J Cancer
2009; 45:1654-1663), endometrial/cervical cancer (Glew S S et al.,
Cancer Res 1992; 52:4009-4016), gastric cancer (Tamori Y et al,
Oncol Rep 2005; 14:873-877), squamous cell carcinoma of the head
and neck (SCCHN) (Han J et al., Head Neck Oncol 2009; 1:27), lung
cancer (McClelland M et al., Am J Pathol 2009; 174:638-646),
glioblastoma (Kitange G J et al., J Neurooncol 2010; 100: 177-186),
malignant lymphoma (Momburg F et al., Int J Cancer 1987;
40:598-603), B cell chronic lymhocytic leukemia (B-CLL) (Narni F et
al., Blood 1986; 68:372-377), non-Hodgkin's lymphoma (NHL),
monocytoid B cell lymphoma (MBCL) (Stroup R et al, Hum Pathol 1992;
23:172-177), hairy-cell leukemia (HCL) (Spiro R C et al., Leuk Res
1984; 8: 55-62), malignant melanoma (Weeraratna A T et al.,
Oncogene 2004; 23:2264-2274), ovarian cancer (Rangel L B et al.,
Cancer Biol Ther 2004; 3:1021-1027), prostate cancer (Meyer-Siegler
K L et al., BMC Cancer 2005; 5:73), pancreatic cancer (Koide N et
al., Clin Cancer Res 2006; 12:2419-2426), renal cancer (Saito T et
al., Cancer Lett 1997; 115:121-127), thymic epithelial neoplasms
(Datta M W et al., Appl Immunohistochem Mol Morphol 2000;
8:210-215), malignant fibrous histiosarcomas (Lazova R et al.,
Cancer 1997; 79:2115-2124), and pituitary adenomas (Rossi M L et
al., Tumori 1990; 76:543-547). CD74 has also been found to be
up-regulated in e.g., gastric epithelium during H. pylori infection
and ulcerative colitis (Beswick, World J Gastroenterol. 2009;
15(23):2855-61).
[0244] Exemplary cells expressing CD74 thus include cancer cells
such as, e.g., cells from NHL, multiple myeloma (MM), ovarian
cancer, breast cancer, pancreatic cancer, prostate cancer, gastric
cancer, colorectal carcinoma and liver cancer.
[0245] In one aspect, the present invention provides methods for
treating or preventing a hematological malignancy, which method
comprises administration of a therapeutically effective amount of a
CD74-specific antibody or ADC of the present invention to a subject
in need thereof, and wherein the hematological malignancy is
selected from a lymphoma, myeloma and/or a leukemia. In one
embodiment, the hematological malignancy is selected from the group
consisting of malignant lymphoma, B cell chronic lymphocytic
leukemia (B-CLL), chronic myeloid leukemia (CML) in blast phase,
NHL, MM, MBCL, HCL and T cell lymphoma.
[0246] In one embodiment, the hematological malignancy is NHL. The
CD74-specific antibodies and ADCs of the present invention can, for
example, be used in the treatment of both indolent and aggressive
forms of NHL. Examples of B cell NHLs include lymphomatoid
granulomatosis, follicular lymphoma, diffuse large B-cell lymphoma,
mantle cell lymphoma, primary effusion lymphoma, intravascular
large B cell lymphoma, mediastinal large B cell lymphoma, heavy
chain diseases (including .gamma., .mu., and .alpha. disease),
lymphomas induced by therapy with immunosuppressive agents, such as
cyclosporine-induced lymphoma, and methotrexate-induced lymphoma.
In one embodiment, the hematological malignancy is multiple
myeloma, such as, e.g., myeloma light chain disease and monoclonal
gammapathy of undetermined significance (MGUS). In other separate
and specific embodiments, the hematological malignancy is malignant
lymphoma, B-CLL (such as, e.g., small lymphocytic lymphoma; SLL),
CML in blast phase, MBCL, or HCL. In one embodiment, the
hematological malignancy is a T cell lymphoma, such as, e.g.,
mycosis fungoides, peripheral T cell lymphomas unspecified,
angioimmunoblastic T cell lymphoma, anaplastic large cell lymphoma
(ALCL), enteropathy-associated T cell lymphoma, or hepatosplenic T
cell lymphoma. In another embodiment, the hematological malignancy
is Hodgkin's lymphoma. In another embodiment, the hematological
malignancy is Waldenstrom's macroglobulinemia. In one embodiment,
the hematological malignancy is CLL, such as B-CLL (e.g., small
lymphocytic lymphoma; SLL).
[0247] In one aspect, the present invention provides methods for
treating or preventing a solid tumor, which method comprises
administration of a therapeutically effective amount of a
CD74-specific antibody or ADC of the present invention to a subject
in need thereof, and wherein the solid tumor is a melanoma,
carcinoma, sarcoma, adenoma and/or a glioma. In one embodiment, the
cancer is selected from the group consisting of breast cancer (such
as, e.g., primary or metastatic breast cancer), colorectal cancer,
endometrial/cervical cancer, gastric cancer, head and neck cancer
(such as, e.g., SCCHN), hepatocellular carcinoma, lung cancer (such
as, e.g., small cell lung cancer or non-small cell lung cancer),
malignant glioma (such as, e.g., anaplastic astrocytoma and
glioblastoma multiforme), malignant melanoma (such as, e.g.,
primary or metastatic melanoma), ovarian cancer (such as, e.g.,
serous, endometrioid or clear cell adenocarcinoma), pancreatic
cancer, prostate cancer, renal cancer, bladder cancer, thymic
cancer (such as, e.g., thymic carcinoma and invasive thymoma),
malignant fibrous histiosarcoma, acoustic schwannoma, pituitary
adenoma, and a soft tissue tumor.
[0248] In one embodiment, the cancer is ovarian cancer. In another
embodiment, the cancer is selected from primary or metastatic
breast cancer. In another embodiment, the cancer is pancreatic
cancer, such as unresectable advanced or metastatic pancreatic
cancer. In another embodiment, the cancer is prostate cancer. In
another embodiment, the cancer is gastric cancer. In another
embodiment, the cancer is colorectal carcinoma, such as metastatic
colorectal carcinoma. In another embodiment, the cancer is
hepatocellular carcinoma. In other separate and specific
embodiments, the cancer is endometrial/cervical cancer, head and
neck cancer, lung cancer, malignant glioma, malignant melanoma,
ovarian cancer, renal cancer, thymic cancer, malignant fibrous
histiosarcoma, acoustic schwannoma, pituitary adenoma, or a soft
tissue tumor.
[0249] In one aspect, the present invention provides methods for
treating or preventing an autoimmune disease, which method
comprises administration of a therapeutically effective amount of a
CD74-specific antibody or ADC of the present invention to a subject
in need thereof. In one embodiment, the autoimmune disease is
selected from an immune-mediated thrombocytopenia (such as acute
idiopathic thrombocytopenic purpura and chronic idiopathic
thrombocytopenic purpura), dermatomyositis, Sjogren's syndrome,
multiple sclerosis, Sydenham's chorea, myasthenia gravis, systemic
lupus erythematosus, lupus nephritis, rheumatic fever,
polyglandular syndromes, bullous pemphigoid, diabetes mellitus,
Henoch-Schonlein purpura, post-streptococcal nephritis, erythema
nodosum, Takayasu's arteritis, Addison's disease, rheumatoid
arthritis, sarcoidosis, ulcerative colitis, erythema multiforme,
IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis,
Goodpasture's syndrome, thromboangitis ubiterans, primary biliary
cinhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma,
chronic active hepatitis, polymyositis/dermatomyositis,
polychondritis, pamphigus vulgaris, Wegener's granulomatosis,
membranous nephropathy, amyofrophic lateral sclerosis, tabes
dorsalis, giant cell arteritis/polymyalgia, pernicious anemia,
rapidly progressive glomerulonephritis and fibrosing
alveolitis.
[0250] In one embodiment, the autoimmune disease is rheumatoid
arthritis. In another embodiment, the autoimmune disease is
systemic sclerosis. In another embodiment, the autoimmune disease
is multiple sclerosis. In another embodiment, the autoimmune
disease is an inflammatory bowel disease, such as, e.g, Crohn's
disease or ulcerative colitis.
[0251] In one embodiment, the invention provides a method of
treatment of any one of the disorders of the above aspects and
embodiments by administration to an individual in need thereof, of
a CD74-specific antibody or ADC of any of the above aspects or
embodiments. The invention also relates to CD74-specific antibodies
or ADCs of the invention for use as a therapeutic, e.g., in the
treatment of cancer or other disorder mentioned herein.
[0252] In an embodiment selection of patients to be treated with
CD74-specific antibodies is based on the level of CD74 expression
in a sample, such as a sample containing tumor cells, or by
detecting CD74-expressing tumors using labeled CD74-specific
antibodies or antibody fragments, e.g., those of the invention.
Exemplary diagnostic assays for determining CD74-expression using
CD74 antibodies or antibody fragment of the invention are described
herein.
[0253] The efficient dosages and dosage regimens for the
CD74-specific antibody or ADC depend on the disease or condition to
be treated and may be determined by the persons skilled in the
art.
[0254] A physician having ordinary skill in the art may readily
determine and prescribe the effective amount of the pharmaceutical
composition required. For example, the physician could start doses
of the CD74-specific antibody employed in the pharmaceutical
composition at levels lower than that required in order to achieve
the desired therapeutic effect and gradually increase the dosage
until the desired effect is achieved. In general, a suitable dose
of a composition of the present invention will be that amount of
the compound which is the lowest dose effective to produce a
therapeutic effect according to a particular dosage regimen. Such
an effective dose will generally depend upon the factors described
above.
[0255] For example, an "effective amount" for therapeutic use may
be measured by its ability to stabilize the progression of disease.
The ability of a compound to inhibit cancer may, for example, be
evaluated in an animal model system predictive of efficacy in human
tumors. Alternatively, this property of a composition may be
evaluated by examining the ability of the compound to inhibit cell
growth or to induce cytotoxicity by in vitro assays known to the
skilled practitioner. A therapeutically effective amount of a
therapeutic compound may decrease tumor size, or otherwise
ameliorate symptoms in a subject. One of ordinary skill in the art
would be able to determine such amounts based on such factors as
the subject's size, the severity of the subject's symptoms, and the
particular composition or route of administration selected.
[0256] An exemplary, non-limiting range for a therapeutically
effective amount of a CD74-specific antibody of the present
invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for
example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for
instance about 0.5, about such as 0.3, about 1, about 3 mg/kg,
about 5 mg/kg or about 8 mg/kg.
[0257] An exemplary, non-limiting range for a therapeutically
effective amount of a CD74-specific ADC of the invention is
0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10
mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg.
[0258] Administration may e.g. be intravenous, intramuscular,
intraperitoneal, or subcutaneous, and for instance administered
proximal to the site of the target.
[0259] Dosage regimens in the above methods of treatment and uses
are adjusted to provide the optimum desired response (e.g., a
therapeutic response). For example, a single bolus may be
administered, several divided doses may be administered over time
or the dose may be proportionally reduced or increased as indicated
by the exigencies of the therapeutic situation.
[0260] In one embodiment, the efficacy-safety window is optimized
by lowering specific toxicity such as for example by lowering the
drug-antibody ratio (DAR) and/or mixing of CD74-specific ADC with
unlabeled CD74-specific antibody.
[0261] In one embodiment, the efficacy of the treatment is
monitored during the therapy, e.g. at predefined points in time. In
one embodiment, the efficacy may be monitored by measuring the
level of CD74 in a sample containing tumor cells, by visualization
of the disease area, or by other diagnostic methods described
further herein, e.g. by performing one or more PET-CT scans, for
example using a labeled CD74-specific antibody, fragment or
mini-antibody derived from the CD74-specific antibody of the
present invention.
[0262] If desired, an effective daily dose of a pharmaceutical
composition may be administered as two, three, four, five, six or
more sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms. In another
embodiment, the CD74-specific antibodies are administered by slow
continuous infusion over a long period, such as more than 24 hours,
in order to minimize any unwanted side effects.
[0263] While it is possible for a compound of the present invention
to be administered alone, it is preferable to administer the
compound as a pharmaceutical composition as described above.
[0264] An effective dose of a CD74-specific antibody or ADC of the
invention may also be administered using a weekly, biweekly or
triweekly dosing period. The dosing period may be restricted to,
e.g., 8 weeks, 12 weeks or until clinical progression has been
established.
[0265] For example, in one embodiment, the CD74-specific antibody
or ADC is administered by infusion in a weekly dosage of between 10
and 500 mg/m.sup.2, such as between 200 and 400 mg/m.sup.2. Such
administration may be repeated, e.g., 1 to 8 times, such as 3 to 5
times. The administration may be performed by continuous infusion
over a period of from 1 to 24 hours, such as of from 1 to 12
hours.
[0266] In another embodiment, the CD74-specific antibody or ADC is
administered by infusion every three weeks in a dosage of between
10 and 500 mg/m.sup.2, such as between 50-200 mg/m.sup.2. Such
administration may be repeated, e.g., 1 to 8 times, such as 3 to 5
times. The administration may be performed by continuous infusion
over a period of from 1 to 24 hours, such as of from 1 to 12
hours.
[0267] In one embodiment, a CD74-specific ADC is administered as a
single dose of about 0.1-10 mg/kg, such as about 1-3 mg/kg, every
week or every third week for up to twelve times, up to eight times,
or until clinical progression. The administration may be performed
by continuous infusion over a period of from 1 to 24 hours, such as
of from 1 to 12 hours. Such regimens may be repeated one or more
times as necessary, for example, after 6 months or 12 months. The
dosage may be determined or adjusted by measuring the amount of
compound of the present invention in the blood upon administration
by for instance taking out a biological sample and using
anti-idiotypic antibodies which target the antigen binding region
of the CD74-specific antibodies of the present invention.
[0268] In one embodiment, the CD74-specific antibodies are
administered as maintenance therapy, such as, e.g., once a week for
a period of six months or more.
[0269] As non-limiting examples, treatment according to the present
invention may be provided as a daily dosage of a compound of the
present invention in an amount of about 0.1-100 mg/kg, such as 0.2,
0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one
of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
or 20 after initiation of treatment, or any combination thereof,
using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or
any combination thereof.
[0270] Parenteral compositions may be formulated in dosage unit
form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited
as unitary dosages for the subjects to be treated; each unit
contains a predetermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the present invention are dictated by and directly
dependent on (a) the unique characteristics of the active compound
and the particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
compound for the treatment of sensitivity in individuals.
Combinations
[0271] The invention also provides for therapeutic applications
where an antibody or ADC of the invention is used in combination
with at least one further therapeutic agent relevant for the
disease or disorder to be treated, as described above. Such
administration may be simultaneous, separate or sequential. For
simultaneous administration the agents may be administered as one
composition or as separate compositions, as appropriate.
[0272] Accordingly, the present invention provides methods for
treating a disorder involving cells expressing CD74 as described
above, which methods comprise administration of a CD74-specific
antibody or ADC of the present invention combined with one or more
additional therapeutic agents. The present invention also provides
for the use of a CD74-specific antibody or ADC of the present
invention for the preparation of a pharmaceutical composition to be
administered with at least one chemotherapeutic agent for treating
such a disorder.
[0273] The further therapeutic agent is typically relevant for the
disorder to be treated. Exemplary therapeutic agents include other
anti-cancer antibodies or ADCs, cytotoxic agents, chemotherapeutic
agents, anti-angiogenic agents, anti-cancer immunogens, cell cycle
control/apoptosis regulating agents, hormonal regulating agents,
and other agents described below.
[0274] In one aspect, the further therapeutic agent is at least one
second antibody or ADC which binds another target such as, e.g.,
CD4, CD5, CD8, CD14, CD15, CD19, CD21, CD22, CD23, CD25, CD30,
CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD80, CD126, B7,
MUC1, tenascin, HM1.24, or HLA-DR. For example, the second antibody
may bind to a B cell antigen, including, but not limited to CD20,
CD19, CD21, CD23, CD38, CD46, CD80, CD138, HLA-DR, CD22, or to
another epitope on CD74. In another embodiment, the second antibody
binds vascular endothelial growth factor A (VEGF-A). In separate
and specific embodiments, the further therapeutic agent is a CD20-
or a CD138-specific antibody.
[0275] In one embodiment, the CD74-specific antibody or ADC of the
invention is for use in combination with a specific therapeutic
antibody, such as veltuzumab, bevacizumab (Avastin.RTM.),
zalutumumab, cetuximab (Erbitux.RTM.), panitumumab (Vectibix.TM.),
ofatumumab (Arzerra.TM.), ocrelizumab, zanolimumab, daratumumab,
ranibizumab (Lucentis.RTM.), Zenapax, Simulect, Remicade, Humira,
Tysabri, Xolair, raptiva, nimotuzumab, rituximab and/or trastuzumab
(Herceptin.RTM.). In one embodiment, the CD74-specific antibody or
ADC of the present invention is administered in combination with a
CD20-specific antibody such as, e.g., veltuzumab, ocrelizumab or
ofatumumab (Arzerra.TM.). In another embodiment, the CD74-specific
antibody or ADC of the present invention is administered in
combination with bevacizumab (Avastin.RTM.).
