U.S. patent application number 15/232039 was filed with the patent office on 2016-11-24 for multimeric complexes with improved in vivo stability, pharmacokinetics and efficacy.
The applicant listed for this patent is IBC Pharmaceuticals, Inc.. Invention is credited to Chien-Hsing Chang, David M. Goldenberg, Edmund A. Rossi.
Application Number | 20160340443 15/232039 |
Document ID | / |
Family ID | 49670522 |
Filed Date | 2016-11-24 |
United States Patent
Application |
20160340443 |
Kind Code |
A1 |
Rossi; Edmund A. ; et
al. |
November 24, 2016 |
Multimeric Complexes with Improved in Vivo Stability,
Pharmacokinetics and Efficacy
Abstract
The present invention concerns multimeric complexes based on
antibody fusion proteins comprising an AD moiety attached to the
C-terminal end of each antibody light chain. The complexes further
comprise effector moities attached to DDD moieties. Two copies of
the DDD moiety form a dimer that binds to the AD moiety. The
complexes may be trimers, pentamers, hexamers or other multimers.
The effector moieties may be selected from a second antibody or
antigen-binding fragment thereof, a cytokine, an interferon, a
toxin, an antigen, a xenoantigen, a hapten, a protamine, a hormone,
an enzyme, a ligand-binding protein, a pro-apoptotic agent and an
anti-angiogenic agent. Surprisingly, attachment of the AD moiety to
the C-terminal end of the antibody light chain results in improved
pharmacokinetics and in vivo stability and efficacy, compared to
homologous complexes wherein the AD moiety is attached to the
antibody heavy chain.
Inventors: |
Rossi; Edmund A.; (Woodland
Park, NJ) ; 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: |
49670522 |
Appl. No.: |
15/232039 |
Filed: |
August 9, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13901737 |
May 24, 2013 |
9446123 |
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15232039 |
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61654310 |
Jun 1, 2012 |
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61662086 |
Jun 20, 2012 |
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61673553 |
Jul 19, 2012 |
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61682531 |
Aug 13, 2012 |
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61693042 |
Aug 24, 2012 |
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61694072 |
Aug 28, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6829 20170801;
C07K 16/2863 20130101; C07K 2317/24 20130101; C07K 2317/55
20130101; A61K 47/6849 20170801; C07K 2317/526 20130101; C07K
16/3007 20130101; A61P 37/00 20180101; A61P 35/00 20180101; C12N
2320/32 20130101; C07K 14/56 20130101; C07K 2319/00 20130101; C07K
2317/622 20130101; C07K 2317/94 20130101; C12N 9/22 20130101; C07K
2317/569 20130101; C07K 16/2887 20130101; C12N 2320/00 20130101;
C07K 16/3092 20130101; C12N 9/12 20130101; A61K 47/6813 20170801;
A61P 9/00 20180101; C07K 2317/30 20130101; C07K 16/2851 20130101;
A61P 25/00 20180101; C12N 15/111 20130101; C12Y 207/11011 20130101;
C12N 15/113 20130101; C07K 2317/32 20130101; C07K 2317/73 20130101;
C12Y 301/27 20130101; C07K 16/2833 20130101; C12N 2310/14 20130101;
C07K 16/44 20130101; A61K 2039/505 20130101; A61K 45/06 20130101;
C07K 16/18 20130101; A61P 31/00 20180101; A61K 47/6815 20170801;
C07K 2317/732 20130101; A61K 47/6817 20170801; C07K 2317/734
20130101; C07K 16/2803 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; C07K 16/28 20060101 C07K016/28; C12N 15/113 20060101
C12N015/113; C07K 14/56 20060101 C07K014/56; C12N 9/12 20060101
C12N009/12; C12N 9/22 20060101 C12N009/22; C07K 16/44 20060101
C07K016/44; C07K 16/18 20060101 C07K016/18 |
Claims
1. A multimeric complex comprising: a) a first fusion protein
comprising (i) a first IgG antibody and (ii) an AD (anchoring
domain) moiety from an AKAP protein attached to the C-terminal end
of each light chain of the antibody; and b) a second fusion protein
comprising (iii) an effector selected from the group consisting of
an antibody fragment, a cytokine, an interferon, a toxin and a
hapten, and (iv) 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) regulatory subunit RII.alpha.;
wherein two copies of the DDD moiety form a dimer that binds to one
copy of the AD moiety to form the complex.
2. The complex of claim 1, wherein the IgG antibody is selected
from the group consisting of hR1 (anti-IGF-1R), hPAM4
(anti-MUC5AC), KC4 (anti-mucin), hA20 (anti-CD20), hA19
(anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2
(anti-CD22), RFB4 (anti-CD22), hMu-9 (anti-CSAp), hL243
(anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7
(anti-TROP-2), hMN-3 (anti-CEACAM6), CC49 (anti-TAG-72), J591
(anti-PSMA), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX),
infliximab (anti-TNF-.alpha.), certolizumab pegol
(anti-TNF-.alpha.), adalimumab (anti-TNF-.alpha.), alemtuzumab
(anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR),
gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20),
panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab
(anti-CD20), GA101 (anti-CD20), trastuzumab (anti-ErbB2),
tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25),
daclizumab (anti-CD25), efalizumab (anti-CD11a), muromonab-CD3
(anti-CD3 receptor), natalizumab (anti-.alpha.4 integrin) and
omalizumab (anti-IgE).
3. The complex of claim 1, wherein the hapten is In-DTPA or
HSG.
4. The complex of claim 1, wherein the antibody fragment is
selected from the group of claim 2 consisting of, a Fab', a Fab, a
Fv, a scFv and a dAb.
5. The complex of claim 1, wherein the toxin is selected from the
group consisting of a bacterial toxin, a plant toxin, ricin, abrin,
alpha toxin, saporin, ribonuclease (RNase), DNase I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria
toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, ranpirnase
(Rap) and Rap (N69Q).
6. The complex according to claim 5, wherein the toxin is
ranpirnase (Rap).
7. The complex of claim 1, wherein the cytokine is selected from
the group consisting of human growth hormone, N-methionyl human
growth hormone, bovine growth hormone, parathyroid hormone,
thyroxin, insulin, proinsulin, relaxin, prorelaxin, follicle
stimulating hormone (FSH), thyroid stimulating hormone (TSH),
luteinizing hormone (LH), hepatic growth factor, prostaglandin,
fibroblast growth factor, prolactin, placental lactogen, OB
protein, tumor necrosis factor-a, tumor necrosis factor-.beta.,
mullerian-inhibiting substance, mouse gonadotropin-associated
peptide, inhibin, activin, vascular endothelial growth factor,
integrin, thrombopoietin (TPO), a nerve growth factor (NGF),
NGF-.beta., platelet-growth factor, a transforming growth factors
(TGF), TGF-.alpha., TGF-.beta., insulin-like growth factor-I,
insulin-like growth factor-II, erythropoietin (EPO), an
osteoinductive factor, an interferon, interferon-.alpha.,
interferon-.beta., interferon-.gamma., interferon-.lamda., a colony
stimulating factors (CSF), macrophage-CSF (M-CSF),
granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF),
interleukin-1 (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, LIF, kit-ligand, FLT-3, angiostatin,
thrombospondin, endostatin, tumor necrosis factor and LT
(lymphotoxin).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] [01] This application is a divisional of U.S. patent
application Ser. No. 13/901,737, filed May 24, 2013, which claimed
the benefit under 35 U.S.C. 119(e) of provisional U.S. Patent
Applications 61/654,310, filed Jun. 1, 2012, 61/662,086, filed Jun.
20, 2012, 61/673,553, filed Jul. 19, 2012, 61/682,531, filed Aug.
13, 2012, 61/693,042, filed Aug. 24, 2012 and 61/694,072, filed
Aug. 28, 2012, each priority application incorporated herein by
reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 3, 2013, is named IBC137US1_SL.txt and is 59,697 bytes in
size.
FIELD
[0003] The present invention relates to compositions and methods of
use of multimeric complexes comprising multiple effector moieties.
Preferably the effector moieties are fusion proteins, each
comprising an anchoring domain (AD) moiety from an A-kinase
anchoring protein (AKAP) or a dimerization and docking domain (DDD)
moiety from a protein kinase A (PKA) regulatory subunit RI.alpha.,
RII.alpha. or RII.beta.. Two copies of the DDD moiety dimerize and
bind to an AD moiety to form the multimeric complex, which may be
trimeric, tetrameric, pentameric, hexameric or multimeric. The
person of ordinary skill will realize that in alternative
embodiments the AD and/or DDD moieties may be attached to an
effector by chemical cross-linking or other known means. The
effectors may be selected from antibodies, antigen-binding antibody
fragments, antigens, cytokines, chemokines, interleukins,
interferons, growth factors, pro-apoptotic agents, anti-angiogenic
agents, toxins, ligand-binding proteins, enzymes, therapeutic
agents or polymers such as polyethylene glycol (PEG). Preferably,
at least one effector is an antibody or antigen-binding antibody
fragment, with an AD moiety attached to the C-terminal end of each
light chain of the antibody or antibody fragment. More preferably,
at least one effector is an IgG antibody. In certain embodiments,
all of the effectors may be antibodies or antibody fragments,
providing bispecific or multispecific antigen-binding complexes.
The subject multimeric complexes are of use for treating a wide
variety of diseases and medical conditions, such as cancer,
autoimmune disease, immune system dysfunction, graft-versus-host
disease, organ transplant rejection, neurologic disease, metabolic
disease, infectious disease or cardiovascular disease.
BACKGROUND
[0004] A significant aspect of recent biomedical research is the
development of increasingly sophisticated antibody-based biologics,
such as bispecific antibodies, immunocytokines, antibody-toxin
conjugates and antibody-drug conjugates. Development of more
complex, and less natural, fusion proteins faces problems with
yield, stability, immunogenicity and pharmacokinetics (Pk). In
particular, immunoconjugates based on antibody fragments, including
single-chain Fv (scFv), Fab, or other Fc-lacking formats
(Kontermann, 2010, Curr Opin Mol Ther 12:176-83), are often
difficult to produce with homogeneity and sufficient yield, lack
Fc-effector functions, and inherently suffer from short circulating
serum half-lives (T.sub.1/2). By comparison, immunoconjugates of
IgG can be produced in high yields, with longer T.sub.1/2 and
in-vivo stability. Further, intact monoclonal antibodies (mAbs)
offer high-avidity bivalent binding with Fc-effector functions,
including antibody-dependent cell-mediated cytotoxicity (ADCC) and
complement-dependent cytotoxicity (CDC). The enhanced Pk of IgG is
attributed to two major factors. Its larger molecular size
(.about.150 kDa) precludes renal clearance, which is responsible
for the rapid elimination of smaller constructs (<60 kDa), such
as scFv, and its dynamic binding to the neonatal Fc receptor (FcRn)
(Kue & Aveson, 2011, MAbs 3:422-30) extends T.sub.1/2.
