U.S. patent application number 15/246089 was filed with the patent office on 2016-12-15 for dock-and-lock (dnl) vaccines for cancer therapy.
The applicant listed for this patent is IBC Pharmaceuticals, Inc.. Invention is credited to Chien-Hsing Chang, David M. Goldenberg.
Application Number | 20160361405 15/246089 |
Document ID | / |
Family ID | 41707458 |
Filed Date | 2016-12-15 |
United States Patent
Application |
20160361405 |
Kind Code |
A1 |
Chang; Chien-Hsing ; et
al. |
December 15, 2016 |
Dock-and-Lock (DNL) Vaccines for Cancer Therapy
Abstract
The present invention concerns methods and compositions for
forming anti-cancer vaccine DNL complexes using dock-and-lock
technology. In preferred embodiments, the anti-cancer vaccine DNL
complex comprises an antibody moiety that binds to dendritic cells,
such as an anti-CD74 antibody or antigen-binding fragment thereof,
attached to an AD (anchoring domain) moiety and a xenoantigen, such
as CD20, attached to a DDD (dimerization and docking domain)
moiety, wherein two copies of the DDD moiety form a dimer that
binds to the AD moiety, resulting in the formation of the DNL
complex. The anti-cancer vaccine DNL complex is capable of inducing
an immune response against xenoantigen expressing cancer cells,
such as CD138.sup.negCD20.sup.+ MM stem cells, and inducing
apoptosis of and inhibiting the growth of or eliminating the cancer
cells.
Inventors: |
Chang; Chien-Hsing;
(Downingtown, PA) ; Goldenberg; David M.;
(Mendham, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IBC Pharmaceuticals, Inc. |
Morris Plains |
NJ |
US |
|
|
Family ID: |
41707458 |
Appl. No.: |
15/246089 |
Filed: |
August 24, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13010993 |
Jan 21, 2011 |
9457072 |
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15246089 |
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12544476 |
Aug 20, 2009 |
7901680 |
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13010993 |
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12396605 |
Mar 3, 2009 |
7858070 |
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12544476 |
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11633729 |
Dec 5, 2006 |
7527787 |
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12396605 |
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11389358 |
Mar 24, 2006 |
7550143 |
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11633729 |
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11391584 |
Mar 28, 2006 |
7521056 |
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11389358 |
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11478021 |
Jun 29, 2006 |
7534866 |
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11391584 |
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60782332 |
Mar 14, 2006 |
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60751196 |
Dec 16, 2005 |
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60751196 |
Dec 16, 2005 |
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60864530 |
Nov 6, 2006 |
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61090487 |
Aug 20, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/001153 20180801;
A61K 47/6897 20170801; A61K 39/001135 20180801; C07K 2317/51
20130101; C07K 2317/526 20130101; A61K 39/001181 20180801; A61K
39/001189 20180801; A61K 39/001103 20180801; A61K 39/001112
20180801; A61K 39/001114 20180801; A61K 39/001162 20180801; A61K
39/001104 20180801; A61K 39/001117 20180801; A61P 43/00 20180101;
B82Y 5/00 20130101; A61K 39/001138 20180801; A61K 39/00114
20180801; A61K 39/001166 20180801; A61P 35/02 20180101; A61K
39/001149 20180801; A61P 35/00 20180101; A61K 39/001194 20180801;
C07K 2317/92 20130101; A61K 39/001109 20180801; A61K 39/001126
20180801; A61K 39/001182 20180801; C12Y 207/11011 20130101; A61K
39/001129 20180801; A61K 2039/625 20130101; C07K 16/2887 20130101;
A61K 39/00117 20180801; A61K 39/001191 20180801; C07K 16/468
20130101; A61K 39/001192 20180801; A61K 39/001186 20180801; A61K
39/001188 20180801; A61K 2039/505 20130101; C07K 2319/30 20130101;
C07K 16/3007 20130101; C07K 2317/55 20130101; A61K 39/001163
20180801; A61K 39/001195 20180801; C07K 2317/24 20130101; C07K
2317/31 20130101; A61K 39/001102 20180801; A61K 39/00113 20180801;
A61K 39/00115 20180801; C07K 2319/00 20130101; A61K 39/001113
20180801; A61K 39/001124 20180801; A61K 2039/6056 20130101; C07K
2317/77 20130101; A61P 37/04 20180101; C07K 16/2833 20130101; A61K
39/001151 20180801; A61K 39/001184 20180801; A61K 39/0011 20130101;
A61K 39/001176 20180801; C07K 16/2803 20130101; C07K 2317/522
20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 39/395 20060101 A61K039/395; C07K 16/28 20060101
C07K016/28 |
Claims
1. A method of treating cancer comprising: a) obtaining a DNL
complex comprising; i) a chimeric, humanized or human anti-CD74
antibody moiety that binds to human CD74, wherein the antibody
moiety is attached to an AD (anchor domain) moiety, wherein the AD
moiety has an amino acid sequence selected from the group
consisting of SEQ ID NO:13; SEQ ID NO:12; SEQ ID NO:19; SEQ ID
NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ
ID NO:25; SEQ ID NO:26; SEQ ID NO:27 and SEQ ID NO:28; and ii) a
mouse xenoantigen moiety selected from the group consisting of
carbonic anhydrase IX, alpha-fetoprotein (AFP), .alpha.-actinin-4,
CD1, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20,
CD21, CD22, CD23, CD25, CD29, CD30, CD33, CD37, CD38, CD40, CD40L,
CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD74, CD79a, CD80, CD83,
CD95, CD126, CD138, CD147, CD154, PSMA, fibronectin, folate
receptor, IL-6R, IL-13R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15,
IL-17, IL-18, IFN-.gamma., IFN-.alpha., IFN-.beta., macrophage
migration inhibitory factor (MIF), mCRP, MIP-1B, MUC1, MUC2, MUC4,
placental growth factor, p53, prostatic acid phosphatase, RANTES,
tenascin, and TNF-.alpha., wherein the xenoantigen moiety is
attached to a DDD (dimerization and docking domain) moiety, wherein
the amino acid sequence of the DDD moiety is residues 1-44 of human
protein kinase A (PKA) RII.alpha. and wherein two copies of the DDD
moiety form a dimer that binds to the AD moiety to form the DNL
complex; and b) administering the complex to a human subject with
cancer to induce an immune response against the cancer.
2. The method of claim 1, wherein the antibody moiety comprises two
heavy chains and each heavy chain is attached at its C-terminal end
to an AD moiety and the complex comprises one antibody moiety and
four xenoantigen moieties.
3. The method of claim 1, wherein the xenoantigen is CD20.
4. The method of claim 1, wherein the amino acid sequence of the
DDD moiety is selected from the group consisting of SEQ ID NO:11
and SEQ ID NO:10.
5. The method of claim 1, wherein the amino acid sequence of the
DDD moiety is SEQ ID NO:11.
6. The method of claim 1, wherein the amino acid sequence of the AD
moiety is SEQ ID NO:13.
7. The method of claim 1, wherein the antibody moiety is a
humanized or chimeric LL1 anti-CD74 antibody or antigen-binding
fragment thereof comprising the light chain variable
complementarity-determining region (CDR) sequences CDR1
(RSSQSLVHRNGNTYLH; SEQ ID NO:1), CDR2 (TVSNRFS; SEQ ID NO:2), and
CDR3 (SQSSHVPPT; SEQ ID NO:3) and the heavy chain variable region
CDR sequences CDR1 (NYGVN; SEQ ID NO:4), CDR2 (WINPNTGEPTFDDDFKG;
SEQ ID NO:5), and CDR3 (SRGKNEAWFAY; SEQ ID NO:6).
8. The method of claim 3, wherein the amino acid sequence of the
CD20 xenoantigen moiety is SEQ ID NO:7.
9. The method of claim 1, wherein the DNL complex is capable of
inducing an immune response against CD138.sup.negCD20.sup.+ MM stem
cells.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/010,993, filed Jan. 21, 2011, which was a divisional of
U.S. patent application Ser. No. 12/544,476 (now issued U.S. Pat.
No. 7,901,680), filed Aug. 20, 2009, which was a
continuation-in-part of U.S. patent application Ser. No. 12/396,605
(now issued U.S. Pat. No. 7,858,070), filed Mar. 3, 2009, which was
a divisional of U.S. patent application Ser. No. 11/633,729 (now
issued U.S. Pat. No. 7,527,787), filed Dec. 5, 2006, which was a
continuation-in-part of U.S. patent application Ser. No. 11/389,358
(now issued U.S. Pat. No. 7,550,143), filed Mar. 24, 2006; Ser. No.
11/391,584 (now issued U.S. Pat. No. 7,521,056), filed Mar. 28,
2006, and Ser. No. 11/478,021 (now issued U.S. Pat. No. 7,534,866),
filed Jun. 29, 2006, and which claimed the benefit under 35 U.S.C.
119(e) of provisional U.S. Patent Application No. 60/782,332, filed
Mar. 14, 2006; 60/751,196, filed Dec. 16, 2005; and No. 60/864,530,
filed Nov. 6, 2006. This application claims the benefit under 35
U.S.C. 119(e) of U.S. Provisional Patent Application Ser. No.
61/090,487, filed Aug. 20, 2008. The entire text of each priority
application is incorporated herein by reference.
BACKGROUND
[0002] Field of the Invention
[0003] The present invention relates to the design and generation
of dendritic cell-based, in vivo antigen targeting vaccines for
therapy of cancer, such as multiple myeloma. In preferred
embodiments the vaccines are generated by the dock-and-lock (DNL)
method, in which effector moieties are attached to anchoring domain
(AD) derived from AKAP proteins and dimerization and docking domain
(DDD) moieties derived from protein kinase A (PKA). DNL complexes
are generated when DDD moieties spontaneously dimerize and bind to
an AD moiety, resulting in a complex with a 2:1 stoichiometry
between DDD and AD-linked effectors. In more preferred embodiments,
the effector moieties comprise a humanized anti-CD74 antibody and a
tumor-associated xenoantigen, such as a CD20 xenoantigen. In most
preferred embodiments, the anti-CD74 antibody is an hLL1 antibody.
The DNL constructs are of use for preparation of pharmaceutical
compositions, for generation of vaccines against cancers, such as
multiple myeloma (MM), and for induction of an immune response
against tumor antigen-expressing cells, such as CD20 positive
cancer cells in patients with multiple myeloma or other
CD20-expressing cancers.
[0004] Related Art
[0005] Multiple myeloma (MM) is a hematological malignancy
characterized by clonal proliferation of neoplastic plasma cells in
the bone marrow. Although responsive to many chemotherapeutic
agents, MM remains largely incurable and the majority of patients
ultimately relapse, due to the existence of a minor population of
MM cancer stem cells that survive standard or high-dose
chemotherapy and are resistant to chemotherapeutic drugs (Reece et
al., Leuk Lymphoma, 2008, 49:1470-85). This small number of MM
cancer stem cells constitutes the minimal residual disease and
causes relapse, eventually leading to the failure of all
treatments. Thus, eradication of MM cancer stem cells may offer a
long-term control or even cure of MM.
[0006] Recently, a small population of clonotypic B cells, that do
not express the characteristic plasma cell surface antigen CD138
but do express the B cell antigen CD20, was identified from both MM
cell lines and primary bone marrow of MM patients (Matsui et al.,
Blood 2004, 103:2332-6). This small population of cells is
resistant to multiple clinical anti-myeloma drugs and is capable of
clonogenic growth in vitro (Matsui et al., Blood 2004, 103:2332-6;
Matsui et al., Cancer Res. 2008, 68:190-7) and in a 3-D culture
model (Kirshner et al., Blood 2008, 112:2935-45), and is capable of
differentiation into MM cells in vitro and in engrafted NOD/SCID
mice during both primary and secondary transplantation (Matsui et
al., Cancer Res. 2008, 68:190-7). It has thus been suggested that
these CD138.sup.negCD20.sup.+ cells represent the putative multiple
myeloma cancer stem cells (Huff and Matsui, J Clin Oncol. 2008,
26:2895-900).
[0007] Like other cancer stem cells, MM cancer stem cells are
refractory to multiple chemotherapeutic drugs and responsible for
tumor re-growth and relapse (Huff and Matsui, J Clin Oncol. 2008,
26:2895-900; Yang and Chang, Cancer Invest. 2008, 26:741-55).
Strategies and approaches that could selectively target and
eradicate cancer stem cells, such as MM stem cells, are needed. Due
to the multiple drug resistance in cancer stem cells, immunotherapy
and vaccination may offer a potential modality to eradicate these
cells, particularly after standard therapies and/or stem cell
transplantation, the time when tumor load is greatly reduced. A
need exists for effective compositions and methods of immunotherapy
and vaccination targeted to treatment of multiple myeloma,
particularly those capable of inducing an immune response against
and inhibiting or eradicating MM cancer stem cells. A further need
exists for effective compositions and methods of immunotherapy and
vaccination targeted to treatment of cancers in general.
SUMMARY OF THE INVENTION
[0008] The present invention discloses methods and compositions for
vaccines against cancer stem cells, such as MM stem cells, that are
prepared using the Dock-and-Lock (DNL) method (Chang et al., 2007,
Clin Cancer Res 13:5586s-91s). The DNL technique has been used to
generate a variety of stable and defined complexes suitable for in
vivo applications. In preferred embodiments, the DNL complexes
comprise an anti-CD74 antibody or antigen binding fragment thereof,
such as the hLL1 antibody, attached to a dimerization and docking
domain (DDD) or anchor domain (AD) moiety. The DDD moieties
spontaneously dimerize and each DDD dimer binds to an AD moiety. In
more preferred embodiments, a complementary AD or DDD moiety is
attached to a CD20 xenoantigen, as described in further detail
below, resulting in formation of DNL complexes comprising anti-CD74
moieties and CD20 xenoantigen moieties. However, the skilled
artisan will realize that depending on the cancer, a different
xenoantigen and/or antibody or antibody fragment may be utilized.
The antibody component directs the DNL complex to antigen
presenting cells (APCs), such as dendritic cells (DCs), while the
xenoantigen component is processed to invoke an immune response
against cells expressing the target antigen.
[0009] Various types of DNL complexes with different structures and
different ratios of target antigen (e.g., CD20) to antibody or
antibody fragment may be constructed and used within the scope of
the claimed methods and compositions, such as those disclosed in
U.S. Pat. No. 7,550,143 (incorporated herein by reference from Col.
28, line 30 through Col. 44, line 28); U.S. Pat. No. 7,521,056
(incorporated herein by reference from Col. 58, line 1 through Col.
Col. 84, line 45); U.S. Pat. No. 7,534,866 (incorporated herein by
reference from Col. 31, line 1 through Col. 36, line 38); U.S. Pat.
No. 7,527,787 (incorporated herein by reference from Col. 61, line
51 through Col. 94, line 65) and U.S. Patent Appl. Publ. No.
2009/006082 (incorporated herein by reference from paragraph [0035]
through paragraph [0097]). DNL complexes comprised of trimeric,
tetrameric, pentameric, hexameric and other structures have been
reported in the above-cited issued patents.
[0010] In most preferred embodiments, the anti-cancer vaccine DNL
construct comprises a humanized, or chimeric LL1 anti-CD74 antibody
or antigen-binding fragment thereof comprising the light chain
variable complementarity-determining region (CDR) sequences CDR1
(RSSQSLVHRNGNTYLH; SEQ ID NO:1), CDR2 (TVSNRFS; SEQ ID NO:2), and
CDR3 (SQSSHVPPT; SEQ ID NO:3) and the heavy chain variable region
CDR sequences CDR1 (NYGVN; SEQ ID NO:4), CDR2 (WINPNTGEPTFDDDFKG;
SEQ ID NO:5), and CDR3 (SRGKNEAWFAY; SEQ ID NO:6). A humanized LL1
(hLL1) anti-CD74 antibody suitable for use in the claimed DNL
complexes is disclosed in U.S. Pat. No. 7,312,318, incorporated
herein by reference from Col. 35, line 1 through Col. 42, line 27
and FIG. 1 through FIG. 4. Alternatively, other anti-CD74
antibodies or antibodies against other APC- or DC-associated
antigens may be utilized.
[0011] The sequences of various CD20 xenoantigens suitable for use
in the anti-cancer vaccine DNL complex are known in the art, such
as the murine CD20 sequence (SEQ ID NO:7). Other CD20 amino acid
sequences of potential use are readily available to the skilled
artisan through such well-known public databases as the NCBI
protein database (see, e.g., NCBI Accession Nos. NP 031667; P19437;
AAA37394; BAE47068; ABA29631; BAD77809). Although the murine CD20
sequence is recited herein, the skilled artisan will realize that
CD20 amino acid sequences are known and readily available from a
wide variety of species and can be incorporated into the
anti-cancer vaccine DNL complex. However, the skilled artisan will
realize that other tumor-associated antigens (TAAs) are known in
the art and may be utilized in the DNL complexes to induce an
immune response against tumors expressing different TAAs. Known
TAAs of potential use include, but are not limited to, carbonic
anhydrase IX, alpha-fetoprotein, .alpha.-actinin-4, A3, antigen
specific for A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen,
CA125, CAMEL, CAP-1, CASP-8/m, CCL19, CCL21, CD1, CD1a, CD2, CD3,
CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21,
CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L,
CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70,
CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154,
CDC27, CDK-4/m, CDKN2A, colon-specific antigen-p (CSAp), CEA
(CEACAM5), CEACAM6, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M,
Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100,
GROB, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its
subunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1),
HSP70-2M, HST-2, Ia, IGF-1R, IFN-.gamma., IFN-.alpha., IFN-.beta.,
IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8,
IL-12, IL-15, IL-17, IL-18, IL-25, insulin growth factor-1 (IGF-1),
KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage
migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2,
NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2,
MUC3, MUC4, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, antigen specific
for PAM-4 antibody, placental growth factor, p53, prostatic acid
phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6, IL-25,
RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72,
tenascin, TRAIL receptors, TNF-.alpha., Tn antigen,
Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-B
fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b,
C5a, C5, an angiogenesis marker, bcl-2, bcl-6, Kras, cMET, an
oncogene marker and an oncogene product (see, e.g., Sensi et al.,
Clin Cancer Res 2006, 12:5023-32; Parmiani et al., J Immunol 2007,
178:1975-79; Novellino et al. Cancer Immunol Immunother 2005,
54:187-207). Xenoantigen amino acid sequences, such as murine
protein amino acid sequences, may be readily obtained from public
databases, such as the NCBI protein database.
[0012] The skilled artisan will further realize that other known
antibodies or antigen-binding fragments thereof may potentially be
incorporated into the anti-cancer vaccine DNL constructs. In
preferred embodiments, the antibody binds to an antigen expressed
by APCs, more preferably dendritic cells. A variety of antigens
associated with dendritic cells are known in the art, including but
not limited to CD209 (DC-SIGN), CD34, CD74, CD205, TLR 2 (toll-like
receptor 2), TLR 4, TLR 7, TLR 9, BDCA-2, BDCA-3, BDCA-4, and
HLA-DR. In preferred embodiments, the target antigen is CD74.
However, other types of target antigen are known to be associated
with dendritic cells and anti-cancer vaccine DNL constructs
incorporating antibodies that target any such alternative antigen
may be utilized in the claimed methods and compositions. In some
embodiments, the anti-cancer vaccine DNL constructs may comprise an
anti-CD74 antibody or antigen-binding fragment thereof and another
anti-dendritic cell antibody or fragment. Exemplary antibodies that
may be utilized in the anti-cancer vaccine DNL constructs include,
but are not limited to, hLL1 (anti-CD74, U.S. Pat. No. 7,312,318)
and hL243 (anti-HLA-DR, U.S. patent application Ser. No.
11/368,296) the Examples section of each incorporated herein by
reference.
[0013] The use of chimeric antibodies is preferred because they
possess human antibody constant region sequences and therefore do
not elicit as strong a human anti-mouse antibody (HAMA) response as
murine antibodies. The use of humanized antibodies is even more
preferred, in order to further reduce the possibility of inducing a
HAMA reaction. As discussed below, techniques for humanization of
murine antibodies by replacing murine framework and constant region
sequences with corresponding human antibody framework and constant
region sequences are well known in the art and have been applied to
numerous murine anti-cancer antibodies. Antibody humanization may
also involve the substitution of one or more human framework amino
acid residues with the corresponding residues from the parent
murine framework region sequences. As also discussed below,
techniques for production of human antibodies are also well known
and such antibodies may be incorporated into the subject
anti-cancer vaccine constructs.
[0014] In certain embodiments, the anti-cancer vaccine DNL
constructs may be administered in combination with at least one
therapeutic agent administered before, simultaneously with or after
the anti-cancer vaccine construct. In preferred embodiments, the
therapeutic agent is administered before the anti-cancer vaccine.