[0276] In one aspect, the invention provides an antibody or ADC of
any of the above aspects or embodiments for the treatment of a
disorder involving CD74-expressing cells, such as cancer, in
combination with at least one chemotherapeutic agent.
[0277] In one embodiment, the chemotherapeutic agent is selected
from an antimetabolite, such as methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, floxuridine (FudR),
3',5'-O-dioleoyl-FudR, fludarabine, 5-fluorouracil, dacarbazine,
hydroxyurea, asparaginase, gemcitabine, cladribine and similar
agents.
[0278] In one embodiment, the chemotherapeutic agent is selected
from an alkylating agent, such as mechlorethamine, thioepa,
chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU),
cyclophosphamide, busulfan, dibromomannitol, streptozotocin,
dacarbazine (DTIC), procarbazine, mitomycin C, and a platinum
derivative such as cisplatin, carboplatin, and similar agents.
[0279] In one embodiment, the chemotherapeutic agent is selected
from an anti-mitotic agent, such as taxanes, for instance
docetaxel, and paclitaxel, and vinca alkaloids, for instance
vindesine, vincristine, vinblastine, and vinorelbine.
[0280] In one embodiment, the chemotherapeutic agent is selected
from a topoisomerase inhibitor, such as topotecan or
irinotecan.
[0281] In one embodiment, the chemotherapeutic agent is selected
from a cytostatic drug, such as etoposide and teniposide.
[0282] In one embodiment, the chemotherapeutic agent is selected
from a growth factor receptor inhibitor, such as an inhibitor of
ErbB1 (EGFR) (such as Iressa, erbitux (cetuximab), tarceva and
similar agents), an inhibitor of ErbB2 (Her2/neu) (such as
herceptin and similar agents) and similar agents.
[0283] In one embodiment, the chemotherapeutic agent is selected
from a tyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec
STI571), Iapatinib, PTK787/ZK222584 and similar agents.
[0284] In one aspect, the present invention provides a method for
treating a disorder involving cells expressing CD74 in a subject,
such as a cancer patient, which method comprises administration of
a therapeutically effective amount of a CD74-specific antibody or
ADC of the present invention and at least one inhibitor of
angiogenesis, neovascularization, and/or other vascularization to a
subject in need thereof.
[0285] Examples of such angiogenesis inhibitors are urokinase
inhibitors, matrix metalloprotease inhibitors (such as marimastat,
neovastat, BAY 12-9566, AG 3340, BMS-275291 and similar agents),
inhibitors of endothelial cell migration and proliferation (such as
TNP-470, squalamine, 2-methoxyestradiol, combretastatins,
endostatin, angiostatin, penicillamine, SCH66336 (Schering-Plough
Corp, Madison, N.J.), R115777 (Janssen Pharmaceutica, Inc,
Titusville, N.J.) and similar agents), antagonists of angiogenic
growth factors (such as such as ZD6474, SU6668, antibodies against
angiogenic agents and/or their receptors (such as VEGF, bFGF, and
angiopoietin-1), thalidomide, thalidomide analogs (such as
CC-5013), Sugen 5416, SU5402, antiangiogenic ribozyme (such as
angiozyme), interferon .alpha. (such as interferon .alpha.2a),
suramin and similar agents), VEGF-R kinase inhibitors and other
inhibitors of angiogenic tyrosine kinases (such as SU011248),
inhibitors of endothelial-specific integrin/survival signaling
(such as vitaxin and similar agents), copper antagonists/chelators
(such as tetrathiomolybdate, captopril and similar agents),
carboxyamido-triazole (CAI), ABT-627, CM101, interleukin-12
(IL-12), IM862, PNU145156E as well as nucleotide molecules
inhibiting angiogenesis (such as antisense-VEGF cDNA, cDNA coding
for angiostatin, cDNA coding for p53 and cDNA coding for deficient
VEGF receptor-2) and similar agents.
[0286] Other examples of such inhibitors of angiogenesis,
neovascularization, and/or other vascularization are
anti-angiogenic heparin derivatives and related molecules (e.g.,
heperinase III), temozolomide, NK4, cyclooxygenase-2 inhibitors,
inhibitors of hypoxia-inducible factor 1, anti-angiogenic soy
isoflavones, oltipraz, fumagillin and analogs thereof, somatostatin
analogues, pentosan polysulfate, tecogalan sodium, dalteparin,
tumstatin, thrombospondin, NM-3, combretastatin, canstatin,
avastatin, antibodies against other relevant targets (such as
anti-alpha-v/beta-3 integrin and anti-kininostatin mAbs) and
similar agents.
[0287] In one embodiment, the therapeutic agent for use in
combination with a CD74-specific antibody or ADC for treating the
disorders as described above is an anti-cancer immunogen, such as a
cancer antigen/tumor-associated antigen (e.g., epithelial cell
adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic
antigen (CEA), tumor-associated glycoprotein 72 (TAG-72), gp100,
Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines
(e.g., human papillomavirus vaccines), tumor-derived heat shock
proteins, and similar agents. A number of other suitable cancer
antigens/tumor-associated antigens known in the art may also or
alternatively be used in such embodiment. Anti-cancer immunogenic
peptides also include anti-idiotypic "vaccines" such as BEC2
anti-idiotypic antibodies (Mitumomab), CeaVac and related
anti-idiotypic antibodies, anti-idiotypic antibody to MG7 antibody,
and other anti-cancer anti-idiotypic antibodies (see for instance
Birebent et al., Vaccine. 21(15), 1601-12 (2003), Li et al., Chin
Med J (Engl). 114(9), 962-6 (2001), Schmitt et al., Hybridoma.
13(5), 389-96 (1994), Maloney et al., Hybridoma. 4(3), 191-209
(1985), Raychardhuri et al., J Immunol. 137(5), 1743-9 (1986), Pohl
et al., Int J Cancer. 50(6), 958-67 (1992), Bohlen et al.,
Cytokines Mol Ther. 2(4), 231-8 (1996) and Maruyama, J Immunol
Methods. 264(1-2), 121-33 (2002)). Such anti-idiotypic antibodies
may optionally be conjugated to a carrier, which may be a synthetic
(typically inert) molecule carrier, a protein (for instance keyhole
limpet hemocyanin (KLH) (see for instance Ochi et al., Eur J
Immunol. 17(11), 1645-8 (1987)), or a cell (for instance a red
blood cell--see for instance Wi et al., J Immunol Methods. 122(2),
227-34 (1989)).
[0288] In one embodiment, the therapeutic agent for use in
combination with a CD74-specific antibody or ADC for treating the
disorders as described above is a cytokine, chemokine or
cytokine/chemokine combination with cancer growth inhibitory
properties. Examples of suitable cytokines and growth factors
include IFN.gamma., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13,
IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF,
IFN.alpha. (e.g., INF.alpha.2b), IFN.beta., GM-CSF, CD40L, Flt3
ligand, stem cell factor, ancestim, and TNF.alpha.. Suitable
chemokines may include Glu-Leu-Arg (ELR)-negative chemokines such
as IP-10, MCP-3, MIG, and SDF-1.alpha. from the human CXC and C-C
chemokine families. Suitable cytokines include cytokine
derivatives, cytokine variants, cytokine fragments, and cytokine
fusion proteins. These and other methods or uses involving
naturally occurring peptide-encoding nucleic acids herein may
alternatively, or additionally, be performed by "gene activation"
and homologous recombination gene upregulation techniques, such as
those described in U.S. Pat. No. 5,968,502, U.S. Pat. No. 6,063,630
and U.S. Pat. No. 6,187,305 and EP 0505500.
[0289] In one embodiment, the therapeutic agent for use in
combination with a CD74-specific antibody or ADC for treating the
disorders as described above may be a cell cycle control/apoptosis
regulator (or "regulating agent"). A cell cycle control/apoptosis
regulator may include molecules that target and modulate cell cycle
control/apoptosis regulators such as (i) cdc-25 (such as NSC
663284), (ii) cyclin-dependent kinases that overstimulate the cell
cycle (such as flavopiridol (L868275, HMR1275),
7-hydroxy-staurosporine (UCN-01, KW-2401), and roscovitine
(R-roscovitine, CYC202)), and (iii) telomerase modulators (such as
BIBR1532, SOT-095, GRN163 and compositions described in for
instance U.S. Pat. No. 6,440,735 and U.S. Pat. No. 6,713,055).
Non-limiting examples of molecules that interfere with apoptotic
pathways include TNF-related apoptosis-inducing ligand
(TRAIL)/apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL
receptors, IFNs, .quadrature. and anti-sense Bcl-2.
[0290] In one embodiment, the therapeutic agent for use in
combination with a CD74-specific antibody or ADC for treating the
disorders as described above is a hormonal regulating agent, such
as agents useful for anti-androgen and anti-estrogen therapy.
Examples of such hormonal regulating agents are tamoxifen,
idoxifene, fulvestrant, droloxifene, toremifene, raloxifene,
diethylstilbestrol, ethinyl estradiol/estinyl, an anti-androgene
(such as flutaminde/eulexin), a progestin (such as such as
hydroxy-progesterone caproate, medroxyprogesterone/provera,
megestrol acepate/megace), an adrenocorticosteroid (such as
hydrocortisone, prednisone), luteinizing hormone-releasing hormone
(and analogs thereof and other LHRH agonists such as buserelin and
goserelin), an aromatase inhibitor (such as anastrazole/arimidex,
aminoglutethimide/cytraden, exemestane), a hormone inhibitor (such
as octreotide/sandostatin) and similar agents.
[0291] In one embodiment, the therapeutic agent for use in
combination with a CD74-specific antibody or ADC for treating the
disorders as described above is an anti-anergic agent (for instance
small molecule compounds, proteins, glycoproteins, or antibodies
that break tolerance to tumor and cancer antigens). Examples of
such compounds are molecules that block the activity of CTLA-4,
such as MDX-010 (ipilimumab, Yervoy.TM.) (Phan et al., PNAS USA
100, 8372 (2003)).
[0292] In one embodiment, the therapeutic agent for use in
combination with a CD74-specific antibody or ADC for treating the
disorders as described above is a tumor suppressor gene-containing
nucleic acid or vector such as a replication-deficient adenovirus
encoding human recombinant wild-type p53/SCH58500, etc.; antisense
nucleic acids targeted to oncogenes, mutated, or deregulated genes;
or siRNA targeted to mutated or deregulated genes. Examples of
tumor suppressor targets include, for example, BRCA1, RB1, BRCA2,
DPC4 (Smad4), MSH2, MLH1, and DCC.
[0293] In one embodiment, a therapeutic agent for use in
combination with a CD74-specific antibody or ADC for treating the
disorders as described above is an anti-cancer nucleic acid.
Exemplary anti-cancer nucleic acids include genasense
(augmerosen/G3139), LY900003 (ISIS 3521), ISIS 2503, OGX-011 (ISIS
112989), LE-AON/LEraf-AON (liposome encapsulated c-raf antisense
oligonucleotide/ISIS-5132), MG98, and other antisense nucleic acids
that target PKCa, clusterin, IGFBPs, protein kinase A, cyclin D1,
or Bcl-2.
[0294] In one embodiment, the therapeutic agent for use in
combination with a CD74-specific antibody or ADC for treating the
disorders as described above is an anti-cancer inhibitory RNA
molecule (see for instance Lin et al., Curr Cancer Drug Targets.
1(3), 241-7 (2001), Erratum in: Curr Cancer Drug Targets. 3(3), 237
(2003), Lima et al., Cancer Gene Ther. 11(5), 309-16 (2004), Grzmil
et al., Int J Oncol. 4(1), 97-105 (2004), Collis et al., Int J
Radiat Oncol Biol Phys. 57(2 Suppl), 5144 (2003), Yang et al.,
Oncogene. 22(36), 5694-701 (2003) and Zhang et al., Biochem Biophys
Res Commun. 303(4), 1169-78 (2003)).
[0295] Compositions and combination administration methods of the
present invention also include the administration of nucleic acid
vaccines, such as naked DNA vaccines encoding such cancer
antigens/tumor-associated antigens (see for instance U.S. Pat. No.
5,589,466, U.S. Pat. No. 5,593,972, U.S. Pat. No. 5,703,057, U.S.
Pat. No. 5,879,687, U.S. Pat. No. 6,235,523, and U.S. Pat. No.
6,387,888). In one embodiment, the combination administration
method and/or combination composition comprises an autologous
vaccine composition. In one embodiment, the combination composition
and/or combination administration method comprises a whole cell
vaccine or cytokine-expressing cell (for instance a recombinant
IL-2 expressing fibroblast, recombinant cytokine-expressing
dendritic cell, and the like) (see for instance Kowalczyk et al.,
Acta Biochim Pol. 50(3), 613-24 (2003), Reilly et al., Methods Mol
Med. 69, 233-57 (2002) and Tirapu et al., Curr Gene Ther. 2(1),
79-89 (2002). Another example of such an autologous cell approach
that may be useful in combination methods of the present invention
is the MyVax.RTM. Personalized Immunotherapy method (previously
referred to as GTOP-99) (Genitope Corporation--Redwood City,
Calif., USA).
[0296] In one embodiment, a CD74-specific antibody or ADC according
to the invention is combined or co-administered with a virus, viral
proteins, or the like. Replication-deficient viruses, that
generally are capable of one or only a few rounds of replication in
vivo, and that are targeted to tumor cells, may for instance be
useful components of such compositions and methods. Such viral
agents may comprise or be associated with nucleic acids encoding
immunostimulants, such as GM-CSF and/or IL-2. Both naturally
oncolytic and such recombinant oncolytic viruses (for instance
HSV-1 viruses, reoviruses, replication-deficient and
replication-sensitive adenovirus, etc.) may be useful components of
such methods and compositions. Accordingly, in one embodiment, the
present invention provides combination compositions and combination
administration methods wherein a CD74-specific antibody is combined
or co-administered with an oncolytic virus. Examples of such
viruses include oncolytic adenoviruses and herpes viruses, which
may or may not be modified viruses (see for instance Shah et al., J
Neurooncol. 65(3), 203-26 (2003), Stiles et al., Surgery. 134(2),
357-64 (2003), Sunarmura et al., Pancreas. 28(3), 326-9 (2004),
Teshigahara et al., J Surg Oncol. 85(1), 42-7 (2004), Varghese et
al., Cancer Gene Ther. 9(12), 967-78 (2002), Wildner et al., Cancer
Res. 59(2), 410-3 (1999), Yamanaka, Int J Oncol. 24(4), 919-23
(2004) and Zwiebel et al., Semin Oncol. 28(4), 336-43 (2001).
[0297] Combination compositions and combination administration
methods of the present invention may also involve "whole cell" and
"adoptive" immunotherapy methods. For instance, such methods may
comprise infusion or re-infusion of immune system cells (for
instance tumor-infiltrating lymphocytes (TILs), such as CD4.sup.+
and/or CD8.sup.+ T cells (for instance T cells expanded with
tumor-specific antigens and/or genetic enhancements),
antibody-expressing B cells or other antibody-producing or
-presenting cells, dendritic cells (e.g., dendritic cells cultured
with a DC-expanding agent such as GM-CSF and/or Flt3-L, and/or
tumor-associated antigen-loaded dendritic cells), anti-tumor NK
cells, so-called hybrid cells, or combinations thereof. Cell
lysates may also be useful in such methods and compositions.
Cellular "vaccines" in clinical trials that may be useful in such
aspects include Canvaxin.TM., APC-8015 (Dendreon), HSPPC-96
(Antigenics), and Melacine.RTM. cell lysates. Antigens shed from
cancer cells, and mixtures thereof (see for instance Bystryn et
al., Clinical Cancer Research Vol. 7, 1882-1887, July 2001),
optionally admixed with adjuvants such as alum, may also be
components in such methods and combination compositions.
[0298] In one embodiment, a CD74-specific antibody or ADC is
delivered to a patient in combination with the application of an
internal vaccination method. Internal vaccination refers to induced
tumor or cancer cell death, such as drug-induced or
radiation-induced, cryo-ablation-induced or radiofrequency
ablation-induced cell death of tumor cells, in a patient, that
typically leads to elicitation of an immune response directed
towards (i) the tumor cells as a whole or (ii) parts of the tumor
cells including (a) secreted proteins, glycoproteins or other
products, (b) membrane-associated proteins or glycoproteins or
other components associated with or inserted in membranes, and/or
(c) intracellular proteins or other intracellular components. An
internal vaccination-induced immune response may be humoral (i.e.
antibody--complement-mediated) or cell-mediated (e.g., the
development and/or increase of endogenous cytotoxic T lymphocytes
that recognize the internally killed tumor cells or parts thereof).
In addition to radiotherapy, non-limiting examples of drugs and
agents that may be used to induce said tumor cell death and
internal vaccination are conventional chemotherapeutic agents,
cell-cycle inhibitors, anti-angiogenesis drugs, monoclonal
antibodies, apoptosis-inducing agents, and signal transduction
inhibitors.