[0005] Multispecific or bispecific antibodies are useful in a
number of biomedical applications. For instance, a bispecific
antibody with binding sites for a tumor cell surface antigen and
for a T-cell surface receptor can direct the lysis of specific
tumor cells by T cells. Bispecific antibodies recognizing gliomas
and the CD3 epitope on T cells have been successfully used in
treating brain tumors in human patients (Nitta, et al. Lancet.
1990; 355:368-371). Numerous methods to produce bispecific
antibodies are known (see, e.g. U.S. Pat. No. 7,405,320).
Bispecific antibodies can be produced by the quadroma method, which
involves the fusion of two different hybridomas, each producing a
monoclonal antibody recognizing a different antigenic site
(Milstein and Cuello. Nature. 1983; 305:537-540). The fused
hybridomas are capable of synthesizing two different heavy chains
and two different light chains, which can associate randomly to
give a heterogeneous population of 10 different antibody structures
of which only one of them, amounting to 1/8 of the total antibody
molecules, will be bispecific, and therefore must be further
purified from the other forms. Fused hybridomas are often less
stable cytogenetically than the parent hybridomas, making the
generation of a production cell line more problematic.
[0006] Another method for producing bispecific antibodies uses
heterobifunctional cross-linkers to chemically tether two different
monoclonal antibodies, so that the resulting hybrid conjugate will
bind to two different targets (Staerz, et al. Nature. 1985;
314:628-631; Perez, et al. Nature. 1985; 316:354-356). Bispecific
antibodies generated by this approach are essentially
heteroconjugates of two IgG molecules, which diffuse slowly into
tissues and are rapidly removed from the circulation. Bispecific
antibodies can also be produced by reduction of each of two
parental monoclonal antibodies to the respective half molecules,
which are then mixed and allowed to reoxidize to obtain the hybrid
structure (Staerz and Bevan. Proc Natl Acad Sci USA. 1986;
83:1453-1457). An alternative approach involves chemically
cross-linking two or three separately purified Fab' fragments using
appropriate linkers. All these chemical methods are undesirable for
commercial development due to high manufacturing cost, laborious
production process, extensive purification steps, low yields
(<20%), and heterogeneous products.
[0007] Other methods include improving the efficiency of generating
hybrid hybridomas by gene transfer of distinct selectable markers
via retrovirus-derived shuttle vectors into respective parental
hybridomas, which are fused subsequently (DeMonte, et al. Proc Natl
Acad Sci USA. 1990, 87:2941-2945); or transfection of a hybridoma
cell line with expression plasmids containing the heavy and light
chain genes of a different antibody. These methods also face the
inevitable purification problems discussed above.
[0008] Discrete V.sub.H and V.sub.L domains of antibodies produced
by recombinant DNA technology may pair with each other to form a
dimer (recombinant Fv fragment) with binding capability (U.S. Pat.
No. 4,642,334). However, such non-covalently associated molecules
are not sufficiently stable under physiological conditions to have
any practical use. Cognate V.sub.H and V.sub.L domains can be
joined with a peptide linker of appropriate composition and length
(usually consisting of more than 12 amino acid residues) to form a
single-chain Fv (scFv) with binding activity. Methods of
manufacturing scFv-based agents of multivalency and
multispecificity by varying the linker length were disclosed in
U.S. Pat. No. 5,844,094, U.S. Pat. No. 5,837,242 and WO 98/44001.
Common problems that have been frequently associated with
generating scFv-based agents of multivalency and multispecificity
are low expression levels, heterogeneous products, instability in
solution leading to aggregates, instability in serum, and impaired
affinity.
[0009] Dock-and-Lock.TM. (DNL.TM.) technology has been used to
produce a variety of immunoconjugates in assorted formats (Rossi et
al., 2012, Bioconjug Chem 23:309-23). Bispecific hexavalent
antibodies (bsHexAbs) based on veltuzumab (anti-CD20) and
epratuzumab (anti-CD22) were constructed by combining a stabilized
(Fab).sub.2 fused to a dimerization and docking domain (DDD) with
an IgG containing an anchor domain (AD) appended at the C-terminus
of each heavy chain (C.sub.H3-AD2-IgG) (Rossi et al., 2009, Blood
113, 6161-71). Compared to mixtures of their parental mAbs, these
Fc-based bsHexAbs, referred to henceforth as "Fc-bsHexAbs", induced
unique signaling events (Gupta et al., 2010, Blood 116:3258-67),
and exhibited potent cytotoxicity in vitro. However, the
Fc-bsHexAbs were cleared from circulation of mice approximately
twice as fast as the parental mAbs (Rossi et al., 2009, Blood 113,
6161-71). Although the Fc-bsHexAbs are highly stable ex vivo, it is
possible that some dissociation occurs in vivo, for example by
intracellular processing. Further, the Fc-bsHexAbs lack CDC
activity.
[0010] Fc-based immunocytokines have also been assembled as DNL.TM.
complexes, comprising two or four molecules of interferon-alpha 2b
(IFN.alpha.2b) fused to the C-terminal end of the C.sub.H3-AD2-IgG
Fc (Rossi et al., 2009, Blood 114:3864-71; Rossi et al., 2010,
Cancer Res 70:7600-09; Rossi et al., 2011, Blood 118:1877-84). The
Fc-IgG-IFN.alpha. maintained high specific activity, approaching
that of recombinant IFN.alpha., and were remarkably potent in vitro
and in vivo against non-Hodgkin lymphoma (NHL) xenografts. The
T.sub.1/2 of the Fc-IgG-IFN.alpha. in mice was longer than
PEGylated IFN.alpha., but half as long as the parental mAbs.
Similar to the Fc-bsHexAbs, the Fc-IgG-IFN.alpha. dissociated in
vivo over time and exhibited diminished CDC, but ADCC was
enhanced.
[0011] A need exists for methods and compositions to generate
improved multimeric complexes with longer T.sub.1/2, better
pharmacokinetic properties, increased in vivo stability and
improved in vivo efficacy.
SUMMARY
[0012] The present invention concerns compositions and methods for
producing improved DNL.TM. complexes with longer T.sub.1/2, better
pharmacokinetic properties and increased in vivo stability. In
preferred embodiments, the improved DNL.TM. complexes comprise IgG
components in which AD moieties are fused to the C-terminal end of
the antibody light chains. Surprisingly, the relocation of the AD
attachment site from the heavy chain to the light chain results in
substantially improved pharmacokinetics, in vivo stability and Fc
effector function, along with increased in vivo efficacy, compared
to the already potent Fc-based counterparts.
[0013] In various embodiments, the subject complexes may be
administered to a subject with a condition, for therapeutic and/or
diagnostic purposes. The skilled artisan will realize that any
condition that may be diagnosed and/or treated with a
multifunctional, bivalent, trivalent, multispecific or bispecific
complex may be treated with the subject compositions. Exemplary
conditions include, but are not limited to, cancer, hyperplasia,
neurodegenerative disease, Alzheimer's disease, cardiovascular
disease, metabolic disease, vasculitis, viral infection, fungal
infection, bacterial infection, diabetic retinopathy, macular
degeneration, autoimmune disease, edema, pulmonary hypertension,
sepsis, myocardial angiogenesis, plaque neovascularization,
restenosis, neointima formation after vascular trauma,
telangiectasia, hemophiliac joints, angiofibroma, fibrosis
associated with chronic inflammation, lung fibrosis, deep venous
thrombosis or wound granulation.
[0014] In particular embodiments, the disclosed methods and
compositions may be of use to treat autoimmune disease, such as
acute idiopathic thrombocytopenic purpura, chronic idiopathic
thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,
myasthenia gravis, systemic lupus erythematosus, lupus nephritis,
rheumatic fever, polyglandular syndromes, bullous pemphigoid, type
1 diabetes, type 2 diabetes, Henoch-Schonlein purpura,
post-streptococcalnephritis, erythema nodosum, Takayasu's
arteritis, Addison's disease, rheumatoid arthritis, multiple
sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme,
IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis,
Goodpasture's syndrome, thromboangitisubiterans, Sjogren's
syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,
thyrotoxicosis, scleroderma, chronic active hepatitis,
polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,
Wegener's granulomatosis, membranous nephropathy, amyotrophic
lateral sclerosis, tabes dorsalis, giant cell
arteritis/polymyalgia, pernicious anemia, rapidly progressive
glomerulonephritis, psoriasis or fibrosing alveolitis.
[0015] In certain embodiments, the complexes may be of use for
therapeutic treatment of cancer. It is anticipated that any type of
tumor and any type of tumor antigen may be targeted. Exemplary
types of cancers that may be targeted include acute lymphoblastic
leukemia, acute myelogenous leukemia, biliary cancer, breast
cancer, cervical cancer, chronic lymphocytic leukemia, chronic
myelogenous leukemia, colorectal cancer, endometrial cancer,
esophageal, gastric, head and neck cancer, Hodgkin's lymphoma, lung
cancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiple
myeloma, renal cancer, ovarian cancer, pancreatic cancer, glioma,
melanoma, liver cancer, prostate cancer, and urinary bladder
cancer.
[0016] Tumor-associated antigens that may be targeted include, but
are not limited to, carbonic anhydrase IX, CCL19, CCL21, CSAp, CD1,
CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19,
IGF-1R, 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, AFP, PSMA, CEACAM5, CEACAM-6, CSAp, B7, ED-B
of fibronectin, Factor H, FHL-1, Flt-3, folate receptor,
GRO-.beta., HMGB-1, hypoxia inducible factor (HIF), HM1.24,
insulin-like growth factor-1 (ILGF-1), 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, IP-10, MAGE, mCRP,
MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac,
macrophage inhibition factor (MIF), antigen bound by the PAM4
antibody, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR,
tenascin, Le(y), RANTES, T101, TAC, Tn antigen,
Thomson-Friedenreich antigens, tumor necrosis antigens,
TNF-.alpha., TRAIL receptor (R1 and R2), VEGFR, EGFR, P1GF, PSA,
PSMA, complement factors C3, C3a, C3b, C5a, C5, and an oncogene
product.
[0017] Exemplary antibodies that may be utilized include, but are
not limited to, hR1 (anti-IGF-1R, U.S. patent application Ser. No.
12/722,645, filed Mar. 12, 2010), hPAM4 (anti-mucin, U.S. Pat. No.
7,282,567), hA20 (anti-CD20, U.S. Pat. No. 7,251,164), hA19
(anti-CD19, U.S. Pat. No. 7,109,304), hIMMU31 (anti-AFP, U.S. Pat.
No. 7,300,655), hLL1 (anti-CD74, U.S. Pat. No. 7,312,318), hLL2
(anti-CD22, U.S. Pat. No. 7,074,403), hMu-9 (anti-CSAp, U.S. Pat.
No. 7,387,773), hL243 (anti-HLA-DR, U.S. Pat. No. 7,612,180),
hMN-14 (anti-CEACAM5, U.S. Pat. No. 6,676,924), hMN-15
(anti-CEACAM6, U.S. Pat. No. 7,541,440), hRS7 (anti-EGP-1, U.S.