However, in alternative embodiments, the therapeutic agent may be
co-administered with or even conjugated to the DNL construct. Any
therapeutic agent known in the art, as discussed in more detail
below, may be utilized in conjunction with an anti-cancer vaccine
DNL construct, including but not limited to radionuclides,
immunomodulators, anti-angiogenic agents, cytokines, chemokines,
growth factors, hormones, drugs, prodrugs, enzymes,
oligonucleotides, siRNAs, pro-apoptotic agents, photoactive
therapeutic agents, cytotoxic agents, chemotherapeutic agents,
toxins, other antibodies or antigen binding fragments thereof.
[0015] In a preferred embodiment, the therapeutic agent is a
cytotoxic agent, such as a drug or a toxin. Also preferred, the
drug is selected from the group consisting of nitrogen mustards,
ethylenimine derivatives, alkyl sulfonates, nitrosoureas,
gemcitabine, triazenes, folic acid analogs, anthracyclines,
taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs,
antibiotics, enzyme inhibitors, epipodophyllotoxins, platinum
coordination complexes, vinca alkaloids, substituted ureas, methyl
hydrazine derivatives, adrenocortical suppressants, hormone
antagonists, endostatin, taxols, camptothecins, SN-38, doxorubicins
and their analogs, antimetabolites, alkylating agents,
antimitotics, anti-angiogenic agents, tyrosine kinase inhibitors,
mTOR inhibitors, heat shock protein (HSP90) inhibitors, proteosome
inhibitors, HDAC inhibitors, pro-apoptotic agents, methotrexate,
CPT-11, and a combination thereof.
[0016] In another preferred embodiment, the therapeutic agent is a
toxin selected from the group consisting of ricin, abrin, alpha
toxin, saporin, ribonuclease (RNase), DNase I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria
toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin and
combinations thereof. Or an immunomodulator selected from the group
consisting of a cytokine, a stem cell growth factor, a lymphotoxin,
a hematopoietic factor, a colony stimulating factor (CSF), an
interferon (IFN), a stem cell growth factor, erythropoietin,
thrombopoietin and a combinations thereof.
[0017] In other preferred embodiments, the therapeutic agent is a
radionuclide selected from the group consisting of .sup.111In,
.sup.177Lu, .sup.212Bi, .sup.213Bi, .sup.211At, .sup.62Cu,
.sup.67Cu, .sup.90Y, .sup.125I, .sup.131I, .sup.32P, .sup.33P,
.sup.47Sc, .sup.111Ag, .sup.67Ga, .sup.142Pr, .sup.153Sm,
.sup.161Tb, .sup.166Dy, .sup.166Ho, .sup.186Re, .sup.188Re,
.sup.189Re, .sup.212Pb, .sup.223Ra, .sup.225Ac, .sup.59Fe,
.sup.75Se, .sup.77As, .sup.89Sr, .sup.99Mo, .sup.105Rh, .sup.109Pd,
.sup.143Pr, .sup.149Pm, .sup.169Er, .sup.194Ir, .sup.198Au,
.sup.199Au, and .sup.211Pb, and combinations thereof. Also
preferred are radionuclides that substantially decay with
Auger-emitting particles. For example, Co-58, Ga-67, Br-80m,
Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161, Os-189m and
Ir-192. Decay energies of useful beta-particle-emitting nuclides
are preferably <1,000 keV, more preferably <100 keV, and most
preferably <70 keV. Also preferred are radionuclides that
substantially decay with generation of alpha-particles. Such
radionuclides include, but are not limited to Dy-152, At-211,
Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217,
Bi-213 and Fm-255. Decay energies of useful alpha-particle-emitting
radionuclides are preferably 2,000-10,000 keV, more preferably
3,000-8,000 keV, and most preferably 4,000-7,000 keV. Additional
potential radioisotopes of use include .sup.11C, .sup.13N,
.sup.15O, .sup.75Br, .sup.198Au, .sup.224Ac, .sup.126I, .sup.133I,
.sup.77Br, .sup.113mIn, .sup.95Ru, .sup.97Ru, .sup.103Ru,
.sup.105Ru, .sup.107Hg, .sup.203Hg, .sup.121mTe, .sup.122mTe,
.sup.125mTe, .sup.165Tm, .sup.167Tm, .sup.168Tm, .sup.197Pt,
.sup.109Pd, .sup.105Rh, .sup.142Pr, .sup.143Pr, .sup.161Tb,
.sup.166Ho, .sup.199Au, .sup.57Co, .sup.58Co, .sup.51Cr, .sup.59Fe,
.sup.75Se, .sup.201Tl, .sup.225Ac, .sup.76Br, .sup.169Yb, and the
like. In other embodiments the therapeutic agent is a photoactive
therapeutic agent selected from the group consisting of chromogens
and dyes.
[0018] Alternatively, the therapeutic agent is an enzyme selected
from the group consisting of malate dehydrogenase, staphylococcal
nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. Such enzymes may be used, for example, in
combination with prodrugs that are administered in relatively
non-toxic form and converted at the target site by the enzyme into
a cytotoxic agent. In other alternatives, a drug may be converted
into less toxic form by endogenous enzymes in the subject but may
be reconverted into a cytotoxic form by the therapeutic enzyme.
[0019] Although in preferred embodiments, the anti-cancer vaccine
DNL complexes are of use for therapy of multiple myeloma, the
skilled artisan will realize that a CD20/anti-CD74 construct may
potentially be of use for other types of diseases, such as other
forms of CD20.sup.+ cancer like B-cell lymphoma, B-cell leukemia,
acute lymphoblastic leukemia, chronic lymphocytic leukemia,
follicular lymphoma, mantle cell lymphoma, small lymphocytic
lymphoma, diffuse B-cell lymphoma, marginal zone lymphoma, Burkitt
lymphoma, Hodgkin's lymphoma or non-Hodgkin's lymphoma. Where a
tumor-associated xenoantigen other than CD20 is used, the skilled
artisan will realize that any type of cancer with an associated TAA
may be targeted using the claimed DNL complexes.
[0020] Still other embodiments relate to DNA sequences encoding
fusion proteins, such as antibody-DDD or xenoantigen-DDD fusion
proteins or antibody-AD or xenoantigen-AD fusion proteins, vectors
and host cells containing the DNA sequences, and methods of making
fusion proteins for the production of anti-cancer vaccine DNL
constructs. Related embodiments include fusion proteins of use for
making anti-cancer vaccine DNL constructs, antibody-DDD or
xenoantigen-DDD fusion proteins or antibody-AD or xenoantigen-AD
fusion proteins. In alternative embodiments, the subunit components
of the DNL complex may be formed by chemical cross-linking of, for
example, an antibody or antibody fragment and a DDD peptide, or a
CD20 xenoantigen and an AD peptide. For particular embodiments, the
fusion protein or chemically cross-linked conjugate may be attached
to a reporter moiety such as a diagnostic agent. A variety of
diagnostic agents are known in the art, such as radionuclides,
contrast agents, fluorescent agents, chemiluminescent agents,
bioluminescent agents, paramagnetic ions, enzymes and photoactive
diagnostic agents.
[0021] Preferably, the diagnostic agent is a radionuclide with an
energy between 20 and 4,000 keV or is a radionuclide selected from
the group consisting of .sup.110In, .sup.111In, .sup.177Lu,
.sup.18F, .sup.52Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga,
.sup.68Ga, .sup.86Y, .sup.90Y, .sup.89Zr, .sup.94mTc, .sup.94Tc,
.sup.99mTc, .sup.120I, .sup.123I, .sup.124I, .sup.125I, .sup.131I,
.sup.154-158Gd, .sup.32P, .sup.11C, .sup.13N, .sup.15O, .sup.186Re,
.sup.188Re, .sup.51Mn, .sup.52mMn, .sup.55Co, .sup.72As, .sup.75Br,
.sup.76Br, .sup.82mRb, .sup.83Sr, or other gamma-, beta-, or
positron-emitters.
[0022] Also preferred, the diagnostic agent is a paramagnetic ion,
such as chromium (III), manganese (II), iron (III), iron (II),
cobalt (II), nickel (II), copper (II), neodymium (III), samarium
(III), ytterbium (III), gadolinium (III), vanadium (II), terbium
(III), dysprosium (III), holmium (III) and erbium (III), or a
radiopaque material, such as barium, diatrizoate, ethiodized oil,
gallium citrate, iocarmic acid, iocetamic acid, iodamide,
iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol,
iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid,
iosulamide meglumine, iosemetic acid, iotasul, iotetric acid,
iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,
ipodate, meglumine, metrizamide, metrizoate, propyliodone, and
thallous chloride.
[0023] In still other embodiments, the diagnostic agent is a
fluorescent labeling compound selected from the group consisting of
fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin,
allophycocyanin, o-phthaldehyde and fluorescamine, a
chemiluminescent labeling compound selected from the group
consisting of luminol, isoluminol, an aromatic acridinium ester, an
imidazole, an acridinium salt and an oxalate ester, or a
bioluminescent compound selected from the group consisting of
luciferin, luciferase and acquorin.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0024] As used herein, the terms "a", "an" and "the" may refer to
either the singular or plural, unless the context otherwise makes
clear that only the singular is meant.
[0025] As used herein, the term "about" means plus or minus ten
percent (10%) of a value. For example, "about 100" would refer to
any number between 90 and 110.
[0026] An antibody refers to a full-length (i.e., naturally
occurring or formed by normal immunoglobulin gene fragment
recombinatorial processes) immunoglobulin molecule (e.g., an IgG
antibody) or an immunologically active, antigen-binding portion of
an immunoglobulin molecule, like an antibody fragment.
[0027] An antibody fragment is a portion of an antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, Fv, scFv and the like.
Regardless of structure, an antibody fragment binds with the same
antigen that is recognized by the intact antibody. Therefore the
term is used synonymously with "antigen-binding antibody fragment."
The term "antibody fragment" also includes isolated fragments
consisting of the variable regions, such as the "Fv" fragments
consisting of the variable regions of the heavy and light chains
and recombinant single chain polypeptide molecules in which light
and heavy variable regions are connected by a peptide linker ("scFv
proteins"). As used herein, the term "antibody fragment" does not
include portions of antibodies without antigen binding activity,
such as Fc fragments or single amino acid residues. Other antibody
fragments, for example single domain antibody fragments, are known
in the art and may be used in the claimed constructs. (See, e.g.,
Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol
Methods 281:161-75, 2003; Maass et al., J Immunol Methods
324:13-25, 2007).
[0028] The term antibody fusion protein may refer to a
recombinantly produced antigen-binding molecule in which one or
more of the same or different single-chain antibody or antibody
fragment segments with the same or different specificities are
linked. Valency of the fusion protein indicates how many binding
arms or sites the fusion protein has to a single antigen or
epitope; i.e., monovalent, bivalent, trivalent or multivalent. The
multivalency of the antibody fusion protein means that it can take
advantage of multiple interactions in binding to an antigen, thus
increasing the avidity of binding to the antigen. Specificity
indicates how many antigens or epitopes an antibody fusion protein
is able to bind; i.e., monospecific, bispecific, trispecific,
multispecific. Using these definitions, a natural antibody, e.g.,
an IgG, is bivalent because it has two binding arms but is
monospecific because it binds to one epitope. Monospecific,
multivalent fusion proteins have more than one binding site for an
epitope but only bind with one epitope. The fusion protein may
comprise a single antibody component, a multivalent or
multispecific combination of different antibody components or
multiple copies of the same antibody component. The fusion protein
may additionally comprise an antibody or an antibody fragment and a
therapeutic agent. Examples of therapeutic agents suitable for such
fusion proteins include immunomodulators and toxins. One preferred
toxin comprises a ribonuclease (RNase), preferably a recombinant
RNase. However, the term is not limiting and a variety of protein
or peptide effectors may be incorporated into a fusion protein. In
another non-limiting example, a fusion protein may comprise an AD
or DDD sequence for producing a DNL construct as discussed
below.
[0029] A chimeric antibody is a recombinant protein that contains
the variable domains including the complementarity determining
regions (CDRs) of an antibody derived from one species, preferably
a rodent antibody, while the constant domains of the antibody
molecule are derived from those of a human antibody. For veterinary
applications, the constant domains of the chimeric antibody may be
derived from that of other species, such as a cat or dog. A
humanized antibody is a recombinant protein in which the CDRs from
an antibody from one species; e.g., a rodent antibody, are
transferred from the heavy and light variable chains of the rodent
antibody into human heavy and light variable domains (e.g.,
framework region sequences). The constant domains of the antibody
molecule are derived from those of a human antibody. In certain
embodiments, a limited number of framework region amino acid
residues from the parent (rodent) antibody may be substituted into
the human antibody framework region sequences.
[0030] A human antibody is, e.g., an antibody obtained from
transgenic mice that have been "engineered" to produce specific
human antibodies in response to antigenic challenge. In this
technique, elements of the human heavy and light chain loci are
introduced into strains of mice derived from embryonic stem cell
lines that contain targeted disruptions of the endogenous murine
heavy chain and light chain loci. The transgenic mice can
synthesize human antibodies specific for particular antigens, and
the mice can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described by Green et al., Nature Genet. 7:13 (1994),
Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.
Immun. 6:579 (1994). A fully human antibody also can be constructed
by genetic or chromosomal transfection methods, as well as phage
display technology, all of which are known in the art. See for
example, McCafferty et al., Nature 348:552-553 (1990) for the
production of human antibodies and fragments thereof in vitro, from
immunoglobulin variable domain gene repertoires from unimmunized
donors. In this technique, antibody variable domain genes are
cloned in-frame into either a major or minor coat protein gene of a
filamentous bacteriophage, and displayed as functional antibody
fragments on the surface of the phage particle. Because the
filamentous particle contains a single-stranded DNA copy of the
phage genome, selections based on the functional properties of the
antibody also result in selection of the gene encoding the antibody
exhibiting those properties. In this way, the phage mimics some of
the properties of the B cell. Phage display can be performed in a
variety of formats, for review, see e.g. Johnson and Chiswell,
Current Opinion in Structural Biology 3:5564-571 (1993). Human
antibodies may also be generated by in vitro activated B cells. See
U.S. Pat. Nos. 5,567,610 and 5,229,275, the Examples section of
which are incorporated herein by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1A. Specific binding of hLL1 on human blood DC subsets,
B cells, and monocytes. Gating strategy for the different APC
subsets.
[0032] FIG. 1B. Specific binding of hLL1 on human blood DC subsets,
B cells, and monocytes. CD74 expression in APCs.
[0033] FIG. 1C. Specific binding of hLL1 on human blood DC subsets,
B cells, and monocytes. The binding efficiency of hLL1 on the
cells. The numbers represent mean fluorescence intensity.
[0034] FIG. 2A. CD74 expression in and binding efficiency of hLL1
with human monocyte-derived immature vs mature DCs. The human
monocyte-derived DCs (day 5 after culture in the presence of
hGM-CSF and hIL-4) were stained with FITC-labeled anti-CD74
antibody or AlexaFluor488-labeled hLL1, in combination with the
staining with fluorescence-labeled mAbs against HLA-DR and CD83.
The HLA-DR-positive cells are gated and analyzed. (A) CD74
expression in immature and LPS-matured DCs.
[0035] FIG. 2B. CD74 expression in and binding efficiency of hLL1
with human monocyte-derived immature vs mature DCs. hLL1 binding
with immature vs LPS-matured DCs.
[0036] FIG. 2C. CD74 expression in and binding efficiency of hLL1
with human monocyte-derived immature vs mature DCs. Comparison of
expression of CD83, HLA-DR, CD74 and hLL1 binding in immature and
mature DCs. CD74 expression in and binding efficiency of hLL1 with
human monocyte-derived immature vs mature DCs.
[0037] FIG. 3A. Side-by-side comparison of the cytotoxic effect of
hLL1 on B cell malignant Daudi cells and normal DCs. Comparison of
the effect of hLL1 on Daudi and DCs.
[0038] FIG. 3B. Side-by-side comparison of the cytotoxic effect of
hLL1 on B cell malignant Daudi cells and normal DCs. Effect of hLL1
on cell viability of DCs in an extended doses.
[0039] FIG. 3C. Side-by-side comparison of the cytotoxic effect of
hLL1 on B cell malignant Daudi cells and normal DCs. The cytotoxic
effect of hLL1 on Daudi cells.
[0040] FIG. 3D. Side-by-side comparison of the cytotoxic effect of
hLL1 on B cell malignant Daudi cells and normal DCs. The
microscopic image shows no effect of hLL1 on DC viability.
[0041] FIG. 4A. Moderate enhancement of DC constitutive maturation
by hLL1. The HLA-DR positive cell populations were gated from day 5
DCs derived from human monocytes in the presence of hGM-CSF and
hIL-4. The expression of antigen-presenting molecule HLA-DR,
costimulatory molecule CD54 and CD86 was measured by flow
cytometry.
[0042] FIG. 4B. Moderate enhancement of DC constitutive maturation
by hLL1. Expression levels of antigen-presenting molecule HLA-DR,
costimulatory molecule CD54 and CD86.
[0043] FIG. 5A. No significant influence of hLL1 on DC-mediated T
cell proliferation. The hLL1-treated DCs were co-cultured with CF
SE-labeled allogeneic PBMCs for 8 days. The expanded T cells were
stained with Percp-conjugated mAb against CD4. The cell
proliferation of total T cells, CD4+ and CD4- T cells were
analyzed.
[0044] FIG. 5B. No significant influence of hLL1 on DC-mediated T
cell proliferation. The hLL1-treated DCs were co-cultured with CF
SE-labeled allogeneic PBMCs for 11 days. The expanded T cells were
stained with Percp-conjugated mAb against CD4. The cell
proliferation of total T cells, CD4+ and CD4- T cells were
analyzed.
[0045] FIG. 6C. Polarization of naive CD4+ T cells by hLL1-treated
DCs favoring the differentiation toward Th1 effector cells. Naive
CD4+ T cells isolated from human PBMCs using the depletion column
with magnetic beads (MACS) were co-cultured with hLL1-treated
allogeneic DCs. After different time points (day 11, 13, 18), the
cells were harvested, stimulated with PMA and ionomycin, and
analyzed with intracellular cytokine staining with
fluorescence-labeled hIFN-gamma and hIL-4 antibodies. Th1/Th2/Th0
cells populations were gated and analyzed. The flow cytokine
production in T cells stimulated by hLL1-treated DCs or by
GAH-cross-linked hLL1-treated DCs was determined. The data of Th1
responses in two donors, in the absence or presence of
cross-linking by GAH, at different days after DC/T coculture, are
shown.
[0046] FIG. 6D. Polarization of naive CD4+ T cells by hLL1-treated
DCs favoring the differentiation toward Th1 effector cells. The
dose-effect curve for increasing Th1 populations by hLL1.
[0047] Vaccines for Therapy of Multiple Myeloma and Other
Cancers
[0048] CD20 is normally expressed in cells of B cell lineage. It
was recently reported that CD20 is expressed in a small population
of MM cells isolated from MM cell lines or clinical specimens,
which do not express the characteristic plasma cell surface antigen
CD138 but have a highly clonogenic potential and are resistant to
multiple clinical anti-myeloma drugs (Matsui et al., Blood 2004,
103:2332-6; Matsui et al., Cancer Res. 2008, 68:190-7). These
CD2O+CD138-cells are capable of clonogenic growth in vitro and in a
3-D culture model (Kirshner et al., Blood 2008, 112:2935-45), and
of differentiation into MM cells in vitro and in the engrafted
NOD/SCID mouse model during both primary and secondary
transplantation. It has thus been suggested that these
CD138.sup.negCD20.sup.+ cells represent the putative multiple
myeloma cancer stem cells.
[0049] Immunization with Xenoantigen as a Means for Breaking Immune
Tolerance for Cancer Immunotherapy.
[0050] Many tumor-associated Ags (TAAs) represent tissue
differentiation Ags which are not inherently immunogenic. T cells
that recognize these TAAs/self-Ags with high avidity are either
clonally deleted in the thymus or anergized in the periphery.
However, immunization with xenoantigen has been shown to be capable
of overcoming the immune tolerance against the homologous self-Ag.
In a phase I clinical trial, eleven of 21 prostate cancer patients
immunized with dendritic cells pulsed with recombinant mouse PAP
developed type I T-cell proliferative responses to the homologous
self-Ag, and 6 patients had clinical stabilization of their
previously progressing prostate cancer (Fong et al., J Immunol.
2001, 167(12):7150-6). These results demonstrate that xenoantigen
immunization can break tolerance to a self-Ag in humans, resulting
in a clinically significant antitumor effect.
[0051] CD20 as a Target for Immunotherapy and Vaccination Against
MM.
[0052] As stated above, CD20 is a hallmark of MM cancer stem cells.
As a self-antigen which is expressed on normal B cells at most
stages of differentiation, it is theoretically difficult to be
targeted by vaccine strategies due to immune tolerance. However,
successful vaccination has been achieved by a xenogeneic DNA
vaccine against CD20 in a tumor challenge model of B-cell lymphoma.
Although autoimmunity against B cells could be induced by a vaccine
targeting CD20, it should not cause a large problem because the B
cell pool is not a vital and critical tissue and can be replenished
from its lineage progenitor. Based on these considerations, a
therapeutic vaccine targeting CD20 would be effective in selective
eradication of MM cancer stem cells.