[0299] Examples of other anti-cancer agents, which may be relevant
as therapeutic agents for use in combination with a CD74-specific
antibody or ADC for treating the disorders as described above are
differentiation inducing agents, retinoic acid analogues (such as
all trans retinoic acid, 13-cis retinoic acid and similar agents),
vitamin D analogues (such as seocalcitol and similar agents),
inhibitors of ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRalpha,
PDGFRbeta, Flk2, Flt4, FGFR1, FGFR2, FGFR3, FGFR4, TRKA, TRKC,
c-met, Ron, Sea, Tie, Tie2, Eph, Ret, Ros, Alk, LTK, PTK7 and
similar agents.
[0300] Examples of other anti-cancer agents, which may be relevant
as therapeutic agents for use in combination with a CD74-specific
antibody or ADC for treating the disorders as described above are
cathepsin B, modulators of cathepsin D dehydrogenase activity,
glutathione-S-transferase (such as glutacylcysteine synthetase and
lactate dehydrogenase), and similar agents.
[0301] Examples of other anti-cancer agents, which may be relevant
as therapeutic agents for use in combination with a CD74-specific
antibody for treating the disorders as described above are
estramustine and epirubicin.
[0302] Examples of other anti-cancer agents, which may be relevant
as therapeutic agents for use in combination with a CD74-specific
antibody for treating the disorders as described above are a HSP90
inhibitors like 17-(Allylamino)-17-demethoxygeldanamycin,
antibodies directed against a tumor antigen such as PSA, CA125,
KSA, etc., integrins like integrin inhibitors of VCAM and similar
agents.
[0303] Examples of other anti-cancer agents, which may be relevant
as therapeutic agents for use in combination with a CD74-specific
antibody or ADC for treating the disorders as described above are
calcineurin-inhibitors (such as valspodar, PSC 833 and other MDR-1
or p-glycoprotein inhibitors), TOR-inhibitors (such as sirolimus,
everolimus and rapamcyin) and inhibitors of "lymphocyte homing"
mechanisms (such as FTY720), and agents with effects on cell
signaling such as adhesion molecule inhibitors (for instance
anti-LFA, etc.).
[0304] In one embodiment, a CD74-specific antibody or ADC may be
administered in connection with the delivery of one or more agents
that promote access of the CD74-specific antibody or combination
composition to the interior of a tumor. Such methods may for
example be performed in association with the delivery of a relaxin,
which is capable of relaxing a tumor (see for instance U.S. Pat.
No. 6,719,977). In one embodiment, a CD74-specific antibody or ADC
of the present invention may be bonded to a cell penetrating
peptide (CPP). Cell penetrating peptides and related peptides (such
as engineered cell penetrating antibodies) are described in for
instance Zhao et al., J Immunol Methods. 254(1-2), 137-45 (2001),
Hong et al., Cancer Res. 60(23), 6551-6 (2000). Lindgren et al.,
Biochem J. 377(Pt 1), 69-76 (2004), Buerger et al., J Cancer Res
Clin Oncol. 129(12), 669-75 (2003), Pooga et al., FASEB J. 12(1),
67-77 (1998) and Tseng et al., Mol Pharmacol. 62(4), 864-72
(2002).
[0305] In yet another embodiment, the CD74-specific antibody or ADC
is administered in conjunction with a CD74 up-regulating agent,
such as, e.g., IFN.gamma. or inactivated H. pylori.
[0306] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD74 in a
subject, which method comprises administration of a therapeutically
effective amount of a CD74-specific antibody or ADC and at least
one anti-inflammatory, immunosuppressive and/or immunomodulatory
agent to a subject in need thereof.
[0307] In one embodiment such an anti-inflammatory agent may be
selected from aspirin and other salicylates, Cox-2 inhibitors (such
as rofecoxib and celecoxib), NSAIDs (such as ibuprofen, fenoprofen,
naproxen, sulindac, diclofenac, piroxicam, ketoprofen, diflunisal,
nabumetone, etodolac, oxaprozin, and indomethacin), anti-IL-6R
antibodies, anti-IL-8 antibodies (e.g. antibodies described in
WO2004058797, such as 10F8), anti-IL-15 antibodies (e.g. antibodies
described in WO03017935 and WO2004076620), anti-IL-15 receptor Abs,
anti-CD4 antibodies (e.g. zanolimumab), anti-CD11a antibodies
(e.g., efalizumab), anti-alpha-4/beta-1 integrin (VLA4) antibodies
(e.g. natalizumab), CTLA4-Ig for the treatment of inflammatory
diseases, prednisolone, prednisone, disease modifying antirheumatic
drugs (DMARDs) such as methotrexate, hydroxychloroquine,
sulfasalazine, pyrimidine synthesis inhibitors (such as
leflunomide), IL-1 receptor blocking agents (such as anakinra),
TNF-.alpha. blocking agents (such as etanercept, infliximab, and
adalimumab) and similar agents.
[0308] In one embodiment, such an immunosuppressive and/or
immunomodulatory agent may be selected from cyclosporine,
azathioprine, mycophenolic acid, mycophenolate mofetil,
corticosteroids such as prednisone, methotrexate, gold salts,
sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine,
15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin,
tacrolimus (FK-506), thymopentin, thymosin-.alpha. and similar
agents.
[0309] In one embodiment, such an immunosuppressive and/or
immunomodulatory agent may be selected from immunosuppressive Abs,
such as antibodies binding to p75 of the IL-2 receptor, antibodies
against CD25 (e.g. those described in WO2004045512, such as AB1,
AB7, AB11, and AB12), antibodies against thymocyte globulin, or
antibodies binding to for instance MHC, CD2, CD3 (such as, e.g.,
OKT3), CD4, CD7, CD28, B7, CD40, CD45, IFN.gamma., TNF-.alpha.,
IL-4, IL-5, IL-6R, IL-7, IL-8, IL-10, CD11a, or CD58, or antibodies
binding to their respective receptor(s) or ligand(s).
[0310] In one embodiment, such an immunosuppressive and/or
immunomodulatory agent may be selected from soluble IL-15R, IL-10,
B7 molecules (B7-1, B7-2, variants thereof, and fragments thereof),
ICOS, and OX40, an inhibitor of a negative T cell regulator (such
as an antibody against CTLA4) and similar agents.
[0311] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD74 in a
subject, which method comprises administration of a therapeutically
effective amount of a CD74-specific antibody or ADC and an
anti-C3b(i) antibody to a subject in need thereof.
[0312] In one embodiment, a therapeutic agent for use in
combination with CD74-specific antibodies or ADCs for treating the
disorders as described above may be selected from histone
deacetylase inhibitors (for instance phenylbutyrate) and/or DNA
repair agents (for instance DNA repair enzymes and related
compositions such as dimericine).
[0313] Methods of the present invention for treating a disorder as
described above comprising administration of a therapeutically
effective amount of a CD74-specific antibody or ADC may also
comprise anti-cancer directed photodynamic therapy (for instance
anti-cancer laser therapy--which optionally may be practiced with
the use of photosensitizing agent, see, for instance Zhang et al.,
J Control Release. 93(2), 141-50 (2003)), anti-cancer sound-wave
and shock-wave therapies (see for instance Kambe et al., Hum Cell.
10(1), 87-94 (1997)), and/or anti-cancer nutraceutical therapy (see
for instance Roudebush et al., Vet Clin North Am Small Anim Pract.
34(1), 249-69, viii (2004) and Rafi, Nutrition. 20(1), 78-82
(2004). Likewise, a CD74-specific antibody may be used for the
preparation of a pharmaceutical composition for treating a disorder
as described above to be administered with anti-cancer directed
photodynamic therapy (for instance anti-cancer laser therapy--which
optionally may be practiced with the use of photosensitizing
agent), anti-cancer sound-wave and shock-wave therapies, and/or
anti-cancer nutraceutical therapy.
[0314] In one embodiment, the present invention provides a method
for treating a disorder involving cells expressing CD74 in a
subject, which method comprises administration of a therapeutically
effective amount of a CD74-specific antibody or ADC of the present
invention, and radiotherapy to a subject in need thereof.
[0315] In one embodiment, the present invention provides a method
for treating or preventing cancer, which method comprises
administration of a therapeutically effective amount of a
CD74-specific antibody or ADC of the present invention, and
radiotherapy to a subject in need thereof.
[0316] In one embodiment, the present invention provides the use of
a CD74-specific antibody or ADC of the present invention, for the
preparation of a pharmaceutical composition for treating cancer to
be administered in combination with radiotherapy.
[0317] Radiotherapy may comprise radiation or associated
administration of radiopharmaceuticals to a patient. The source of
radiation may be either external or internal to the patient being
treated (radiation treatment may, for example, be in the form of
external beam radiation therapy (EBRT) or brachytherapy (BT)).
Radioactive elements that may be used in practicing such methods
include, e.g., radium, cesium-137, iridium-192, americium-241,
gold-198, cobalt-57, copper-67, technetium-99, iodide-123,
iodide-131, and indium-111.
[0318] In a further embodiment, the present invention provides a
method for treating or preventing cancer, which method comprises
administration to a subject in need thereof of a therapeutically
effective amount of a CD74-specific antibody or ADC of the present
invention, in combination with surgery.
[0319] As described above, a pharmaceutical composition of the
present invention may be administered in combination therapy, i.e.,
combined with one or more agents relevant for the disease or
condition to be treated either as separate pharmaceutical
compositions or with a compound of the present invention
co-formulated with one or more additional therapeutic agents as
described above. Such combination therapies may require lower
dosages of the compound of the present invention and/or the
co-administered agents, thus avoiding possible toxicities or
complications associated with the various monotherapies.
[0320] In one embodiment, the further therapeutic agent for a
particular therapeutic use is selected from the following: [0321] A
CD20-specific antibody, particularly for treatment of a
hematological malignancy such as, e.g., B-CLL or follicular
lymphoma; [0322] A CD138-specific antibody, particularly for
treatment of a hematological malignancy such as, e.g., myeloma;
[0323] A CD38-specific antibody, particularly for treatment of a
hematological malignancy such as, e.g., myeloma or CLL; [0324]
Melphalan (or melphalan hydrochloride) for treatment of a
hematological malignancy such as, e.g., myeloma; [0325] An
anti-VEGF-A antibody such as, e.g., bevacizumab, particularly for
treatment of a cancer such as, e.g., breast cancer; [0326]
Lenalidomide or bortezomib, particularly for treatment of a
hematological malignancy such as, e.g., myeloma; [0327]
Fluorouracil or gemticabine, particularly for treatment of a cancer
such as, e.g., pancreatic cancer; [0328] Irinotecan, particularly
for treatment of cancer such as, e.g., colorectal cancer, and
[0329] Cisplatin or other platinum-derivative, particularly for
treatment of a cancer such as, e.g., SCCHN.
Diagnostic Applications
[0330] The CD74-specific antibodies of the invention may also be
used for diagnostic purposes, using a composition comprising a
CD74-specific antibody as described herein. Accordingly, the
invention provides diagnostic methods and compositions using the
CD74-specific antibodies described herein. Such methods and
compositions can be used for purely diagnostic purposes, such as
detecting or identifying a disease involving CD74-expressing cells,
as well as for monitoring of the progress of therapeutic
treatments, monitoring disease progression, assessing status after
treatment, monitoring for recurrence of disease, evaluating risk of
developing a disease, and the like.
[0331] In one aspect, the CD74-specific antibodies of the present
invention are used ex vivo, such as in diagnosing a disease in
which cells expressing CD74 are indicative of disease or involved
in the pathogenesis, by detecting levels of CD74 or levels of cells
which express CD74 on their cell surface in a sample taken from a
patient. This may be achieved, for example, by contacting the
sample to be tested, optionally along with a control sample, with
the CD74-specific antibody under conditions that allow for binding
of the antibody to CD74. Complex formation can then be detected
(e.g., using an ELISA). When using a control sample along with the
test sample, the level of CD74-specific antibody or CD74-specific
antibody-CD74 complex is analyzed in both samples and a
statistically significant higher level of CD74-specific antibody or
CD74-specific antibody-CD74 complex in the test sample indicates a
higher level of CD74 in the test sample compared with the control
sample.
[0332] Examples of conventional immunoassays in which CD74-specific
antibodies of the present invention can be used include, without
limitation, ELISA, RIA, FACS assays, plasmon resonance assays,
chromatographic assays, tissue immunohistochemistry, Western blot,
and/or immunoprecipitation.
[0333] In one embodiment, the invention relates to a method for
detecting the presence of CD74 antigen, or a cell expressing CD74,
in a sample comprising: [0334] contacting the sample with a
CD74-specific antibody of the invention under conditions that allow
for binding of the CD74-specific antibody to CD74 in the sample;
and [0335] analyzing whether a complex has been formed. Typically,
the sample is a biological sample.
[0336] In one embodiment, the sample is a tissue sample known or
suspected of containing CD74 antigen and/or cells expressing CD74.
For example, in situ detection of CD74 expression may be
accomplished by removing a histological specimen from a patient,
and providing the antibody of the present invention to such a
specimen. The antibody may be provided by applying or by overlaying
the antibody to the specimen, which is then detected using suitable
means. It is then possible to determine not only the presence of
CD74 or CD74-expressing cells, but also the distribution of CD74 or
CD74-expressing cells in the examined tissue (e.g., in the context
of assessing the spread of cancer cells). Using the present
invention, those of ordinary skill will readily perceive that any
of a wide variety of histological methods (such as staining
procedures) may be modified in order to achieve such in situ
detection.
[0337] In the above assays, the CD74-specific antibody can be
labeled with a detectable substance to allow CD74-bound antibody to
be detected. Alternatively, bound (primary) CD74-specific antibody
may be detected by a secondary antibody which is labeled with a
detectable substance and which binds to the primary antibody.
[0338] The level of CD74 in a sample can also be estimated by a
competition immunoassay utilizing CD74 standards labeled with a
detectable substance and an unlabeled CD74-specific antibody. In
this type of assay, the biological sample, the labeled CD74
standard(s) and the CD74-specific antibody are combined, and the
amount of labeled CD74 standard bound to the unlabeled
CD74-specific antibody is determined. The amount of CD74 in the
biological sample is inversely proportional to the amount of
labeled CD74 standard bound to the CD74-specific antibody.
[0339] Suitable labels for the CD74-specific antibody, secondary
antibody and/or CD74 standard used in in vitro diagnostic
techniques include, without limitation, various enzymes, prosthetic
groups, fluorescent materials, luminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
and acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride and
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.125I, .sup.131I, .sup.35S, and .sup.3H.
[0340] In one aspect, the CD74-specific antibodies of the invention
are used in the in vivo imaging of CD74-expressing tissues such as
tumors. For in vivo methods, antibody fragments such as, e.g.,
(Fab').sub.2, Fab and Fab' fragments, are particularly advantageous
because of their rapid distribution kinetics.
[0341] In vivo imaging can be performed by any suitable technique.
For example, a CD74-specific antibody (such as, e.g., a fragment)
labeled with .sup.99Tc, .sup.131I, .sup.111In or other gamma-ray
emitting isotope may be used to image CD74-specific antibody
accumulation or distribution in CD74-expressing tissues such as
tumors with a gamma scintillation camera (e.g., an Elscint Apex
409ECT device), typically using low-energy, high resolution
collimator or a low-energy all-purpose collimator. Alternatively,
labeling with .sup.89Zr, .sup.76Br, .sup.18F or other
positron-emitting radionuclide may be used to image CD74-specific
antibody or antibody fragment distribution in tumors using positron
emission tomography (PET). The images obtained by the use of such
techniques may be used to assess biodistribution of CD74 in a
patient, mammal, or tissue, for example in the context of using
CD74 as a biomarker for the presence of cancer cells. Variations on
this technique may include the use of magnetic resonance imaging
(MRI) to improve imaging over gamma camera techniques. Conventional
immunoscintigraphy methods and principles are described in, e.g.,
Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging
And Therapy (Plenum Press 1988), Chase, "Medical Applications of
Radioisotopes," in Remington's Pharmaceutical Sciences, 18th
Edition, Gennaro et al., (eds.), pp. 624-652 (Mack Publishing Co.,
1990), and Brown, "Clinical Use of Monoclonal Antibodies," in
Biotechnology And Pharmacy 227-49, Pezzuto et al., (eds.) (Chapman
& Hall 1993). Moreover, such images may also, or alternatively,
serve as the basis for surgical techniques to remove tumors.
Furthermore, such in vivo imaging techniques may allow for the
identification and localization of a tumor in a situation where a
patient is identified as having a tumor (due to the presence of
other biomarkers, metastases, etc.), but the tumor cannot be
identified by traditional analytical techniques. All of these
methods are features of the present invention.
[0342] The in vivo imaging and other diagnostic methods provided by
the present invention are particularly useful in the detection of
micrometastases in a human patient (e.g., a patient not previously
diagnosed with cancer or a patient in a period of
recovery/remission from a cancer).