Pat. No. 7,238,785) and hMN-3 (anti-CEACAM6, U.S. Pat. No.
7,541,440) the Examples section of each cited patent or application
incorporated herein by reference. The skilled artisan will realize
that this list is not limiting and that any known antibody may be
used, as discussed in more detail below.
[0018] In other embodiments, the subject complexes may be of use to
treat subjects infected with pathogenic organisms, such as
bacteria, viruses or fungi. Exemplary fungi that may be treated
include Microsporum, Trichophyton, Epidermophyton, Sporothrix
schenckii, Cryptococcus neoformans, Coccidioides immitis,
Histoplasma capsulatum, Blastomyces dermatitidis or Candida
albican. Exemplary viruses include human immunodeficiency virus
(HIV), herpes virus, cytomegalovirus, rabies virus, influenza
virus, human papilloma virus, hepatitis B virus, hepatitis C virus,
Sendai virus, feline leukemia virus, Reo virus, polio virus, human
serum parvo-like virus, simian virus 40, respiratory syncytial
virus, mouse mammary tumor virus, Varicella-Zoster virus, Dengue
virus, rubella virus, measles virus, adenovirus, human T-cell
leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps
virus, vesicular stomatitis virus, Sindbis virus, lymphocytic
choriomeningitis virus or blue tongue virus. Exemplary bacteria
include Bacillus anthracis, Streptococcus agalactiae, Legionella
pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria
gonorrhoeae, Neisseria meningitidis, Pneumococcus spp., Hemophilis
influenzae B, Treponema pallidum, Lyme disease spirochetes,
Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus,
Mycobacterium tuberculosis or a Mycoplasma.
[0019] Complexes of use for infection may comprise, for example,
binding sites for one or more antigenic determinant on a pathogen,
and may be conjugated or attached to a therapeutic agent for the
pathogen, for example an anti-viral, antibiotic or anti-fungal
agent. Alternatively, a stably tethered conjugate may comprise a
first binding site for a pathogen antigen and a second binding site
for a hapten or carrier that is attached to one or more therapeutic
agents. Therapeutic agents of use against infectious organisms that
may be conjugated to, incorporated into or targeted to bind to the
subject complexes include, but are not limited to, acyclovir,
albendazole, amantadine, amikacin, amoxicillin, amphotericin B,
ampicillin, aztreonam, azithromycin, bacitracin, bactrim,
BATRAFEN.RTM., bifonazole, carbenicillin, caspofungin, cefaclor,
cefazolin, cephalosporins, cefepime, ceftriaxone, cefotaxime,
chloramphenicol, cidofovir, CIPRO.RTM., clarithromycin, clavulanic
acid, clotrimazole, cloxacillin, doxycycline, econazole,
erythrocycline, erythromycin, flagyl, fluconazole, flucytosine,
foscamet, furazolidone, ganciclovir, gentamycin, imipenem,
isoniazid, itraconazole, kanamycin, ketoconazole, lincomycin,
linezolid, meropenem, miconazole, minocycline, naftifine, nalidixic
acid, neomycin, netilmicin, nitrofurantoin, nystatin, oseltamivir,
oxacillin, paromomycin, penicillin, pentamidine,
piperacillin-tazobactam, rifabutin, rifampin, rimantadine,
streptomycin, sulfamethoxazole, sulfasalazine, tetracycline,
tioconazole, tobramycin, tolciclate, tolnaftate, trimethoprim
sulfamethoxazole, valacyclovir, vancomycin, zanamir, and
zithromycin.
[0020] In various embodiments, the complexes may comprise one or
more toxins, such as a bacterial toxin, a plant toxin, ricin,
abrin, a ribonuclease (RNase), DNase I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria
toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, onconase,
ranpirnase (Rap), Rap (N69Q) or PE38. In a preferred embodiment,
the complex may comprise an IgG antibody attached to two AD
moieties and four copies of a toxin, such as ranpirnase, attached
to DDD moieties. Such complexes have been demonstrated to be highly
efficacious, with improved toxocity and phamacokinetic properties
(see, e.g., U.S. patent application Ser. No. 12/871,345, the
Examples section of which is incorporated herein by reference).
[0021] The subject complexes may also comprise an immunomodulator
selected from the group consisting of a cytokine, a chemokine, a
stem cell growth factor, a lymphotoxin, an hematopoietic factor, a
colony stimulating factor (CSF), an interferon, erythropoietin,
thrombopoietin, tumor necrosis factor-a (TNF), TNF-.beta.,
granulocyte-colony stimulating factor (G-CSF), granulocyte
macrophage-colony stimulating factor (GM-CSF), interferon-.alpha.,
interferon-.beta., interferon-.gamma., interferon-.lamda., stem
cell growth factor designated "S1 factor", human growth hormone,
N-methionyl human growth hormone, bovine growth hormone,
parathyroid hormone, thyroxine, insulin, proinsulin, relaxin,
prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating
hormone (TSH), luteinizing hormone (LH), hepatic growth factor,
prostaglandin, fibroblast growth factor, prolactin, placental
lactogen, OB protein, mullerian-inhibiting substance, mouse
gonadotropin-associated peptide, inhibin, activin, vascular
endothelial growth factor, integrin, NGF-.beta., platelet-growth
factor, TGF-.alpha., TGF-.beta., insulin-like growth factor-I,
insulin-like growth factor-II, macrophage-CSF (M-CSF), 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, FLT-3, angiostatin, thrombospondin, endostatin and
lymphotoxin. In certain preferred embodiments, the complex may
comprise an antibody or antibody fragment attached to two AD
moieties and four copies of an immunomodulator, such as a cytokine,
each attached to a DDD moiety. Cytokine complexes are disclosed,
for example, in U.S. Pat. Nos. 7,906,118 and 8,034,3522, the
Examples section of each incorporated herein by reference.
[0022] Chemokines of use that may be incorporated in a subject
complex include, but are not limited to, RANTES, MCAF, MIP1-alpha,
MIP1-beta and IT-10.
[0023] Anti-angiogenic agents of use that may be incorporated in a
subject complex include, but are not limited to, angiostatin,
baculostatin, canstatin, maspin, anti-VEGF antibodies or peptides,
anti-placental growth factor antibodies or peptides, anti-Flk-1
antibodies, anti-Flt-1 antibodies or peptides, 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, thalidomide,
pentoxifylline, genistein, TNP-470, endostatin, paclitaxel,
accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470,
platelet factor 4 or minocycline.
[0024] In still other embodiments, one or more therapeutic agents,
such as aplidin, azaribine, anastrozole, azacytidine, bleomycin,
bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin,
10-hydroxycamptothecin, carmustine, celebrex, chlorambucil,
cisplatin, irinotecan (CPT-11), SN-38, carboplatin, cladribine,
cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin,
daunomycin glucuronide, daunorubicin, dexamethasone,
diethylstilbestrol, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),
cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin
glucuronide, ethinyl estradiol, estramustine, etoposide, etoposide
glucuronide, etoposide phosphate, floxuridine (FUdR),
3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,
fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone
caproate, hydroxyurea, idarubicin, ifosfamide, L-asparaginase,
leucovorin, lomustine, mechlorethamine, medroprogesterone acetate,
megestrol acetate, melphalan, mercaptopurine, 6-mercaptopurine,
methotrexate, nitoxantrone, mithramycin, mitomycin, mitotane,
phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin,
PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol,
testosterone propionate, thalidomide, thioguanine, thiotepa,
teniposide, topotecan, uracil mustard, velcade, vinblastine,
vinorelbine, vincristine, an antisense oligonucleotide, an
interference RNA, or a combination thereof, may be conjugated to or
incorporated into a complex.
[0025] In embodiments involving pretargeting, the complex may
comprise a first antibody or fragment thereof that binds to an
antigen associated with a diseased cell, tissue or pathogen, while
a second antibody or fragment thereof may bind to a hapten on a
targetable construct. Following administration of the complex and
localization to a disease-associated cell, tissue, or pathogen, a
targetable construct may be added to bind to the localized complex.
Optionally, a clearing agent may be administered to clear
non-localized complexes from circulation before administration of
the targetable construct. These methods are known in the art and
described in U.S. Pat. No. 4,624,846, WO 92/19273, and Sharkey et
al., Int. J. Cancer 51: 266 (1992). An exemplary targetable
construct may have a structure of
X-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Y)-NH.sub.2, where the compound
includes a hard acid cation chelator at X or Y, and a soft acid
cation chelator at remaining X or Y; and wherein the compound
further comprises at least one diagnostic or therapeutic cation,
and/or one or more chelated or chemically bound therapeutic agent,
diagnostic agent, or enzyme.
[0026] Various embodiments may concern complexes that are of use to
induce apoptosis of diseased cells. Further details may be found in
U.S. Patent Application Publication No. 20050079184, the entire
text of which is incorporated herein by reference. Such structures
may comprise a first and/or second precursor with binding affinity
for an antigen selected from the group consisting of CD2, CD3, CD8,
CD10, CD21, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD48,
CD52, CD55, CD59, CD70, CD74, CD80, CD86, CD138, CD147, HLA-DR,
CEA, CSAp, CA-125, TAG-72, EFGR, HER2, HER3, HER4, IGF-1R, c-Met,
PDGFR, MUC1, MUC2, MUC3, MUC4, TNFR1, TNFR2, NGFR, Fas (CD95), DR3,
DR4, DRS, DR6, VEGF, PIGF, ED-B fibronectin, tenascin, PSMA, PSA,
carbonic anhydrase IX, and IL-6. In more particular embodiments, a
complex of use to induce apoptosis may comprise monoclonal
antibodies, Fab fragments, chimeric, humanized or human antibodies
or fragments. In preferred embodiments, the complex may comprise
combinations of anti-CD74 X anti-CD20, anti-CD74 X anti-CD22,
anti-CD22 X anti-CD20, anti-CD20 X anti-HLA-DR, anti-CD19 X
anti-CD20, anti-CD20 X anti-CD80, anti-CD2 X anti-CD25, anti-CD8 X
anti-CD25, and anti-CD2 X anti-CD147. In more preferred
embodiments, the chimeric, humanized or human antibodies or
antibody fragments may be derived from the variable domains of LL2
(anti-CD22), LL1 (anti-CD74) or A20 (anti-CD20).
[0027] Various embodiments may concern complexes and methods of use
for treating inflammatory and immune-dysregulatory diseases,
infectious diseases, pathologic angiogenesis or cancer. In this
application the complexes bind to two different targets selected
from the group consisting of (A) proinflammatory effectors of the
innate immune system, (B) coagulation factors, (C) complement
factors and complement regulatory proteins, and (D) targets
specifically associated with an inflammatory or
immune-dysregulatory disorder or with a pathologic angiogenesis or
cancer, wherein the latter target is not (A), (B), or (C). At least
one of the targets is (A), (B) or (C). Suitable combinations of
targets are described in U.S. Provisional Application No.