[0053] Monoclonal Anti-CD20 Antibody as a Potential Modality for
Eradication of MM Stem Cells.
[0054] The discovery of CD20+ MM progenitor cells has prompted
several small clinical trials to test the efficacy of rituximab, an
anti-CD20 monoclonal antibody, in MM patients. As reviewed by
Kapoor et al. (Br J Haematol. 2008, 141:135-48), anti-CD20 therapy
with rituximab elicits a partial response in approximately 10% of
CD20+ patients with multiple myeloma. In addition, there is
preliminary evidence of disease stabilization in 50-57% of CD20+
patients for a period of 10-27 months (Kapoor et al., (Br J
Haematol. 2008, 141:135-48). Furthermore, a case report by Bergua
et al. (Leukemia. 2008, 22:1082-3) where rituximab was used in
combination with chemotherapy demonstrated no minimal residual
disease found after treatment, either in immunophenotype, bone
marrow aspiration or biopsy, and the CD20+ plasma cells
disappeared. These results justify large scale clinical trials to
establish the role of this strategy in the treatment of myeloma.
The vaccine approach, due to its induction of CTL response, would
be expected to supplement the monoclonal antibody therapy against
CD20 MM stem cells.
[0055] In Vivo Targeting of Antigens to Dendritic Cells and Other
Antigen-Presenting Cells as an Efficient Strategy for Vaccination
and Breaking Immune Tolerance.
[0056] As the professional antigen-presenting cells, dendritic
cells (DCs) play a pivotal role in orchestrating innate and
adaptive immunity, and have been harnessed to create effective
vaccines (Vulink et al., Adv Cancer Res. 2008, 99:363-407; O'Neill
et al., Mol Biotechnol. 2007, 36:131-41). In vivo targeting of
antigens to DCs represents a promising approach for DC-based
vaccination, as it can bypass the laborious and expensive ex vivo
antigen loading and culturing, and facilitate large-scale
application of DC-based immunotherapy (Tacken et al., Nat Rev
Immunol. 2007, 7:790-802). More significantly, in vivo DC targeting
vaccination is more efficient in eliciting anti-tumor immune
response, and more effective in controlling tumor growth in animal
models (Kretz-Rommel et al., J Immunother 2007, 30:715-726). In
addition to DCs, B cells are another type of potent
antigen-presenting cells capable of priming Th1/Th2 cells (Morris
et al, J Immunol. 1994, 152:3777-3785; Constant, J Immunol. 1999,
162:5695-5703) and activating CD8 T cells via cross-presentation
(Heit et al., J Immunol. 2004, 172:1501-1507; Yan et al., Int
Immunol. 2005, 17:869-773). It was recently reported that in vivo
targeting of antigens to B cells breaks immune tolerance of MUC1
(Ding et al., Blood 2008, 112:2817-25).
[0057] CD74 as a Potential Receptor for Targeting Vaccination.
[0058] Some receptors expressed on DCs have been used as the
targets for in vivo antigen targeting, such as the mannose receptor
(He et al., J. Immunol 2007, 178, 6259-6267; Ramakrishna et al., J.
Immunol. 2004, 172, 2845-2852) CD205 (Bonifaz et al., J Exp Med.
2004, 199:815-24), DC-SIGN (Tacken et al., Blood 2005,
106:1278-85), and LOX1 (Deineste et al., Immunity 2002, 17,
353-362), etc. CD74 is a type II integral membrane protein
essential for proper MHC II folding and targeting of MHC II-CD74
complex to the endosomes (Stein et al., Clin Cancer Res. 2007,
13:5556s-5563s; Matza et al., Trends Immunol. 2003, 24(5):264-8).
CD74 expression is not restricted to DCs, but is found in almost
all antigen-presenting cells (Freudenthal et al., Proc Natl Acad
Sci USA. 1990, 87:7698-702; Clark et al., J Immunol. 1992,
148(11):3327-35). The wide expression of CD74 in APCs may offer
some advantages over sole expression in myeloid DCs, as targeting
of antigens to other APCs like B cells has been reported to break
immune tolerance (Ding et al., Blood 2008, 112:2817-25), and
targeting to plasmacytoid DCs cross-presents antigens to naive CD8
T cells. More importantly, CD74 is also expressed in follicular DCs
(Clark et al., J Immunol. 1992, 148(11):3327-35), a DC subset
critical for antigen presentation to B cells (Tew et al., Immunol
Rev. 1997, 156:39-52). This expression profile makes CD74 an
excellent candidate for in vivo targeting vaccination.
[0059] Humanized Anti-CD74 Monoclonal Antibody hLL1 as a Novel
Targeting Tool with Dock-and-Lock Technology Platform.
[0060] The DNL technology, discussed in more detail below, provides
a means to link virtually any selected effector moieties into a
covalent or noncovalent complex (Goldenberg et al., J Nucl Med.
2008, 49:158-63; Rossi et al., Proc Natl Acad Sci USA. 2006,
103(18):6841-6). The DNL method has generated several trivalent,
bispecific, binding proteins containing Fab fragments reacting with
carcinoembryonic antigen (CEA), and has been successfully used in
improved cancer imaging and radioimmunotherapy through a
pretargeting strategy (Goldenberg et al., J Nucl Med. 2008,
49:158-63).
[0061] hLL1 is a humanized monoclonal antibody against human CD74
(Leung et al., Mol Immunol. 1995, 32:1416-1427; Losman et al.,
Cancer 1997, 80:2660-2666; Stein et al., Blood 2004, 104:3705-11).
This MAb, in the presence of cross-linking by a second antibody,
exhibits cytotoxicity against B cell malignancies. The naked hLL1
is also capable of controlling tumor growth in a MM mouse model.
However, our recent data demonstrate that hLL1, in the presence or
absence of cross-linking, has no cytotoxicity against human
monocyte-derived DCs. But, our preliminary data shows hLL1 could
efficiently bind different subsets of blood DCs and B cells. It
also could moderately induce DC maturation and polarize naive T
cell differentiation toward Th1 effector cells, suggesting it has
some adjuvant activity and may be a good candidate for use as a
targeting tool. This makes it possible and feasible to construct a
DNL-based tumor vaccine targeted to APCs through the DNL-carried
hLL1 antibody.
[0062] Immunotherapy for Selective Elimination of Cancer Stem
Cells.
[0063] Cancer stem cells are capable of self-renewal, possess the
ability for unlimited proliferation, and are resistant to multiple
therapeutic approaches. A pressing and interesting question is
raised if cancer stem cells are sensitive to immunotherapy. In the
case of leukemia, it was reported that CD8(+) minor
histocompatibility antigen-specific cytotoxic T lymphocyte clones
could eliminate human acute myeloid leukemia stem cells (Bonnet et
al., Proc Natl Acad Sci U.S.A. 1999, 96:8639-8644). More recently,
Rosinski et al. (Blood 2008, 111:4817-26) reported that
DDX36-encoded H-Y epitope is expressed by leukemic stem cells and
can be recognized by the DDX36-specific CTLs, which can prevent
engraftment of human acute leukemia in NOD/SCID mice (Rosinski et
al. Blood 2008, 111:4817-26). Another report indicates that
engraftment of mHA myeloid leukemia stem cells in
NOD/SCID.gamma.c.sup.null mice was completely inhibited by in vitro
preincubation with the mHA-specific CTL clone (Kawase et al., Blood
2007, 110:1055-63). These results highlight the prospects that
immunotherapy would be a potentially effective approach for
selective elimination of cancer stem cells including MM stem cells,
which would be required for achieving long-term control or even
cure of this malignancy.
[0064] Dock and Lock (DNL) Method
[0065] The DNL method exploits specific protein/protein
interactions that occur between the regulatory (R) subunits of
cAMP-dependent protein kinase (PKA) and the anchoring domain (AD)
of A-kinase anchoring proteins (AKAPs) (Baillie et al., FEBS
Letters. 2005; 579:3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.
2004; 5:959). PKA, which plays a central role in one of the best
studied signal transduction pathways triggered by the binding of
the second messenger cAMP to the R subunits, was first isolated
from rabbit skeletal muscle in 1968 (Walsh et al., J. Biol. Chem.
1968; 243:3763). The structure of the holoenzyme consists of two
catalytic subunits held in an inactive form by the R subunits
(Taylor, J. Biol. Chem. 1989; 264:8443). Isozymes of PKA are found
with two types of R subunits (RI and MI), and each type has a and
isoforms (Scott, Pharmacol. Ther. 1991; 50:123). The R subunits
have been isolated only as stable dimers and the dimerization
domain has been shown to consist of the first 44 amino-terminal
residues (Newlon et al., Nat. Struct. Biol. 1999; 6:222). Binding
of cAMP to the R subunits leads to the release of active catalytic
subunits for a broad spectrum of serine/threonine kinase
activities, which are oriented toward selected substrates through
the compartmentalization of PKA via its docking with AKAPs (Scott
et al., J. Biol. Chem. 1990; 265; 21561)
[0066] Since the first AKAP, microtubule-associated protein-2, was
characterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA.
1984; 81:6723), more than 50 AKAPs that localize to various
sub-cellular sites, including plasma membrane, actin cytoskeleton,
nucleus, mitochondria, and endoplasmic reticulum, have been
identified with diverse structures in species ranging from yeast to
humans (Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The
AD of AKAPs for PKA is an amphipathic helix of 14-18 residues (Carr
et al., J. Biol. Chem. 1991; 266:14188). The amino acid sequences
of the AD are quite varied among individual AKAPs, with the binding
affinities reported for RII dimers ranging from 2 to 90 nM (Alto et
al., Proc. Natl. Acad. Sci. USA. 2003; 100:4445). Interestingly,
AKAPs will only bind to dimeric R subunits. For human RII.alpha.,
the AD binds to a hydrophobic surface formed by the 23
amino-terminal residues (Colledge and Scott, Trends Cell Biol.
1999; 6:216). Thus, the dimerization domain and AKAP binding domain
of human RII.alpha. are both located within the same N-terminal 44
amino acid sequence (Newlon et al., Nat. Struct. Biol. 1999; 6:222;
Newlon et al., EMBO J. 2001; 20:1651), which is termed the DDD
herein.
[0067] DDD of Human RII.alpha. and AD of AKAPs as Linker
Modules
[0068] We have developed a platform technology to utilize the DDD
of human RII.alpha. and the AD of a AKAPs as an excellent pair of
linker modules for docking any two entities, referred to hereafter
as A and B, into a noncovalent complex, which could be further
locked into a stably tethered structure through the introduction of
cysteine residues into both the DDD and AD at strategic positions
to facilitate the formation of disulfide bonds. The general
methodology of the "dock-and-lock" approach is as follows. Entity A
is constructed by linking a DDD sequence to a precursor of A,
resulting in a first component hereafter referred to as a. Because
the DDD sequence would effect the spontaneous formation of a dimer,
A would thus be composed of a.sub.2. Entity B is constructed by
linking an AD sequence to a precursor of B, resulting in a second
component hereafter referred to as b. The dimeric motif of DDD
contained in a.sub.z will create a docking site for binding to the
AD sequence contained in b, thus facilitating a ready association
of a.sub.2 and b to form a binary, trimeric complex composed of
a.sub.2b. This binding event is made irreversible with a subsequent
reaction to covalently secure the two entities via disulfide
bridges, which occurs very efficiently based on the principle of
effective local concentration because the initial binding
interactions should bring the reactive thiol groups placed onto
both the DDD and AD into proximity (Chmura et al., Proc. Natl.
Acad. Sci. USA. 2001; 98:8480) to ligate site-specifically.
[0069] In preferred embodiments, the anti-cancer vaccine DNL
constructs are based on a variation of the a.sub.2b structure, in
which each heavy chain of an anti-CD74 antibody or F(ab').sub.2 or
F(ab).sub.2 antibody fragment, such as an hLL1 antibody or
fragment, is attached at its C-terminal end to one copy of an AD
moiety. Since there are two heavy chains per antibody or fragment,
there are two AD moieties per antibody or fragment. A CD20
xenoantigen is attached to a complementary DDD moiety. After
dimerization of DDD moieties, each DDD dimer binds to one of the AD
moieties attached to the IgG antibody or F(ab').sub.2 or
F(ab).sub.2 fragment, resulting in a stoichiometry of four CD20
xenoantigens per IgG or F(ab').sub.2 or F(ab).sub.2 unit. However,
the skilled artisan will realize that alternative complexes may be
utilized, such as attachment of the CD20 to the AD sequence and
attachment of the anti-CD74 MAb or fragment to the DDD moiety,
resulting in a different stoichiometry of effector moieties. For
example, by attaching a DDD sequence to the C-terminal end of each
heavy chain of an IgG antibody or F(ab').sub.2 fragment, and
attaching an AD sequence to the CD20 xenoantigen, a DNL complex may
be constructed that comprises one CD20 molecule and one anti-CD74
antibody or fragment.
[0070] By attaching the DDD and AD away from the functional groups
of the two precursors, such site-specific ligations are expected to
preserve the original activities of the two precursors. This
approach is modular in nature and potentially can be applied to
link, site-specifically and covalently, a wide range of
substances.
[0071] In preferred embodiments, as illustrated in the Examples
below, the effector moiety is a protein or peptide, which can be
linked to a DDD or AD unit to form a fusion protein or peptide. A
variety of methods are known for making fusion proteins, including
nucleic acid synthesis, hybridization and/or amplification to
produce a synthetic double-stranded nucleic acid encoding a fusion
protein of interest. Such double-stranded nucleic acids may be
inserted into expression vectors for fusion protein production by
standard molecular biology techniques (see, e.g. Sambrook et al.,
Molecular Cloning, A laboratory manual, 2.sup.nd Ed, Cold Spring
Harbor Press, Cold Spring Harbor, N.Y., 1989). In such preferred
embodiments, the AD and/or DDD moiety may be attached to either the
N-terminal or C-terminal end of an effector protein or peptide.
However, the skilled artisan will realize that the site of
attachment of an AD or DDD moiety to an effector moiety may vary,
depending on the chemical nature of the effector moiety and the
part(s) of the effector moiety involved in its physiological
activity. For example, although an AD or DDD moiety may be attached
to either the N- or C-terminal end of an antibody or antibody
fragment while retaining antigen-binding activity, attachment to
the C-terminal end positions the AD or DDD moiety farther from the
antigen-binding site and appears to result in a stronger binding
interaction (e.g., Chang et al., Clin Cancer Res 2007,
13:5586s-91s). Site-specific attachment of a variety of effector
moieties may be also performed using techniques known in the art,
such as the use of bivalent cross-linking reagents and/or other
chemical conjugation techniques.
[0072] Antibodies and Antibody Fragments
[0073] In various embodiments, antibodies or antigen-binding
fragments of antibodies may be incorporated into the anti-cancer
vaccine DNL complex. Antigen-binding antibody fragments are well
known in the art, such as F(ab').sub.2, F(ab).sub.2, Fab', Fab, Fv,
scFv and the like, and any such known fragment may be used. As used
herein, an antigen-binding antibody fragment refers to any fragment
of an antibody that binds with the same antigen that is recognized
by the intact or parent antibody. Techniques for preparing AD
and/or DDD conjugates of virtually any antibody or fragment of
interest are known (e.g., U.S. Pat. No. 7,527,787).
[0074] An antibody or fragment thereof may be used which is not
conjugated to a therapeutic agent--referred to as a "naked"
antibody or fragment thereof. In alternative embodiments,
antibodies or fragments may be conjugated to one or more
therapeutic and/or diagnostic agents. A wide variety of such
therapeutic and diagnostic agents are known in the art, as
discussed in more detail below, and any such known therapeutic or
diagnostic agent may be used.
[0075] Techniques for preparing monoclonal antibodies against
virtually any target antigen, such as CD74, are well known in the
art. See, for example, Kohler and Milstein, Nature 256:495 (1975),
and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1,
pages 2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly, monoclonal
antibodies can be obtained by injecting mice with a composition
comprising an antigen, removing the spleen to obtain B-lymphocytes,
fusing the B-lymphocytes with myeloma cells to produce hybridomas,
cloning the hybridomas, selecting positive clones which produce
antibodies to the antigen, culturing the clones that produce
antibodies to the antigen, and isolating the antibodies from the
hybridoma cultures.
[0076] MAbs can be isolated and purified from hybridoma cultures by
a variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography.
See, for example, Coligan at pages 2.7.1-2.7.12 and pages
2.9.1-2.9.3. Also, see Baines et al., "Purification of
Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10,
pages 79-104 (The Humana Press, Inc. 1992). After the initial
raising of antibodies to the immunogen, the antibodies can be
sequenced and subsequently prepared by recombinant techniques.
Humanization and chimerization of murine antibodies and antibody
fragments are well known to those skilled in the art. The use of
antibody components derived from humanized, chimeric or human
antibodies obviates potential problems associated with the
immunogenicity of murine constant regions.
[0077] Chimeric Antibodies
[0078] A chimeric antibody is a recombinant protein in which the
variable regions of a human antibody have been replaced by the
variable regions of, for example, a mouse antibody, including the
complementarity-determining regions (CDRs) of the mouse antibody.
Chimeric antibodies exhibit decreased immunogenicity and increased
stability when administered to a subject. General techniques for
cloning murine immunoglobulin variable domains are disclosed, for
example, in Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833
(1989). Techniques for constructing chimeric antibodies are well
known to those of skill in the art. As an example, Leung et al.,
Hybridoma 13:469 (1994), produced an LL2 chimera by combining DNA
sequences encoding the V.sub..kappa. and V.sub.H domains of murine
LL2, an anti-CD22 monoclonal antibody, with respective human
.kappa. and IgG.sub.1 constant region domains.
[0079] Humanized Antibodies
[0080] Techniques for producing humanized MAbs are well known in
the art (see, e.g., Jones et al., Nature 321:522 (1986), Riechmann
et al., Nature 332:323 (1988), Verhoeyen et al., Science 239:1534
(1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992),
Sandhu, Crit. Rev. Biotech. 12:437 (1992), and Singer et al., J.
Immun. 150:2844 (1993)). A chimeric or murine monoclonal antibody
may be humanized by transferring the mouse CDRs from the heavy and
light variable chains of the mouse immunoglobulin into the
corresponding variable domains of a human antibody. The mouse
framework regions (FR) in the chimeric monoclonal antibody are also
replaced with human FR sequences. As simply transferring mouse CDRs
into human FRs often results in a reduction or even loss of
antibody affinity, additional modification might be required in
order to restore the original affinity of the murine antibody. This
can be accomplished by the replacement of one or more human
residues in the FR regions with their murine counterparts to obtain
an antibody that possesses good binding affinity to its epitope.
See, for example, Tempest et al., Biotechnology 9:266 (1991) and
Verhoeyen et al., Science 239:1534 (1988). Generally, those human
FR amino acid residues that differ from their murine counterparts
and are located close to or touching one or more CDR amino acid
residues would be candidates for substitution.
[0081] A humanized LL1 (hLL1) anti-CD74 antibody is disclosed in
U.S. Pat. No. 7,312,318, incorporated herein by reference from Col.
35, line 1 through Col. 42, line 27 and FIG. 1 through FIG. 4.
[0082] Human Antibodies
[0083] Methods for producing fully human antibodies using either
combinatorial approaches or transgenic animals transformed with
human immunoglobulin loci are known in the art (e.g., Mancini et
al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005,
Comb. Chem. High Throughput Screen. 8:117-26; Brekke and Loset,
2003, Curr. Opin. Phamacol. 3:544-50). A fully human antibody also
can be constructed by genetic or chromosomal transfection methods,
as well as phage display technology, all of which are known in the
art. See for example, McCafferty et al., Nature 348:552-553 (1990).
Such fully human antibodies are expected to exhibit even fewer side
effects than chimeric or humanized antibodies and to function in
vivo as essentially endogenous human antibodies. In certain
embodiments, the claimed methods and procedures may utilize human
antibodies produced by such techniques.
[0084] In one alternative, the phage display technique may be used
to generate human antibodies (e.g., Dantas-Barbosa et al., 2005,
Genet. Mol. Res. 4:126-40). Human antibodies may be generated from
normal humans or from humans that exhibit a particular disease
state, such as cancer (Dantas-Barbosa et al., 2005). The advantage
to constructing human antibodies from a diseased individual is that
the circulating antibody repertoire may be biased towards
antibodies against disease-associated antigens.
[0085] In one non-limiting example of this methodology,
Dantas-Barbosa et al. (2005) constructed a phage display library of
human Fab antibody fragments from osteosarcoma patients. Generally,
total RNA was obtained from circulating blood lymphocytes (Id.).
Recombinant Fab were cloned from the .mu., .gamma. and .kappa.
chain antibody repertoires and inserted into a phage display
library (Id.). RNAs were converted to cDNAs and used to make Fab
cDNA libraries using specific primers against the heavy and light
chain immunoglobulin sequences (Marks et al., 1991, J Mol. Biol.