[0343] In one embodiment, the present invention provides an in vivo
imaging method wherein a CD74-specific antibody of the present
invention is conjugated to a detection-promoting radio-opaque
agent, the conjugated antibody is administered to a host, such as
by injection into the bloodstream, and the presence and location of
the labeled antibody in the host is assayed. Through this technique
and any other diagnostic method provided herein, the present
invention provides a method for screening for the presence of
disease-related cells in a human patient or a biological sample
taken from a human patient and/or for assessing the distribution of
CD74-specific antibody prior to CD74-specific ADC therapy.
[0344] For diagnostic imaging, radioisotopes may be bound to a
CD74-specific antibody either directly or indirectly by using an
intermediary functional group. Useful intermediary functional
groups include chelators, such as ethylenediaminetetraacetic acid
and diethylenetriaminepentaacetic acid (see for instance U.S. Pat.
No. 5,057,313).
[0345] In addition to radioisotopes and radio-opaque agents,
diagnostic methods may be performed using CD74-specific antibodies
that are conjugated to dyes (such as with the biotin-streptavidin
complex), contrast agents, fluorescent compounds or molecules and
enhancing agents (e.g. paramagnetic ions) for magnetic resonance
imaging (MRI) (see, e.g., U.S. Pat. No. 6,331,175, which describes
MRI techniques and the preparation of antibodies conjugated to a
MRI enhancing agent). Such diagnostic/detection agents may be
selected from agents for use in MRI, and fluorescent compounds. In
order to load a CD74-specific antibody with radioactive metals or
paramagnetic ions, it may be necessary to react it with a reagent
having a long tail to which a multiplicity of chelating groups are
attached for binding the ions. Such a tail may be a polymer such as
a polylysine, polysaccharide, or another derivatized or
derivatizable chain having pendant groups to which may be bound
chelating groups such as, e.g., porphyrins, polyamines, crown
ethers, bisthiosemicarbazones, polyoximes, and like groups known to
be useful for this purpose. Chelates may be coupled to
CD74-specific antibodies using standard chemistries.
[0346] Thus, the present invention provides a diagnostic
CD74-specific antibody, wherein the CD74-specific antibody is
conjugated to a contrast agent (such as for magnetic resonance
imaging, computed tomography, or ultrasound contrast-enhancing
agent) or a radionuclide that may be, for example, a gamma-, beta-,
alpha-, Auger electron-, or positron-emitting isotope.
[0347] In a further aspect, the invention relates to a kit for
detecting the presence of CD74 antigen or a cell expressing CD74,
in a sample, comprising: [0348] A CD74-specific antibody or ADC of
the invention; and [0349] Instructions for use of the kit.
[0350] In one embodiment, the present invention provides a kit for
diagnosis of cancer comprising a container comprising a
CD74-specific Ab, and one or more reagents for detecting binding of
the CD74-specific antibody to CD74. Reagents may include, for
example, fluorescent tags, enzymatic tags, or other detectable
tags. The reagents may also include secondary or tertiary
antibodies or reagents for enzymatic reactions, wherein the
enzymatic reactions produce a product that may be visualized. In
one embodiment, the present invention provides a diagnostic kit
comprising one or more CD74-specific Abs, of the present invention
in labeled or unlabeled form in suitable container(s), reagents for
the incubations for an indirect assay, and substrates or
derivatizing agents for detection in such an assay, depending on
the nature of the label. Control reagent(s) and instructions for
use also may be included.
[0351] Diagnostic kits may also be supplied for use with a
CD74-specific Ab, such as a conjugated/labeled CD74-specific Ab,
for the detection of the presence of CD74 in a tissue sample or
host. In such diagnostic kits, as well as in kits for therapeutic
uses described elsewhere herein, a CD74-specific antibody typically
may be provided in a lyophilized form in a container, either alone
or in conjunction with additional antibodies specific for a target
cell or peptide. Typically, a pharmaceutically acceptable carrier
(e.g., an inert diluent) and/or components thereof, such as a Tris,
phosphate, or carbonate buffer, stabilizers, preservatives,
biocides, inert proteins, e.g., serum albumin, or the like, also
are included (typically in a separate container for mixing) and
additional reagents (also typically in separate container(s)). In
certain kits, a secondary antibody capable of binding to the
CD74-specific Ab, which typically is present in a separate
container, is also included. The second antibody is typically
conjugated to a label and formulated in a manner similar to the
CD74-specific antibody of the present invention. Using the methods
described above and elsewhere herein, CD74-specific antibodies may
be used to define subsets of cancer/tumor cells and characterize
such cells and related tumor tissues.
Anti-Idiotypic Antibodies
[0352] In a further aspect, the invention relates to an
anti-idiotypic antibody which binds to a CD74-specific antibody of
the invention as described herein.
[0353] An anti-idiotypic (Id) antibody is an antibody which
recognizes unique determinants generally associated with the
antigen-binding site of an antibody. An anti-Id antibody may be
prepared by immunizing an animal of the same species and genetic
type as the source of a CD74-specific monoclonal antibody with the
monoclonal antibody to which an anti-Id is being prepared. The
immunized animal typically can recognize and respond to the
idiotypic determinants of the immunizing antibody by producing an
antibody to these idiotypic determinants (the anti-Id antibody).
Such antibodies are described in for instance U.S. Pat. No.
4,699,880. Such antibodies are further features of the present
invention.
[0354] An anti-Id antibody may also be used as an "immunogen" to
induce an immune response in yet another animal, producing a
so-called anti-anti-Id antibody. An anti-anti-Id antibody may be
epitopically identical to the original mAb, which induced the
anti-Id antibody. Thus, by using antibodies to the idiotypic
determinants of a mAb, it is possible to identify other clones
expressing antibodies of identical specificity. Anti-Id antibodies
may be varied (thereby producing anti-Id antibody variants) and/or
derivatized by any suitable technique, such as those described
elsewhere herein with respect to CD74-specific antibodies of the
present invention. For example, a monoclonal anti-Id antibody may
be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and
used to immunize BALB/c mice. Sera from these mice typically will
contain anti-anti-Id antibodies that have the binding properties
similar, if not identical, to an original/parent CD74-specific
antibody.
[0355] The present invention is further illustrated by the
following examples which should not be construed as further
limiting.
EXAMPLES
Example 1
Construction of CD74v1 and -v2, His-CD74v1 and -v2 and
CD74del2-36v1 and -v2 Expression Vectors
[0356] The encoding sequences for human CD74 variant 1 (CD74v1)
(identical to Genbank sequence NP_001020330) and human CD74 variant
2 (CD74v2) (identical to Genbank sequence AAV383110330) were made
synthetically and fully codon optimized (GeneArt, Regensburg,
Germany). The constructs were cloned in the mammalian expression
vector pEE13.4 (Lonza Biologics, Slough, UK). These constructs were
named pEE13.4CD74v1 and pEE13.4CD74v2. To enhance the expression
level of CD74 on the cell surface, the cytoplasmic ER retention
signal (aa2-36) was removed as described (Khalil H et al., J Cell
Sci 2005; 118: 4679-4687). To this end, new constructs were made by
amplifying the CD74-encoding regions from pEE13.4CD74v1 and
pEE13.4CD74v2, and removing the aa2-36 encoding regions in the
process. These PCR fragments were recloned in pEE13.4 and fully
sequenced to confirm the correctness of the new constructs. These
expression vectors were named pEE13.4CD74v1del2-36 and
pEE13.4CD74v2del2-36.
[0357] The encoding regions for the extracellular domains of CD74v1
(aa 73-296) and -v2 (aa 73-232) were amplified by PCR from
pEE13.4CD74v1 and pEE13.4CD74v2, in the process introducing the
encoding region for a hexameric N-terminal His tag. The PCR
fragments were cloned in mammalian expression vector pEE12.4 (Lonza
Biologics) containing the encoding region of an efficient signal
peptide (HMM38 [Barash S et al., Biochem Biophys Res Commun 2002;
294: 835-842). The expression vectors were fully sequenced and
named pEE12.4SPHisCD74v1 and pEE12.4SPHisCD74v2. The resulting
proteins were named HisCD74v1 and HisCD74v2.
[0358] The protein sequences of CD74 variants are shown in FIG.
1.
Example 2
Transient Expression in HEK-293F Cells and in CHO-S Cells
[0359] Freestyle.TM. 293-F (a HEK-293 subclone adapted to
suspension growth and chemically defined Freestyle medium;
HEK-293F) cells were obtained from Invitrogen and transfected with
pEE13.4CD74v1, pEE13.4CD74v2, pEE13.4CD74del2-36v1,
pEE13.4CD74del2-36v2, pEE12.4SPHisCD74v1 or pEE12.4SPHisCD74v2,
using 293fectin (Invitrogen) according to the manufacturer's
instructions.
[0360] Cell culture supernatants, in the case of pEE12.4SPHisCD74v1
or pEE12.4CD74v2, were harvested and soluble HisCD74v1 or HisCD74v2
was purified by metal affinity chromatography, as described
below.
[0361] In the case of pEE13.4CD74v1, pEE13.4CD74v2,
pEE13.4CD74v1del2-36 or pEE13.4CD74v2del2-36, cells were harvested
1-2 days post transfection and used in subsequent assays. These
cells were named TH2013-CD74v1, TH2013-CD74v2, TH2013-CD74v1del2-36
and TH2013-CD74v2del2-36.
[0362] A suspension-adapted CHO-K1SV cell line (CHO-S, Invitrogen)
was transfected with pEE13.4CD74v1, pEE13.4CD74v2,
pEE13.4CD74v1del2-36 or pEE13.4CD74v2del2-36, according to the
manufacturer's protocol using the CHO-Max reagent (Invitrogen).
Transfected CHO-S cells were harvested 1-2 days post transfection
and used in subsequent assays. These cells were named
TC2013-CD74v1, TC2013-CD74v2, TC2013-CD74v1del2-36 and
TC2013-CD74v2del2-36.
[0363] In the case of antibody expression, the appropriate heavy
chain and light chain vectors, as described in Example 9, were
co-expressed in HEK-293F cells as described supra.
Example 3
Stable Expression in NSO Cells
[0364] The pEE13.4CD74v1del2-36 plasmid was transfected in NSO
cells (Lonza Biologics). Cells were selected for stable integration
of the expression vector by culture in glutamine-free cell culture
medium in the presence of 25 .mu.M methylsulphoximine (MSX) as
described (Bebbington C R et al., Biotechnology (N Y) 1992;
10:169-175). Cells expressing CD74 were pooled and used as a
semi-stable population or individual stable clones were selected
and used. These cells were named N2013del-v1-012.
Example 4
PURIFICATION OF HIS-Tagged CD74
[0365] HisCD74v1 and HisCD74v2 were expressed in HEK-293F cells.
The His-tag in the proteins enables purification with immobilized
metal affinity chromatography. In this process, a chelator fixed
onto the chromatographic resin is charged with Co.sup.2+ cations.
CD74ECDHis-containing supernatant is incubated with the resin in
batch mode (i.e. solution). His-tagged protein binds strongly to
the resin beads, while other proteins present in the culture
supernatant do not bind strongly. After incubation, the beads are
retrieved from the supernatant and packed into a column. The column
is washed in order to remove weakly bound proteins. The strongly
bound CD74ECDHis proteins are then eluted with a buffer containing
imidazole, which competes with the binding of His to Co.sup.2+. The
eluent is removed from the protein by buffer exchange on a
desalting column.
Example 5
Immunization Procedure of Transgenic Mice
[0366] Antibodies HuMab-CD74-005, -006, -008 and -011 were derived
from the immunizations of HCo17 HuMAb mice (human monoclonal
antibody; Medarex Inc., San Jose, Calif., USA) which have four
genetic modifications. These mice were made transgenic for the
human Ig heavy and human Ig kappa light chain and double knock out
for the mouse heavy and mouse kappa light chain loci. These
disruptions prevent the expression of any antibodies that are
completely murine. Different strains were used; HCo12,
HCo12-BALB/c, HCo17 and HCo20. These differ in the number of human
VH (variable region of heavy chain) and VL (variable region of
light chain) genes. HCo12-BALB/c mice were derived by crossbreeding
with KCo5-BALB/c (kappa light chain transgenic) mice.
[0367] Six different immunogens were used for the immunizations:
TH2013-CD74v1del2-36, TH2013-CD74v2del2-36, N2013del-v1-012,
SU-DHL-4 cells (Human B cell lymphoma cell line) and HisCD74v1 or
HisCD74v2 coupled to the carrier protein KLH (Keyhole Limpet
Hemocyanin). Mice were immunized every fortnight, alternating with
5.times.10.sup.6 cells or with 15 .mu.g of protein. Eight
immunizations were performed in total, four intraperitoneal (IP)
and four subcutaneous (SC).
[0368] Antibodies -005, -006 and -008 were obtained from
immunization of an HCo17 mouse with 5.times.10.sup.6
TH2013-CD74v1del2-36 cells IP, alternated with 15 .mu.g HisCD74v2
SC. The first immunization was performed IP, with cells in complete
Freund's adjuvant (CFA; Difco Laboratories, Detroit, Mich., USA),
the following immunizations in incomplete Freund's adjuvant (IFA)
(protein, SC) or in PBS (cells, IP).
[0369] Antibody -011 was obtained from the immunization of an HCo17
mouse with 5.times.10.sup.6 TH2013-CD74v1del2-36 cells IP,
alternated with 15 .mu.g HisCD74v1 SC. The first immunization was
performed with protein in CFA (IP), the following immunizations in
IFA (protein, SC) or PBS (cells, IP).
[0370] When serum titers were found to be sufficient (dilution of
serum of 1/50 or lower found positive in antigen-specific screening
assay as described in Example 6 on at least two sequential,
biweekly, screening events), mice were additionally boosted twice
intravenously (IV) with 10 .mu.g HisCD74 protein in 100 .mu.L PBS,
four and three days before fusion.
Example 6
Homogeneous Antigen-Specific Screening Assay
[0371] Mouse sera and hybridoma supernatants were analyzed in a
high throughput screening (HTS) Fluorometric Micro Volume Assay
Technology (FMAT assay; Applied Biosystems, Foster City, Calif.,
USA) for the presence of anti-CD74 antibodies. In this assay,
TC2013-CD74v1del2-36 and TC2013-CD74v2del2-36 cells were used to
detect human anti-CD74 antibodies. Wild type CHO-S cells were used
to measure non-specific binding. Samples were added to the cells to
allow binding to CD74. Subsequently, binding of HuMab was detected
using a fluorescent conjugate (Goat anti-human IgG-Cy5; Jackson
Immunoresearch). Mouse anti-human CD74 antibody (Becton Dickinson;
IgG2a, .kappa.; clone M-B741), labeled with Alexa-647, as described
below, was used as a positive control and mouse-chrompure (Jackson
Immunoresearch) labeled with Alexa-647 was used as a negative
control.
[0372] Antibodies were labeled with Alexa Fluor.RTM. 647 Dye
(Molecular Probes), hereinafter "Alexa-647", using the following
procedure:
[0373] An antibody solution of 1 mg/mL IgG was prepared in 0.1 M
sodiumcarbonate buffer pH 9.0 (NaHCO.sub.3, Riedel de Haen, cat.
no. 31437;). Alexa-647 was prepared freshly, by adding 100 .mu.L
DMSO (Sigma, cat. no. D2438) and 900 .mu.L 0.1 M sodiumcarbonate
buffer pH 9.0 to one vial (Alexa Fluor.RTM. 647 carboxylic acid,
succinimidyl ester (1 mg/vial), Molecular Probes, Leiden, The
Netherlands, cat. no. A-20006). A five-times molar excess of
Alexa-647, calculated as indicated below, was added to the IgG
solution and incubated, while rotating, in the dark at RT for 1
hour. After labeling, unbound Alexa-647 was removed, using a PD-10
column (Amersham Biosciences, cat. no. 17-0851-01), with Tris
buffer pH 8.0 (50 mM Tris [Trizma base, Sigma, cat. no. T-6066];
100 mM NaCl [Riedel de Haen, cat. no. 31437]; 0.01% sodium azide
[NaN.sub.3, Riedel de Haen, cat. no. 13412]). The amount of
Alexa-647 to be added to the IgG solution was calculated using the
formula:
Volume Alexa-647 to be added(in .mu.L)=(IgG conc(mg/mL)/MW
IgG(Da)*ratio*volume*MW Alexa-647*1,000.
[0374] MW IgG=150,000 Da; ratio is the molar excess of Alexa-647 to
be used; volume is the volume of the sample to be labeled (in mL);
MW Alexa-647=1250 Da.
[0375] Protein concentration (IgG) and degree of labeling (D.O.L.)
were determined by measuring OD 280 nm and 650 nm on an Ultrospec
2100 Pro (Amersham Biosciences). IgG concentration (mg/mL) was
calculated using the formula:
IgG concentration=[A.sub.280-(0.03*A.sub.650)]/IgG extinction
coefficient.
[0376] D.O.L. was calculated using the formula:
D.O.L.=A.sub.650/239,000/[A.sub.280-(0.03*A.sub.650)/(IgG
extinction coefficient*MW IgG)].