60/634,076, filed Dec. 8, 2004, entitled "Methods and Compositions
for Immunotherapy and Detection of Inflammatory and
Immune-Dysregulatory Disease, Infectious Disease, Pathologic
Angiogenesis and Cancer," the contents of which are incorporated
herein in their entirety.
[0028] The proinflammatory effector of the innate immune system to
which the binding molecules may bind may be a proinflammatory
effector cytokine, a proinflammatory effector chemokine or a
proinflammatory effector receptor. Suitable proinflammatory
effector cytokines include MIF, HMGB-1 (high mobility group box
protein 1), TNF-.alpha., IL-1, IL-4, IL-5, IL-6, IL-8, IL-12,
IL-15, and IL-18. Examples of proinflammatory effector chemokines
include CCL19, CCL21, IL-8, MCP-1, RANTES, MIP-1A, MIP-1B, ENA-78,
MCP-1, IP-10, GROB, and Eotaxin. Proinflammatory effector receptors
include IL-4R (interleukin-4 receptor), IL-6R (interleukin-6
receptor), IL-13R (interleukin-13 receptor), IL-15R (interleukin-15
receptor) and IL-18R (interleukin-18 receptor).
[0029] The complex also may react with at least one coagulation
factor, particularly tissue factor (TF) or thrombin. In other
embodiments, the complex reacts with at least one complement factor
or complement regulatory protein. In preferred embodiments, the
complement factor is selected from the group consisting of C3, C5,
C3a, C3b, and C5a. When the binding molecule reacts specifically
with a complement regulatory protein, the complement regulatory
protein preferably is selected from the group consisting of CD46,
CD55, CD59 and mCRP.
[0030] In certain embodiments, any therapeutic protein or peptide
known in the art may be attached to an AD or DDD sequence and used
as an effector in the claimed methods and compositions. A large
number of such therapeutic proteins or peptides are known, and are
described for example, in U.S. Patent Application Publication No.
20060084794, "Albumin fusion proteins," filed Nov. 2, 2005,
incorporated herein by reference in its entirety. Table 1 of
20060084794, which lists various known exemplary therapeutic
proteins or peptides of use, including exemplary identifiers,
patent reference numbers and preferred indications, is specifically
incorporated herein by reference in its entirety. Additional
therapeutic proteins or peptides of use are disclosed, for example,
in U.S. Pat. No. 6,309,633, incorporated herein by reference in its
entirety, and may include but are not limited to
adrenocorticotropic hormone, ebiratide, angiotensin, angiotensin
II, asparaginase, atrial natriuretic peptides, atrial sodium
diuretic peptides, bacitracin, beta-endorphins, blood coagulation
factors VII, VIII and IX, blood thymic factor, bone morphogenic
factor, bone morphogenic protein, bradykinin, caerulein, calcitonin
gene related polypeptide, calcitonins, CCK-8, cell growth factors,
EGF, TGF-alpha, TGF-beta, acidic FGF, basic FGF, chemokines,
cholecystokinin, cholecystokinin-8, cholecystokinin-pancreozymin,
colistin, colony-stimulating factors, GMCSF, MCSF,
corticotropin-releasing factor, cytokines, desmopressin, dipeptide,
dismutase, dynorphin, eledoisin, endorphins, endothelin,
endothelin-antagonistic peptides, endotherins, enkephalins,
epidermal growth factor, erythropoietin, follicle-stimulating
hormone, gallanin, gastric inhibitory polypeptide,
gastrin-releasing polypeptide, gastrins, G-CSF, glucagon,
glutathione peroxidase, glutathio-peroxidase, gonadotropin,
gramicidin, gramicidines, growth factor, growth hormone-releasing
factor, growth hormones, h-ANP, hormone releasing hormone, human
chorionic gonadotrophin, human chorionic gonadotrophin (3-chain,
human placental lactogen, insulin, insulin-like growth factors,
IGF-I, IGF-II, interferons, interleukins, intestinal polypeptide,
kallikrein, kyotorphin, luliberin, luteinizing hormone, luteinizing
hormone-releasing hormone, lysozyme chloride,
melanocyte-stimulating hormone, melanophore stimulating hormone,
mellitin, motilin, muramyl, muramyldipeptide, nerve growth factor,
nerve nutrition factors, NT-3, NT-4, CNTF, GDNF, BDNF, neuropeptide
Y, neurotensin, oxytocin, pancreastatin, pancreatic polypeptide,
pancreozymin, parathyroid hormone, pentagastrin, polypeptide YY,
pituitary adenyl cyclase-activating polypeptides, platelet derived
growth factor, polymixin B, prolactin, protein synthesis
stimulating polypeptide, PTH-related protein, relaxin, renin,
secretin, serum thymic factor, somatomedins, somatostatins,
substance P, superoxide, superoxide dismutase, taftsin,
tetragastrin, thrombopoietin, thymic humoral factor, thymopoietin,
thymosin, thymostimulin, thyroid hormone releasing hormone,
thyroid-stimulating hormone, thyrotropin releasing hormone TRH,
trypsin, tuftsin, tumor growth factor, tumor necrosis factor,
tyrocidin, urogastrone, urokinase, vasoactive intestinal
polypeptide, vasopressins, and functional equivalents.
[0031] In other preferred embodiments, the complexes may comprise
an antibody or fragment thereof that binds to an antigen on an
antigen-presenting cell (APC), such as a dendritic cell (DC)
antigen, attached to a xenoantigen or a mutagenized or chemically
modified antigen (see, e.g., U.S. Pat. No. 7,901,680, U.S. Ser. No.
12/754,740; the Examples section of each incorporated herein by
reference). Such complexes are of use to induce an immune response
against the selected target antigen, for example a tumor-associated
antigen. Exemplary DC antigens that may be targeted include, but
are 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 other embodiments, the DC targeting antibody
or fragment thereof may be attached to a pathogen-associated
antigen to induce an immune response against pathogens responsible
for infectious disease. An exemplary embodiment is disclosed for
pox-virus associated antigens in U.S. patent application Ser. No.
12/915,515, the Examples section of which is incorporated herein by
reference.
[0032] In alternative embodiments, the DNL.TM. complex may comprise
an antibody or antigen-binding antibody fragment attached to an
oligonucleotide carrier moiety, such as a protamine, dendrimer or
other polymer, of use to deliver interference RNA species such as
siRNA. Exemplary complexes of use for such purposes are disclosed
in U.S. patent application Ser. No. 12/964,021, filed Dec. 9, 2010,
the Examples section of which is incorporated herein by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The following drawings form part of the present
specification and are included to further demonstrate certain
embodiments of the present invention. The embodiments may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0034] FIG. 1. DNL.TM. modules and conjugates. (a) C.sub.k-AD2-IgG
(C.sub.k-AD2-IgG-epratuzumab or C.sub.k-AD2-IgG-veltuzumab), an
IgG-AD2 module with an AD2 fused to the carboxyl-terminal end of
each kappa light chain. (b) Dimeric C.sub.H1-DDD2-Fab-veltuzumab, a
Fab-DDD module with DDD2 fused to the carboxyl-terminal end of the
F.sub.d chain. (c) 22*-(20)-(20), a bsHexAb comprising
C.sub.k-AD2-IgG-epratuzumab and two dimeric
C.sub.H1-DDD2-Fab-veltuzumab modules. (d) 22-(20)-(20), a bsHexAb
comprising C.sub.H3-AD2-IgG-epratuzumab and two dimeric
C.sub.H1-DDD2-Fab-veltuzumab modules. (e) A dimeric
IFN.alpha.2b-DDD2 module with DDD2 fused to the carboxyl-terminal
end of IFN.alpha.2b via a flexible peptide linker. (f) 20*-2b, an
immunocytokine with tetrameric IFN.alpha.2b constructed with
C.sub.k-AD2-IgG-veltuzumab fused with two dimeric IFN.alpha.2b-DDD2
modules. (g) 20-2b, an IgG-IFN.alpha. with tetrameric IFN.alpha.2b
constructed with C.sub.H3-AD2-IgG fused with two dimeric
IFN.alpha.2b-DDD2 modules. Variable (V) and constant (C) domains of
IgG heavy (H) and light (L) chains are represented as ovals. The
DDD2 and AD2 peptides are shown as dark and light helices,
respectively, with the locations indicated for the reactive
sulfhydryl groups (SH) and the "locking" disulfide bridges
indicated as lines joining the helices.
[0035] FIG. 2. Pharmacokinetics of 22*-(20)-(20) and 22-(20)-(20)
in mice and rabbits. In this and other Figures, use of an asterisk
(*) indicates a DNL.TM. complex comprising light-chain conjugated
AD moieties, while the absence of an asterisk indicates a DNL.TM.
complex comprising heavy-chain conjugated AD moieties. At the
indicated intervals, the concentration of the intact molecules in
serum samples was measured using a bispecific ELISA. Animals were
administered 22*-(20)-(20) (circles), 22-(20)-(20) (squares) or
epratuzumab (triangles) subcutaneously. Mice were terminally bled,
while rabbits were serially bled. (a) Groups of 3 mice (mean.+-.SD)
were dosed with 1.0 mg of bsHexAb or an equal molar amount of
epratuzumab (0.41 mg). (b) Groups of 4 mice were dosed with 0.5 mg
of bsAb. Individual data points are plotted with non-linear
regression analysis using a one-phase exponential decay model with
Prism software. (c,d) Rabbits were administered 18 mg (6 mg/kg) of
bsAb. The Pk curves are shown for the individual animals (c) and
the mean.+-.SD of each group (d).
[0036] FIG. 3. Pharmacokinetics and in vivo stability of 20*-2b and
20-2b in mice. Groups of 3 mice were administered 1.0 mg of 20*-2b
(circles) or 20-2b (squares) by subcutaneous (a) or intravenous
(b,c,d) injection. (c) For each data point, obtained from
individual animals, the serum concentration of the intact
IgG-IFN.alpha. and total IgG (veltuzumab) was measured using
bispecific (solid line) or veltuzumab-specific (dashed line) ELISA
formats. (d) For each data point the % intact IgG-IFN.alpha. was
calculated by dividing the serum concentration measured with the
bispecific ELISA by that determined with the veltuzumab-specific
assay, and multiplying the quotient by 100. The % intact
IgG-IFN.alpha. was plotted vs. hours post injection, and the
dissociation rate was determined by linear regression analysis
(.+-.SD).
[0037] FIG. 4. Effector functions. In vitro CDC was compared among
(a) 20*-2b, 20-2b and veltuzumab, and (b) 22*-(20)-(20),
22-(20)-(20), veltuzumab and epratuzumab. (c) In vitro ADCC was
compared among 22*-(20)-(20), 22-(20)-(20), veltuzumab and
epratuzumab. mAb hMN-14 was used as a non-binding isotype control
for each experiment.
[0038] FIG. 5. In vivo efficacy with disseminated Daudi Burkitt
lymphoma xenografts. (a) Groups of 8 SCID mice were inoculated with
Daudi by IV injection. On day 7, mice were administered a single
dose of 1.0 or 0.25 .mu.g of 20*-2b or 20-2b by SC injection.