222:581-97). Library construction was performed according to
Andris-Widhopf et al. (2000, In: Phage Display Laboratory Manual,
Barbas et al. (eds), 1.sup.st edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22). The
final Fab fragments were digested with restriction endonucleases
and inserted into the bacteriophage genome to make the phage
display library. Such libraries may be screened by standard phage
display methods, as known in the art (see, e.g., Pasqualini and
Ruoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart.
J. Nucl. Med. 43:159-162).
[0086] Phage display can be performed in a variety of formats, for
their review, see e.g. Johnson and Chiswell, Current Opinion in
Structural Biology 3:5564-571 (1993). Human antibodies may also be
generated by in vitro activated B-cells. See U.S. Pat. Nos.
5,567,610 and 5,229,275, incorporated herein by reference in their
entirety. The skilled artisan will realize that these techniques
are exemplary and any known method for making and screening human
antibodies or antibody fragments may be utilized.
[0087] In another alternative, transgenic animals that have been
genetically engineered to produce human antibodies may be used to
generate antibodies against essentially any immunogenic target,
using standard immunization protocols. Methods for obtaining human
antibodies from transgenic mice are disclosed by Green et al.,
Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994),
and Taylor et al., Int. Immun. 6:579 (1994). A non-limiting example
of such a system is the XenoMouse.RTM. (e.g., Green et al., 1999,
J. Immunol. Methods 231:11-23) from Abgenix (Fremont, Calif.). In
the XenoMouse.RTM. and similar animals, the mouse antibody genes
have been inactivated and replaced by functional human antibody
genes, while the remainder of the mouse immune system remains
intact.
[0088] The XenoMouse.RTM. was transformed with germline-configured
YACs (yeast artificial chromosomes) that contained portions of the
human IgH and Igkappa loci, including the majority of the variable
region sequences, along accessory genes and regulatory sequences.
The human variable region repertoire may be used to generate
antibody producing B-cells, which may be processed into hybridomas
by known techniques. A XenoMouse.RTM. immunized with a target
antigen will produce human antibodies by the normal immune
response, which may be harvested and/or produced by standard
techniques discussed above. A variety of strains of XenoMouse.RTM.
are available, each of which is capable of producing a different
class of antibody. Transgenically produced human antibodies have
been shown to have therapeutic potential, while retaining the
pharmacokinetic properties of normal human antibodies (Green et
al., 1999). The skilled artisan will realize that the claimed
compositions and methods are not limited to use of the
XenoMouse.RTM. system but may utilize any transgenic animal that
has been genetically engineered to produce human antibodies.
[0089] Antibody Fragments
[0090] Antibody fragments which recognize specific epitopes can be
generated by known techniques. Antibody fragments are antigen
binding portions of an antibody, such as F(ab).sub.2, Fab',
F(ab).sub.2, Fab, Fv, sFv and the like. F(ab').sub.2 fragments can
be produced by pepsin digestion of the antibody molecule and Fab'
fragments can be generated by reducing disulfide bridges of the
F(ab').sub.2 fragments. Alternatively, Fab' expression libraries
can be constructed (Huse et al., 1989, Science, 246:1274-1281) to
allow rapid and easy identification of monoclonal Fab' fragments
with the desired specificity. F(ab).sub.2 fragments may be
generated by papain digestion of an antibody and Fab fragments
obtained by disulfide reduction.
[0091] A single chain Fv molecule (scFv) comprises a VL domain and
a VH domain. The VL and VH domains associate to form a target
binding site. These two domains are further covalently linked by a
peptide linker (L). Methods for making scFv molecules and designing
suitable peptide linkers are described in U.S. Pat. No. 4,704,692,
U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, "Single Chain
Fvs." FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker,
"Single Chain Antibody Variable Regions," TIBTECH, Vol 9:132-137
(1991).
[0092] Techniques for producing single domain antibodies are also
known in the art, as disclosed for example in Cossins et al. (2006,
Prot Express Purif 51:253-259). Single domain antibodies (VHH) may
be obtained, for example, from camels, alpacas or llamas by
standard immunization techniques. (See, e.g., Muyldermans et al.,
TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75,
2003; Maass et al., J Immunol Methods 324:13-25, 2007). The VHH may
have potent antigen-binding capacity and can interact with novel
epitopes that are inaccessible to conventional VH-VL pairs.
(Muyldermans et al., 2001). Alpaca serum IgG contains about 50%
camelid heavy chain only IgG antibodies (HCAbs) (Maass et al.,
2007). Alpacas may be immunized with known antigens, such as
TNF-.alpha., and VHHs can be isolated that bind to and neutralize
the target antigen (Maass et al., 2007). PCR primers that amplify
virtually all alpaca VHH coding sequences have been identified and
may be used to construct alpaca VHH phage display libraries, which
can be used for antibody fragment isolation by standard biopanning
techniques well known in the art (Maass et al., 2007).
[0093] An antibody fragment can be prepared by proteolytic
hydrolysis of the full length antibody or by expression in E. coli
or another host of the DNA coding for the fragment. An antibody
fragment can be obtained by pepsin or papain digestion of full
length antibodies by conventional methods. These methods are
described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and
4,331,647 and references contained therein. Also, see Nisonoff et
al., Arch Biochem. Biophys. 89:230 (1960); Porter, Biochem. J.
73:119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1,
page 422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10
and 2.10-2.10.4.
[0094] Known Antibodies
[0095] In certain embodiments antibodies against other antigenic
targets besides CD74 may be incorporated into the anti-cancer
vaccine DNL complex. A wide variety of antibodies against
tumor-associated antigens are known and may be obtained from
commercial sources. For example, a number of antibody secreting
hybridoma lines are available from the American Type Culture
Collection (ATCC, Manassas, Va.). See, e.g., U.S. Pat. Nos.
7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;
7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;
7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;
6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;
6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;
6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;
6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;
6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;
6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;
6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;
6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;
6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;
6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;
6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;
6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;
6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;
6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;
6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;
6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310;
6,444,206' 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;
6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;
6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;
6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;
6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;
6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;
5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;
5,677,136; 5,587,459; 5,443,953, 5,525,338. These are exemplary
only and a wide variety of other antibodies and their hybridomas
are known in the art. The skilled artisan will realize that
antibody sequences or antibody-secreting hybridomas against almost
any tumor-associated antigen may be obtained by a simple search of
the ATCC, NCBI and/or USPTO databases for antibodies against a
selected disease-associated target of interest. The antigen binding
domains of the cloned antibodies may be amplified, excised, ligated
into an expression vector, transfected into an adapted host cell
and used for protein production, using standard techniques well
known in the art.
[0096] Amino Acid Substitutions
[0097] In certain embodiments, the disclosed methods and
compositions may involve production and use of proteins or peptides
with one or more substituted amino acid residues. For example, as
discussed in the working Examples below the sequences of the AD
and/or DDD moieties may be varied to improve DNL complex formation
and/or in vivo stability of the DNL complexes. In other
embodiments, the structural, physical and/or therapeutic
characteristics of native, chimeric, humanized or human antibodies
may be optimized by replacing one or more amino acid residues. For
example, it is well known in the art that the functional
characteristics of humanized antibodies may be improved by
substituting a limited number of human framework region (FR) amino
acids with the corresponding FR amino acids of the parent murine
antibody. This is particularly true when the framework region amino
acid residues are in close proximity to the CDR residues.
[0098] In other cases, the therapeutic properties of an antibody,
such as binding affinity for the target antigen, the dissociation-
or off-rate of the antibody from its target antigen, or even the
effectiveness of induction of CDC (complement-dependent
cytotoxicity) or ADCC (antibody dependent cellular cytotoxicity) by
the antibody, may be optimized by a limited number of amino acid
substitutions.
[0099] The skilled artisan will be aware that, in general, amino
acid substitutions typically involve the replacement of an amino
acid with another amino acid of relatively similar properties
(i.e., conservative amino acid substitutions). The properties of
the various amino acids and effect of amino acid substitution on
protein structure and function have been the subject of extensive
study and knowledge in the art.
[0100] For example, the hydropathic index of amino acids may be
considered (Kyte & Doolittle, 1982, J. Mol. Biol.,
157:105-132). The relative hydropathic character of the amino acid
contributes to the secondary structure of the resultant protein,
which in turn defines the interaction of the protein with other
molecules. Each amino acid has been assigned a hydropathic index on
the basis of its hydrophobicity and charge characteristics (Kyte
& Doolittle, 1982), these are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and arginine (-4.5). In making conservative substitutions,
the use of amino acids whose hydropathic indices are within .+-.2
is preferred, within .+-.1 are more preferred, and within .+-.0.5
are even more preferred.
[0101] Amino acid substitution may also take into account the
hydrophilicity of the amino acid residue (e.g., U.S. Pat. No.
4,554,101). Hydrophilicity values have been assigned to amino acid
residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0);
glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine
(+0.2); glycine (0); threonine (-0.4); proline (-0.5+-0.1); alanine
(-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3);
valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). Replacement of amino acids
with others of similar hydrophilicity is preferred.
[0102] Other considerations include the size of the amino acid side
chain. For example, it would generally not be preferred to replace
an amino acid with a compact side chain, such as glycine or serine,
with an amino acid with a bulky side chain, e.g., tryptophan or
tyrosine. The effect of various amino acid residues on protein
secondary structure is also a consideration. Through empirical
study, the effect of different amino acid residues on the tendency
of protein domains to adopt an alpha-helical, beta-sheet or reverse
turn secondary structure has been determined and is known in the
art (see, e.g., Chou & Fasman, 1974, Biochemistry, 13:222-245;
1978, Ann. Rev. Biochem., 47:251-276; 1979, Biophys. J.,
26:367-384).
[0103] Based on such considerations and extensive empirical study,
tables of conservative amino acid substitutions have been
constructed and are known in the art. For example arginine and
lysine; glutamate and aspartate; serine and threonine; glutamine
and asparagine; and valine, leucine and isoleucine. Alternatively
Ala (A) leu, ile, val; Arg (R) gln, asn, lys; Asn (N) his, asp,
lys, arg, gln; Asp (D) asn, glu; Cys (C) ala, ser; Gln (Q) glu,
asn; Glu (E) gln, asp; Gly (G) ala; His (H) asn, gln, lys, arg; Ile
(I) val, met, ala, phe, leu; Leu (L) val, met, ala, phe, ile; Lys
(K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val, ile,
ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr;
Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.
[0104] Other considerations for amino acid substitutions include
whether or not the residue is located in the interior of a protein
or is solvent exposed. For interior residues, conservative
substitutions would include Asp and Asn; Ser and Thr; Ser and Ala;
Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile;
Leu and Met; Phe and Tyr; Tyr and Trp. (See, e.g., PROWL website at
rockefeller.edu) For solvent exposed residues, conservative
substitutions would include Asp and Asn; Asp and Glu; Glu and Gln;
Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser;
Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile;
Ile and Val; Phe and Tyr. (See, e.g., PROWL website at
rockefeller.edu) Various matrices have been constructed to assist
in selection of amino acid substitutions, such as the PAM250
scoring matrix, Dayhoff matrix, Grantham matrix, McLachlan matrix,
Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,
Jones matrix, Rao matrix, Levin matrix and Risler matrix (See,
e.g., PROWL website at rockefeller.edu)
[0105] In determining amino acid substitutions, one may also
consider the existence of intermolecular or intramolecular bonds,
such as formation of ionic bonds (salt bridges) between positively
charged residues (e.g., His, Arg, Lys) and negatively charged
residues (e.g., Asp, Glu) or disulfide bonds between nearby
cysteine residues.
[0106] Methods of substituting any amino acid for any other amino
acid in an encoded protein sequence are well known and a matter of
routine experimentation for the skilled artisan, for example by the
technique of site-directed mutagenesis or by synthesis and assembly
of oligonucleotides encoding an amino acid substitution and
splicing into an expression vector construct. (E.g., Sambrook et
al., Molecular Cloning, A laboratory manual, 2.sup.nd Ed, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., 1989.)
[0107] Therapeutic Agents
[0108] In certain embodiments, therapeutic agents such as cytotoxic
agents, anti-angiogenic agents, pro-apoptotic agents, antibiotics,
hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins,
enzymes or other agents may be used as adjunct therapies to the
anti-cancer vaccine DNL complexes described herein. Drugs of use
may possess a pharmaceutical property selected from the group
consisting of antimitotic, antikinase, alkylating, antimetabolite,
antibiotic, alkaloid, anti-angiogenic, pro-apoptotic agents and
combinations thereof.
[0109] Exemplary drugs of use may include 5-fluorouracil, aplidin,
azaribine, anastrozole, anthracyclines, bendamustine, bleomycin,
bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin,
carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,
chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan
(CPT-11), SN-38, carboplatin, cladribine, camptothecans,
cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin,
daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),
cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin
glucuronide, estramustine, epidophyllotoxin, estrogen receptor
binding agents, etoposide (VP 16), etoposide glucuronide, etoposide
phosphate, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO),
fludarabine, flutamide, farnesyl-protein transferase inhibitors,
gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase,
lenolidamide, leucovorin, lomustine, mechlorethamine, melphalan,
mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,
mithramycin, mitomycin, mitotane, navelbine, nitrosurea,
plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341,
raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide
(an aqueous form of DTIC), transplatinum, thalidomide, thioguanine,
thiotepa, teniposide, topotecan, uracil mustard, vinorelbine,
vinblastine, vincristine and vinca alkaloids.
[0110] Toxins of use may include ricin, abrin, alpha toxin,
saporin, ribonuclease (RNase), e.g., onconase, DNase I,
Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,
diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas
endotoxin.
[0111] In certain embodiments, a therapeutic agent may be an
immunomodulator. An immunomodulator is an agent that when present,
alters, suppresses or stimulates the body's immune system.
Immunomodulators of use may include a cytokine, a stem cell growth
factor, a lymphotoxin, a hematopoietic factor, a colony stimulating
factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin
and a combination thereof. Specifically useful are lymphotoxins
such as tumor necrosis factor (TNF), hematopoietic factors, such as
interleukin (IL), colony stimulating factor, such as
granulocyte-colony stimulating factor (G-CSF) or granulocyte
macrophage-colony stimulating factor (GM-CSF), interferon, such as
interferons-.alpha., -.beta. or -.gamma., and stem cell growth
factor, such as that designated "S1 factor".
[0112] In various embodiments, the therapeutic agent may include
one or more cytokines, such as lymphokines, monokines, growth
factors and traditional polypeptide hormones. Included among the
cytokines are growth hormones such as human growth hormone,
N-methionyl human growth hormone, and bovine growth hormone;
parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;
prorelaxin; glycoprotein hormones such as follicle stimulating
hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing
hormone (LH); placenta growth factor (PlGF), hepatic growth factor;
prostaglandin, fibroblast growth factor; prolactin; placental
lactogen, OB protein; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth
factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-.alpha., -.beta., and -.gamma.;
colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14,
IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3,
angiostatin, thrombospondin, endostatin, tumor necrosis factor
(TNF, such as TNF-.alpha.) and LT. Chemokines of use may include
RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.
[0113] Anti-angiogenic agents include angiostatin, baculostatin,
canstatin, maspin, anti-VEGF antibodies, anti-P1GF peptides and
antibodies, anti-vascular growth factor antibodies, anti-Flk-1
antibodies, anti-Flt-1 antibodies and peptides, anti-Kras
antibodies, anti-cMET antibodies, anti-MIF (macrophage
migration-inhibitory factor) antibodies, laminin peptides,
fibronectin peptides, plasminogen activator inhibitors, tissue
metalloproteinase inhibitors, interferons, interleukin-12, IP-10,
Gro-.beta., thrombospondin, 2-methoxyoestradiol, proliferin-related
protein, carboxiamidotriazole, CM101, Marimastat, pentosan
polysulphate, angiopoietin-2, interferon-alpha, herbimycin A,
PNU145156E, 16K prolactin fragment, Linomide (roquinimex),
thalidomide, pentoxifylline, genistein, TNP-470, endostatin,
paclitaxel, accutin, angiostatin, cidofovir, vincristine,
bleomycin, AGM-1470, platelet factor 4 or minocycline may be of
use.
[0114] Other useful therapeutic agents may comprise
oligonucleotides, especially antisense oligonucleotides that
preferably are directed against oncogenes and oncogene products,
such as bcl-2 or p53. A preferred form of therapeutic
oligonucleotide is siRNA.
[0115] Diagnostic Agents
[0116] Diagnostic agents may be selected from the group consisting
of a radionuclide, a radiological contrast agent, a paramagnetic
ion, a metal, a fluorescent label, a chemiluminescent label, an
ultrasound contrast agent and a photoactive agent. Such diagnostic
agents are well known and any such known diagnostic agent may be
used. Non-limiting examples of diagnostic agents may include a
radionuclide such as .sup.110In, .sup.111In, .sup.177Lu, .sup.18F,
.sup.52Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga,
.sup.86Y, .sup.90Y, .sup.89Zr, .sup.94mTc, .sup.94Tc, .sup.99mTc,
.sup.120I, .sup.123I, .sup.124I, .sup.125I, .sup.131I,
.sup.154-158Gd, .sup.32P, .sup.11C, .sup.13N, .sup.15O, .sup.186Re,
.sup.188Re, .sup.51Mn, .sup.52mMn, .sup.55Co, .sup.72AS, .sup.75Br,
.sup.76Br, .sup.82mRb, .sup.83Sr, or other gamma-, beta-, or
positron-emitters. Paramagnetic ions of use may include chromium
(III), manganese (II), iron (III), iron (II), cobalt (II), nickel
(II), copper (II), neodymium (III), samarium (III), ytterbium
(III), gadolinium (III), vanadium (II), terbium (III), dysprosium
(III), holmium (III) or erbium (III). Metal contrast agents may
include lanthanum (III), gold (III), lead (II) or bismuth (III).
Ultrasound contrast agents may comprise liposomes, such as gas
filled liposomes. Radiopaque diagnostic agents may be selected from
compounds, barium compounds, gallium compounds, and thallium
compounds. A wide variety of fluorescent labels are known in the
art, including but not limited to fluorescein isothiocyanate,
rhodamine, phycoerytherin, phycocyanin, allophycocyanin,
o-phthaldehyde and fluorescamine. Chemiluminescent labels of use
may include luminol, isoluminol, an aromatic acridinium ester, an
imidazole, an acridinium salt or an oxalate ester.
[0117] Immunoconjugates
[0118] In certain embodiments, the anti-cancer vaccine DNL
construct may be conjugated to one or more therapeutic or
diagnostic agents. The therapeutic agents do not need to be the
same but can be different, e.g. a drug and a radioisotope. For
example, .sup.131I can be incorporated into a tyrosine of an
antibody or fusion protein and a drug attached to an epsilon amino
group of a lysine residue. Therapeutic and diagnostic agents also
can be attached, for example to reduced SH groups and/or to
carbohydrate side chains. Many methods for making covalent or
non-covalent conjugates of therapeutic or diagnostic agents with
antibodies or fusion proteins are known in the art and any such
known method may be utilized.
[0119] A therapeutic or diagnostic agent can be attached at the
hinge region of a reduced antibody component via disulfide bond
formation. Alternatively, such agents can be attached using a
heterobifunctional cross-linker, such as N-succinyl
3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer
56:244 (1994). General techniques for such conjugation are
well-known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN
CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al.,
"Modification of Antibodies by Chemical Methods," in MONOCLONAL
ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages
187-230 (Wiley-Liss, Inc. 1995); Price, "Production and
Characterization of Synthetic Peptide-Derived Antibodies," in
MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge
University Press 1995). Alternatively, the therapeutic or
diagnostic agent can be conjugated via a carbohydrate moiety in the
Fc region of the antibody. The carbohydrate group can be used to
increase the loading of the same agent that is bound to a thiol
group, or the carbohydrate moiety can be used to bind a different
therapeutic or diagnostic agent.
[0120] Methods for conjugating peptides to antibody components via
an antibody carbohydrate moiety are well-known to those of skill in
the art. See, for example, Shih et al., Int. J. Cancer 41:832
(1988); Shih et al., Int. J. Cancer 46:1101 (1990); and Shih et
al., U.S. Pat. No. 5,057,313, incorporated herein in their entirety
by reference. The general method involves reacting an antibody
component having an oxidized carbohydrate portion with a carrier
polymer that has at least one free amine function. This reaction
results in an initial Schiff base (imine) linkage, which can be
stabilized by reduction to a secondary amine to form the final
conjugate.
[0121] The Fc region may be absent if the antibody used as the
antibody component of the immunoconjugate is an antibody fragment.
However, it is possible to introduce a carbohydrate moiety into the
light chain variable region of a full length antibody or antibody
fragment. See, for example, Leung et al., J. Immunol. 154:5919
(1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995), Leung et
al., U.S. Pat. No. 6,254,868, incorporated herein by reference in
their entirety. The engineered carbohydrate moiety is used to
attach the therapeutic or diagnostic agent.