239,000 is the extinction coefficient of Alexa-647 at
.lamda..sub.max in cm.sup.-1M.sup.-1; 0.03 is the correction factor
(A.sub.280 free dye/A.sub.max free dye) (both provided by the
manufacturer). Bovine serum albumin (BSA; Sigma, cat. no. A 2934)
was added from a 10% (w/v) solution to a final concentration of
0.1% (w/v) and labeled antibodies were stored at 5.degree. C. In a
few fusion screens, in addition to the anti-human IgG-Cy5.5, to
detect human antibodies, an anti-mouse IgG Cy5.5-labeled conjugate
was used, to detect specific chimeric antibodies. Samples were
scanned using an Applied Biosystems 8200 Cellular Detection System
(8200 CDS) and `counts.times.fluorescence` was used as
read-out.
Example 7
HuMab Hybridoma-Generation
[0377] HuMAb mice with sufficient (defined as above)
antigen-specific titer development were sacrificed and the spleen
and lymph nodes flanking the abdominal aorta and caval vein were
collected. Fusion of splenocytes and lymph node cells to a mouse
myeloma cell line was done by electrofusion using a CEEF 50
Electrofusion System (Cyto Pulse Sciences, Glen Burnie, Md., USA),
essentially according to the manufacturer's instructions. Fused
cells were seeded fusion medium containing 10% Fetal Clone I Bovine
serum (Perbio), 1 mM sodium pyruvate (Cambrex), 0.5 U/mL
penicillin, 0.5 U/mL streptomycin (Cambrex), 50 .mu.M
2-mercaptoethanol (Invitrogen), 600 ng/mL interleukin 6 (IL-6)
(Strathmann), 1.times.HAT (Sigma) and 0.5 mg/mL kanamycin
(Invitrogen) in HyQ mADCF-Mab (Perbio). After ten days, supernatant
was harvested and cells were refreshed with harvest medium,
containing 10% Fetal Clone I Bovine serum, 0.5 U/mL penicillin, 0.5
U/mL streptomycin, 600 ng/mL IL-6 and 1.times.proHT (Cambrex) in
HyQ mADCF-Mab. Supernatants of the hybridoma cultures were screened
by primary FMAT screening assays on TC2013-CD74v1del2-36 cells and
TC2013-CD74v2del2-36 cells to detect hybridomas producing human (or
chimeric) anti-CD74 antibodies as described supra. The 60 best
primary wells were seeded in semisolid medium made from 40%
CloneMedia (Genetix, Hampshire, UK) and 60% HyQ 2.times. complete
medium (Hyclone, Waltham, USA). From each primary well, two wells
of a Genetix black 6-well plate were seeded. From each well, 33 sub
clones were picked, using the ClonePix system (Genetix). The sub
clones were picked in harvest medium. After seven days, the
supernatants of the sub clones were screened again for
CD74-specific human IgG binding and human IgG concentration was
measured using Octet (Fortebio, Menlo Park, USA). From each primary
well, the best sub clone was expanded in expansion medium
containing only 600 ng/mL IL-6, 0.5 U/mL penicillin, 0.5 U/mL
streptomycin and 1.times.proHT. The sub clones were expanded from
one 96-well plate well to one 24-well plate well to four 24-well
plate wells to six 6-well plate wells to Hyperflask (small scale
production). Supernatants of the hyperflasks were screened for
CD74-specific human IgG binding. Clones derived by this process
were designated PC2013.
Example 8
Mass-Spectrometry of Purified Antibodies
[0378] Small aliquots of 0.8 mL antibody-containing supernatant
from 6-well or Hyperflask stage were purified using PhyTip columns
containing Protein G resin (PhyNexus Inc., San Jose, USA) on a
Sciclone ALH 3000 workstation (Caliper Lifesciences, Hopkinton,
USA). The PhyTip columns were used according to manufacturer's
instructions, but buffers were replaced. PBS (B.Braun, Medical
B.V., Oss, Netherlands) was used as Binding Buffer and 0.1M
Glycine-HCl pH 2.7 (Fluka Riedel-de Haen, Buchs, Germany) was used
as Elution Buffer. After purification, samples were neutralized
with 2M Tris-HCl pH 9.0 (Sigma-Aldrich, Zwijndrecht, Netherlands).
Alternatively, in some cases larger volumes of culture supernatant
were purified using Protein A affinity column chromatography.
[0379] After purification, samples were placed in a 384-well plate
(Waters, 100 uL square-well plate, art#186002631). Samples were
de-glycosylated with N-glycosidase F (Roche cat no 11365177001) at
37.degree. C., 0/N. DTT (15 mg/mL) was added (1 .mu.L/well) and
incubated at 37.degree. C. for 1 h. Samples (5 or 6 .mu.L) were
desalted on an Acquity UPLC.TM. (Waters, Milford, USA) with a
BEH300 C18, 1.7 .mu.m, 2.1.times.50 mm column at 60.degree. C. MQ
water and LC-MS grade acetonitrile (Biosolve, cat no 01204101,
Valkenswaard, The Netherlands), both with 0.1% formic acid (Fluka,
cat no 56302, Buchs, Germany), were used as Eluens A and B.
Time-of-flight electrospray ionization mass spectra were recorded
on-line on a micrOTOF.TM. mass spectrometer (Bruker, Bremen,
Germany) operating in the positive ion mode. Prior to analysis, a
900-3000 m/z scale was calibrated with ES tuning mix (Agilent
Technologies, Santa Clara, USA). Mass spectra were deconvoluted
with DataAnalysis.TM. software v3.4 (Bruker) using the Maximal
Entropy algorithm searching for molecular weights between 5 and 80
kDa.
[0380] After deconvolution the resulting heavy and light chain
masses for all samples were compared in order to find duplicate
antibodies. In the comparison of the heavy chains the possible
presence of C-terminal lysine variants was taken into account. This
resulted in a list of unique antibodies, where unique is defined as
a unique combination of heavy and light chains. In case duplicate
antibodies were found, results from other tests were used to decide
which material was used to continue experiments with.
[0381] Mass spectrometry analysis of the molecular weights of heavy
and light chains of 41 anti-CD74 hybridomas yielded 18 unique
antibodies (unique heavy chain/light chain combination).
Example 9
Sequence Analysis of the CD74-Specific HuMab Variable Domains and
Cloning in Expression Vectors
[0382] Total RNA of the anti-CD74 HuMab antibodies was prepared
from 5.times.10.sup.6 hybridoma cells and 5'-RACE-Complementary DNA
(cDNA) was prepared from 100 ng total RNA, using the SMART RACE
cDNA Amplification kit (Clontech), according to the manufacturer's
instructions. V.sub.H (variable region of heavy chain) and V.sub.L
(variable region of light chain) coding regions were amplified by
PCR. Amplified PCR products of antibodies 006, 008 and 011 were
cloned into the pCR-Blunt II-TOPO vector (Invitrogen) using the
Zero Blunt PCR cloning kit (Invitrogen).
[0383] Amplified V.sub.H and V.sub.L PCR products of antibody 005
were cloned in pcDNA3.3 vectors (Invitrogen) encoding G1f and.
Kappa constant domains. For each HuMab, 16 V.sub.L clones and 8
V.sub.H clones were sequenced. Clones with predicted heavy and
light chain mass in agreement with the mass of the
hybridoma-derived material of the same antibody (as determined by
mass spectrometry) were selected for further study and expression.
The resulting sequences are shown in the Sequence Listing and FIG.
2 herein.
[0384] Table 1 and Table 2 (below) give an overview of antibody
sequence information and most homologous germline sequences.
TABLE-US-00004 TABLE 1 Heavy chain homologies V-GENE V-REGION
J-GENE D-GENE CDR-IMGT Ab and allele identity, % (nt) and allele
and allele lengths 005 IGHV3-30-3*01 100.0 (288/288 nt) IGHJ4*02
IGHD3-10*01 8.8.17 006 IGHV3-30-3*01 98.6 (284/288 nt) IGHJ4*02
IGHD3-16*02 8.8.17 008 IGHV3-30-3*01 100.0 (288/288 nt) IGHJ4*02
IGHD3-16*02 8.8.17 011 IGHV3-33*01 99.7 (287/288 nt) IGHJ6*02
IGHD3-10*01 8.8.16
TABLE-US-00005 TABLE 2 Light chain homologies V-GENE V-REGION
J-GENE CDR-IMGT Ab and allele identity % (nt) and allele lengths
005 IGKV1D-16*01 99.6 (278/279 nt) IGKJ4*01 6.3.9 006 IGKV1D-16*01
100.0 (279/279 nt) IGKJ4*01 6.3.9 008 IGKV1D-16*01 100.0 (279/279
nt) IGKJ4*01 6.3.9 011 IGKV1D-16*01 100.0 (279/279 nt) IGKJ4*01
6.3.9
TABLE-US-00006 TABLE 3 References to the sequence listing:
VH-region SEQ ID No: 7 VH 005 SEQ ID No: 8 VH 005, CDR1 SEQ ID No:
9 VH 005, CDR2 SEQ ID No: 10 VH 005, CDR3 SEQ ID No: 11 VH 006 SEQ
ID No: 12 VH 006, CDR1 SEQ ID No: 13 VH 006, CDR2 SEQ ID No: 14 VH
006, CDR3 SEQ ID No: 15 VH 008 SEQ ID No: 16 VH 008, CDR1 SEQ ID
No: 17 VH 008, CDR2 SEQ ID No: 18 VH 008, CDR3 SEQ ID No: 19 VH 011
SEQ ID No: 20 VH 011, CDR1 SEQ ID No: 21 VH 011, CDR2 SEQ ID No: 22
VH 011, CDR3 VL-region SEQ ID No: 23 VL 005 SEQ ID No: 24 VL 005,
CDR1 (=VL 011, VL 006, and VL 008 CDR1) AAS VL 005, CDR2 (=VL 011,
VL 006, and VL 008 CDR2) SEQ ID No: 25 VL 005, CDR3 (=VL 011, VL
006, and VL 008 CDR3) SEQ ID No: 26 VL 006 = VL 008 = VL 011
Example 10
Purification of Antibodies
[0385] Culture supernatant was filtered over 0.2 .mu.m dead-end
filters, loaded on 5 mL Protein A columns (rProtein A FF, Amersham
Bioscience) and eluted with 0.1 M citric acid-NaOH, pH 3. The
eluate was immediately neutralized with 2M Tris-HCl, pH 9 and
dialyzed to 12.6 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4
(B.Braun), 0/N. After dialysis, samples were sterile-filtered over
0.2 .mu.m dead-end filters. Purity was determined by SDS-PAGE and
concentration was measured by nephelometry and absorbance at 280
nm. Purified antibodies were aliquoted and stored at -80.degree. C.
Once thawed, purified antibody aliquots were kept at 4.degree. C.
Mass spectrometry was performed to identify the molecular mass of
the antibody heavy and light chains expressed by the hybridomas as
described in Example 9.
Example 11
Binding of CD74-Specific HuMab Antibodies to Recombinant
Extracellular Domain of Two CD74 Isoforms, Determined by ELISA, and
to Cellular CD74 on Raji Cells, Determined by FACS
[0386] Binding of anti-CD74 HuMab antibodies to two isoforms of
CD74 was measured by ELISA (coated recombinant extracellular domain
of CD74) and to cellular CD74 on Raji cells (ATCC, Manassas, Va.)
by FACS analysis.
[0387] ELISA plates (Greiner BioOne) were coated 0/N at 4.degree.
C. with 2 .mu.g/mL, 100 .mu.L per well, recombinant CD74v1 or
CD74v2 in PBS (B. Braun Melsungen AG). Sequences and production of
the isoforms were described supra. ELISA wells were washed three
times with PBS containing 0.05% Tween-20 (PBST), emptied, and
blocked with 1% (w/v) BSA fraction V (Roche) in PBS at RT for 1 h
while shaking (300 rpm), and emptied. Subsequently, 100 .mu.L
anti-CD74 HuMab antibodies were added in serial dilutions in 0.2%
(w/v) BSA fraction V in PBST (assay buffer) and incubated while
shaking at RT for 90 min. ELISA plates were washed three times with
PBST, emptied, and bound HuMab antibodies were detected using
HRP-conjugated goat-anti human IgG (100 .mu.L; 1:5,000; Peroxidase
Affinipure Goat anti-human IgG, F(ab')2 Fragment Specific [min X
Bov,Hrs,Ms Sr Prot]; Jackson Immunoresearch) in assay buffer and
incubated while shaking at RT for 90 min. Plates were washed three
times with PBST, emptied, and incubated with 100 .mu.L ABTS
solution (50 ml ABTS buffer [Roche] and one ABTS tablet [50 mg;
Roche]). After incubation in the dark at RT for 30 min, the
reaction was stopped by addition of 100 .mu.L per well oxalic acid
(2% [w/v]). Plates were measured at OD 405 nm in an ELISA reader
(Biotek Instruments, EL808 Absorbance Microplate Reader).
[0388] For FACS analysis, 10.sup.5 cells in 100 .mu.L FACS buffer
(PBS supplemented with 0.1% BSA and 0.02% sodium azide) were seeded
per well in 96-well round-bottom plates. Cells were spun down (1200
rpm, 4.degree. C., 5 min) and supernatant was discarded. Serially
diluted anti-CD74 HuMab antibodies were added (100 .mu.L) and
incubated on ice for 1 h. Cells were washed with FACS buffer,
supernatants were discarded, and 100 .mu.L of R-Phycoerythrin
labeled goat anti-human IgG (R-Phycoerythrin AffiniPure
F(ab').sub.2 Frag Gt Anti-Human IgG, Fc.gamma. Frag Spec [min X
Bov,Hrs,Ms Sr Prot]; Jackson Immunoresearch), diluted 1:100 in FACS
buffer, was added. After 1 h on ice (in the dark), cells were
washed once in FACS buffer, supernatant was discarded, and specific
binding of the HuMab antibodies was detected by flow cytometry on a
FACS Canto II (BD Biosciences).
[0389] Isotype control Ab IgG1-b12 was used as a negative control.
Binding curves were analyzed using non-linear regression (sigmoidal
dose-response with variable slope) using GraphPad Prism 5 software
(GraphPad Software, San Diego, Calif., USA).
[0390] FIG. 3 shows that HuMab-CD74-006 and -011 bound with high
affinity (EC.sub.50 values between 210 and 344 ng/mL) to both
isoforms of the CD74 extracellular domain. HuMab-CD74-008 bound
with intermediate affinity (EC.sub.50 between 759 and 1391 ng/mL)
to both isoforms.
[0391] FIG. 4 shows that HuMab-CD74-006 and -011 also bound with
high affinity (EC.sub.50 between 150 and 200 ng/mL) to cellular
CD74 expressed by Raji cells. HuMab-CD74-008 and -005 bound to
cellular CD74 with intermediate affinity (EC.sub.50 values could
not be determined because maximum binding was not reached).
[0392] Table 4 shows EC.sub.50 values of CD74-specific HuMab
antibodies for binding to the extracellular domain of CD74v1 and
CD74v2 by ELISA and to cellular CD74 by FACS on Raji cells.
TABLE-US-00007 TABLE 4 Overview of EC.sub.50 values for binding of
CD74 specific HuMab antibodies to the extracellular domain of
CD74v1 and CD74v2, determined by ELISA, and to cellular CD74 on
Raji cells, determined by FACS. All HuMab antibodies were produced
by transiently co-transfecting HEK293F cells with relevant heavy
and light chain expression vectors (as described supra). EC.sub.50
(ELISA) HuMab-CD74- CD74v1 CD74v2 EC.sub.50 (FACS) 005 nt nt nd 006
321 210 196 008 1391 759 nd 011 344 245 151 EC.sub.50 values are in
ng/mL. nd--could not be calculated. nt--not tested.
Example 12
Cross-Reactivity of Anti-CD74 HuMab Antibodies to Cynomolgus Monkey
Tissues
[0393] The capacity of CD74-specific HuMab antibodies to bind to
cynomolgus CD74 was tested by immunohistochemistry.
Immunohistochemistry with anti-CD74 HuMab antibodies was performed
on frozen human tonsil and cynomolgus lymph node tissue, with
anticipated CD74 expression on (follicular) B lymphocytes and
macrophages. Frozen tissue sections were cut (4-6 .mu.m thickness)
and fixated in acetone. HuMab antibodies were complexed with
fluorescein-isothiocyanate (FITC) by incubation with goat
anti-human IgG(Fc)-FITC (Fab) (Protos) (1:1 ratio with Humab).
Prior to HuMab staining (1 .mu.g/mL), tissues were blocked for
endogenous biotin, peroxidase (PO) and immunoglobulins.
HuMab-Fab-FITC complex was detected by subsequent incubations with
rabbit anti-FITC (Invitrogen) (diluted 1:1000) and PO-conjugated
goat anti-rabbit IgG (Powervision, [rb IgG]-PO; undiluted). PO
activity was visualized with amino-ethyl-carbazole (AEC) as
substrate, resulting in a red color, and nuclei were visualized
with hematoxylin (blue). Tissue-stainings were examined with light
microscopy (Axioskop-2 plus), converted to digital pictures by an
Axiocam-camera and stored as digital pictures.