Control groups were administered 0.6 .mu.g of veltuzumab or saline.
(b) Groups of 10 SCID mice were inoculated by IV injection on day
0. On days 1 and 5, mice were administered high (1 mg) or low (10
.mu.g) doses of 22*-(20)-(20) or 22-(20)-(20) by SC injection.
Control groups were administered a high dose of epratuzumab, high
dose epratuzumab plus C.sub.H1-DDD2-veltuzumab or saline.
Statistical significance was determined by log-rank analysis of
Kaplan-Myer survival plots.
[0039] FIG. 6. CD20 binding. Daudi cells were incubated with
increasing concentrations of PE-labeled 20*-2b or veltuzumab and
binding was evaluated by flow cytometry. MFI, mean fluorescence
intensity.
[0040] FIG. 7. In vitro cytotoxicity. Daudi cells were incubated
with increasing concentrations of 20*-2b (circles) or 20-2b
(squares) for three days before quantification of the relative
viable cell density.
[0041] FIG. 8. In vivo dissociation of 22*-(20)-(20) and
22-(20)-(20) in mice. For each data point, obtained from individual
animals, the serum concentration of the intact bsHexAb and total
IgG (epratuzumab) was measured using bispecific or
epratuzumab-specific ELISA. The % intact bsHexAb was calculated by
dividing the serum concentration measured with the bispecific ELISA
by that determined with the epratuzumab-specific assay, and
multiplying the quotient by 100. The % intact IgG-IFN.alpha. was
plotted vs. hours post injection, and the dissociation rate was
determined by linear regression analysis. Error bars, standard
deviation.
[0042] FIG. 9. In vivo dissociation of 22*-(20)-(20) and
22-(20)-(20) in rabbits. For each data point, the serum
concentration of the intact bsHexAb and total IgG was measured
using bispecific or epratuzumab-specific ELISA. The % intact
bsHexAb was calculated by dividing the serum concentration measured
with the bispecific ELISA by that determined with the
epratuzumab-specific assay, and multiplying the quotient by 100. No
dissociation was observed in rabbits. Error bars, standard
deviation.
[0043] FIG. 10. Ex vivo depletion of Raji and normal B cells from
whole blood. Heparinized whole blood was incubated with 5 nM of the
indicated mAb for two days at 37.degree. C. before FACS
analysis.
[0044] FIG. 11. Schematic representation of a C.sub.k-AD2-IgG-pdHL2
expression vector.
[0045] FIG. 12. Schematic representation of a C.sub.k-AD2-IgG-pGSHL
expression vector.
DETAILED DESCRIPTION
Definitions
[0046] Unless otherwise specified, "a" or "an" means "one or
more".
[0047] As used herein, the terms "and" and "or" may be used to mean
either the conjunctive or disjunctive. That is, both terms should
be understood as equivalent to "and/or" unless otherwise
stated.
[0048] A "therapeutic agent" is an atom, molecule, or compound that
is useful in the treatment of a disease. Examples of therapeutic
agents include antibodies, antibody fragments, peptides, drugs,
toxins, enzymes, nucleases, hormones, immunomodulators, antisense
oligonucleotides, small interfering RNA (siRNA), chelators, boron
compounds, photoactive agents, dyes, and radioisotopes.
[0049] A "diagnostic agent" is an atom, molecule, or compound that
is useful in diagnosing a disease. Useful diagnostic agents
include, but are not limited to, radioisotopes, dyes (such as with
the biotin-streptavidin complex), contrast agents, fluorescent
compounds or molecules, and enhancing agents (e.g., paramagnetic
ions) for magnetic resonance imaging (MM).
[0050] An "antibody" as used herein 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 (i.e., specifically
binding) portion of an immunoglobulin molecule, like an antibody
fragment. An "antibody" includes monoclonal, polyclonal,
bispecific, multispecific, murine, chimeric, humanized and human
antibodies.
[0051] A "naked antibody" is an antibody or antigen binding
fragment thereof that is not attached to a therapeutic or
diagnostic agent. The Fc portion of an intact naked antibody can
provide effector functions, such as complement fixation and ADCC
(see, e.g., Markrides, Pharmacol Rev 50:59-87, 1998). Other
mechanisms by which naked antibodies induce cell death may include
apoptosis. (Vaswani and Hamilton, Ann Allergy Asthma Immunol 81:
105-119, 1998.)
[0052] An "antibody fragment" is a portion of an intact antibody
such as F(ab').sub.2, F(ab).sub.2, Fab', Fab, Fv, sFv, scFv, dAb
and the like. Regardless of structure, an antibody fragment binds
with the same antigen that is recognized by the full-length
antibody. For example, antibody fragments include isolated
fragments consisting of the variable regions, such as the "Fv"
fragments consisting of the variable regions of the heavy and light
chains or recombinant single chain polypeptide molecules in which
light and heavy variable regions are connected by a peptide linker
("scFv proteins"). "Single-chain antibodies", often abbreviated as
"scFv" consist of a polypeptide chain that comprises both a V.sub.H
and a V.sub.L domain which interact to form an antigen-binding
site. The V.sub.H and V.sub.L domains are usually linked by a
peptide of 1 to 25 amino acid residues. Antibody fragments also
include diabodies, triabodies and single domain antibodies
(dAb).
[0053] 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.
[0054] 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,
including human framework region (FR) sequences. The constant
domains of the antibody molecule are derived from those of a human
antibody.
[0055] A "human antibody" is an antibody obtained from transgenic
mice that have been genetically engineered to produce specific
human antibodies in response to antigenic challenge. In this
technique, elements of the human heavy and light chain locus are
introduced into strains of mice derived from embryonic stem cell
lines that contain targeted disruptions of the endogenous heavy
chain and light chain loci. The transgenic mice can synthesize
human antibodies specific for human 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, e.g.,
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 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).
[0056] As used herein, the term "antibody fusion protein" is a
recombinantly produced antigen-binding molecule in which an
antibody or antibody fragment is linked to another protein or
peptide, such as the same or different antibody or antibody
fragment or a DDD or AD peptide. 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.
[0057] A "multispecific antibody" is an antibody that can bind
simultaneously to at least two targets that are of different
structure, e.g., two different antigens, two different epitopes on
the same antigen, or a hapten and/or an antigen or epitope. A
"multivalent antibody" is an antibody that can bind simultaneously
to at least two targets that are of the same or different
structure. Valency indicates how many binding arms or sites the
antibody has to a single antigen or epitope; i.e., monovalent,
bivalent, trivalent or multivalent. The multivalency of the
antibody 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 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. Multispecific,
multivalent antibodies are constructs that have more than one
binding site of different specificity.
[0058] A "bispecific antibody" is an antibody that can bind
simultaneously to two targets which are of different structure.
Bispecific antibodies (bsAb) and bispecific antibody fragments
(bsFab) may have at least one arm that specifically binds to, for
example, a B cell, T cell, myeloid-, plasma-, and mast-cell antigen
or epitope and at least one other arm that specifically binds to a
targetable conjugate that bears a therapeutic or diagnostic agent.
A variety of bispecific antibodies can be produced using molecular
engineering.
[0059] A "xenoantigen" is an antigen that is found in more than one
species. When used herein in a vaccine to induce an immune response
in a subject, the xenoantigen is from a species that is different
from the subject. For example, a xenoantigen that is of use in a
human vaccine may be from a mouse, a rat, a rabbit or another
non-human species.
[0060] An antibody or immunotoxin preparation, or a composition
described herein, is said to be administered in a "therapeutically
effective amount" if the amount administered is physiologically
significant. An agent is physiologically significant if its
presence results in a detectable change in the physiology of a
recipient subject. In particular embodiments, an antibody
preparation is physiologically significant if its presence invokes
an antitumor response or mitigates the signs and symptoms of an
autoimmune disease state. A physiologically significant effect
could also be the evocation of a humoral and/or cellular immune
response in the recipient subject leading to growth inhibition or
death of target cells.
[0061] Antibodies
[0062] Techniques for preparing monoclonal antibodies against
virtually any target antigen 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.
[0063] 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).
[0064] 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.
[0065] Chimeric Antibodies
[0066] 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.
[0067] Humanized Antibodies
[0068] 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.
[0069] Human Antibodies
[0070] 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.
[0071] 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.
[0072] 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).
[0073] 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.
[0074] 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.
[0075] 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.
[0076] Antibody Cloning and Production
[0077] Various techniques, such as production of chimeric or
humanized antibodies, may involve procedures of antibody cloning
and construction. The antigen-binding V.sub..kappa. (variable light
chain) and V.sub.H (variable heavy chain) sequences for an antibody
of interest may be obtained by a variety of molecular cloning
procedures, such as RT-PCR, 5'-RACE, and cDNA library screening.
The V genes of an antibody from a cell that expresses a murine
antibody can be cloned by PCR amplification and sequenced. To
confirm their authenticity, the cloned V.sub.L and V.sub.H genes
can be expressed in cell culture as a chimeric Ab as described by
Orlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)).
Based on the V gene sequences, a humanized antibody can then be
designed and constructed as described by Leung et al. (Mol.
Immunol., 32: 1413 (1995)).
[0078] cDNA can be prepared from any known hybridoma line or
transfected cell line producing a murine antibody by general
molecular cloning techniques (Sambrook et al., Molecular Cloning, A
laboratory manual, 2.sup.nd Ed (1989)). The V.sub..kappa. sequence
for the antibody may be amplified using the primers VK1BACK and
VK1FOR (Orlandi et al., 1989) or the extended primer set described
by Leung et al. (BioTechniques, 15: 286 (1993)). The V.sub.H
sequences can be amplified using the primer pair VH1BACK/VH1FOR
(Orlandi et al., 1989) or the primers annealing to the constant
region of murine IgG described by Leung et al. (Hybridoma, 13:469
(1994)). Humanized V genes can be constructed by a combination of
long oligonucleotide template syntheses and PCR amplification as
described by Leung et al. (Mol. Immunol., 32: 1413 (1995)).
[0079] PCR products for V.sub..kappa. can be subcloned into a
staging vector, such as a pBR327-based staging vector, VKpBR, that
contains an Ig promoter, a signal peptide sequence and convenient
restriction sites. PCR products for V.sub.H can be subcloned into a
similar staging vector, such as the pBluescript-based VHpBS.
Expression cassettes containing the V.sub..kappa. and V.sub.H
sequences together with the promoter and signal peptide sequences
can be excised from VKpBR and VHpBS and ligated into appropriate
expression vectors, such as pKh and pG1g, respectively (Leung et
al., Hybridoma, 13:469 (1994)). The expression vectors can be
co-transfected into an appropriate cell and supernatant fluids
monitored for production of a chimeric, humanized or human
antibody. Alternatively, the V.sub..kappa. and V.sub.H expression
cassettes can be excised and subcloned into a single expression
vector, such as pdHL2, as described by Gillies et al. (J. Immunol.
Methods 125:191 (1989) and also shown in Losman et al., Cancer,
80:2660 (1997)).