[0122] In some embodiments, a chelating agent may be attached to an
antibody, antibody fragment or fusion protein and used to chelate a
therapeutic or diagnostic agent, such as a radionuclide. Exemplary
chelators include but are not limited to DTPA (such as Mx-DTPA),
DOTA, TETA, NETA or NOTA. Methods of conjugation and use of
chelating agents to attach metals or other ligands to proteins are
well known in the art (see, e.g., U.S. patent application Ser. No.
12/112,289, incorporated herein by reference in its entirety).
[0123] In certain embodiments, radioactive metals or paramagnetic
ions may be attached to proteins or peptides by reaction with a
reagent having a long tail, to which may be attached a multiplicity
of chelating groups for binding ions. Such a tail can be a polymer
such as a polylysine, polysaccharide, or other derivatized or
derivatizable chains having pendant groups to which can be bound
chelating groups such as, e.g., ethylenediaminetetraacetic acid
(EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins,
polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and
like groups known to be useful for this purpose.
[0124] Chelates may be directly linked to antibodies or peptides,
for example as disclosed in U.S. Pat. No. 4,824,659, incorporated
herein in its entirety by reference. Particularly useful
metal-chelate combinations include 2-benzyl-DTPA and its monomethyl
and cyclohexyl analogs, used with diagnostic isotopes in the
general energy range of 60 to 4,000 keV, such as .sup.125I,
.sup.131I, .sup.123I, .sup.124I, .sup.62Cu, .sup.64Cu, .sup.18F,
.sup.111In, .sup.67Ga, .sup.68Ga, .sup.99mTc, .sup.94mTc, .sup.11C,
.sup.13N, .sup.15O, .sup.76Br for radioimaging. The same chelates,
when complexed with non-radioactive metals, such as manganese, iron
and gadolinium are useful for MM. Macrocyclic chelates such as
NOTA, DOTA, and TETA are of use with a variety of metals and
radiometals, most particularly with radionuclides of gallium,
yttrium and copper, respectively. Such metal-chelate complexes can
be made very stable by tailoring the ring size to the metal of
interest. Other ring-type chelates such as macrocyclic polyethers,
which are of interest for stably binding nuclides, such as
.sup.223Ra for RAIT are encompassed.
[0125] More recently, methods of .sup.18F-labeling of use in PET
scanning techniques have been disclosed, for example by reaction of
F-18 with a metal or other atom, such as aluminum. The .sup.18F-Al
conjugate may be complexed with chelating groups, such as DOTA,
NOTA or NETA that are attached directly to antibodies or used to
label targetable constructs in pre-targeting methods. Such F-18
labeling techniques are disclosed in U.S. patent application Ser.
No. 12/112,289, filed Apr. 30, 2008, the entire text of which is
incorporated herein by reference.
[0126] Methods of Therapeutic Treatment
[0127] Various embodiments concern methods of treating a cancer,
such as multiple myeloma, in a subject, such as a mammal, including
humans, domestic or companion pets, such as dogs and cats. The
methods may comprise administering to a subject a therapeutically
effective amount of an anti-cancer vaccine DNL construct. In
preferred embodiments, the anti-cancer vaccine DNL construct
comprises an anti-CD74 antibody or fragment thereof and a CD20
xenoantigen, as described in further detail in the Examples
below.
[0128] The administration of anti-cancer vaccine DNL construct can
be supplemented by administering concurrently or sequentially a
therapeutically effective amount of another antibody that binds to
or is reactive with another antigen on the surface of the target
cell. Preferred additional MAbs comprise at least one humanized,
chimeric or human MAb selected from the group consisting of a MAb
reactive with CD209 (DC-SIGN), CD34, CD74, CD205, TLR 2 (toll-like
receptor 2), TLR 4, TLR 7, TLR 9, BDCA-2, BDCA-3, BDCA-4 and
HLA-DR. Various antibodies of use are known to those of skill in
the art, as discussed above. See, for example, Ghetie et al.,
Cancer Res. 48:2610 (1988); Hekman et al., Cancer Immunol.
Immunother. 32:364 (1991); Longo, Curr. Opin. Oncol. 8:353 (1996),
U.S. Pat. Nos. 5,798,554; 6,187,287; 6,306,393; 6,676,924;
7,109,304; 7,151,164; 7,230,084; 7,230,085; 7,238,785; 7,238,786;
7,282,567; 7,300,655; 7,312,318; and U.S. Patent Application Publ.
Nos. 20080131363; 20080089838; 20070172920; 20060193865;
20060210475; 20080138333; and 20080146784, the Examples section of
each cited patent or application incorporated herein by
reference.
[0129] In alternative embodiments an antibody or fragment thereof
against another dendritic cell antigen, such as CD209 (DC-SIGN),
CD34, CD205, TLR 2 (toll-like receptor 2), TLR 4, TLR 7, TLR 9,
BDCA-2, BDCA-3, BDCA-4 or HLA-DR, may be substituted for the
anti-CD74 antibody in the DNL complex. Such antibodies may be
obtained from public sources like the American Type Culture
Collection or from commercial antibody vendors. For example,
antibodies against CD209(DC-SIGN), CD34, BDCA-2, TLR2, TLR 4, TLR 7
and TLR 9 may be purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, Calif.). Antibodies against CD205 and BDCA-3 may be
purchased from Miltenyi Biotec Inc. (Auburn, Calif.). Numerous
other commercial sources of antibodies are known to the skilled
artisan.
[0130] The anti-cancer vaccine DNL construct therapy can be further
supplemented with the administration, either concurrently or
sequentially, of at least one therapeutic agent. Therapeutic agents
used for the treatment of multiple myeloma include dexamethasone,
thalidomide/dexamethasone, cyclophosphamide, VAD (vincristine,
doxorubicin and dexamethasone), DVd (DOXIL.RTM. (PEGylated
doxorubicin), vincristine and reduced schedule dexamethasone),
BCNU, melphalan, carmustine, bortezomib (VELCADE.RTM.) prednisone
and corticosteroids. The individual therapeutic agents may be used
alone or in various combinations known in the art, such as CP
(cyclophosphamide, prednisone), CT (cyclophosphamide, thalidomide),
VBMCP (vincristine, BCNU, melphalan, cyclophosphamide, melphalan),
VMCP (vincristine, melphalan, cyclophosphamide, prednisone),
DT-PACE (dexamethasone, thalidomide, cisplatin, doxorubicin,
cyclophosphamide, etoposide), MPT (melphalan, prednisone,
thalidomide), CVAD (cyclophosphamide and VAD), EDAP (etoposide,
dexamethasone, ara-C, cisplatin) MTD (melphalan, thalidomide,
dexamethasone), VT (VELCADE.RTM., thalidomide), VDT (VELCADE.RTM.,
doxorubicin, thalidomide), VADT (VELCADE.RTM., adriamycin,
thalidomide, dexamethasone) or DCEP (dexamethasone,
cyclophosphamide, etoposide, cisplatin).
[0131] Chemotherapeutic treatment of multiple myeloma prior to stem
cell transplantation is referred to as induction therapy. Certain
of the chemotherapeutic agents listed herein are more suitable for
induction therapy than others. Examples of chemotherapeutic
treatments of use for induction therapy for MM include
dexamethasone, thalidomide/dexamethasone, cyclophosphamide, VAD and
DVd. Because MM is often resistant to chemotherapeutic treatment,
administration of therapeutic agents may occur at higher doses than
are used in conventional chemotherapy. Such high-dose chemotherapy
usually results in bone marrow toxicity and is often used in
conjunction with stem cell transplantation. Dosages and schedules
for chemotherapeutic treatment of MM are well known in the an and
any such known dosage and/or schedule may be utilized in
conjunction with administration of the anti-cancer vaccine DNL
construct.
[0132] Where the DNL vaccine is used for other types of cancers
besides MM, other chemotherapeutic regimens are known. For example,
"CVB" (1.5 g/m.sup.2 cyclophosphamide, 200-400 mg/m.sup.2
etoposide, and 150-200 mg/m.sup.2 carmustine) is a regimen used to
treat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51:18
(1993). Other suitable combination chemotherapeutic regimens are
well-known to those of skill in the art. See, for example, Freedman
et al., "Non-Hodgkin's Lymphomas," in CANCER MEDICINE, VOLUME 2,
3rd Edition, Holland et al. (eds.), pages 2028-2068 (Lea &
Febiger 1993). As an illustration, first generation
chemotherapeutic regimens for treatment of intermediate-grade
non-Hodgkin's lymphoma (NHL) include C-MOPP (cyclophosphamide,
vincristine, procarbazine and prednisone) and CHOP
(cyclophosphamide, doxorubicin, vincristine, and prednisone). A
useful second generation chemotherapeutic regimen is m-BACOD
(methotrexate, bleomycin, doxorubicin, cyclophosphamide,
vincristine, dexamethasone and leucovorin), while a suitable third
generation regimen is MACOP-B (methotrexate, doxorubicin,
cyclophosphamide, vincristine, prednisone, bleomycin and
leucovorin). Chemotherapeutic agents of use against other types of
cancers include, but are not limited to, 5-fluorouracil, aplidin,
azaribine, anastrozole, anthracyclines, bendamustine, bleomycin,
bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin,
carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,
chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan
(CPT-11), SN-38, carboplatin, cladribine, camptothecans,
cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin,
daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),
cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin
glucuronide, estramustine, epidophyllotoxin, estrogen receptor
binding agents, etoposide (VP 16), etoposide glucuronide, etoposide
phosphate, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO),
fludarabine, flutamide, farnesyl-protein transferase inhibitors,
gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase,
lenolidamide, leucovorin, lomustine, mechlorethamine, melphalan,
mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,
mithramycin, mitomycin, mitotane, navelbine, nitrosurea, phenyl
butyrate, plicomycin, procarbazine, paclitaxel, pentostatin,
PSI-341, raloxifene, semustine, streptozocin, tamoxifen, taxol,
temazolomide (an aqueous form of DTIC), transplatinum, thalidomide,
thioguanine, thiotepa, teniposide, topotecan, uracil mustard,
vinorelbine, vinblastine, vincristine and vinca alkaloids.
[0133] Formulations
[0134] The anti-cancer vaccine DNL construct can be formulated
according to known methods to prepare pharmaceutically useful
compositions, whereby the anti-cancer vaccine DNL construct is
combined in a mixture with a pharmaceutically suitable excipient.
Sterile phosphate-buffered saline is one example of a
pharmaceutically suitable excipient. Other suitable excipients are
well-known to those in the art. See, for example, Ansel et al.,
PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition
(Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S
PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company
1990), and revised editions thereof.
[0135] The anti-cancer vaccine can be formulated for intravenous
administration via, for example, bolus injection or continuous
infusion. Formulations for injection can be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient can
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0136] Additional pharmaceutical methods may be employed to control
the duration of action of the anti-cancer vaccine. Control release
preparations can be prepared through the use of polymers to complex
or adsorb the anti-cancer vaccine DNL construct. For example,
biocompatible polymers include matrices of poly(ethylene-co-vinyl
acetate) and matrices of a polyanhydride copolymer of a stearic
acid dimer and sebacic acid. Sherwood et al., Bio/Technology
10:1446 (1992). The rate of release from such a matrix depends upon
the molecular weight of the anti-cancer vaccine DNL construct, the
amount of anti-cancer vaccine within the matrix, and the size of
dispersed particles. Saltzman et al., Biophys. J. 55:163 (1989);
Sherwood et al., supra. Other solid dosage forms are described in
Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY
SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),
REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing
Company 1990), and revised editions thereof.
[0137] The anti-cancer vaccine DNL construct may also be
administered to a mammal subcutaneously or even by other parenteral
routes. Moreover, the administration may be by continuous infusion
or by single or multiple boluses. Preferably, the anti-cancer
vaccine is administered as a single or multiple boluses via
subcutaneous injection.
[0138] Generally, the dosage of an administered anti-cancer vaccine
DNL construct for humans will vary depending upon such factors as
the patient's age, weight, height, sex, general medical condition
and previous medical history. It may be desirable to provide the
recipient with a dosage of anti-cancer vaccine DNL construct that
is in the range of from about 1 mg/kg to 25 mg/kg as a single
administration, although a lower or higher dosage also may be
administered as circumstances dictate. A dosage of 1-20 mg/kg for a
70 kg patient, for example, is 70-1,400 mg, or 41-824 mg/m.sup.2
for a 1.7-m patient. The dosage may be repeated as needed for
induction of an immune response.
[0139] In preferred embodiments, the vaccine DNL constructs are of
use for therapy of cancer. Examples of cancers include, but are not
limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma,
and leukemia, myeloma, or lymphoid malignancies. More particular
examples of such cancers are noted below and include: squamous cell
cancer (e.g., epithelial squamous cell cancer), Ewing sarcoma,
Wilms tumor, astrocytomas, lung cancer including small-cell lung
cancer, non-small cell lung cancer, adenocarcinoma of the lung and
squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer including
gastrointestinal cancer, pancreatic cancer, glioblastoma
multiforme, cervical cancer, ovarian cancer, liver cancer, bladder
cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors,
medullary thyroid cancer, differentiated thyroid carcinoma, breast
cancer, ovarian cancer, colon cancer, rectal cancer, endometrial
cancer or uterine carcinoma, salivary gland carcinoma, kidney or
renal cancer, prostate cancer, vulvar cancer, anal carcinoma,
penile carcinoma, as well as head-and-neck cancer. The term
"cancer" includes primary malignant cells or tumors (e.g., those
whose cells have not migrated to sites in the subject's body other
than the site of the original malignancy or tumor) and secondary
malignant cells or tumors (e.g., those arising from metastasis, the
migration of malignant cells or tumor cells to secondary sites that
are different from the site of the original tumor).
[0140] Other examples of cancers or malignancies include, but are
not limited to: Acute Childhood Lymphoblastic Leukemia, Acute
Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid
Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular
Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic
Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Lymphoma,
Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult
Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related
Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile
Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain
Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter,
Central Nervous System (Primary) Lymphoma, Central Nervous System
Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical
Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood
(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,
Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma,
Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma,
Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's
Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and
Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood
Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal
and Supratentorial Primitive Neuroectodermal Tumors, Childhood
Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft
Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma,
Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon
Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell
Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer,
Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine
Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ
Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female
Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric
Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors,
Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell
Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's
Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal
Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell
Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal
Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer,
Lymphoproliferative Disorders, Macroglobulinemia, Male Breast
Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma,
Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck
Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic
Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma
Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia,
Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and
Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,
Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell
Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer,
Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma,
Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant
Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian
Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic
Cancer, Paraproteinemias, Polycythemia vera, Parathyroid Cancer,
Penile Cancer, Pheochromocytoma, Pituitary Tumor, Primary Central
Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer,
Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer,
Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,
Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung
Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck
Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal
and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma,
Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and
Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic
Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer,
Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and
Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's
Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
[0141] The methods and compositions described and claimed herein
may be used to treat malignant or premalignant conditions and to
prevent progression to a neoplastic or malignant state, including
but not limited to those disorders described above. Such uses are
indicated in conditions known or suspected of preceding progression
to neoplasia or cancer, in particular, where non-neoplastic cell
growth consisting of hyperplasia, metaplasia, or most particularly,
dysplasia has occurred (for review of such abnormal growth
conditions, see Robbins and Angell, Basic Pathology, 2d Ed., W. B.
Saunders Co., Philadelphia, pp. 68-79 (1976)).
[0142] Dysplasia is frequently a forerunner of cancer, and is found
mainly in the epithelia. It is the most disorderly form of
non-neoplastic cell growth, involving a loss in individual cell
uniformity and in the architectural orientation of cells. Dysplasia
characteristically occurs where there exists chronic irritation or
inflammation. Dysplastic disorders which can be treated include,
but are not limited to, anhidrotic ectodermal dysplasia,
anterofacial dysplasia, asphyxiating thoracic dysplasia,
atriodigital dysplasia, bronchopulmonary dysplasia, cerebral
dysplasia, cervical dysplasia, chondroectodermal dysplasia,
cleidocranial dysplasia, congenital ectodermal dysplasia,
craniodiaphysial dysplasia, craniocarpotarsal dysplasia,
craniometaphysial dysplasia, dentin dysplasia, diaphysial
dysplasia, ectodermal dysplasia, enamel dysplasia,
encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia,
dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,
epithelial dysplasia, faciodigitogenital dysplasia, familial
fibrous dysplasia of jaws, familial white folded dysplasia,
fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous
dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal
dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic
dysplasia, mammary dysplasia, mandibulofacial dysplasia,
metaphysial dysplasia, Mondini dysplasia, monostotic fibrous
dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia,
oculoauriculovertebral dysplasia, oculodentodigital dysplasia,
oculovertebral dysplasia, odontogenic dysplasia,
opthalmomandibulomelic dysplasia, periapical cemental dysplasia,
polyostotic fibrous dysplasia, pseudoachondroplastic
spondyloepiphysial dysplasia, retinal dysplasia, septo-optic
dysplasia, spondyloepiphysial dysplasia, and ventriculoradial
dysplasia.
[0143] Additional pre-neoplastic disorders which can be treated
include, but are not limited to, benign dysproliferative disorders
(e.g., benign tumors, fibrocystic conditions, tissue hypertrophy,
intestinal polyps or adenomas, and esophageal dysplasia),
leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar
cheilitis, and solar keratosis.
[0144] In preferred embodiments, the method of the invention is
used to inhibit growth, progression, and/or metastasis of cancers,
in particular those listed above.
[0145] Additional hyperproliferative diseases, disorders, and/or
conditions include, but are not limited to, progression, and/or
metastases of malignancies and related disorders such as leukemia
(including acute leukemias (e.g., acute lymphocytic leukemia, acute
myelocytic leukemia (including myeloblastic, promyelocytic,
myelomonocytic, monocytic, and erythroleukemia)) and chronic
leukemias (e.g., chronic myelocytic (granulocytic) leukemia and
chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,
Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, and solid
tumors including, but not limited to, sarcomas and carcinomas such
as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, emangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma.
[0146] Kits
[0147] Various embodiments may concern kits containing components
suitable for treating or diagnosing diseased tissue in a patient.
Exemplary kits may contain at least one or more anti-cancer vaccine
constructs as described herein. If the composition containing
components for administration is not formulated for delivery via
the alimentary canal, such as by oral delivery, a device capable of
delivering the kit components through some other route may be
included. One type of device, for applications such as parenteral
delivery, is a syringe that is used to inject the composition into
the body of a subject. Inhalation devices may also be used. In
certain embodiments, a therapeutic agent may be provided in the
form of a prefilled syringe or autoinjection pen containing a
sterile, liquid formulation or lyophilized preparation.
[0148] The kit components may be packaged together or separated
into two or more containers. In some embodiments, the containers
may be vials that contain sterile, lyophilized formulations of a
composition that are suitable for reconstitution. A kit may also
contain one or more buffers suitable for reconstitution and/or
dilution of other reagents. Other containers that may be used
include, but are not limited to, a pouch, tray, box, tube, or the
like. Kit components may be packaged and maintained sterilely
within the containers. Another component that can be included is
instructions to a person using a kit for its use.
[0149] Expression Vectors
[0150] Still other embodiments may concern DNA sequences comprising
a nucleic acid encoding a anti-cancer vaccine construct, or its
constituent fusion proteins. Fusion proteins may comprise an
anti-CD74 antibody or CD20 xenoantigen attached to a different
peptide or protein, such as the AD and DDD peptides utilized for
DNL construct formation as discussed in more detail in the Examples
below. Alternatively the encoded fusion proteins may comprise a DDD
or AD moiety attached to a different antibody or xenoantigen.
[0151] Various embodiments relate to expression vectors comprising
the coding DNA sequences. The vectors may contain sequences
encoding the light and heavy chain constant regions and the hinge
region of a human immunoglobulin to which may be attached chimeric,
humanized or human variable region sequences. The vectors may
additionally contain promoters that express the encoded protein(s)
in a selected host cell, enhancers and signal or leader sequences.
Vectors that are particularly useful are pdHL2 or GS. More
preferably, the light and heavy chain constant regions and hinge
region may be from a human EU myeloma immunoglobulin, where
optionally at least one of the amino acid in the allotype positions
is changed to that found in a different IgG1 allotype, and wherein
optionally amino acid 253 of the heavy chain of EU based on the EU
number system may be replaced with alanine. See Edelman et al.,
Proc. Natl. Acad. Sci USA 63:78-85 (1969). In other embodiments, an
IgG1 sequence may be converted to an IgG4 sequence.
[0152] The skilled artisan will realize that methods of genetically
engineering expression constructs and insertion into host cells to
express engineered proteins are well known in the art and a matter
of routine experimentation. Host cells and methods of expression of
cloned antibodies or fragments have been described, for example, in
U.S. patent application Ser. No. 11/187,863, filed Jul. 25, 2005;
Ser. No. 11/253,666, filed Oct. 20, 2005 and Ser. No. 11/487,215,
filed Jul. 14, 2006, the Examples section of each incorporated
herein by reference.
Examples
[0153] The following examples are provided to illustrate, but not
to limit, the claims of the present invention.