[0394] FIG. 5 shows that HuMab-CD74-006 and -011 showed
cross-reactivity with cynomolgus CD74, as shown by staining of
macrophages and follicular B cells (staining for isotype control is
negative). The extent of cross-reactivity with cynomolgus CD74 was
less for HuMab-CD74-006 than for -011, as shown by less intensive
staining of cynomolgus tissue as compared with human tissue.
Example 13
Induction of ADCC and CDC
[0395] Induction of ADCC by CD74-specific HuMab antibodies was
tested in a .sup.51Cr release assay. Briefly, Raji cells were
labeled with 100 .mu.Ci .sup.51Cr and used as target cells.
Peripheral blood mononuclear cells, isolated from buffy coats, were
used as effector cells. Target cells were pre-incubated with
anti-CD74 HuMab antibodies (RT, 30 min) and effector cells were
added, resulting in an effector to target ratio of 100:1, and
incubated at 37.degree. C., 5% CO.sub.2. O/N. .sup.51Cr release in
the supernatant was measured in a gamma counter. No significant
induction of ADCC by anti-CD74 HuMab antibodies was detected.
[0396] Induction of CDC by anti-CD74 HuMab antibodies was tested
using propidium iodide method. Briefly, Raji cells were
pre-incubated with anti-CD74 HuMab antibodies (RT, 15 min) and
normal human serum was added, to a final concentration of 20%, and
incubated at 37.degree. C., 5% CO.sub.2 for 45 min. Plates were put
on ice to stop the reaction. Propidium iodide was added and cells
were analyzed by FACS analysis. No significant induction of CDC by
anti-CD74 HuMab antibodies was detected.
Example 14
Antibody-Mediated Internalization and Cell Killing by Anti-CD74
HuMab Antibodies in an Anti-Kappa-ETA' Assay
[0397] To evaluate the suitability of the anti-CD74 HuMab
antibodies for an antibody-drug conjugate approach, a generic in
vitro cell-based killing assay using kappa-directed
pseudomonas-exotoxin A (anti-kappa-ETA') was developed. In this
assay, a construct consisting of a high affinity anti-human kappa
light chain domain antibody and a truncated form of the
pseudomonas-exotoxin A plus a KDEL retention motif was used. Upon
internalization, the anti-kappa-domain-antibody-ETA' construct
undergoes proteolysis and disulfide-bond reduction, separating the
catalytic and the binding domain. The catalytic domain is believed
to be transported from the Golgi system to the endoplasmic
reticulum via the KDEL retention motif, and subsequently
translocated to the cytosol where it inhibits protein synthesis and
induces apoptosis (Kreitman R J. BioDrugs 2009; 23(1): 1-13).
[0398] Antibody-mediated internalization and cell killing by the
toxin was tested for different anti-CD74 HuMab antibodies with Raji
cells. The number of CD74 molecules expressed on the Raji cell
surface was estimated to be 10.sup.4 molecules per cell, using
QIFIKIT.RTM. method (Dako, Glostrup, Denmark). 10.sup.4 cells per
well in cell culture medium were seeded in 96-well tissue culture
plates (Greiner Bio-one). Plates were incubated at 37.degree. C.
for 1 h, to let cells settle down. To identify anti-CD74 HuMab
antibodies that enable internalization of and killing by the toxin,
a fixed concentration (1 .mu.g/mL final concentration in the wells)
of anti-kappa-ETA', that did not induce non-specific cell death in
the absence of antibody, was pre-incubated for 30 min with a
titrated amount of anti-CD74 HuMab antibodies before addition to
the cells. After three days, the amount of viable cells was
quantified with AlamarBlue (BioSource International, San Francisco,
US), added in 10 .mu.L per well according to the manufacturer's
instructions. After incubation at 37.degree. C. for 4 h,
fluorescence was monitored using the EnVision 2101 Multilabel
reader (PerkinElmer, Turku, Finland) with standard AlamarBlue
settings. An isotype control antibody (IgG1-b12), pre-incubated
with anti-kappa-ETA', was used as a negative control. Staurosporine
(Sigma-Aldrich) was used as a control for determining background
signal and added to cells at a final concentration of 1
.mu.g/mL.
[0399] Percentage viability was calculated as follows:
(FL.sub.treated-FL.sub.background)(FL.sub.control-FL.sub.background).tim-
es.100%. [0400] FL.sub.control=fluorescence from untreated wells
[0401] FL.sub.background=fluorescence from staurosporine-treated
wells.
[0402] FIG. 6 and Table 5 show that all
anti-kappa-ETA'-pre-incubated anti-CD74 HuMab antibodies were able
to kill Raji cells in a dose-dependent manner. Anti-kappa-ETA'
pre-incubated HuMab-CD74-006, -011, -005 and -008 induced efficient
killing (EC.sub.50 between 25 and 250 .mu.g/mL and minimal
percentage viability left between 0 and 15). Anti-kappa-ETA'
pre-incubated control mAb IgG1-b12 did not induce cell killing.
TABLE-US-00008 TABLE 5 Overview of EC.sub.50 values and percentages
of cell viability left after treatment of Raji cells with
anti-kappa-ETA'-pre-incubated anti-CD74 HuMab antibodies. Data
shown are EC.sub.50 values (in .mu.g/mL) and minimal percentages
viability of Raji cells treated with anti-kappa-ETA' pre-incubated
anti-CD74 HuMab antibodies, measured in one representative
experiment. Antibody (HuMab-CD74-) % viability EC.sub.50 005 3.24
120 006 1.50 57 008 14.65 247 011 0.47 25 IgG1-b12 85.89
n.d..sup.a) .sup.a)Could not be calculated.
Example 15
Preparation of CD74-Specific ADCs
[0403] HuMab-CD74-005, HuMab-CD74-006, HuMab-CD74-011 and the
negative control IgG1-b12 were produced transiently in HEK-293F
cells. The antibodies were purified by Protein A chromatography
according to standard procedures, finally yielding approximately
263 mg purified HuMab-CD74-005, 165 mg HuMab-CD74-006 and 720 mg
HuMab-CD74-011. The amount of conjugated antibody obtained is shown
in Table 6. The drug-linker vcMMAE or mcMMAF was alkylated to the
cysteines of the reduced antibodies according to procedures
described in literature (Sun et al. (2005) Bioconjugate Chem. 16:
1282-1290; McDonagh et al., (2006) Protein Eng. Design Sel. 19:
299-307; Alley et al., (2008) Bioconjugate Chem. 19: 759-765). The
reaction was quenched by the addition of an excess of
N-acetylcysteine. Any residual unconjugated drug was removed by
diafiltration and the final CD74-specific antibody drug conjugates
were formulated in PBS.
[0404] The CD74-specific antibody drug conjugates were subsequently
analyzed for concentration (by absorbance at 280 nm), the
drug-to-antibody ratio (`DAR`) by reverse phase chromatography
(RP-HPLC) and hydrophobic interaction chromatography (HIC), the
amount of unconjugated drug (by reverse phase chromatography), the
percentage aggregation (by size-exclusion chromatography, SEC-HPLC)
and the endotoxin levels (by Limulus Amebocyte Lysate (LAL)
endotoxin test). The results are shown in Table 7.
TABLE-US-00009 TABLE 6 Amount of ADC obtained HuMab-CD74-
Linker-drug Amount of ADC (mg) 005 vcMMAE 94 005 mcMMAF 91 006
vcMMAE 63 006 mcMMAF 60 011 vcMMAE 276 011 mcMMAF 293 b12 vcMMAE
174 b12 mcMMAF 245
TABLE-US-00010 TABLE 7 Analysis of antibody-drug conjugates
HuMab-CD74- HuMab- 005 CD74-006 HuMab-CD74-011 IgG1-b12 Assay
vcMMAE mcMMAF vcMMAE mcMMAF vcMMAE mcMMAF vcMMAE mcMMAF
Concentration 7.2 6.4 6.4 6.2 8.2 8.1 6.6 9.1 (mg/mL) DAR by RP-
3.9 3.9 4.0 3.7 3.8 --* 3.2 3.9 HPLC DAR by HIC 4.0 4.0 4.0 4.1 3.9
3.9 3.3 4.0 % unconjugated <0.5 <0.5 <0.5 <0.5 <0.5
<0.5 <0.5 <0.5 drug % aggregate 1.3 1.2 0.7 0.3 0.7 0.3
0.8 1.0 by SEC-HPLC Endotoxin 0.199 0.152 0.101 0.085 0.200 0.083
0.078 0.104 (EU/mg) *DAR could not be assigned because of
co-elution of peaks
Example 16
Binding of Anti-CD74 ADCs to Recombinant Extracellular Domain of
CD74v1, Determined by ELISA
[0405] Binding of CD74-specific ADCs to CD74 was measured by ELISA
(coated recombinant extracellular domain of CD74v1) and compared
with binding of unconjugated CD74-specific HuMab antibodies.
[0406] ELISA plates (Greiner BioOne) were coated with 2 .mu.g/mL,
100 .mu.L per well, recombinant CD74ECDHis in PBS (B. Braun
Melsungen AG) at 4.degree. C., 0/N. ELISA plates were emptied and
blocked with 200 .mu.L/well PBS containing 0.05% Tween-20 (PBST)
while shaking (300 rpm), at RT for 1 hour, washed three times with
300 .mu.L PBST and emptied. Subsequently, 100 .mu.L anti-CD74 ADCs
or unconjugated CD74-specific HuMab antibodies were added in serial
dilutions in PBST and incubated while shaking at RT for 2 hours.
ELISA plates were washed with PBST and emptied. Bound anti-CD74
ADCs and unconjugated HuMab antibodies were detected by addition of
HRP-conjugated mouse-anti human IgG1 (100 .mu.L; 0.015 .mu.g/mL;
Sanquin; # M1328) in PBST and incubation while shaking, at RT for 2
hours. Plates were washed with PBST, emptied and incubated with 100
.mu.L ABTS solution (50 ml ABTS buffer [Roche] and one ABTS tablet
[50 mg; Roche]). After incubation in the dark while shaking, at RT
for 30 min, the reaction was stopped by incubation with 100 .mu.L
per well oxalic acid (2% [w/v]; Riedel de Haen) in the dark while
shaking, for 10 min. Plates were measured at OD 405 nm in an ELISA
reader (Biotek Instruments, EL808 Absorbance Microplate
Reader).
[0407] IgG1-b12, an antibody binding to a non-related antigen, was
used as a negative control (both unconjugated as well as in ADC
format). Binding curves were analyzed by non-linear regression
(sigmoidal dose-response with variable slope) using GraphPad Prism
5 software (GraphPad Software, San Diego, Calif., USA).
[0408] All anti-CD74 HuMab antibodies and ADCs bound within a
similar range to the CD74v1 extracellular domain in an ELISA
(EC.sub.50 values between 0.02 and 0.04 .mu.g/mL), as demonstrated
by the binding curves in FIG. 7. Table 8 shows EC.sub.50 values of
CD74-specific HuMab antibodies and ADCs for binding to the
extracellular domain of CD74.
TABLE-US-00011 TABLE 8 Overview of EC.sub.50 values for binding of
CD74-specific HuMab antibodies and ADCs to the extracellular domain
of CD74v1, determined by ELISA. EC.sub.50 values are in .mu.g/mL.
Data shown are mean EC.sub.50 values calculated from four
independent experiments. EC.sub.50 (ELISA) HuMab-CD74- Unconjugated
vcMMAE mcMMAF 005 0.03 0.04 0.04 006 0.02 0.03 0.02 011 0.02 0.03
0.02
Example 17
Binding of CD74-Specific ADCs to Surface-Expressed CD74, Determined
by FACS Analysis on Daudi Cells
[0409] Binding of anti-CD74 ADCs to surface-expressed CD74 was
measured by FACS analysis on Daudi cells and compared with binding
of unconjugated anti-CD74 HuMab antibodies.
[0410] 1.times.10.sup.5 Daudi cells in 100 .mu.L PBS containing
0.1% bovine serum albumin (BSA) (Roche, cat. no. 10735086001) and
0.02% sodium azide (Sigma-Aldrich, cat. no. 13412) (FACS buffer)
were seeded per well in 96-well round-bottom plates (Greiner
bio-one, cat. no. 650101). Cells were spun down (1200 rpm,
4.degree. C., 3 min) and supernatant was discarded. Serially
diluted anti-CD74 HuMab antibodies or ADCs were added (100 .mu.L)
and incubated on ice for 30 min. Cells were washed twice with 150
.mu.L FACS buffer and 100 .mu.L rabbit anti-human IgG-FITC (cat.
nr. F0185, Dako), diluted 1:100 in FACS buffer, was added. After 30
min on ice (in the dark), cells were washed twice in 150 .mu.L FACS
buffer and specific binding of the HuMab antibodies and ADCs was
detected by flow cytometry on a FACS Canto II (BD Biosciences).
[0411] Isotype control antibody IgG1-b12, an antibody binding to a
non-related antigen, was used as a negative control (both
unconjugated and as an ADC). Binding curves were analyzed using
non-linear regression (sigmoidal dose-response with variable slope)
using GraphPad Prism 5 software (GraphPad Software, San Diego,
Calif., USA).
[0412] FIG. 8 shows binding curves and Tables 9 and 10 show
EC.sub.50 values and maximal mean fluorescence intensities for
binding to surface-expressed CD74 of anti-CD74 HuMab antibodies and
ADCs. All but one conjugated anti-CD74 HuMab antibodies bound to
surface-expressed CD74 on Daudi cells with an affinity similar to
the corresponding unconjugated HuMab antibodies. The
vcMMAE-conjugate of HuMab-CD74-005 bound with higher affinity
(lower EC.sub.50 value) than the unconjugated HuMab. HuMab-CD74-005
and its mcMMAF-conjugate bound with lower affinity than
HuMab-CD74-006 and -011 and their conjugates. Maximal binding was
lower for vcMMAE-conjugated HuMab-CD74-006 and -011 than for the
corresponding unconjugated HuMab antibodies.
TABLE-US-00012 TABLE 9 Overview of EC.sub.50 values for binding of
CD74-specific HuMab antibodies and ADCs to surface-expressed CD74,
determined by FACS analysis on Daudi cells. EC.sub.50 values are in
.mu.g/mL. Data shown are mean EC.sub.50 values calculated from
three independent experiments. EC.sub.50 (FACS) HuMab-CD74-
Unconjugated vcMMAE mcMMAF 005 1.27 0.26 1.05 006 0.04 0.03 0.03
011 0.05 0.05 0.05
TABLE-US-00013 TABLE 10 Overview of mean fluoresencence intensities
at 10 .mu.g/mL of CD74-specific HuMab antibodies and ADCs,
determined by FACS analysis on Daudi cells. Data shown are mean
maximal MFI values as measured at 10 .mu.g/mL of HuMab-CD74 mAbs
and ADCs. Mean maximal MFI values were calculated from three
independent experiments. Maximal binding (FACS) HuMab-CD74-
Unconjugated vcMMAE mcMMAF 005 2784 2863 2526 006 4599 3277 4050
011 5782 3791 5330
Example 18
Antibody-Mediated Internalization and Cell Killing by Anti-CD74
ADCs in an In Vitro Killing Assay
[0413] To determine the capacity of anti-CD74 ADCs to induce
cytotoxicity, an in vitro cell-based killing assay was
performed.
[0414] Cell killing of four cell lines was tested for the different
anti-CD74 ADCs. All cell lines were obtained from American Tissue
Culture Collection (ATCC, Manassas, Va., USA): Raji (cat. no.
CCL-86), Daudi (cat. no. CCL-213), M4A4 (cat. no. CRL-2914; derived
from the human cell line MDA MB 435) and NCI-H747 cells (cat. no.
CCL-252, derived from colorectal adenocarcinoma metastasis). Cells
were seeded in optimal concentration (Raji: 1.times.10.sup.4
cells/well; Daudi: 1.times.10.sup.3 cells/well, M4A4:
2.times.10.sup.3 cells/well, NCI-H747 3.times.10.sup.3 cells/well)
in 100 .mu.L cell culture medium (for Daudi and Raji; RPMI 1640
[Lonza, cat. no. BE12-115F] supplemented with 10% Cosmic Calf Serum
[Perbio Science Nederland B.V., cat. no. SH30087.04], 2 mM
L-glutamin [Lonza, cat. no. BE17-605F] and 1 mM Sodium Pyruvate
[Lonza, cat. no. BE13-115E]; for NCI-H747: RPMI 1640 supplemented
with 10% Cosmic Calf Serum, 1 mM Sodium Pyruvate, 0.15% Sodium
Bicarbonate [Lonza, cat. no. BE17-613E] and 0.5% Glucose [Sigma,
cat. no. G8769]; and for M4A4: DMEM [Lonza, cat. no. BE12-709F]
supplemented with 10% Cosmic Calf Serum) in 96-well tissue culture
plates (Greiner Bio-one) and allowed to adhere. Serial dilutions of
anti-CD74 ADCs were added and incubated at 37.degree. C. for three
(Raji, Daudi) or five (M4A4, NCI-H747) days. The amount of viable
cells was quantified with AlamarBlue (cat. nr. DAL1100, BioSource
International, San Francisco, US), according to the manufacturer's
instructions. Fluorescence was monitored using the EnVision 2101
Multilabel reader (PerkinElmer, Turku, Finland) with standard
AlamarBlue settings. IgG1-b12 (an antibody binding to a non-related
antigen) ADCs were used as negative controls. Staurosporine (Sigma,
# S6942) was used to induce maximal cell killing. The amount of
CD74 molecules on cell lines was determined by QIFIKIT.RTM. (Dako,
Glostrup, Denmark), according to the manufacturer's instructions,
using mouse IgG1 anti-CD74 (clone By2; Santa Cruz, cat. no.