[0080] In an alternative embodiment, expression vectors may be
transfected into host cells that have been pre-adapted for
transfection, growth and expression in serum-free medium. Exemplary
cell lines that may be used include the Sp/EEE, Sp/ESF and Sp/ESF-X
cell lines (see, e.g., U.S. Pat. Nos. 7,531,327; 7,537,930 and
7,608,425; the Examples section of each of which is incorporated
herein by reference). These exemplary cell lines are based on the
Sp2/0 myeloma cell line, transfected with a mutant Bcl-EEE gene,
exposed to methotrexate to amplify transfected gene sequences and
pre-adapted to serum-free cell line for protein expression.
[0081] Antibody Fragments
[0082] 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.
[0083] 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).
[0084] Techniques for producing single domain antibodies (DABs) are
also known in the art, as disclosed for example in Cossins et al.
(2006, Prot Express Purif 51:253-259), incorporated herein by
reference.
[0085] 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), incorporated herein by reference.
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 inacessible 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). In certain embodiments, anti-pancreatic
cancer VHH antibody fragments may be utilized in the claimed
compositions and methods.
[0086] 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.
[0087] Antibody Allotypes
[0088] Immunogenicity of therapeutic antibodies is associated with
increased risk of infusion reactions and decreased duration of
therapeutic response (Baert et al., 2003, N Engl J Med 348:602-08).
The extent to which therapeutic antibodies induce an immune
response in the host may be determined in part by the allotype of
the antibody (Stickler et al., 2011, Genes and Immunity 12:213-21).
Antibody allotype is related to amino acid sequence variations at
specific locations in the constant region sequences of the
antibody. The allotypes of IgG antibodies containing a heavy chain
.gamma.-type constant region are designated as Gm allotypes (1976,
J Immunol 117:1056-59).
[0089] For the common IgG1 human antibodies, the most prevalent
allotype is G1m1 (Stickler et al., 2011, Genes and Immunity
12:213-21). However, the G1m3 allotype also occurs frequently in
Caucasians (Stickler et al., 2011). It has been reported that G1m1
antibodies contain allotypic sequences that tend to induce an
immune response when administered to non-G1m1 (nG1m1) recipients,
such as G1m3 patients (Stickler et al., 2011). Non-G1m1 allotype
antibodies are not as immunogenic when administered to G1m1
patients (Stickler et al., 2011).
[0090] The human G1m1 allotype comprises the amino acids aspartic
acid at Kabat position 356 and leucine at Kabat position 358 in the
CH3 sequence of the heavy chain IgG1. The nG1m1 allotype comprises
the amino acids glutamic acid at Kabat position 356 and methionine
at Kabat position 358. Both G1m1 and nG1m1 allotypes comprise a
glutamic acid residue at Kabat position 357 and the allotypes are
sometimes referred to as DEL and EEM allotypes. A non-limiting
example of the heavy chain constant region sequences for G1m1 and
nG1m1 allotype antibodies is shown for the exemplary antibodies
rituximab (SEQ ID NO:85) and veltuzumab (SEQ ID NO:86).
TABLE-US-00001 Rituximab heavy chain variable region sequence (SEQ
ID NO: 85) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable
region (SEQ ID NO: 86)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0091] Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence
variations characteristic of IgG allotypes and their effect on
immunogenicity. They reported that the G1m3 allotype is
characterized by an arginine residue at Kabat position 214,
compared to a lysine residue at Kabat 214 in the G1m17 allotype.
The nG1m1,2 allotype was characterized by glutamic acid at Kabat
position 356, methionine at Kabat position 358 and alanine at Kabat
position 431. The G1m1,2 allotype was characterized by aspartic
acid at Kabat position 356, leucine at Kabat position 358 and
glycine at Kabat position 431. In addition to heavy chain constant
region sequence variants, Jefferis and Lefranc (2009) reported
allotypic variants in the kappa light chain constant region, with
the Km1 allotype characterized by valine at Kabat position 153 and
leucine at Kabat position 191, the Km1,2 allotype by alanine at
Kabat position 153 and leucine at Kabat position 191, and the Km3
allotype characterized by alanine at Kabat position 153 and valine
at Kabat position 191.
[0092] With regard to therapeutic antibodies, veltuzumab and
rituximab are, respectively, humanized and chimeric IgG1 antibodies
against CD20, of use for therapy of a wide variety of hematological
malignancies and/or autoimmune diseases. Table 1 compares the
allotype sequences of rituximab vs. veltuzumab. As shown in Table
1, rituximab (G1m17,1) is a DEL allotype IgG1, with an additional
sequence variation at Kabat position 214 (heavy chain CH1) of
lysine in rituximab vs. arginine in veltuzumab. It has been
reported that veltuzumab is less immunogenic in subjects than
rituximab (see, e.g., Morchhauser et al., 2009, J Clin Oncol
27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &
Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed
to the difference between humanized and chimeric antibodies.
However, the difference in allotypes between the EEM and DEL
allotypes likely also accounts for the lower immunogenicity of
veltuzumab.
TABLE-US-00002 TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy
chain position and associated allotypes 214 356/358 431 Complete
allotype (allotype) (allotype) (allotype) Rituximab G1m17,1 K 17
D/L 1 A -- Veltuzumab G1m3 R 3 E/M -- A --
[0093] In order to reduce the immunogenicity of therapeutic
antibodies in individuals of nG1m1 genotype, it is desirable to
select the allotype of the antibody to correspond to the G1m3
allotype, characterized by arginine at Kabat 214, and the nG1m1,2
null-allotype, characterized by glutamic acid at Kabat position
356, methionine at Kabat position 358 and alanine at Kabat position
431. Surprisingly, it was found that repeated subcutaneous
administration of G1m3 antibodies over a long period of time did
not result in a significant immune response. In alternative
embodiments, the human IgG4 heavy chain in common with the G1m3
allotype has arginine at Kabat 214, glutamic acid at Kabat 356,
methionine at Kabat 359 and alanine at Kabat 431. Since
immunogenicity appears to relate at least in part to the residues
at those locations, use of the human IgG4 heavy chain constant
region sequence for therapeutic antibodies is also a preferred
embodiment. Combinations of G1m3 IgG1 antibodies with IgG4
antibodies may also be of use for therapeutic administration.
[0094] Known Antibodies
[0095] In various embodiments, the claimed methods and compositions
may utilize any of a variety of antibodies known in the art.
Antibodies of use may be commercially obtained from a number of
known sources. For example, a variety of antibody secreting
hybridoma lines are available from the American Type Culture
Collection (ATCC, Manassas, Va.). A large number of antibodies
against various disease targets, including but not limited to
tumor-associated antigens, have been deposited at the ATCC and/or
have published variable region sequences and are available for use
in the claimed methods and compositions. 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, the Examples section of
each of which is incorporated herein by reference. 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 disease-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 (see, e.g., U.S. Pat. Nos.
7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples section
of each of which is incorporated herein by reference).
[0096] Particular antibodies that may be of use for therapy of
cancer within the scope of the claimed methods and compositions
include, but are not limited to, LL1 (anti-CD74), LL2 and RFB4
(anti-CD22), RS7 (anti-epithelial glycoprotein-1 (EGP-1)), PAM4 and
KC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA,
also known as CD66e), Mu-9 (anti-colon-specific antigen-p), Immu 31
(an anti-alpha-fetoprotein), TAG-72 (e.g., CC49), Tn, J591 or
HuJ591 (anti-PSMA (prostate-specific membrane antigen)),
AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250
(anti-carbonic anhydrase IX), hL243 (anti-HLA-DR), alemtuzumab
(anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR),
gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20);
panitumumab (anti-EGFR); rituximab (anti-CD20); tositumomab
(anti-CD20); GA101 (anti-CD20); and trastuzumab (anti-ErbB2). Such
antibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;
5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;
6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;
7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;
7,612,180; 7,642,239; and U.S. Patent Application Publ. No.
20040202666 (now abandoned); 20050271671; and 20060193865; the
Examples section of each incorporated herein by reference.)
Specific known antibodies of use include hPAM4 (U.S. Pat. No.
7,282,567), hA20 (U.S. Pat. No. 7,251,164), hA19 (U.S. Pat. No.
7,109,304), hIMMU31 (U.S. Pat. No. 7,300,655), hLL1 (U.S. Pat. No.
7,312,318), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S. Pat. No.
7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat. No.
6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. patent
application Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785),
hMN-3 (U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent
application Ser. No. 11/983,372, deposited as ATCC PTA-4405 and
PTA-4406) and D2/B (WO 2009/130575) the text of each recited patent
or application is incorporated herein by reference with respect to
the Figures and Examples sections.
[0097] Anti-TNF-.alpha. antibodies are known in the art and may be
of use to treat immune diseases, such as autoimmune disease, immune
dysfunction (e.g., graft-versus-host disease, organ transplant
rejection) or diabetes. Known antibodies against TNF-.alpha.
include the human antibody CDP571 (Ofei et al., 2011, Diabetes
45:881-85); murine antibodies MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI,
M302B and M303 (Thermo Scientific, Rockford, Ill.); infliximab
(Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,
Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and
many other known anti-TNF-.alpha. antibodies may be used in the
claimed methods and compositions. Other antibodies of use for
therapy of immune dysregulatory or autoimmune disease include, but
are not limited to, anti-B-cell antibodies such as veltuzumab,
epratuzumab, milatuzumab or hL243; tocilizumab (anti-IL-6
receptor); basiliximab (anti-CD25); daclizumab (anti-CD25);
efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3 receptor);
anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-.alpha.4
integrin) and omalizumab (anti-IgE).
[0098] Type-1 and Type-2 diabetes may be treated using known
antibodies against B-cell antigens, such as CD22 (epratuzumab),
CD74 (milatuzumab), CD19 (hA19), CD20 (veltuzumab) or HLA-DR
(hL243) (see, e.g., Winer et al., 2011, Nature Med 17:610-18).
Anti-CD3 antibodies also have been proposed for therapy of type 1
diabetes (Cernea et al., 2010, Diabetes Metab Rev 26:602-05).
[0099] Macrophage migration inhibitory factor (MIF) is an important
regulator of innate and adaptive immunity and apoptosis. It has
been reported that CD74 is the endogenous receptor for MIF (Leng et
al., 2003, J Exp Med 197:1467-76). The therapeutic effect of
antagonistic anti-CD74 antibodies on MIF-mediated intracellular
pathways may be of use for treatment of a broad range of disease
states, such as cancers of the bladder, prostate, breast, lung,
colon and chronic lymphocytic leukemia (e.g., Meyer-Siegler et al.,
2004, BMC Cancer 12:34; Shachar & Haran, 2011, Leuk Lymphoma
52:1446-54); autoimmune diseases such as rheumatoid arthritis and
systemic lupus erythematosus (Morand & Leech, 2005, Front
Biosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma
52:1446-54); kidney diseases such as renal allograft rejection
(Lan, 2008, Nephron Exp Nephrol. 109:e79-83); and numerous
inflammatory diseases (Meyer-Siegler et al., 2009, Mediators
Inflamm epub Mar. 22, 2009; Takahashi et al., 2009, Respir Res
10:33; Milatuzumab (hLL1) is an exemplary anti-CD74 antibody of
therapeutic use for treatment of MIF-mediated diseases.