Example 1
Preparation of Dock-and-Lock (DNL) Constructs
[0154] DDD and AD Fusion Proteins
[0155] The DNL technique can be used to make dimers, trimers,
tetramers, hexamers, etc. comprising virtually any antibodies or
fragments thereof or other effector moieties. For certain preferred
embodiments, IgG antibodies, F(ab').sub.2 antibody fragments and
xenoantigens, such as CD20 xenoantigens, may be produced as fusion
proteins containing either a dimerization and docking domain (DDD)
or anchoring domain (AD) sequence. Although in preferred
embodiments the DDD and AD moieties are produced as fusion
proteins, the skilled artisan will realize that other methods of
conjugation, such as chemical cross-linking, may be utilized within
the scope of the claimed methods and compositions.
[0156] DNL constructs may be formed by combining, for example, an
Fab-DDD fusion protein of an anti-CD74 antibody with a CD20-AD
fusion protein. Alternatively, constructs may be made that combine
IgG-AD fusion proteins with CD20-DDD fusion proteins. The technique
is not limiting and any protein or peptide of use may be produced
as an AD or DDD fusion protein for incorporation into a DNL
construct. Where chemical cross-linking is utilized, the AD and DDD
conjugates are not limited to proteins or peptides and may comprise
any molecule that may be cross-linked to an AD or DDD sequence
using any cross-linking technique known in the art.
[0157] Independent transgenic cell lines may be developed for each
DDD or AD fusion protein. Once produced, the modules can be
purified if desired or maintained in the cell culture supernatant
fluid. Following production, any DDD-fusion protein module can be
combined with any AD-fusion protein module to generate a DNL
construct. For different types of constructs, different AD or DDD
sequences may be utilized. Exemplary DDD and AD sequences are
provided below.
TABLE-US-00001 DDD1: (SEQ ID NO: 10)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2: (SEQ ID NO: 11)
CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1: (SEQ ID NO: 12)
QIEYLAKQIVDNAIQQA AD2: (SEQ ID NO: 13) CGQIEYLAKQIVDNAIQQAGC
[0158] Expression Vectors
[0159] The plasmid vector pdHL2 has been used to produce a number
of antibodies and antibody-based constructs. See Gillies et al., J
Immunol Methods (1989), 125:191-202; Losman et al., Cancer (Phila)
(1997), 80:2660-6. The di-cistronic mammalian expression vector
directs the synthesis of the heavy and light chains of IgG. The
vector sequences are mostly identical for many different IgG-pdHL2
constructs, with the only differences existing in the variable
domain (VH and VL) sequences. Using molecular biology tools known
to those skilled in the art, these IgG expression vectors can be
converted into Fab-DDD or Fab-AD expression vectors. To generate
Fab-DDD expression vectors, the coding sequences for the hinge, CH2
and CH3 domains of the heavy chain are replaced with a sequence
encoding the first 4 residues of the hinge, a 14 residue Gly-Ser
linker and the first 44 residues of human RII.alpha. (referred to
as DDD1). To generate Fab-AD expression vectors, the sequences for
the hinge, CH2 and CH3 domains of IgG are replaced with a sequence
encoding the first 4 residues of the hinge, a 15 residue Gly-Ser
linker and a 17 residue synthetic AD called AKAP-IS (referred to as
AD1), which was generated using bioinformatics and peptide array
technology and shown to bind RII.alpha. dimers with a very high
affinity (0.4 nM). See Alto, et al. Proc. Natl. Acad. Sci., U.S.A
(2003), 100:4445-50.
[0160] Two shuttle vectors were designed to facilitate the
conversion of IgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1
expression vectors, as described below.
[0161] Preparation of CH1
[0162] The CH1 domain was amplified by PCR using the pdHL2 plasmid
vector as a template. The left PCR primer consisted of the upstream
(5') end of the CH1 domain and a SacII restriction endonuclease
site, which is 5' of the CH1 coding sequence. The right primer
consisted of the sequence coding for the first 4 residues of the
hinge (PKSC (SEQ ID NO:29) followed by four glycines and a serine,
with the final two codons (GS) comprising a Bam HI restriction
site. The 410 bp PCR amplimer was cloned into the PGEMT.RTM. PCR
cloning vector (PROMEGA.RTM., Inc.) and clones were screened for
inserts in the T7 (5') orientation.
[0163] Construction of (G.sub.4S).sub.2DDD1 ((G.sub.4S).sub.2
Disclosed as SEQ ID NO:14)
[0164] A duplex oligonucleotide, designated (G.sub.4S).sub.2DDD1
((G.sub.4S).sub.2 disclosed as SEQ ID NO:14), was synthesized by
Sigma GENOSYS.RTM. (Haverhill, UK) to code for the amino acid
sequence of DDD1 preceded by 11 residues of the linker peptide,
with the first two codons comprising a BamHI restriction site. A
stop codon and an EagI restriction site are appended to the 3'end.
The encoded polypeptide sequence is shown below.
TABLE-US-00002 (SEQ ID NO: 15)
GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRL REARA
[0165] Two oligonucleotides, designated RIIA1-44 top and RIIA1-44
bottom, that overlap by 30 base pairs on their 3' ends, were
synthesized (Sigma GENOSYS.RTM.) and combined to comprise the
central 154 base pairs of the 174 bp DDD1 sequence. The
oligonucleotides were annealed and subjected to a primer extension
reaction with Taq polymerase. Following primer extension, the
duplex was amplified by PCR. The amplimer was cloned into
PGEMT.RTM. and screened for inserts in the T7 (5') orientation.
[0166] Construction of (G.sub.4S).sub.2 AD1 ((G.sub.4S).sub.2
disclosed as SEQ ID NO:14)
[0167] A duplex oligonucleotide, designated (G.sub.4S).sub.2-AD1
((G.sub.4S).sub.2 disclosed as SEQ ID NO:14), was synthesized
(Sigma GENOSYS.RTM.) to code for the amino acid sequence of AD1
preceded by 11 residues of the linker peptide with the first two
codons comprising a BamHI restriction site. A stop codon and an
EagI restriction site are appended to the 3' end. The encoded
polypeptide sequence is shown below.
TABLE-US-00003 (SEQ ID NO: 16) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA
[0168] Two complimentary overlapping oligonucleotides encoding the
above peptide sequence, designated AKAP-IS Top and AKAP-IS Bottom,
were synthesized and annealed. The duplex was amplified by PCR. The
amplimer was cloned into the PGEMT.RTM. vector and screened for
inserts in the T7 (5') orientation.
[0169] Ligating DDD.sub.1 with CH1
[0170] A 190 bp fragment encoding the DDD1 sequence was excised
from PGEMT.RTM. with BamHI and NotI restriction enzymes and then
ligated into the same sites in CH1-PGEMT.RTM. to generate the
shuttle vector CH1-DDD1-PGEMT.RTM..
[0171] Ligating AD1 with CH1
[0172] A 110 bp fragment containing the AD1 sequence was excised
from PGEMT.RTM. with BamHI and NotI and then ligated into the same
sites in CH1-PGEMT.RTM. to generate the shuttle vector
CH1-AD1-PGEMT.RTM..
[0173] Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors
[0174] With this modular design either CH1-DDD1 or CH1-AD1 can be
incorporated into any IgG construct in the pdHL2 vector. The entire
heavy chain constant domain is replaced with one of the above
constructs by removing the SacII/EagI restriction fragment
(CH1-CH3) from pdHL2 and replacing it with the SacII/EagI fragment
of CH1-DDD1 or CH1-AD1, which is excised from the respective pGemT
shuttle vector.
[0175] Construction of h679-Fd-AD1-pdHL2
[0176] h679-Fd-AD1-pdHL2 is an expression vector for production of
h679 Fab with AD1 coupled to the carboxyl terminal end of the CH1
domain of the Fd via a flexible Gly/Ser peptide spacer composed of
14 amino acid residues. A pdHL2-based vector containing the
variable domains of h679 was converted to h679-Fd-AD1-pdHL2 by
replacement of the SacII/EagI fragment with the CH1-AD1 fragment,
which was excised from the CH1-AD1-SV3 shuttle vector with SacII
and EagI.
[0177] Construction of C-DDD1-Fd-hMN-14-pdHL2
[0178] C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for
production of a stable dimer that comprises two copies of a fusion
protein C-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at
the carboxyl terminus of CH1 via a flexible peptide spacer. The
plasmid vector hMN-14(I)-pdHL2, which has been used to produce
hMN-14 IgG, was converted to C-DDD1-Fd-hMN-14-pdHL2 by digestion
with SacII and EagI restriction endonucleases to remove the CH1-CH3
domains and insertion of the CH1-DDD1 fragment, which was excised
from the CH1-DDD1-SV3 shuttle vector with SacII and EagI.
[0179] The same technique has been utilized to produce plasmids for
Fab expression of a wide variety of known antibodies, such as hLL1,
hLL2, hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others.
Generally, the antibody variable region coding sequences were
present in a pdHL2 expression vector and the expression vector was
converted for production of an AD- or DDD-fusion protein as
described above.
[0180] Construction of C-DDD2-Fd-hMN-14-pdHL2
[0181] C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for
production of C-DDD2-Fab-hMN-14, which possesses a dimerization and
docking domain sequence of DDD2 appended to the carboxyl terminus
of the Fd of hMN-14 via a 14 amino acid residue Gly/Ser peptide
linker. The fusion protein secreted is composed of two identical
copies of hMN-14 Fab held together by non-covalent interaction of
the DDD2 domains.
[0182] The expression vector was engineered as follows. Two
overlapping, complimentary oligonucleotides, which comprise the
coding sequence for part of the linker peptide (GGGGSGGGCG, SEQ ID
NO:17) and residues 1-13 of DDD2, were made synthetically. The
oligonucleotides were annealed and phosphorylated with T4 PNK,
resulting in overhangs on the 5' and 3' ends that are compatible
for ligation with DNA digested with the restriction endonucleases
BamHI and PstI, respectively.
[0183] The duplex DNA was ligated with the shuttle vector
CH1-DDD1-PGEMT.RTM., which was prepared by digestion with BamHI and
PstI, to generate the shuttle vector CH1-DDD2-PGEMT.RTM.. A 507 bp
fragment was excised from CH1-DDD2-PGEMT.RTM. with SacII and EagI
and ligated with the IgG expression vector hMN-14(I)-pdHL2, which
was prepared by digestion with SacII and EagI. The final expression
construct was designated C-DDD2-Fd-hMN-14-pdHL2. Similar techniques
have been utilized to generated DDD2-fusion proteins of the Fab
fragments of a number of different humanized antibodies.
[0184] Construction of h679-Fd-AD2-pdHL2
[0185] h679-Fd-AD2-pdHL2 is an expression vector for the production
of h679-Fab-AD2, which possesses an anchoring domain sequence of
AD2 appended to the carboxyl terminal end of the CH1 domain via a
14 amino acid residue Gly/Ser peptide linker. AD2 has one cysteine
residue preceding and another one following the anchor domain
sequence of AD1.
[0186] The expression vector was engineered as follows. Two
overlapping, complimentary oligonucleotides which comprise the
coding sequence for AD2 and part of the linker sequence, were made
synthetically. The oligonucleotides were annealed and
phosphorylated with T4 PNK, resulting in overhangs on the 5' and 3'
ends that are compatible for ligation with DNA digested with the
restriction endonucleases BamHI and SpeI, respectively.
[0187] The duplex DNA was ligated into the shuttle vector
CH1-AD1-PGEMT.RTM., which was prepared by digestion with BamHI and
SpeI, to generate the shuttle vector CH1-AD2-PGEMT.RTM.. A 429 base
pair fragment containing CH1 and AD2 coding sequences was excised
from the shuttle vector with SacII and EagI restriction enzymes and
ligated into h679-pdHL2 vector that prepared by digestion with
those same enzymes. The final expression vector is
h679-Fd-AD2-pdHL2.
[0188] Generation of TF2 Trimeric DNL Construct
[0189] A trimeric DNL construct designated TF2 was obtained by
reacting C-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2
was generated with >90% yield as follows. Protein L-purified
C-DDD2-Fab-hMN-14 (200 mg) was mixed with h679-Fab-AD2 (60 mg) at a
1.4:1 molar ratio. The total protein concentration was 1.5 mg/ml in
PBS containing 1 mM EDTA. Subsequent steps involved TCEP reduction,
HIC chromatography, DMSO oxidation, and IMP 291 affinity
chromatography. Before the addition of TCEP, SE-HPLC did not show
any evidence of a.sub.2b formation. Addition of 5 mM TCEP rapidly
resulted in the formation of a.sub.2b complex consistent with a 157
kDa protein expected for the binary structure. TF2 was purified to
near homogeneity by IMP 291 affinity chromatography (not shown).
IMP 291 is a synthetic peptide containing the HSG hapten to which
the 679 Fab binds (Rossi et al., 2005, Clin Cancer Res
11:7122s-29s). SE-HPLC analysis of the IMP 291 unbound fraction
demonstrated the removal of a.sub.4, a.sub.2 and free kappa chains
from the product (not shown).
[0190] Non-reducing SDS-PAGE analysis demonstrated that the
majority of TF2 exists as a large, covalent structure with a
relative mobility near that of IgG (not shown). Reducing SDS-PAGE
shows that any additional bands apparent in the non-reducing gel
are product-related (not shown), as only bands representing the
constituent polypeptides of TF2 were evident (not shown). However,
the relative mobilities of each of the four polypeptides were too
close to be resolved. MALDI-TOF mass spectrometry (not shown)
revealed a single peak of 156,434 Da, which is within 99.5% of the
calculated mass (157,319 Da) of TF2.
[0191] The functionality of TF2 was determined by BIACORE.RTM.
assay. TF2, C-DDD1-hMN-14+h679-AD1 (used as a control sample of
noncovalent a.sub.2b complex), or C-DDD2-hMN-14+h679-AD2 (used as a
control sample of unreduced a.sub.2 and b components) were diluted
to 1 .mu.g/ml (total protein) and passed over a sensorchip
immobilized with HSG. The response for TF2 was approximately
two-fold that of the two control samples, indicating that only the
h679-Fab-AD component in the control samples would bind to and
remain on the sensorchip. Subsequent injections of WI2 IgG, an
anti-idiotype antibody for hMN-14, demonstrated that only TF2 had a
DDD-Fab-hMN-14 component that was tightly associated with
h679-Fab-AD as indicated by an additional signal response. The
additional increase of response units resulting from the binding of
WI2 to TF2 immobilized on the sensorchip corresponded to two fully
functional binding sites, each contributed by one subunit of
C-DDD2-Fab-hMN-14. This was confirmed by the ability of TF2 to bind
two Fab fragments of WI2 (not shown).
Example 2
C.sub.H3-AD2-IgG Expression Vectors
[0192] A plasmid shuttle vector was produced to facilitate the
conversion of any IgG-pdHL2 vector into a C.sub.H3-AD2-IgG-pdHL2
vector. The gene for the Fc (C.sub.H2 and C.sub.H3 domains) was
amplified by PCR using the pdHL2 vector as a template and the
following oligonucleotide primers:
TABLE-US-00004 Fc BglII Left (SEQ ID NO: 8)
AGATCTGGCGCACCTGAACTCCTG Fc Bam- EcoRI Right (SEQ ID NO: 9)
GAATTCGGATCCTTTACCCGGAGACAGGGAGAG.
[0193] The amplimer was cloned in the pGemT PCR cloning vector
(Promega). The Fc insert fragment was excised from pGemT with Xba I
and Bam HI and ligated with AD2-pdHL2 vector that was prepared by
digesting h679-Fab-AD2-pdHL2 (Rossi et al., Proc Natl Acad Sci USA
2006, 103:6841-6) with Xba I and Bam HI, to generate the shuttle
vector Fc-AD2-pdHL2. To convert IgG-pdHL2 expression vectors to a
C.sub.H3-AD2-IgG-pdHL2 expression vectors, an 861 bp BsrG I/Nde I
restriction fragment was excised from the former and replaced with
a 952 bp BsrG I/Nde I restriction fragment excised from the
Fc-AD2-pdHL2 vector. The following is a partial list of
C.sub.H3-AD2-IgG-pdHL2 expression vectors that have been generated
and used for the production of recombinant humanized IgG-AD2
modules:
[0194] C.sub.H3-AD2-IgG-hA20 (anti-CD20)
[0195] C.sub.H3-AD2-IgG-hLL2 (anti-CD22)
[0196] C.sub.H3-AD2-IgG-hL243 (anti-HLA-DR)
[0197] C.sub.H3-AD2-IgG-hLL1 (anti-CD74)
[0198] C.sub.H3-AD2-IgG-hR1 (anti-IGF-1R)
[0199] C.sub.H3-AD2-IgG-h734 (anti-Indium-DTPA).
Example 3
Production of C.sub.H3-AD2-IgG
[0200] Transfection and Selection of Stable C.sub.H3-AD2-IgG
Secreting Cell Lines
[0201] All cell lines were grown in Hybridoma SFM (Invitrogen,
Carlsbad Calif.). C.sub.H3-AD2-IgG-pdHL2 vectors (30 .mu.g) were
linearized by digestion with Sal I restriction endonuclease and
transfected into Sp2/0-Ag14 (2.8.times.10.sup.6 cells) by
electroporation (450 volts, 25 .mu.F). The pdHL2 vector contains
the gene for dihydrofolate reductase allowing clonal selection as
well as gene amplification with methotrexate (MTX).
[0202] Following transfection, the cells were plated in 96-well
plates and transgenic clones were selected in media containing 0.2
.mu.M MTX. Clones were screened for C.sub.H3-AD2-IgG productivity
by a sandwich ELISA using 96-well microtitre plates coated with
specific anti-idiotype MAbs. Conditioned media from the putative
clones were transferred to the micro-plate wells and detection of
the fusion protein was accomplished with horseradish
peroxidase-conjugated goat anti-human IgG F(ab').sub.2 (Jackson
ImmunoResearch Laboratories, West Grove, Pa.). Wells giving the
highest signal were expanded and ultimately used for
production.
[0203] Production and Purification of C.sub.H3-AD2-IgG Modules
[0204] For production of the fusion proteins, roller bottle
cultures were seeded at 2.times.10.sup.5 cells/ml and incubated in
a roller bottle incubator at 37.degree. C. under 5% CO.sub.2 until
the cell viability dropped below 25% (.about.10 days). Culture
broth was clarified by centrifugation, filtered, and concentrated
up to 50-fold by ultrafiltration. For purification of
C.sub.H3-AD2-IgG modules, concentrated supernatant fluid was loaded
onto a Protein-A (MAB Select) affinity column. The column was
washed to baseline with PBS and the fusion proteins were eluted
with 0.1 M Glycine, pH 2.5.
Example 4
Generation of DDD2-mCD20(136-178) and Construction of
DDD2-mCD20(136-178)-pdHL2
[0205] DDD2-mCD20(136-178)-pdHL2 is the expression vector for
DDD2-mCD20(136-178), which comprises
DDD2-linker-mCD20(136-178)-HHHHHH (HHHHHH disclosed as SEQ ID
NO:30). The extracellular domain of mouse CD20 (mCD20) is referred
to as mCD20(136-178), comprising amino acid residues 136 to 178 of
the sequence shown below:
TABLE-US-00005 (SEQ ID NO: 18)
TLSHFLKMRRLELIQTSKPYVDIYDCEPSNSSEKNSPSTQYCN
[0206] The amino acid sequence of mouse CD20 xenoantigen is shown
below.
TABLE-US-00006 (SEQ ID NO: 7)
MSGPFPAEPTKGPLAMQPAPKVNLKRTSSLVGPTQSFFMRESKALGAVQI
MNGLFHITLGGLLMIPTGVFAPICLSVWYPLWGGIMYIISGSLLAAAAEK
TSRKSLVKAKVIMSSLSLFAAISGIILSIMDILNIVITLSHFLKMRRLEL
IQTSKPYVDIYDCEPSNSSEKNSPSTQYCNSIQSVFLGILSAMLISAFFQ
KLVTAGIVENEWKRIVICTRSKSNVVLLSAGEKNEQTIKMKEDIELSGVS
SQPKNEEEIDIPVQEEEEEEAEINFPAPPQEQESLPVENEIAP
[0207] The DNA segment comprising the nucleotide sequence of
mCD20(136-178) flanked by BamH1 and Xho1 restriction sites is
obtained by PCR using a full length murine CD20 cDNA clone as
template and the two primers shown below:
TABLE-US-00007 Upstream primer: BamHI_mCD20 primer (30-mer) (SEQ ID
NO: 31) 5'-GGATCCACACTTTCTCATTTTTTAAAAATG Downstream primer: XhoI
mCD20 primer (30-mer) (SEQ ID NO: 32)
5'-CTCGAGGTTACAGTACTGTGTAGATGGGGA
[0208] The PCR amplimer (141 bp) is cloned into the PGEMT.RTM.
vector (PROMEGA.RTM.). A DDD2-pdHL2 mammalian expression vector,
for example, N-DDD2-hG-CSF-His-pdHL2, is prepared for ligation with
the amplimer by digestion with XbaI and Bam HI restriction
endonucleases. The mCD20-amplimer is excised from PGEMT.RTM. with
XbaI and Bam HI and ligated into the DDD2-pdHL2 vector to generate
the expression vector DDD2-mCD20(136-178)-pdHL2.