SC-20062) and isotype control (CLB, cat. no. M1415) antibody. Both
antibodies were used at a concentration of 10 .mu.g/mL. It was
determined that Raji and Daudi cells express .about.20,000; and
M4A4 cells .about.11,000 CD74 molecules on the cell surface.
[0415] FIG. 9 and Table 11 show that all anti-CD74 ADCs were able
to kill Raji, Daudi and M4A4 cells in a dose-dependent manner.
IC.sub.50 values for all conjugates were about 5-12 times higher on
M4A4 cells (i.e. lower efficacy), expressing about six-fold lower
levels of CD74. NCI-H747 cells were only killed at the highest dose
of ADCs tested (10 .mu.g/mL). For HuMab-CD74-006 and -011 mcMMAF
conjugates were slightly more efficient in inducing killing of
Daudi and Raji cells than vcMMEA conjugates (-006: on Daudi cells
three-fold lower and on Raji cells five-fold lower IC.sub.50; -011:
on Daudi cells two-fold lower and on Raji cells four-fold lower
IC.sub.50).
TABLE-US-00014 TABLE 11 Overview of IC.sub.50 values and
percentages of cell killing induced by anti-CD74 ADCs. Data shown
are mean IC.sub.50 values (in .mu.g/mL) and mean maximal
percentages kill (at a concentration of 10 .mu.g/mL) of the
indicated cell lines treated with anti-CD74 ADCs. Data were
calculated from three independent experiments. Percentage of cell
killing (% kill) was calculated as follows: (MFI.sub.untreated -
MFI.sub.anti-CD74 ADC-treated)/(MFI.sub.untreated -
MFI.sub.stauroporine-treated) .times. 100%. Raji Daudi M4A4
NCI-H747 ADC % kill IC.sub.50 % kill IC.sub.50 % kill IC.sub.50 %
kill IC.sub.50 005-vcMMAE 88 0.11 85 0.08 100 0.56 41 7.48
005-mcMMAF 92 0.05 90 0.03 97 0.38 5 N.A..sup.a) 006-vcMMAE 90 0.05
85 0.03 100 0.28 49 6.12 006-mcMMAF 93 0.01 93 0.01 97 0.12 21
N.A..sup.a) 011-vcMMAE 89 0.04 86 0.02 100 0.32 40 7.91 011-mcMMAF
92 0.01 92 0.01 96 0.10 17 N.A..sup.a) .sup.a)Could not be
calculated since plateau level of curve was not reached.
Example 19
Therapeutic Treatment of Daudi Tumor Xenografts in SCID Mice with
CD74-Specific ADCs
[0416] The in vivo efficacy of HuMab-CD74-011 ADCs was determined
in established intravenous (i.v.) Daudi (Burkitt's lymphoma)
xenograft tumors in SCID mice.
[0417] Daudi cells were transfected by electroporation with gWIZ
luciferase (Aldevron, Fargo, N. Dak., USA) and pPur vector (BD
Biosciences, Alphen a/d Rijn, The Netherlands) in a 4:1 ratio.
After 48 h, puromycin was added for selection of a stably
transfected clone (Daudi-luc). Daudi-luc #1E3 cells were cultured
in RPMI supplemented with 10% cosmic calf serum (cat. no.
SH30087.04, Hyclone), 1% penicillin/streptomycin (cat. no.
DE17-603, Cambrex, Germany), 1% sodium pyruvate and 1 .mu.g/mL
puromycin (cat. no. P-8833, Sigma, Zwijndrecht, The Netherlands).
2.5.times.10.sup.6 Daudi-luc tumor cells in 100 .mu.L PBS were
injected i.v. in the tail vein of female SCID mice. Mice were
imaged directly after tumor inoculation, followed by imaging at
weekly intervals starting on day 14. For imaging, mice were
anesthetized using isoflurane, followed by intraperitoneal (i.p.)
administration of 2.5 mg D-luciferin (acid form, cat.no. BT11-1000;
Biothema, Haninge, Sweden) in 200 .mu.L 10 mg/mL TRIS (cat.no.
T60666-1 kg, Sigma). Bioluminescence imaging (BLI), from the back
side (dorsal view), started 10 min after administration of
D-luciferin, 5 min exposure time, on a Biospace Imager. Black and
white images were made for anatomical reference. Mice were treated
twice weekly with 60 .mu.g (.about.3 mg/kg) HuMab-CD74-011 and
control antibody (IgG1-b12), both as ADC and as unconjugated IgG1,
in 100 .mu.L PBS from day 21 after tumor inoculation, four times in
total. Before treatment, mice were divided in groups of seven mice
each, each group having equal average BLI signals and equal
variances.
[0418] FIG. 10 shows that both HuMab-CD74-011-vcMMAE and -mcMMAF
were effective in reducing the size of i.v. Daudi-luc tumors. As
shown in FIG. 10, there was an apparent tendency for a higher tumor
growth inhibition in the case of unconjugated HuMab-CD74-011 as
compared to the control antibody group, although the differences
were not significant.
Example 20
Therapeutic Treatment of Raji Tumor Xenografts in SCID Mice with
Anti-CD74 ADCs
[0419] The in vivo efficacy of anti-CD74 ADCs was also determined
in an i.v. Raji xenograft tumor model in SCID mice.
[0420] Raji cells were transfected by electroporation with gWIZ
luciferase (Aldevron, Fargo, N. Dak., USA) and pPur vector (BD
Biosciences, Alphen a/d Rijn, The Netherlands) in a 4:1 ratio.
After 48 h, puromycin was added for selection of a stably
transfected clone (Raji-luc). Raji-luc #2D1 cells were cultured in
RPMI supplemented with 10% cosmic calf serum (cat. no. SH30087.04,
Hyclone), 1% penicillin/streptomycin (cat. no. DE17-603, Cambrex,
Germany), 1% sodium pyruvate and 1 .mu.g/mL puromycin (cat. no.
P-8833, Sigma, Zwijndrecht, The Netherlands). 2.5.times.10.sup.6
Raji-luc tumor cells in 100 .mu.L PBS were injected i.v. in the
tail vein of female SCID mice. Mice were imaged directly after
tumor inoculation, followed by imaging twice weekly from day 7
onwards. For imaging, mice were anesthetized using isoflurane,
followed by i.p. administration of 2.5 mg D-luciferin (acid form,
cat.no. BT11-1000; Biothema, Haninge, Sweden) in 200 .mu.L 10 mg/mL
TRIS (cat.no. T60666-1 kg, Sigma). Bioluminescence imaging (BLI),
from the back side (dorsal view), started 10 min after
administration of D-luciferin, 5 min exposure time, on a Biospace
Imager. Black and white images were made for anatomical reference.
Mice were treated twice weekly with 60 .mu.g (.about.3 mg/kg)
HuMab-CD74-011 or control antibody (IgG1-b12), both as ADC and as
unconjugated IgG, in 100 .mu.L PBS, from day 11 after tumor
inoculation, four times in total. Before treatment, mice were
divided in groups of seven mice each, each group having equal
average BLI signals and equal variances.
[0421] FIG. 11 shows that both HuMab-CD74-011-vcMMAE and -mcMMAF
eliminated virtually all Raji-luc tumors. As shown in FIG. 11,
there was an apparent tendency for a higher tumor growth inhibition
in the case of unconjugated HuMab-CD74-011 as compared to the
control antibody group, although the differences were not
significant.
Example 21
Therapeutic Treatment of Raji Tumor Xenografts in SCID Mice with
Anti-CD74 ADCs
[0422] The in vivo efficacy of anti-CD74 ADCs was also determined
in established subcutaneous (s.c.) Raji (Burkitt's lymphoma)
xenograft tumors in SCID mice.
[0423] 5.times.10.sup.6 Raji-luc #2D1 tumor cells (obtained as
described Example 20) in 200 .mu.L PBS were injected s.c. in the
right flank of female SCID mice, followed by two injections with
anti-CD74 ADCs or controls (IgG1-b12; both as ADC and as
unconjugated IgG1), one when tumor sizes were on average .about.400
mm.sup.3, on day 17, and the other four days later, on day 21 (per
injection 60 .mu.g/mouse, .about.3 mg/kg, in 100 .mu.L,
intraperitoneally). Before the first treatment, mice with tumor
growth were divided into groups with equal tumor volume
distribution. Tumor volume was determined at least two times per
week. Tumor volumes (mm.sup.3) were calculated from caliper (PLEXX)
measurements as: 0.52.times.(length).times.(width).sup.2.
[0424] FIG. 12 shows that all anti-CD74 ADCs effectively reduced
the size of established s.c. Raji-luc tumors. Tumors in mice
treated with IgG1-b12, both as ADC and unconjugated, continued to
grow.
Example 22
Therapeutic Treatment of M4A4 Tumor Xenografts in SCID Mice with
Anti-CD74 ADCs
[0425] The in vivo efficacy of anti-CD74 ADCs was also determined
in established subcutaneous (s.c.) M4A4 xenograft tumors in SCID
mice. M4A4 melanoma cells (cat. no. CRL-2914; American Tissue
Culture Collection, ATCC; derived from the human cell line
MDA-MB-435) were cultured in DMEM (cat. no. BE12-709F, Cambrex,
Germany) containing 10% cosmic calf serum (cat. no. SH30087.04,
Hyclone, The Netherlands) and 1% penicillin/streptomycin (cat. no.
DE17-603, Cambrex, Germany). 10.sup.7 M4A4 tumor cells in 200 .mu.L
PBS were injected s.c. in the right flank of female SCID mice,
followed by four injections with anti-CD74 ADCs or controls
(IgG1-b12; both as ADC and as unconjugated IgG1), starting when
tumor sizes were .about.200 mm.sup.3: day 11, day 14, day 18 and
day 21 (per injection 60 .mu.g/mouse, .about.3 mg/kg, in 100 .mu.L,
intraperitoneally). Before the first treatment, mice were divided
in groups with equal average tumor volume and equal variance in
tumor volume. Tumor volume was determined at least two times per
week. Tumor volumes (mm.sup.3) were calculated from caliper (PLEXX)
measurements as: 0.52.times.(length).times.(width).sup.2.
[0426] FIG. 13 shows that, whereas all anti-CD74 ADCs inhibited
tumor growth of established s.c. M4A4 tumors, the vcMMAE conjugates
strongly reduced the tumor size. Compared with unconjugated
IgG1-b12, the ADCs of IgG1-b12 slightly inhibited tumor growth.
Example 23
Determination of the Off-Rate of Anti-CD74 HuMab Antibodies on
Daudi Cells
[0427] This Example describes determination of the off-rates of
anti-CD74 HuMab antibodies in binding to Daudi cells.
[0428] Antibodies were labeled with Alexa Fluor.RTM. 488 Dye
(Molecular Probes), hereinafter "Alexa-488", using the following
procedure:
[0429] An antibody solution of 1 mg/mL IgG was prepared in 0.1 M
sodiumcarbonate buffer pH 9.0 (NaHCO.sub.3, Riedel de Haen, cat.
no. 31437;). Alexa-488 was prepared freshly, by adding 100 .mu.L
DMSO (Sigma, cat. no. D2438) to one vial (Alexa Fluor.RTM. 488
carboxylic acid, succinimidyl ester (1 mg/vial), Molecular Probes,
Leiden, The Netherlands, cat. no. A-20000). A 25-times molar excess
of Alexa-488, calculated as indicated below, was added to the IgG
solution and incubated, while rotating, in the dark at RT for 1
hour. After labeling, unbound Alexa-488 was removed, using a PD-10
column (Amersham Biosciences, cat. no. 17-0851-01), with Tris
buffer pH 8.0 (50 mM Tris [Trizma base, Sigma, cat. no. T-6066];
100 mM NaCl [Riedel de Haen, cat. no. 31437]; 0.01% sodium azide
[NaN.sub.3, Riedel de Haen, cat. no. 13412]). The amount of
Alexa-488 to be added to the IgG solution was calculated using the
formula:
Volume Alexa-488 to be added(in .mu.L)=(IgG conc(mg/mL)/MW
IgG(Da)*ratio*volume*MW Alexa-488*100.
[0430] MW IgG=150,000 Da; ratio is the molar excess of Alexa-488 to
be used; volume is the volume of the sample to be labeled (in mL);
MW Alexa-488=643 Da.
[0431] Protein concentration (IgG) and degree of labeling (D.O.L.)
were determined by measuring OD 280 nm and 495 nm on an Ultrospec
2100 Pro (Amersham Biosciences). IgG concentration (mg/mL) was
calculated using the formula:
IgG concentration=[A.sub.280-(0.11*A.sub.495)]/IgG extinction
coefficient.
[0432] D.O.L. was calculated using the formula:
D.O.L.=A.sub.495/71,000/[A.sub.280-(0.11*A.sub.495)/(IgG extinction
coefficient*MW IgG)].
71,000 is the extinction coefficient of Alexa-488 at
.lamda..sub.max in cm.sup.-11M.sup.-1; 0.11 is the correction
factor (A.sub.280 free dye/.lamda..sub.max free dye) (both provided
by the manufacturer).
[0433] Bovine serum albumin (BSA; Sigma, cat. no. A 2934) was added
from a 10% (w/v) solution to a final concentration of 0.1% (w/v)
and labeled antibodies were stored at 5.degree. C.
[0434] Daudi cells were incubated with Alexa-488-labeled anti-CD74
HuMab antibodies. Daudi cells were washed twice with ice cold PBS.
10.sup.5 cells per well in ice-cold FACS buffer were seeded in
96-well round-bottom tissue culture plates (Greiner Bio-one). 0.5
.mu.g/mL (HuMab-CD74-006 and -011; final concentration) or 1
.mu.g/mL (HuMab-CD74-008; final concentration) Alexa-488-labeled
anti-CD74 HuMab was added in ice-cold FACS buffer. After incubation
on ice for 30 min, 50 .mu.g/mL (HuMab-CD74-006 and -011; final
concentration) or 100 .mu.g/mL (HuMab-CD74-008; final
concentration) unlabeled anti-CD74 HuMab was added and incubated on
ice for different time intervals ranging from 15 to 180 min. Total
incubation time with unlabeled antibody is indicated below the
graphs. To determine maximal binding, Daudi cells were incubated
with Alexa-488-labeled HuMab antibodies on ice for 30 min. As a
negative control, cells were incubated with isotype control
antibody IgG1-b12 (0.5 .mu.g/mL final concentration), followed by
unlabeled IgG1-b12 (50 .mu.g/mL final concentration). After
antibody incubation, cells were washed once in FACS buffer and
bound Alexa--labeled anti-CD74 HuMab antibodies were detected by
flow cytometry on a FACS Canto II (BD Biosciences).
[0435] FIG. 14 shows that off-rates of HuMab-CD74-006 and -008
measured at 0.degree. C. were quite rapid (half of the bound
Alexa-488-labeled antibodies were replaced with unlabeled
antibodies within .about.3 and .about.4 min at 0.degree. C., K
values were 0.24 and 0.20 min.sup.-1; [K=k.sub.off]), whereas
off-rate of -011 measured at 0.degree. C. was a little bit slower
(half of the bound Alexa 488 labeled antibodies were replaced with
unlabeled antibodies within .about.10 min at 0.degree. C., K value
was 0.07 min.sup.-1).
Example 24
Internalization and Accumulation of Anti-CD74 HuMab Antibodies
[0436] To determine whether anti-CD74 HuMab antibodies are suitable
for an antibody-drug conjugate approach, internalization and
accumulation of antibodies was studied by FACS analysis after
incubation of different anti-CD74 HuMab antibodies with Daudi
cells. 10.sup.5 cells per well in cell culture medium were seeded
in 96-well round-bottom tissue culture plates (Greiner Bio-one). 10
.mu.g/mL (final concentration) Alexa-488-labeled anti-CD74 HuMab
antibodies were added at different time points and incubated at
4.degree. C. (to measure binding to cell surface expressed CD74) or
at 37.degree. C. (to measure binding and internalization). Total
incubation time with antibody is indicated below the graphs.