[0100] The complement system is a complex cascade involving
proteolytic cleavage of serum glycoproteins often activated by cell
receptors. The "complement cascade" is constitutive and
non-specific but it must be activated in order to function.
Complement activation results in a unidirectional sequence of
enzymatic and biochemical reactions. In this cascade, a specific
complement protein, C5, forms two highly active, inflammatory
byproducts, C5a and CSb, which jointly activate white blood cells.
This in turn evokes a number of other inflammatory byproducts,
including injurious cytokines, inflammatory enzymes, and cell
adhesion molecules. Together, these byproducts can lead to the
destruction of tissue seen in many inflammatory diseases. This
cascade ultimately results in induction of the inflammatory
response, phagocyte chemotaxis and opsonization, and cell
lysis.
[0101] The complement system can be activated via two distinct
pathways, the classical pathway and the alternate pathway. Most of
the complement components are numbered (e.g., C1, C2, C3, etc.) but
some are referred to as "Factors." Some of the components must be
enzymatically cleaved to activate their function; others simply
combine to form complexes that are active. Active components of the
classical pathway include C1q, C1r, C1s, C2a, C2b, C3a, C3b, C4a,
and C4b. Active components of the alternate pathway include C3a,
C3b, Factor B, Factor Ba, Factor Bb, Factor D, and Properdin. The
last stage of each pathway is the same, and involves component
assembly into a membrane attack complex. Active components of the
membrane attack complex include C5a, C5b, C6, C7, C8, and C9n.
[0102] While any of these components of the complement system can
be targeted by an antibody complex, certain of the complement
components are preferred. C3a, C4a and C5a cause mast cells to
release chemotactic factors such as histamine and serotonin, which
attract phagocytes, antibodies and complement, etc. These form one
group of preferred targets. Another group of preferred targets
includes C3b, C4b and C5b, which enhance phagocytosis of foreign
cells. Another preferred group of targets are the predecessor
components for these two groups, i.e., C3, C4 and C5. C5b, C6, C7,
C8 and C9 induce lysis of foreign cells (membrane attack complex)
and form yet another preferred group of targets.
[0103] Complement C5a, like C3a, is an anaphylatoxin. It mediates
inflammation and is a chemotactic attractant for induction of
neutrophilic release of antimicrobial proteases and oxygen
radicals. Therefore, C5a and its predecessor C5 are particularly
preferred targets. By targeting C5, not only is C5a affected, but
also C5b, which initiates assembly of the membrane-attack complex.
Thus, C5 is another preferred target. C3b, and its predecessor C3,
also are preferred targets, as both the classical and alternate
complement pathways depend upon C3b. Three proteins affect the
levels of this factor, C1 inhibitor, protein H and Factor I, and
these are also preferred targets according to the invention.
Complement regulatory proteins, such as CD46, CD55, and CD59, may
be targets to which the antibody complexes bind.
[0104] Coagulation factors also are preferred targets, particularly
tissue factor and thrombin. Tissue factor is also known also as
tissue thromboplastin, CD142, coagulation factor III, or factor
III. Tissue factor is an integral membrane receptor glycoprotein
and a member of the cytokine receptor superfamily. The ligand
binding extracellular domain of tissue factor consists of two
structural modules with features that are consistent with the
classification of tissue factor as a member of type-2 cytokine
receptors. Tissue factor is involved in the blood coagulation
protease cascade and initiates both the extrinsic and intrinsic
blood coagulation cascades by forming high affinity complexes
between the extracellular domain of tissue factor and the
circulating blood coagulation factors, serine proteases factor VII
or factor VIIa. These enzymatically active complexes then activate
factor IX and factor X, leading to thrombin generation and clot
formation.
[0105] Tissue factor is expressed by various cell types, including
monocytes, macrophages and vascular endothelial cells, and is
induced by IL-1, TNF-.alpha. or bacterial lipopolysaccharides.
Protein kinase C is involved in cytokine activation of endothelial
cell tissue factor expression. Induction of tissue factor by
endotoxin and cytokines is an important mechanism for initiation of
disseminated intravascular coagulation seen in patients with
Gram-negative sepsis. Tissue factor also appears to be involved in
a variety of non-hemostatic functions including inflammation,
cancer, brain function, immune response, and tumor-associated
angiogenesis. Thus, antibody complexes that target tissue factor
are useful not only in the treatment of coagulopathies, but also in
the treatment of sepsis, cancer, pathologic angiogenesis, and other
immune and inflammatory dysregulatory diseases according to the
invention. A complex interaction between the coagulation pathway
and the cytokine network is suggested by the ability of several
cytokines to influence tissue factor expression in a variety of
cells and by the effects of ligand binding to the receptor. Ligand
binding (factor VIIa) has been reported to give an intracellular
calcium signal, thus indicating that tissue factor is a true
receptor.
[0106] Thrombin is the activated form of coagulation factor II
(prothrombin); it converts fibrinogen to fibrin. Thrombin is a
potent chemotaxin for macrophages, and can alter their production
of cytokines and arachidonic acid metabolites. It is of particular
importance in the coagulopathies that accompany sepsis. Numerous
studies have documented the activation of the coagulation system
either in septic patients or following LPS administration in animal
models. Despite more than thirty years of research, the mechanisms
of LPS-induced liver toxicity remain poorly understood. It is now
clear that they involve a complex and sequential series of
interactions between cellular and humoral mediators. In the same
period of time, gram-negative systemic sepsis and its sequalae have
become a major health concern, attempts to use monoclonal
antibodies directed against LPS or various inflammatory mediators
have yielded only therapeutic failures. antibody complexes that
target both thrombin and at least one other target address the
clinical failures in sepsis treatment.
[0107] In other embodiments, the antibody complexes bind to a MHC
class I, MHC class II or accessory molecule, such as CD40, CD54,
CD80 or CD86. The antibody complex also may bind to a T-cell
activation cytokine, or to a cytokine mediator, such as
NF-.kappa.B.
[0108] In certain embodiments, one of the two different targets may
be a cancer cell receptor or cancer-associated antigen,
particularly one that is selected from the group consisting of
B-cell lineage antigens (CD19, CD20, CD21, CD22, CD23, etc.), VEGF,
VEGFR, EGFR, carcinoembryonic antigen (CEA), placental growth
factor (P1GF), tenascin, HER-2/neu, EGP-1, EGP-2, CD25, CD30, CD33,
CD38, CD40, CD45, CD52, CD74, CD80, CD138, NCA66, CEACAM1, CEACAM6
(carcinoembryonic antigen-related cellular adhesion molecule 6),
MUC1, MUC2, MUC3, MUC4, MUC16, IL-6, a-fetoprotein (AFP), A3,
CA125, colon-specific antigen-p (CSAp), folate receptor, HLA-DR,
human chorionic gonadotropin (HCG), Ia, EL-2, insulin-like growth
factor (IGF) and IGF receptor, KS-1, Le(y), MAGE, necrosis
antigens, PAM-4, prostatic acid phosphatase (PAP), Pr1, prostate
specific antigen (PSA), prostate specific membrane antigen (PSMA),
S100, T101, TAC, TAG72, TRAIL receptors, and carbonic anhydrase
IX.
[0109] Targets associated with sepsis and immune dysregulation and
other immune disorders include MIF, IL-1, IL-6, IL-8, CD74, CD83,
and C5aR. Antibodies and inhibitors against C5aR have been found to
improve survival in rodents with sepsis (Huber-Lang et al., FASEB J
2002; 16:1567-1574; Riedemann et al., J Clin Invest 2002;
110:101-108) and septic shock and adult respiratory distress
syndrome in monkeys (Hangen et al., J Surg Res 1989; 46:195-199;
Stevens et al., J Clin Invest 1986; 77:1812-1816). Thus, for
sepsis, one of the two different targets preferably is a target
that is associated with infection, such as LPS/C5a. Other preferred
targets include HMGB-1, tissue factor, CD14, VEGF, and IL-6, each
of which is associated with septicemia or septic shock. Preferred
antibody complexes are those that target two or more targets from
HMGB-1, tissue factor and MIF, such as MIF/tissue factor, and
HMGB-1/tissue factor.
[0110] In still other embodiments, one of the different targets may
be a target that is associated with graft versus host disease or
transplant rejection, such as MIF (Lo et al., Bone Marrow
Transplant, 30(6):375-80 (2002)). One of the different targets also
may be one that associated with acute respiratory distress
syndrome, such as IL-8 (Bouros et al., PMC Pulm Med, 4(1):6 (2004),
atherosclerosis or restenosis, such as MIF (Chen et al.,
Arterioscler Thromb Vasc Biol, 24(4):709-14 (2004), asthma, such as
IL-18 (Hata et al., Int Immunol, Oct. 11, 2004 Epub ahead of
print), a granulomatous disease, such as TNF-.alpha. (Ulbricht et
al., Arthritis Rheum, 50(8):2717-8 (2004), a neuropathy, such as
carbamylated EPO (erythropoietin) (Leist et al., Science
305(5681):164-5 (2004), or cachexia, such as IL-6 and
TNF-.alpha..
[0111] Other targets include C5a, LPS, IFN-gamma, B7; CD2, CD4,
CD14, CD18, CD11a, CD11b, CD11c, CD14, CD18, CD27, CD29, CD38,
CD40L, CD52, CD64, CD83, CD147, CD154. Activation of mononuclear
cells by certain microbial antigens, including LPS, can be
inhibited to some extent by antibodies to CD18, CD11b, or CD11c,
which thus implicate .beta..sub.2-integrins (Cuzzola et al., J
Immunol 2000; 164:5871-5876; Medvedev et al., J Immunol 1998; 160:
4535-4542). CD83 has been found to play a role in giant cell
arteritis (GCA), which is a systemic vasculitis that affects
medium- and large-size arteries, predominately the extracranial
branches of the aortic arch and of the aorta itself, resulting in
vascular stenosis and subsequent tissue ischemia, and the severe
complications of blindness, stroke and aortic arch syndrome (Weyand
and Goronzy, N Engl J Med 2003; 349:160-169; Hunder and Valente,
In: Inflammatory Diseases of Blood Vessels. G. S. Hoffman and C. M.
Weyand, eds, Marcel Dekker, New York, 2002; 255-265). Antibodies to
CD83 were found to abrogate vasculitis in a SCID mouse model of
human GCA (Ma-Krupa et al., J Exp Med 2004; 199:173-183),
suggesting to these investigators that dendritic cells, which
express CD83 when activated, are critical antigen-processing cells
in GCA. In these studies, they used a mouse anti-CD83 MAb (IgG1
clone HB15e from Research Diagnostics). CD154, a member of the TNF
family, is expressed on the surface of CD4-positive T-lymphocytes,
and it has been reported that a humanized monoclonal antibody to
CD154 produced significant clinical benefit in patients with active
systemic lupus erythematosus (SLE) (Grammar et al., J Clin Invest
2003; 112:1506-1520). It also suggests that this antibody might be
useful in other autoimmune diseases (Kelsoe, J Clin Invest 2003;
112:1480-1482). Indeed, this antibody was also reported as
effective in patients with refractory immune thrombocytopenic
purpura (Kuwana et al., Blood 2004; 103:1229-1236).