[0209] Transfection and Screen to Obtain Clones Expressing
DDD2-mCD20(136-178)
[0210] The vector DDD2-mCD20(136-178) is linearized by digestion
with SalI enzyme and stably transfected into SpESF myeloma cells by
electroporation (see, e.g., U.S. Pat. No. 7,537,930, the Examples
section of which is incorporated herein by reference). A number of
clones are found to have detectable levels of DDD2-mCD20(136-178)
by ELISA, from which the best producing clone is selected and
subsequently amplified with increasing methotrexate (MTX)
concentrations from 0.1 to 0.8 .mu.M over five weeks. At this
stage, it is sub-cloned by limiting dilution and the highest
producing sub-clone is expanded.
[0211] The clone is expanded to 34 roller bottles containing a
total of 20 L of serum-free Hybridoma SFM with 0.8 .mu.M MTX and
allowed to reach terminal culture. The supernatant fluid is
clarified by centrifugation and filtered (0.2 .mu.M). The filtrate
is diafiltered into 1.times. Binding buffer (10 mM imidazole, 0.5 M
NaCl, 50 mM NaH.sub.2PO.sub.4, pH 7.5) and concentrated to 310 mL
in preparation for purification by immobilized metal affinity
chromatography (IMAC). The concentrate is loaded onto a 30-mL
Ni-NTA column, which is washed with 500 mL of 0.02% Tween 20 in
1.times. binding buffer and then 290 mL of 30 mM imidazole, 0.02%
Tween 20, 0.5 M NaCl, 50 mM NaH.sub.2PO.sub.4, pH 7.5. The product
is eluted with 110 mL of 250 mM imidazole, 0.02% Tween 20, 150 mM
NaCl, 50 mM NaH.sub.2PO.sub.4, pH 7.5. The purity of
DDD2-mCD20(136-178) is assessed by SDS-PAGE under reducing
conditions.
Example 5
Generation of 74-mCD20 DNL Vaccine Comprising hLL1 IgG Linked to
Four Copies of mCD20(136-178)
[0212] C.sub.H3-AD2-IgG-hLL1 (anti-CD74) is produced as described
in Examples 2 and 3. The construct comprises an AD2 moiety attached
to the C-terminal end of each heavy chain of the hLL1 IgG.
DDD2-mCD20(136-178) is produced as described in Example 4. A DNL
reaction is performed by mixing hLL1 IgG-AD2 and
DDD2-mCD20(136-178) in PBS containing 1 mM reduced glutathione. On
the next day oxidized glutathione is added to a final concentration
of 2 mM and the reaction mixture is purified on a Protein A column
24 h later. In this embodiment, two copies of the DDD2-mCD20 are
attached to each AD2 moiety, resulting in a DNL complex comprising
one hLL1 IgG moiety and four mCD20 xenoantigen moieties.
[0213] In an alternative embodiment, the Fab of hLL1 is linked to
DDD2 and the mCD20(136-178) to AD2. Formation of a DNL construct as
described above results in the formation of an MM vaccine,
designated hLL1-F(ab).sub.2-mCD20(136-178), which comprises a
single mCD20(136-178) attached to two Fab moieties of hLL1. The
generation of AD2-mCD20(136-178) is described in Example 6.
[0214] Administration of 74-mCD20(136-178) or
hLL1-F(ab).sub.2-mCD20(136-178) to subjects with MM induces an
immune response against CD138.sup.negCD20.sup.+.gtoreq.putative MM
stem cells. The immune response is effective to reduce or eliminate
MM disease cells in the subjects.
Example 6
Generation of Recombinant AD2-mCD20(136-178)
[0215] AD2-mCD20(136-178)-pdHL2 is the expression vector for
recombinant AD2-mCD20(136-178), which comprises
AD2-linker-mCD20(136-178)-HHHHHH (HHHHHH disclosed as SEQ ID
NO:30). The DNA segment comprising the nucleotide sequence of
mCD20(136-178) flanked by Bgl2 and Eag1 restriction sites is
obtained by PCR using a full length murine CD20 cDNA clone as
template and the two primers shown below:
TABLE-US-00008 Upstream primer: Bgl2_mCD20 primer (30-mer) (SEQ ID
NO: 33) 5'-AGATCTACACTTTCTCATTTTTTAAAAATG Downstream primer:
Eag1_mCD20 primer (48-mer) (SEQ ID NO: 34) 5'
CGGCCGTCAGTGGTGGTGGTGGTGGTGGTTACAGTACTGTGTAGATGG
[0216] The PCR amplimer (162 bp) is cloned into the PGEMT.RTM.
vector (PROMEGA.RTM.). An AD2-pdHL2 mammalian expression vector,
for example, N-AD2-hTransferrin-His-pdHL2, is prepared for ligation
with the amplimer by digestion with Bgl2 and Eag1 restriction
endonucleases. The mCD20-amplimer is excised from PGEMT.RTM. with
Bgl2 and Eag1 and ligated into the AD2-pdHL2 vector to generate the
expression vector AD2-mCD20(136-178)-pdHL2. Clones expressing
AD2-mCD20(136-178) are obtained as described in Example 4 and
AD2-mCD20(136-178) is purified from culture supernatants using
Ni-select.
Example 7
AD and DDD Sequence Variants
[0217] In certain preferred embodiments, the AD and DDD sequences
incorporated into the DNL complexes comprise the amino acid
sequences of AD2 (SEQ ID NO:13) and DDD2 (SEQ ID NO:11), as
described above. However, in alternative embodiments sequence
variants of the AD and/or DDD moieties may be utilized in
construction of the cytokine-MAb DNL complexes. The
structure-function relationships of the AD and DDD domains have
been the subject of investigation. (See, e.g., Burns-Hamuro et al.,
2005, Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem
276:17332-38; Alto et al., 2003, Proc Natl Acad Sci USA
100:4445-50; Hundsrucker et al., 2006, Biochem J 396:297-306;
Stokka et al., 2006, Biochem J 400:493-99; Gold et al., 2006, Mol
Cell 24:383-95; Kinderman et al., 2006, Mol Cell 24:397-408.)
[0218] For example, Kinderman et al. (2006) examined the crystal
structure of the AD-DDD binding interaction and concluded that the
human DDD sequence contained a number of conserved amino acid
residues that were important in either dimer formation or AKAP
binding, underlined in SEQ ID NO:35 below. (See FIG. 1 of Kinderman
et al., 2006.) The skilled artisan will realize that in designing
sequence variants of the DDD sequence, one would desirably avoid
changing any of the underlined residues, while conservative amino
acid substitutions might be made for residues that are less
critical for dimerization and AKAP binding.
[0219] Human DDD Sequence from Protein Kinase a
TABLE-US-00009 (SEQ ID NO: 35)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
[0220] Alto et al. (2003) performed a bioinformatic analysis of the
AD sequence of various AKAP proteins to design an RII selective AD
sequence called AKAP-IS (SEQ ID NO:12), with a binding constant for
DDD of 0.4 nM. The AKAP-IS sequence was designed as a peptide
antagonist of AKAP binding to PKA. Residues in the AKAP-IS sequence
where substitutions tended to decrease binding to DDD are
underlined in SEQ ID NO:12 below.
[0221] AKAP-IS Sequence
TABLE-US-00010 (SEQ ID NO: 12) QIEYLAKQIVDNAIQQA
[0222] Similarly, Gold (2006) utilized crystallography and peptide
screening to develop a SuperAKAP-IS sequence (SEQ ID NO:19),
exhibiting a five order of magnitude higher selectivity for the RII
isoform of PKA compared with the RI isoform. Underlined residues
indicate the positions of amino acid substitutions, relative to the
AKAP-IS sequence, that increased binding to the DDD moiety of
RII.alpha.. In this sequence, the N-terminal Q residue is numbered
as residue number 4 and the C-terminal A residue is residue number
20. Residues where substitutions could be made to affect the
affinity for RII.alpha. were residues 8, 11, 15, 16, 18, 19 and 20
(Gold et al., 2006). It is contemplated that in certain alternative
embodiments, the SuperAKAP-IS sequence may be substituted for the
AKAP-IS AD moiety sequence to prepare cytokine-MAb DNL constructs.
Other alternative sequences that might be substituted for the
AKAP-IS AD sequence are shown in SEQ ID NO:20-22. Substitutions
relative to the AKAP-IS sequence are underlined. It is anticipated
that, as with the AKAP-IS sequence shown in SEQ ID NO:19, the AD
moiety may also include the additional N-terminal residues cysteine
and glycine and C-terminal residues glycine and cysteine.
[0223] SuperAKAP-IS
TABLE-US-00011 (SEQ ID NO: 19) QIEYVAKQIVDYAIHQA (SEQ ID NO: 20)
QIEYKAKQIVDHAIHQA (SEQ ID NO: 21) QIEYHAKQIVDHAIHQA (SEQ ID NO: 22)
QIEYVAKQIVDHAIHQA
[0224] Alternative AKAP Sequences
[0225] Stokka et al. (2006) also developed peptide competitors of
AKAP binding to PKA, shown in SEQ ID NO:23-25. The peptide
antagonists were designated as Ht31 (SEQ ID NO:23), RIAD (SEQ ID
NO:24) and PV-38 (SEQ ID NO:25). The Ht-31 peptide exhibited a
greater affinity for the RII isoform of PKA, while the RIAD and
PV-38 showed higher affinity for RI.
TABLE-US-00012 Ht31 (SEQ ID NO: 23) DLIEEAASRIVDAVIEQVKAAGAY RIAD
(SEQ ID NO: 24) LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 25)
FEELAWKIAKMIWSDVFQQC
[0226] Hundsrucker et al. (2006) developed still other peptide
competitors for AKAP binding to PKA, with a binding constant as low
as 0.4 nM to the DDD of the RII form of PKA. The sequences of
various AKAP antagonistic peptides is provided in Table 1 of
Hundsrucker et al. (incorporated herein by reference). Residues
that were highly conserved among the AD domains of different AKAP
proteins are indicated below by underlining with reference to the
AKAP IS sequence (SEQ ID NO:12). The residues are the same as
observed by Alto et al. (2003), with the addition of the C-terminal
alanine residue. (See FIG. 4 of Hundsrucker et al. (2006),
incorporated herein by reference.) The sequences of peptide
antagonists with particularly high affinities for the RII DDD
sequence are shown in SEQ ID NO:26-28.
TABLE-US-00013 AKAP-IS (SEQ ID NO: 12) QIEYLAKQIVDNAIQQA
AKAP7.delta.-wt-pep (SEQ ID NO: 26) PEDAELVRLSKRLVENAVLKAVQQY
AKAP7.delta.-L304T-pep (SEQ ID NO: 27) PEDAELVRTSKRLVENAVLKAVQQY
AKAP7.delta.-L308D-pep (SEQ ID NO: 28)
PEDAELVRLSKRDVENAVLKAVQQY
[0227] Carr et al. (2001) examined the degree of sequence homology
between different AKAP-binding DDD sequences from human and
non-human proteins and identified residues in the DDD sequences
that appeared to be the most highly conserved among different DDD
moieties. These are indicated below by underlining with reference
to the human PKA RII.alpha. DDD sequence of SEQ ID NO:35. Residues
that were particularly conserved are further indicated by italics.
The residues overlap with, but are not identical to those suggested
by Kinderman et al. (2006) to be important for binding to AKAP
proteins.
TABLE-US-00014 (SEQ ID NO: 35)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
[0228] The skilled artisan will realize that in general, those
amino acid residues that are highly conserved in the DDD and AD
sequences from different proteins are ones that it may be preferred
to remain constant in making amino acid substitutions, while
residues that are less highly conserved may be more easily varied
to produce sequence variants of the AD and/or DDD sequences
described herein.
[0229] The skilled artisan will realize that these and other amino
acid substitutions in the antibody moiety or linker portions of the
DNL constructs may be utilized to enhance the therapeutic and/or
pharmacokinetic properties of the resulting DNL constructs.
Example 8
Effects of hLL1 on DCs--Efficient Binding of hLL1 with Different
Subsets of APCs
[0230] Early studies demonstrated that CD74 is expressed in most
antigen-presenting cells including blood DCs, B cells, monocytes.
To further characterize the expression profile of CD74 in APCs, we
examined the expression of CD74 in different subsets of human PBMCs
and in vitro monocyte-derived DCs. Using the gating strategy that
is shown in FIG. 1A, we found all of the blood DC subsets, the
myeloid DC1 (MDC1) and DC2 (MDC2), and plasmacytoid DC (PDC)
expressed CD74, with MDC2 expressing the highest level of CD74
(FIG. 1B). CD74 was also expressed in monocyte-derived immature DCs
at much higher level than in LPS-matured DCs (FIG. 2A). Consistent
with the CD74 expression profiles, hLL1 bound efficiently with
blood DC subsets, B cells, monocytes, and monocyte-derived immature
DCs (FIG. 1C, FIG. 2B), but not LPS-matured DCs (FIG. 2B, FIG. 2C).
The binding efficiency of hLL1 in these APC subsets correlates well
with their CD74 expression levels. These data provide the basis for
in vivo targeting of antigen to APCs using hLL1 as the targeting
vehicle by Dock-and-lock technology.
[0231] Cytotoxic Effect of hLL1 on CD74-Expressing Malignant B
Cells but not on Normal DCs
[0232] Since CD74 is highly expressed in immature DCs, with which
hLL1 binds efficiently, as shown in FIG. 1A and FIG. 1B, we
wondered if hLL1 has the same cytotoxicity in DCs, as it does a in
CD74-expressing B cell lymphoma, which was shown previously (Stein
et al., Blood 2004, 104:3705-11). To this end, the effects of hLL1
on the cell viability of B cell malignancy Daudi cells and human
monocyte-derived DCs were side-by-side compared using an MTS assay
and microscope imaging. The results demonstrated that hLL1, in the
presence of GAH (goat anti-human antibody), the second antibody for
hLL1 cross-linking, significantly reduced cell viability of Daudi
cells but not DCs (FIG. 3A), which normally expressed high level of
CD74 as shown above. The microscopic imaging showed that Daudi
cells treated with hLL1 crosslinked with GAH became clumped and
condensed, while the DCs maintained normal morphology after the
same treatment (FIG. 3C, FIG. 3D). The cytotoxicity against Daudi
cells by hLL1 cross-linked with GAH was consistent with the earlier
study by Stein et al. (2004) showing that hLL1 was cytotoxic to B
cell malignancies in vitro and in vivo. The lack of cytotoxicity of
hLL1 plus GAH on DCs was further demonstrated in apoptosis assay,
which showed that the hypodiploid nuclei populations were not
influenced by hLL1 cross-linked with GAH (not shown).
[0233] To further confirm the lack of cytotoxicity of hLL1 on DCs,
we performed apoptosis assay using flow cytometry. The nuclei from
hLL1 treated immature DCs were obtained and stained with PI for
flow cytometry analysis. The PI+ particles were gated first, and
the debris was excluded by gating out the SSC-low particles. The
resulting gated nuclei were analyzed for apoptosis by measuring
hypodiploid nuclei population (FIG. 2A). The results demonstrate
that hLL1 had no influence on DC apoptosis in both donors (FIG. 2B,
FIG. 2C), in the presence or absence of a second mAb (20 .mu.g/ml)
for cross-linking (GAH, F(ab').sub.2 GAH IgG Fc.gamma.-specific).
These data demonstrated that hLL1, unlike its cytotoxic effect on B
cell malignancies, has little cytotoxicity against normal dendritic
cells which also express CD74 surface antigen.
[0234] Moderate Enhancement of DC Constitutive Maturation by
hLL1
[0235] Human IgG can interact with DCs through FcR ligation and has
opposing effects on DC maturation depending on which subtype(s) of
FcR is involved. hLL1, as a humanized IgG, may interact with human
DCs not only through CD74 but also through FcR expressed on DCs.
For this reason, we speculated that hLL1 may influence DC functions
through interaction with CD74 or FcR, or both. To investigate this,
we tested the effect of hLL1 on DC constitutive maturation during
in vitro culture of monocytes in the presence of hGM-CSF and
hIL-4.
[0236] Since DC maturation is usually reflected by its
morphological change, we also examined if hLL1 treatment has any
effect on DC morphology. As shown in FIG. 3B, DCs treated with
hLL1, at different doses for various days, in the absence or
presence of GAH cross-linking, appeared healthy and intact. The
hLL1-treated DCs exhibited some minor morphological changes
featured with fiber-like cells, which are similar to but less
obvious than LPS-treated DCs (not shown).
[0237] As mature DCs differ from immature DCs mainly in the
upregulation of antigen-presenting and costimulatory molecule
expression, altered cytokine production, and enhanced T-cell
stimulatory ability, we then investigated if hLL1 has any effect on
the expression level of antigen-presenting molecule HLA-DR and
costimulatory molecules CD54 and CD86 in DCs (FIG. 4). The results
show that hLL1 could upregulate HLA-DR, CD54, and CD86 in a
dose-dependent manner within the range of hLL1 concentrations at
0.05-5 ug/ml (FIG. 4A). However, the effect was not strong, as the
expression of HLA-DR and costimulatory molecules, CD54 and CD86,
were only 10% upregulated at 5 .mu.g/ml hLL1 compared to 0 ug/ml
(FIG. 4B). At the highest concentration (50 .mu.g/ml), the
expression of HLA-DR, CD54 and CD86 was not further upregulated but
slightly reduced, compared to hLL1 at 5 .mu.g/ml (FIG. 4B). These
results indicate that hLL1, although not potently, could enhance
the constitutive maturation of DCs.
[0238] No Significant Influence on T Cell Expansion by hLL1-Treated
DCs
[0239] The functional difference between immature DCs and mature
DCs is that mature DCs have a stronger capacity to stimulate T cell
proliferation and expansion. Since hLL1 could enhance the
constitutive maturation by upregulating the expression of HLA-DR,
CD54 and CD86 expression in DCs (FIG. 4B), we determined whether
this DC-maturing effect could be reflected by an enhanced T cell
expansion by DCs. As shown in FIG. 5, DCs treated with hLL1 at 0.05
to 50 .mu.g/ml did not influence the DC-mediated T cell expansion,
including total T cells, CD4+ and CD4- T cells (FIG. 5). This
result suggests that hLL1-enhanced DC constitutive maturation was
not strong enough to be translated into an enhanced T cell
stimulatory ability.
[0240] Polarization of Naive CD4+ T Cells Toward Th1 Effector Cells
by hLL1-Treated DCs
[0241] However, DCs have another important function: the
polarization of naive CD4 T cells to differentiate into different
effector cells, Th1, Th2, Th17, as well as newly defined Th17-1
cells. Th1 cells are critical for cellular immunity against
intracellular pathogens and cancers, whereas induction of Th2 cells
is responsible for humoral immunity. The IL-17-producing Th17 and
Th17-1 cells are other polarized cell populations which have
multiple functions in immunity to certain pathogens and autoimmune
inflammation. The polarization of these effector cells is largely
mediated through DC-secreted cytokines, the so-called "signal 3",
that DCs provide to T cells in the DC/T cell synapse. The CD4+
naive T cells can differentiate into Th1, Th2 and Th0 cells which
mediate different effector functions, among which the Th1 effector
cells play an essential role in maintaining CTL response against
cancer and infectious diseases. We have shown that hLL1 at 0.05 to
50 .mu.g/ml could enhance DC constitutive maturation in a weak but
dose-dependent manner, but DCs treated with these concentrations of
hLL1 didn't influence the DC-mediated T cell expansion (FIG. 5). We
were then interested if the hLL1-treated DCs could influence the
polarization of CD4+ naive T cells. As shown in FIG. 5,
hLL1-treated DCs polarized the CD4+ naive T cells to differentiate
toward more Th1 effector cells and fewer Th2 and Tnp cells. These
results indicate that DCs can be functionally modulated by hLL1. As
Th1 plays a crucial role in adaptive immunity against tumor and
infectious diseases, hLL1 may have an adjuvant-like activity when
used in vaccination.
Example 9
In Vitro Properties of 74-mCD20--Induction of hCD20-Specific
Immunity by 74-mCD20 in Human PBMCs
[0242] CD20 is a self antigen normally expressed on B cells, which
is theoretically difficult to target by vaccine strategies due to
immune tolerance. However, specific T-cell immune response to CD20
has been achieved in tumor bearing mice by vaccination with a
minigene encoding the extracellular domain of human CD20 (Palomba
et al., Clin Cancer Res 2005; 11:370-9), or a conjugate comprising
the extracellular domain of human CD20 and a carrier protein with
QS21 adjuvant (Roberts et al., Blood 2002; 99:3748-55). Several
other reports have also demonstrated the feasibility of using
xenoantigens to break immune tolerance, as shown for MUC1 in animal
models (Ding et al., Blood 2008; 112:2817-25; Soares et al., J
Immunol 2001; 166:6555-63) as well as in patients (Ramanathan et
al., Cancer Immunol Immunother 2005; 54:254-64). To test whether
74-mCD20 could successfully induce hCD20-specific immunity and
overcome the immune tolerance of CD20, the following experiment is
performed.