Internalization and accumulation at 37.degree. C. was also tested
using Raji and M4A4 cells with a final concentration of 3 .mu.g/mL
Alexa-488-labeled anti-CD74 HuMab antibodies. After incubation with
antibody, cells were put on ice and plates were washed twice with
FACS buffer. Cell-associated labeled antibodies were detected by
flow cytometry on a FACS Canto II (BD Biosciences).
[0437] FIG. 15A shows that only low levels of binding of
Alexa-488-labeled anti-CD74 HuMab antibodies to the cell surface of
Daudi were detected after incubation at 4.degree. C. at any time
point. Therefore, the observed fluorescence intensities measured
after incubation at 37.degree. C. represent internalized antibody.
FIG. 15B shows that all anti-CD74 HuMab antibodies tested were
internalized, but with different efficacies. Internalization was
most rapid for HuMab-CD74-011, slower for HuMab-CD74-006 and most
slow for HuMab-CD74-008. The same was observed for internalization
and accumulation in Raji cells (15C) and M4A4 cells (15D).
Example 25
Prophylactic Treatment of Daudi Tumor Xenografts in SCID Mice with
Anti CD74 HuMab Antibodies
[0438] The in vivo efficacy of anti-CD74 HuMab antibodies was
determined in an intravenous (i.v.) Daudi (Burkitt's lymphoma)
xenograft tumor model in SCID mice. Daudi cells were transfected by
electroporation with gWIZ luciferase (Aldevron, Fargo, N. Dak.,
USA) and pPur vector (BD Biosciences, Alphen a/d Rijn, The
Netherlands) in a 4:1 ratio. After 48 h, puromycin was added for
selection of a stably transfected clone (Daudi-luc). Daudi luc #1E3
cells were cultured in RPMI supplemented with 10% cosmic calf serum
(cat. no. SH30087.04, Hyclone), 1% penicillin/streptomycin (cat.
no. DE17 603, Cambrex, Germany), 1% sodium pyruvate and 1 .mu.g/mL
puromycin (cat. no. P 8833, Sigma, Zwijndrecht, The Netherlands).
2.5.times.10.sup.6 Daudi luc tumor cells in 100 .mu.L PBS were
injected i.v. in the tail vein of female SCID mice (7 mice per
group). Mice were treated at the day of tumor inoculation with 100
.mu.g (.about.5 mg/kg) HuMab-CD74-005, -006 or -011 or control
antibody (IgG1 b12), in 200 .mu.L PBS, intraperitoneally (i.p.).
Mice were imaged directly after tumor inoculation, followed by
imaging at weekly intervals starting on day 14. For imaging, mice
were anesthetized using isoflurane, followed by i.p. administration
of 2.5 mg D luciferin (acid form, cat.no. BT11 1000; Biothema,
Haninge, Sweden) in 200 .mu.L 10 mg/mL TRIS (cat.no. T60666-1 kg,
Sigma). Bioluminescence imaging (BLI), from the back side (dorsal
view), started 10 min after administration of D luciferin, 5 min
exposure time, on a Biospace Imager. Black and white images were
made for anatomical reference.
[0439] FIG. 16 shows that all tested anti-CD74 HuMab antibodies
almost completely prevented the outgrowth of i.v. Daudi luc
tumors.
EQUIVALENTS
[0440] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims. Any and all combination of embodiments disclosed
in dependent claims is also contemplated to be within the scope of
the invention.
Sequence CWU 1
1
271296PRTArtificial SequenceSynthetic partial sequence 1Met His Arg
Arg Arg Ser Arg Ser Cys Arg Glu Asp Gln Lys Pro Val 1 5 10 15 Met
Asp Asp Gln Arg Asp Leu Ile Ser Asn Asn Glu Gln Leu Pro Met 20 25
30 Leu Gly Arg Arg Pro Gly Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala
35 40 45 Leu Tyr Thr Gly Phe Ser Ile Leu Val Thr Leu Leu Leu Ala
Gly Gln 50 55 60 Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly
Arg Leu Asp Lys 65 70 75 80 Leu Thr Val Thr Ser Gln Asn Leu Gln Leu
Glu Asn Leu Arg Met Lys 85 90 95 Leu Pro Lys Pro Pro Lys Pro Val
Ser Lys Met Arg Met Ala Thr Pro 100 105 110 Leu Leu Met Gln Ala Leu
Pro Met Gly Ala Leu Pro Gln Gly Pro Met 115 120 125 Gln Asn Ala Thr
Lys Tyr Gly Asn Met Thr Glu Asp His Val Met His 130 135 140 Leu Leu
Gln Asn Ala Asp Pro Leu Lys Val Tyr Pro Pro Leu Lys Gly 145 150 155
160 Ser Phe Pro Glu Asn Leu Arg His Leu Lys Asn Thr Met Glu Thr Ile
165 170 175 Asp Trp Lys Val Phe Glu Ser Trp Met His His Trp Leu Leu
Phe Glu 180 185 190 Met Ser Arg His Ser Leu Glu Gln Lys Pro Thr Asp
Ala Pro Pro Lys 195 200 205 Val Leu Thr Lys Cys Gln Glu Glu Val Ser
His Ile Pro Ala Val His 210 215 220 Pro Gly Ser Phe Arg Pro Lys Cys
Asp Glu Asn Gly Asn Tyr Leu Pro 225 230 235 240 Leu Gln Cys Tyr Gly
Ser Ile Gly Tyr Cys Trp Cys Val Phe Pro Asn 245 250 255 Gly Thr Glu
Val Pro Asn Thr Arg Ser Arg Gly His His Asn Cys Ser 260 265 270 Glu
Ser Leu Glu Leu Glu Asp Pro Ser Ser Gly Leu Gly Val Thr Lys 275 280
285 Gln Asp Leu Gly Pro Val Pro Met 290 295 2232PRTArtificial
sequenceSynthetic partial sequence 2Met His Arg Arg Arg Ser Arg Ser
Cys Arg Glu Asp Gln Lys Pro Val 1 5 10 15 Met Asp Asp Gln Arg Asp
Leu Ile Ser Asn Asn Glu Gln Leu Pro Met 20 25 30 Leu Gly Arg Arg
Pro Gly Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala 35 40 45 Leu Tyr
Thr Gly Phe Ser Ile Leu Val Thr Leu Leu Leu Ala Gly Gln 50 55 60
Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys 65
70 75 80 Leu Thr Val Thr Ser Gln Asn Leu Gln Leu Glu Asn Leu Arg
Met Lys 85 90 95 Leu Pro Lys Pro Pro Lys Pro Val Ser Lys Met Arg
Met Ala Thr Pro 100 105 110 Leu Leu Met Gln Ala Leu Pro Met Gly Ala
Leu Pro Gln Gly Pro Met 115 120 125 Gln Asn Ala Thr Lys Tyr Gly Asn
Met Thr Glu Asp His Val Met His 130 135 140 Leu Leu Gln Asn Ala Asp
Pro Leu Lys Val Tyr Pro Pro Leu Lys Gly 145 150 155 160 Ser Phe Pro
Glu Asn Leu Arg His Leu Lys Asn Thr Met Glu Thr Ile 165 170 175 Asp
Trp Lys Val Phe Glu Ser Trp Met His His Trp Leu Leu Phe Glu 180 185
190 Met Ser Arg His Ser Leu Glu Gln Lys Pro Thr Asp Ala Pro Pro Lys
195 200 205 Glu Ser Leu Glu Leu Glu Asp Pro Ser Ser Gly Leu Gly Val
Thr Lys 210 215 220 Gln Asp Leu Gly Pro Val Pro Met 225 230
3261PRTArtificial sequenceSynthetic partial sequence 3Met Pro Gly
Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala Leu Tyr Thr 1 5 10 15 Gly
Phe Ser Ile Leu Val Thr Leu Leu Leu Ala Gly Gln Ala Thr Thr 20 25
30 Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr Val
35 40 45 Thr Ser Gln Asn Leu Gln Leu Glu Asn Leu Arg Met Lys Leu
Pro Lys 50 55 60 Pro Pro Lys Pro Val Ser Lys Met Arg Met Ala Thr
Pro Leu Leu Met 65 70 75 80 Gln Ala Leu Pro Met Gly Ala Leu Pro Gln
Gly Pro Met Gln Asn Ala 85 90 95 Thr Lys Tyr Gly Asn Met Thr Glu
Asp His Val Met His Leu Leu Gln 100 105 110 Asn Ala Asp Pro Leu Lys
Val Tyr Pro Pro Leu Lys Gly Ser Phe Pro 115 120 125 Glu Asn Leu Arg
His Leu Lys Asn Thr Met Glu Thr Ile Asp Trp Lys 130 135 140 Val Phe
Glu Ser Trp Met His His Trp Leu Leu Phe Glu Met Ser Arg 145 150 155
160 His Ser Leu Glu Gln Lys Pro Thr Asp Ala Pro Pro Lys Val Leu Thr
165 170 175 Lys Cys Gln Glu Glu Val Ser His Ile Pro Ala Val His Pro
Gly Ser 180 185 190 Phe Arg Pro Lys Cys Asp Glu Asn Gly Asn Tyr Leu
Pro Leu Gln Cys 195 200 205 Tyr Gly Ser Ile Gly Tyr Cys Trp Cys Val
Phe Pro Asn Gly Thr Glu 210 215 220 Val Pro Asn Thr Arg Ser Arg Gly
His His Asn Cys Ser Glu Ser Leu 225 230 235 240 Glu Leu Glu Asp Pro
Ser Ser Gly Leu Gly Val Thr Lys Gln Asp Leu 245 250 255 Gly Pro Val
Pro Met 260 4197PRTArtificial sequenceSynthetic partial sequence
4Met Pro Gly Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala Leu Tyr Thr 1
5 10 15 Gly Phe Ser Ile Leu Val Thr Leu Leu Leu Ala Gly Gln Ala Thr
Thr 20 25 30 Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
Leu Thr Val 35 40 45 Thr Ser Gln Asn Leu Gln Leu Glu Asn Leu Arg
Met Lys Leu Pro Lys 50 55 60 Pro Pro Lys Pro Val Ser Lys Met Arg
Met Ala Thr Pro Leu Leu Met 65 70 75 80 Gln Ala Leu Pro Met Gly Ala
Leu Pro Gln Gly Pro Met Gln Asn Ala 85 90 95 Thr Lys Tyr Gly Asn
Met Thr Glu Asp His Val Met His Leu Leu Gln 100 105 110 Asn Ala Asp
Pro Leu Lys Val Tyr Pro Pro Leu Lys Gly Ser Phe Pro 115 120 125 Glu
Asn Leu Arg His Leu Lys Asn Thr Met Glu Thr Ile Asp Trp Lys 130 135
140 Val Phe Glu Ser Trp Met His His Trp Leu Leu Phe Glu Met Ser Arg
145 150 155 160 His Ser Leu Glu Gln Lys Pro Thr Asp Ala Pro Pro Lys
Glu Ser Leu 165 170 175 Glu Leu Glu Asp Pro Ser Ser Gly Leu Gly Val
Thr Lys Gln Asp Leu 180 185 190 Gly Pro Val Pro Met 195
5230PRTArtificial sequenceSynthetic partial sequence with His-tag
5His His His His His His Gln Gln Gln Gly Arg Leu Asp Lys Leu Thr 1
5 10 15 Val Thr Ser Gln Asn Leu Gln Leu Glu Asn Leu Arg Met Lys Leu
Pro 20 25 30 Lys Pro Pro Lys Pro Val Ser Lys Met Arg Met Ala Thr
Pro Leu Leu 35 40 45 Met Gln Ala Leu Pro Met Gly Ala Leu Pro Gln
Gly Pro Met Gln Asn 50 55 60 Ala Thr Lys Tyr Gly Asn Met Thr Glu
Asp His Val Met His Leu Leu 65 70 75 80 Gln Asn Ala Asp Pro Leu Lys
Val Tyr Pro Pro Leu Lys Gly Ser Phe 85 90 95 Pro Glu Asn Leu Arg
His Leu Lys Asn Thr Met Glu Thr Ile Asp Trp 100 105 110 Lys Val Phe
Glu Ser Trp Met His His Trp Leu Leu Phe Glu Met Ser 115 120 125 Arg
His Ser Leu Glu Gln Lys Pro Thr Asp Ala Pro Pro Lys Val Leu 130 135
140 Thr Lys Cys Gln Glu Glu Val Ser His Ile Pro Ala Val His Pro Gly
145 150 155 160 Ser Phe Arg Pro Lys Cys Asp Glu Asn Gly Asn Tyr Leu
Pro Leu Gln 165 170 175 Cys Tyr Gly Ser Ile Gly Tyr Cys Trp Cys Val
Phe Pro Asn Gly Thr 180 185 190 Glu Val Pro Asn Thr Arg Ser Arg Gly
His His Asn Cys Ser Glu Ser 195 200 205 Leu Glu Leu Glu Asp Pro Ser
Ser Gly Leu Gly Val Thr Lys Gln Asp 210 215 220 Leu Gly Pro Val Pro
Met 225 230 6166PRTArtificial sequenceSynthetic partial sequence
with His-tag 6His His His His His His Gln Gln Gln Gly Arg Leu Asp
Lys Leu Thr 1 5 10 15 Val Thr Ser Gln Asn Leu Gln Leu Glu Asn Leu
Arg Met Lys Leu Pro 20 25 30 Lys Pro Pro Lys Pro Val Ser Lys Met
Arg Met Ala Thr Pro Leu Leu 35 40 45 Met Gln Ala Leu Pro Met Gly
Ala Leu Pro Gln Gly Pro Met Gln Asn 50 55 60 Ala Thr Lys Tyr Gly
Asn Met Thr Glu Asp His Val Met His Leu Leu 65 70 75 80 Gln Asn Ala
Asp Pro Leu Lys Val Tyr Pro Pro Leu Lys Gly Ser Phe 85 90 95 Pro
Glu Asn Leu Arg His Leu Lys Asn Thr Met Glu Thr Ile Asp Trp 100 105
110 Lys Val Phe Glu Ser Trp Met His His Trp Leu Leu Phe Glu Met Ser
115 120 125 Arg His Ser Leu Glu Gln Lys Pro Thr Asp Ala Pro Pro Lys
Glu Ser 130 135 140 Leu Glu Leu Glu Asp Pro Ser Ser Gly Leu Gly Val
Thr Lys Gln Asp 145 150 155 160 Leu Gly Pro Val Pro Met 165
7124PRTHomo sapiens 7Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Ser Gly Arg Tyr Tyr Gly Ser Gly Ser Tyr Ser Ser Tyr Phe Asp
100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
88PRTHomo sapiens 8Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 98PRTHomo
sapiens 9Ile Ser Tyr Asp Gly Ser Asn Lys 1 5 1017PRTHomo sapiens
10Ala Ser Gly Arg Tyr Tyr Gly Ser Gly Ser Tyr Ser Ser Tyr Phe Asp 1
5 10 15 Tyr 11124PRTHomo sapiens 11Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val
Ile Ser Tyr Asp Gly Ser Ile Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Phe
Tyr Cys 85 90 95 Ala Arg Gly Arg Glu Ile Thr Ser Gln Asn Ile Val
Ile Leu Leu Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Thr Ser 115 120 128PRTHomo sapiens 12Gly Phe Thr Phe Ser Ser Tyr
Ala 1 5 138PRTHomo sapiens 13Ile Ser Tyr Asp Gly Ser Ile Lys 1 5
1417PRTHomo sapiens 14Ala Arg Gly Arg Glu Ile Thr Ser Gln Asn Ile
Val Ile Leu Leu Asp 1 5 10 15 Tyr 15124PRTHomo sapiens 15Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Glu Ile Thr
Ser Gln Asn Ile Val Ile Leu Leu Asp 100 105 110 Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 168PRTHomo sapiens 16Gly Phe
Thr Phe Ser Ser Tyr Ala 1 5 178PRTHomo sapiens 17Ile Ser Tyr Asp
Gly Ser Asn Lys 1 5 1817PRTHomo sapiens 18Ala Arg Gly Arg Glu Ile
Thr Ser Gln Asn Ile Val Ile Leu Leu Asp 1 5 10 15 Tyr 19123PRTHomo
sapiens 19Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Asp
Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Gly Gly Thr Leu Val Arg Gly Ala Met Tyr Gly Thr Asp Val 100 105 110
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 208PRTHomo
sapiens 20Gly Phe Thr Phe Ser Ser Tyr Gly 1 5 218PRTHomo sapiens
21Ile Trp Tyr Asp Gly Ser Asn Lys 1 5 2216PRTHomo sapiens 22Ala Arg
Gly Gly Thr Leu Val Arg Gly Ala Met Tyr Gly Thr Asp Val 1 5 10 15
23107PRTHomo sapiens 23Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Phe Gln Gln Lys
Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 246PRTHomo
sapiens 24Gln Gly Ile Ser Ser Trp 1 5 259PRTHomo sapiens 25Gln Gln
Tyr Asn Ser Tyr Pro Leu Thr 1 5 26107PRTHomo sapiens 26Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro
Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
273PRTHomo sapiens 27Ala Ala Ser 1
* * * * *
References