[0243] Human DCs are generated from PBMCs by culturing for 5 days
in the presence of hGM-CSF and hIL-4. The immature DCs are loaded
with 74-mCD20, and matured by LPS plus IFN-gamma. The mature DCs
are used to stimulate autologous PBMCs for 10 days. Restimulation
with the same loaded DCs is performed twice weekly. After the last
restimulation, the T cells are tested for their antigen specificity
by measuring cytokine response (IFN-gamma) upon stimulation by
sorted CD20-positive MM cancer stem cells. The CD20-negative MM
cells are used as a control. The T cells show a positive reaction
to CD20-positive MM cancer stem cells but not to control
CD20-negative MM cells.
[0244] Specific Binding, Internalization and Intracellular Location
of 74-mCD20 in Various Antigen Presenting Cells In Vitro
[0245] Our preliminary data have shown that hLL1 efficiently and
specifically binds with different APCs, including myeloid DC1 and
myeloid DC2, plasmacytoid DC, B cells and monocytes. In order to
confirm that 74-mCD20 has the same efficiency and specificity in
binding with APCs as hLL1 alone, the following experiment is
performed.
[0246] 74-mCD20 and the control M1-mCD20 (comprising the anti-MUC1
antibody hPAM4 linked to four copies of mCD20) are used. Binding
assays are performed as follows. Briefly, 15 .mu.g of 74-mCD20 or
M1-mCD20 are labeled with a ZENON.TM. ALEXA FLUOR.RTM. 488 human
IgG labeling kit (INVITROGEN.RTM.) following the manufacturer's
instructions. The labeled preparations are used to stain the human
PBMCs as described below.
[0247] Human PBMCs isolated from buffy coat using FICOLL-PAQUE.TM.
are treated with human FcR blocking Reagent (Miltenyi Biotec, 1:20
dilution) at 4.degree. C. for 10 min. The washed cells are stained
with specifically labeled mAbs and analyzed by flow cytometry
(FACSCALIBUR.RTM.). The labeled mAbs used for the study include
FITC-labeled anti-CD74 mAb ALEXA FLUOR.RTM. 488-labeled 74-mCD20;
ALEXA FLUOR.RTM. 488-labeled M1-mCD20; PE-conjugated anti-CD19 mAb
(for B cells); PE-conjugated anti-CD14 mAb (for monocytes); and
APC-conjugated mAb to BDCA-1 (for MDC1), BDCA-2 (for PDC), or
BDCA-3 (for MDC2). A gating strategy is used for identification of
B cells, monocytes, MDC1, MDC2, and PDC. Data were analyzed by
FlowJo software for mean fluorescence intensity and positive cell
populations expressing the surface markers.
[0248] To see if 74-mCD20 is internalized to endosomes for further
processing to MHC class II presentation and MHC class I
cross-presentation, the following experiment is performed. 74-mCD20
or M1-mCD20 is mixed with human PBMCs, and incubated at 4.degree.
C. for 1 hr, followed by extensive washing. The cells are then
transferred to 37.degree. C., fixed at different time points (0,
15, 30, or 45 min) and stained with ALEXA FLUOR.RTM.-labeled
anti-human IgG secondary antibody with or without prior
permeabilization. The mean fluorescence is determined by flow
cytometry, and the amount of internalized antibody is calculated by
subtracting the mean fluorescence in fixed cells (surface bound)
from that recorded with fixed and permeabilized cells (internalized
and surface bound) at various time points.
[0249] The results show that the 74-mCD20 DNL complex has the same
efficiency and specificity in binding with APCs as hLL1 alone.
Example 10
Induction of hCD20-Specific Immune Responses by 74-mCD20 In
Vivo
[0250] Intrahepatic injection of CD34+ human cord blood cells (HLA
A1 healthy donor) into irradiated newborn Rag2-/-.gamma.c-/- mice
is performed to generate the animal model for a reconstituted human
adaptive immune system including human T, B, and DC cells, and
structured primary and secondary lymphoid organs (Huff et al., J
Clin Oncol. 2008, 26:2895-900; Yang and Chang, Cancer Invest. 2008,
26:741-55). These mice are called Hu-Rag2-/-.gamma.c-/-mice.
[0251] To assess the immune responses induced by 74-mCD20, human
CD34+ cells reconstituted in Rag2-/-.gamma.c-/- mice are immunized
weekly for three times with 74-mCD20 or M1-mCD20(50 .mu.g per
mouse), in combination with or without CpG (50 .mu.g per mouse) for
in vivo DC maturation. Five days after the last immunization,
splenocytes of each animal are isolated and restimulated with
HLA-matched MM cancer stem cells for cytokine (IFN-gamma)
production, as assessed by intracellular cytokine staining with
flow cytometry. The specific cytotoxicity against MM cancer stem
cells is assessed by a calcein AM release assay with MM cancer stem
cells as the target cells. The CD20+ MM cancer stem cells are
isolated from the MM cell line RPMI18226 using magnetic beads. The
stem cell property is verified by staining with aldehyde
dehydrogenase. The results indicate that 74-mCD20 is capable of
inducing an anti-hcd20 specific immune response in vivo.
Example 11
Therapeutic Potential of 74-mCD20 Against MM Cancer Stem Cells: In
Vivo Evaluation by hPBMC/NOD/SCID Mouse Model or Adoptive
Transfer
[0252] The best way for in vivo evaluation of the therapeutic
effect of 74-mCD20 is to immunize an animal model that can support
both the growth of MM and the development of a human adaptive
immune system. Since human CD34+ cell-reconstituted
Rag2-/-.gamma.c-/- mice are immune-competent, which may not support
MM growth, the hPBMC/NOD/SCID mouse model is used to test the
therapeutic effect of 74-mCD20 against MM stem cells. The NOD/SCID
mice have been used for engraftment of clonogenic multiple myeloma
stem cells by Matsui et al. (Blood 2004, 103:2332-6; Cancer Res
2008, 68:190-7).
[0253] The NOD/SCID mice are also used for evaluating the
therapeutic effect by co-engraftment of tumor cells and hPBMC. By
carefully adjusting the cell numbers infused, this model can
support both tumor growth and hPBMC engraftment, and has been used
for testing the effect of an in vivo vaccine targeting DC-SIGN.
[0254] Four to six-week-old female NOD/SCID mice (Jackson
Laboratories, Barr Harbor, Me.) are irradiated with 300 cGy (84
cGy/min using a 137Cs gamma irradiator). 12-16 h later, sorted
CD20+ MM cancer stem cells (2 million) are injected via dorsal tail
vein. Meanwhile, a mixture of human PBMCs (3 million), immature DC
(30,000) and the DNL vaccine is injected into the mice
subcutaneously. At certain time points (days), mice are
euthanatized and bone marrow is harvested from the long bones and
the engraftment and therapeutic efficacy are determined by staining
for human CD138.sup.+ MM cells.
[0255] In order to further evaluate the therapeutic potential of
74-mCD20, an alternative method by adoptive transfer is used to
test the vaccine-elicited cytotoxicity against MM stem cells. The
human CD34+ cell-reconstituted Rag2-/-.gamma.c-/- mice are
immunized with 74-mCD20 as described above. The splenocytes are
harvested and injected via the tail vein into NOD/SCID mice
engrafted with CD20+ MM cancer stem cells. At certain time points
(days), mice are euthanatized and bone marrow is harvested from the
long bones and the engraftment and therapeutic efficacy are
determined by staining for human CD138+MM cells. The results
confirm that 74-mCD20 is capable of inducing an immune response
against CD20.sup.+ MM stem cells in vivo.
Example 12
Generation of DDD2-mPAP and DNL Vaccine Complex
[0256] A DDD2 conjugated PAP xenoantigen is generated from murine
prostatic acid phosphatase according to the method of Example 4.
The efficacy of dendritic cell based vaccination with a PAP
xenoantigen has been previously disclosed (Fong et al. J Immunol
2001, 167:7150-56). A DDD2-mPAP-pdHL2 expression vector is
constructed as described in Example 4 and the DDD2-mPAP xenoantigen
fusion protein is expressed in cell culture according to Example 4.
The murine prostatic acid phosphatase sequence is disclosed, for
example, in the NCBI database at Accession No. AAF23171. A
DDD2-mPAP-6His fusion protein is expressed and purified by
immobilized metal affinity chromatography (IMAC) as described in
Example 4.
[0257] A DNL construct comprising one copy of C.sub.H3-AD2-IgG-hLL1
(anti-CD74) and four copies of DDD2-mPAP is prepared according to
the methods of Example 5. The hLL1 IgG moiety comprises an AD2
sequence attached to the C-terminal end of each heavy chain of the
hLL1 IgG. A DNL reaction is performed by mixing hLL1 IgG-AD2 and
DDD2-mPAP in PBS containing 1 mM reduced glutathione. On the next
day oxidized glutathione is added to a final concentration of 2 mM
and the reaction mixture is purified on a Protein A column 24 h
later. Two copies of the DDD2-mPAP are attached to each AD2 moiety,
resulting in a DNL complex comprising one hLL1 IgG moiety and four
mPAP xenoantigen moieties.
[0258] Administration of DNL vaccine anti-CD74-mPAP to subjects
with prostate cancer induces an immune response against PAP
expressing prostatic cancer stem cells. The immune response is
effective to reduce or eliminate prostatic cancer cells in the
subjects.
Example 13
Generation of DDD2-mEGFR and DNL Vaccine Complex
[0259] A DDD2 conjugated EGFR xenoantigen is generated from murine
EGFR according to the method of Example 4. The efficacy of EGFR
xenoantigen at inducing a humoral immune response has been
previously disclosed (Fang et al. Int J Mol Med 2009, 23:181-88). A
DDD2-mEGFR-pdHL2 expression vector comprising the extracellular
domain of murine EGFR is constructed as described in Example 4 and
the DDD2-mEGFR xenoantigen fusion protein is expressed in cell
culture according to Example 4. The murine EGFR sequence is
disclosed, for example, in the NCBI database at Accession No.
AAG43241. A DDD2-mEGFR-6His fusion protein is expressed and
purified by immobilized metal affinity chromatography (IMAC) as
described in Example 4.
[0260] A DNL construct comprising one copy of C.sub.H3-AD2-IgG-hLL1
(anti-CD74) and four copies of DDD2-mEGFR is prepared according to
the methods of Example 5. The hLL1 IgG moiety comprises an AD2
sequence attached to the C-terminal end of each heavy chain of the
hLL1 IgG. A DNL reaction is performed by mixing hLL1 IgG-AD2 and
DDD2-mEGFR in PBS containing 1 mM reduced glutathione. On the next
day oxidized glutathione is added to a final concentration of 2 mM
and the reaction mixture is purified on a Protein A column 24 h
later. Two copies of the DDD2-mEGFR are attached to each AD2
moiety, resulting in a DNL complex comprising one hLL1 IgG moiety
and four mEGFR xenoantigen moieties.
[0261] Administration of DNL vaccine anti-CD74-mEGFR to subjects
with EGFR-expressing NSCLC induces an immune response against
EGFR-expressing cancer stem cells. The immune response is effective
to reduce or eliminate EGFR positive cancer cells in the
subjects.
[0262] The skilled artisan will realize that DNL-based vaccines
incorporating xenoantigen moieties corresponding to a wide variety
of tumor-associated antigens may be constructed and utilized
according to the techniques described herein.
[0263] All of the COMPOSITIONS and METHODS disclosed and claimed
herein can be made and used without undue experimentation in light
of the present disclosure. While the compositions and methods have
been described in terms of preferred embodiments, it is apparent to
those of skill in the art that variations maybe applied to the
COMPOSITIONS and METHODS and in the steps or in the sequence of
steps of the METHODS described herein without departing from the
concept, spirit and scope of the invention. More specifically,
certain agents that are both chemically and physiologically related
may be substituted for the agents described herein while the same
or similar results would be achieved. All such similar substitutes
and modifications apparent to those skilled in the art are deemed
to be within the spirit, scope and concept of the invention as
defined by the appended claims.
Sequence CWU 1
1
35116PRTArtificial SequenceDescription of Artificial Sequence
Syntheticpeptide 1Arg Ser Ser Gln Ser Leu Val His Arg Asn Gly Asn
Thr Tyr Leu His 1 5 10 15 27PRTArtificial SequenceDescription of
Artificial Sequence Syntheticpeptide 2Thr Val Ser Asn Arg Phe Ser 1
5 39PRTArtificial SequenceDescription of Artificial Sequence
Syntheticpeptide 3Ser Gln Ser Ser His Val Pro Pro Thr 1 5
45PRTArtificial SequenceDescription of Artificial Sequence
Syntheticpeptide 4Asn Tyr Gly Val Asn 1 5 517PRTArtificial
SequenceDescription of Artificial Sequence Syntheticpeptide 5Trp
Ile Asn Pro Asn Thr Gly Glu Pro Thr Phe Asp Asp Asp Phe Lys 1 5 10
15 Gly 611PRTArtificial SequenceDescription of Artificial Sequence
Syntheticpeptide 6Ser Arg Gly Lys Asn Glu Ala Trp Phe Ala Tyr 1 5
10 7291PRTMus sp. 7Met Ser Gly Pro Phe Pro Ala Glu Pro Thr Lys Gly
Pro Leu Ala Met 1 5 10 15 Gln Pro Ala Pro Lys Val Asn Leu Lys Arg
Thr Ser Ser Leu Val Gly 20 25 30 Pro Thr Gln Ser Phe Phe Met Arg
Glu Ser Lys Ala Leu Gly Ala Val 35 40 45 Gln Ile Met Asn Gly Leu
Phe His Ile Thr Leu Gly Gly Leu Leu Met 50 55 60 Ile Pro Thr Gly
Val Phe Ala Pro Ile Cys Leu Ser Val Trp Tyr Pro 65 70 75 80 Leu Trp
Gly Gly Ile Met Tyr Ile Ile Ser Gly Ser Leu Leu Ala Ala 85 90 95
Ala Ala Glu Lys Thr Ser Arg Lys Ser Leu Val Lys Ala Lys Val Ile 100
105 110 Met Ser Ser Leu Ser Leu Phe Ala Ala Ile Ser Gly Ile Ile Leu
Ser 115 120 125 Ile Met Asp Ile Leu Asn Met Thr Leu Ser His Phe Leu
Lys Met Arg 130 135 140 Arg Leu Glu Leu Ile Gln Thr Ser Lys Pro Tyr
Val Asp Ile Tyr Asp 145 150 155 160 Cys Glu Pro Ser Asn Ser Ser Glu
Lys Asn Ser Pro Ser Thr Gln Tyr 165 170 175 Cys Asn Ser Ile Gln Ser
Val Phe Leu Gly Ile Leu Ser Ala Met Leu 180 185 190 Ile Ser Ala Phe
Phe Gln Lys Leu Val Thr Ala Gly Ile Val Glu Asn 195 200 205 Glu Trp
Lys Arg Met Cys Thr Arg Ser Lys Ser Asn Val Val Leu Leu 210 215 220
Ser Ala Gly Glu Lys Asn Glu Gln Thr Ile Lys Met Lys Glu Glu Ile 225
230 235 240 Ile Glu Leu Ser Gly Val Ser Ser Gln Pro Lys Asn Glu Glu
Glu Ile 245 250 255 Glu Ile Ile Pro Val Gln Glu Glu Glu Glu Glu Glu
Ala Glu Ile Asn 260 265 270 Phe Pro Ala Pro Pro Gln Glu Gln Glu Ser
Leu Pro Val Glu Asn Glu 275 280 285 Ile Ala Pro 290
824DNAArtificial SequenceDescription of Artificial Sequence
Syntheticprimer 8agatctggcg cacctgaact cctg 24933DNAArtificial
SequenceDescription of Artificial Sequence Syntheticprimer
9gaattcggat cctttacccg gagacaggga gag 331044PRTHomo sapiens 10Ser
His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1 5 10
15 Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala
20 25 30 Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40
1145PRTArtificial SequenceDescription of Artificial Sequence
Syntheticpolypeptide 11Cys Gly His Ile Gln Ile Pro Pro Gly Leu Thr
Glu Leu Leu Gln Gly 1 5 10 15 Tyr Thr Val Glu Val Leu Arg Gln Gln
Pro Pro Asp Leu Val Glu Phe 20 25 30 Ala Val Glu Tyr Phe Thr Arg
Leu Arg Glu Ala Arg Ala 35 40 45 1217PRTArtificial
SequenceDescription of Artificial Sequence Syntheticpeptide 12Gln
Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10
15 Ala 1321PRTArtificial SequenceDescription of Artificial Sequence
Syntheticpeptide 13Cys Gly Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val
Asp Asn Ala Ile 1 5 10 15 Gln Gln Ala Gly Cys 20 1410PRTArtificial
SequenceDescription of Artificial Sequence Syntheticpeptide 14Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 1555PRTArtificial
SequenceDescription of Artificial Sequence Syntheticpolypeptide
15Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Ile Gln Ile 1
5 10 15 Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr Thr Val Glu Val
Leu 20 25 30 Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala Val Glu
Tyr Phe Thr 35 40 45 Arg Leu Arg Glu Ala Arg Ala 50 55
1629PRTArtificial SequenceDescription of Artificial Sequence
Syntheticpeptide 16Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gln Ile Glu Tyr 1 5 10 15 Leu Ala Lys Gln Ile Val Asp Asn Ala Ile
Gln Gln Ala 20 25 1710PRTArtificial SequenceDescription of
Artificial Sequence Syntheticpeptide 17Gly Gly Gly Gly Ser Gly Gly
Gly Cys Gly 1 5 10 1843PRTMus sp. 18Thr Leu Ser His Phe Leu Lys Met
Arg Arg Leu Glu Leu Ile Gln Thr 1 5 10 15 Ser Lys Pro Tyr Val Asp
Ile Tyr Asp Cys Glu Pro Ser Asn Ser Ser 20 25 30 Glu Lys Asn Ser
Pro Ser Thr Gln Tyr Cys Asn 35 40 1917PRTArtificial
SequenceDescription of Artificial Sequence Syntheticpeptide 19Gln
Ile Glu Tyr Val Ala Lys Gln Ile Val Asp Tyr Ala Ile His Gln 1 5 10
15 Ala 2017PRTArtificial SequenceDescription of Artificial Sequence
Syntheticpeptide 20Gln Ile Glu Tyr Lys Ala Lys Gln Ile Val Asp His
Ala Ile His Gln 1 5 10 15 Ala 2117PRTArtificial SequenceDescription
of Artificial Sequence Syntheticpeptide 21Gln Ile Glu Tyr His Ala
Lys Gln Ile Val Asp His Ala Ile His Gln 1 5 10 15 Ala
2217PRTArtificial SequenceDescription of Artificial Sequence
Syntheticpeptide 22Gln Ile Glu Tyr Val Ala Lys Gln Ile Val Asp His
Ala Ile His Gln 1 5 10 15 Ala 2324PRTArtificial SequenceDescription
of Artificial Sequence Syntheticpeptide 23Asp Leu Ile Glu Glu Ala
Ala Ser Arg Ile Val Asp Ala Val Ile Glu 1 5 10 15 Gln Val Lys Ala
Ala Gly Ala Tyr 20 2418PRTArtificial SequenceDescription of
Artificial Sequence Syntheticpeptide 24Leu Glu Gln Tyr Ala Asn Gln
Leu Ala Asp Gln Ile Ile Lys Glu Ala 1 5 10 15 Thr Glu
2520PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 25Phe Glu Glu Leu Ala Trp Lys Ile Ala Lys Met Ile
Trp Ser Asp Val 1 5 10 15 Phe Gln Gln Cys 20 2625PRTArtificial
SequenceDescription of Artificial Sequence Syntheticpeptide 26Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 2725PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 27Pro
Glu Asp Ala Glu Leu Val Arg Thr Ser Lys Arg Leu Val Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 2825PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 28Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Asp Val Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 294PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 29Pro
Lys Ser Cys 1 306PRTArtificial SequenceDescription of Artificial
Sequence Synthetic 6xHis tag 30His His His His His His 1 5
3130DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 31ggatccacac tttctcattt tttaaaaatg
303230DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 32ctcgaggtta cagtactgtg tagatgggga
303330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 33agatctacac tttctcattt tttaaaaatg
303448DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 34cggccgtcag tggtggtggt ggtggtggtt acagtactgt
gtagatgg 483544PRTHomo sapiens 35Ser His Ile Gln Ile Pro Pro Gly
Leu Thr Glu Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val Leu Arg
Gln Gln Pro Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu Tyr Phe
Thr Arg Leu Arg Glu Ala Arg Ala 35 40
* * * * *