[0112] In rheumatoid arthritis, a recombinant interleukin-1
receptor antagonist, IL-1Ra or anakinra, has shown activity (Cohen
et al., Ann Rheum Dis 2004; 63:1062-8; Cohen, Rheum Dis Clin North
Am 2004; 30:365-80). An improvement in treatment of these patients,
which hitherto required concomitant treatment with methotrexate, is
to combine anakinra with one or more of the anti-proinflammatory
effector cytokines or anti-proinflammatory effector chemokines (as
listed above). Indeed, in a review of antibody therapy for
rheumatoid arthritis, Taylor (Curr Opin Pharmacol 2003; 3:323-328)
suggests that in addition to TNF, other antibodies to such
cytokines as IL-1, IL-6, IL-8, IL-15, IL-17 and IL-18, are
useful.
[0113] The pharmaceutical composition of the present invention may
be used to treat a subject having a metabolic disease, such
amyloidosis, or a neurodegenerative disease, such as Alzheimer's
disease. Bapineuzumab is in clinical trials for Alzheimer's disease
therapy. Other antibodies proposed for therapy of Alzheimer's
disease include Alz 50 (Ksiezak-Reding et al., 1987, J Biol Chem
263:7943-47), gantenerumab, and solanezumab. Infliximab, an
anti-TNF-.alpha. antibody, has been reported to reduce amyloid
plaques and improve cognition.
[0114] In a preferred embodiment, diseases that may be treated
using the claimed compositions and methods include cardiovascular
diseases, such as fibrin clots, atherosclerosis, myocardial
ischemia and infarction. Antibodies to fibrin (e.g., scFv(59D8);
T2G1s; MH1) are known and in clinical trials as imaging agents for
disclosing said clots and pulmonary emboli, while anti-granulocyte
antibodies, such as MN-3, MN-15, anti-NCA95, and anti-CD15
antibodies, can target myocardial infarcts and myocardial ischemia.
(See, e.g., U.S. Pat. Nos. 5,487,892; 5,632,968; 6,294,173;
7,541,440, the Examples section of each incorporated herein by
reference) Anti-macrophage, anti-low-density lipoprotein (LDL),
anti-MIF, and anti-CD74 (e.g., hLL1) antibodies can be used to
target atherosclerotic plaques. Abciximab (anti-glycoprotein
IIb/IIIa) has been approved for adjuvant use for prevention of
restenosis in percutaneous coronary interventions and the treatment
of unstable angina (Waldmann et al., 2000, Hematol 1:394-408).
Anti-CD3 antibodies have been reported to reduce development and
progression of atherosclerosis (Steffens et al., 2006, Circulation
114:1977-84). Antibodies against oxidized LDL induced a regression
of established atherosclerosis in a mouse model (Ginsberg, 2007, J
Am Coll Cardiol 52:2319-21). Anti-ICAM-1 antibody was shown to
reduce ischemic cell damage after cerebral artery occlusion in rats
(Zhang et al., 1994, Neurology 44:1747-51). Commercially available
monoclonal antibodies to leukocyte antigens are represented by: OKT
anti-T-cell monoclonal antibodies (available from Ortho
Pharmaceutical Company) which bind to normal T-lymphocytes; the
monoclonal antibodies produced by the hybridomas having the ATCC
accession numbers HB44, HB55, HB12, HB78 and HB2; G7E11, W8E7,
NKP15 and G022 (Becton Dickinson); NEN9.4 (New England Nuclear);
and FMC11 (Sera Labs). A description of antibodies against fibrin
and platelet antigens is contained in Knight, Semin. Nucl. Med.,
20:52-67 (1990).
[0115] Other antibodies that may be used include antibodies against
infectious disease agents, such as bacteria, viruses, mycoplasms or
other pathogens. Many antibodies against such infectious agents are
known in the art and any such known antibody may be used in the
claimed methods and compositions. For example, antibodies against
the gp120 glycoprotein antigen of human immunodeficiency virus I
(HIV-1) are known, and certain of such antibodies can have an
immunoprotective role in humans. See, e.g., Rossi et al., Proc.
Natl. Acad. Sci. USA. 86:8055-8058, 1990. Known anti-HIV antibodies
include the anti-envelope antibody described by Johansson et al.
(AIDS. 2006 Oct. 3; 20(15):1911-5), as well as the anti-HIV
antibodies described and sold by Polymun (Vienna, Austria), also
described in U.S. Pat. No. 5,831,034, U.S. Pat. No. 5,911,989, and
Vcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al.,
Antimicrob. Agents Chemother. 2006; 50(5):1773-9, all incorporated
herein by reference.
[0116] Antibodies against malaria parasites can be directed against
the sporozoite, merozoite, schizont and gametocyte stages.
Monoclonal antibodies have been generated against sporozoites
(cirumsporozoite antigen), and have been shown to neutralize
sporozoites in vitro and in rodents (N. Yoshida et al., Science
207:71-73, 1980). Several groups have developed antibodies to T.
gondii, the protozoan parasite involved in toxoplasmosis (Kasper et
al., J. Immunol. 129:1694-1699, 1982; Id., 30:2407-2412, 1983).
Antibodies have been developed against schistosomular surface
antigens and have been found to act against schistosomulae in vivo
or in vitro (Simpson et al., Parasitology, 83:163-177, 1981; Smith
et al., Parasitology, 84:83-91, 1982: Gryzch et al., J. Immunol.,
129:2739-2743, 1982; Zodda et al., J. Immunol. 129:2326-2328, 1982;
Dissous et al., J. immunol., 129:2232-2234, 1982)
[0117] Trypanosoma cruzi is the causative agent of Chagas' disease,
and is transmitted by blood-sucking reduviid insects. An antibody
has been generated that specifically inhibits the differentiation
of one form of the parasite to another (epimastigote to
trypomastigote stage) in vitro, and which reacts with a
cell-surface glycoprotein; however, this antigen is absent from the
mammalian (bloodstream) forms of the parasite (Sher et al., Nature,
300:639-640, 1982).
[0118] Anti-fungal antibodies are known in the art, such as
anti-Sclerotinia antibody (U.S. Pat. No. 7,910,702);
antiglucuronoxylomannan antibody (Zhong and Priofski, 1998, Clin
Diag Lab Immunol 5:58-64); anti-Candida antibodies (Matthews and
Burnie, 2001, 2:472-76); and anti-glycosphingolipid antibodies
(Toledo et al., 2010, BMC Microbiol 10:47).
[0119] Suitable antibodies have been developed against most of the
microorganism (bacteria, viruses, protozoa, fungi, other parasites)
responsible for the majority of infections in humans, and many have
been used previously for in vitro diagnostic purposes. These
antibodies, and newer antibodies that can be generated by
conventional methods, are appropriate for use in the present
invention.
[0120] Immunoconjugates
[0121] In certain embodiments, the antibodies or fragments thereof
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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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. Pat. No. 7,563,433, the
Examples section of which is incorporated herein by reference).
[0126] 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.
[0127] 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.
[0128] 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. Pat. No. 7,563,433, the
Examples section of which is incorporated herein by reference.
[0129] Pre-Targeting
[0130] In certain embodiments, therapeutic agents may be
administered by a pretargeting method, utilizing bispecific or
multispecific antibody complexes. In pretargeting, the bispecific
or multispecific antibody comprises at least one binding arm that
binds to an antigen exhibited by a targeted cell or tissue, while
at least one other binding arm binds to a hapten on a targetable
construct. The targetable construct comprises one or more haptens
and one or more therapeutic and/or diagnostic agents.
[0131] Pre-targeting is a multistep process originally developed to
resolve the slow blood clearance of directly targeting antibodies,
which contributes to undesirable toxicity to normal tissues such as
bone marrow. With pre-targeting, a radionuclide or other diagnostic
or therapeutic agent is attached to a small delivery molecule
(targetable construct) that is cleared within minutes from the
blood. A pre-targeting bispecific or multispecific antibody, which
has binding sites for the targetable construct as well as a target
antigen, is administered first, free antibody is allowed to clear
from circulation and then the targetable construct is
administered.
[0132] Pre-targeting methods are disclosed, for example, in Goodwin
et al., U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med.
29:226, 1988; Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr
et al., J. Nucl. Med. 29:728, 1988; Klibanov et al., J. Nucl. Med.
29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989;
Kalofonos et al., J. Nucl. Med. 31:1791, 1990; Schechter et al.,
Int. J. Cancer 48:167, 1991; Paganelli et al., Cancer Res. 51:5960,
1991; Paganelli et al., Nucl. Med. Commun. 12:211, 1991; U.S. Pat.
No. 5,256,395; Stickney et al., Cancer Res. 51:6650, 1991; Yuan et
al., Cancer Res. 51:3119, 1991; U.S. Pat. Nos. 6,077,499;
7,011,812; 7,300,644; 7,074,405; 6,962,702; 7,387,772; 7,052,872;
7,138,103; 6,090,381; 6,472,511; 6,962,702; and 6,962,702, each
incorporated herein by reference.
[0133] A pre-targeting method of treating or diagnosing a disease
or disorder in a subject may be provided by: (1) administering to
the subject an antibody complex comprising a bispecific antibody or
antibody fragment; (2) optionally administering to the subject a
clearing composition, and allowing the composition to clear the
antibody from circulation; and (3) administering to the subject the
targetable construct, containing one or more chelated or chemically
bound therapeutic or diagnostic agents.
[0134] DOCK-AND-LOCK.TM. (DNL.TM.)
[0135] In preferred embodiments, a bivalent or multivalent antibody
or an antibody complexed to one or more effectors, such as
cytokines, toxins, xenoantigens or siRNA carriers, is formed as a
DOCK-AND-LOCK.TM. (DNL.TM.) complex (see, e.g., U.S. Pat. Nos.
7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,901,680;
7,906,118; 7,981,398; 8,003,111, the Examples section of each of
which is incorporated herein by reference.) Generally, the
technique takes advantage of the specific and high-affinity binding
interactions that occur between a dimerization and docking domain
(DDD) sequence of the regulatory (R) subunits of cAMP-dependent
protein kinase (PKA) and an anchor domain (AD) sequence derived
from any of a variety of AKAP proteins (Baillie et al., FEBS
Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.
2004; 5: 959). The DDD and AD peptides may be attached to any
protein, peptide or other molecule. Because the DDD sequences
spontaneously dimerize and bind to the AD sequence, the technique
allows the formation of complexes between any selected molecules
that may be attached to DDD or AD sequences.
[0136] Although the standard DNL.TM. complex comprises a trimer
with two DDD-linked molecules attached to one AD-linked molecule,
variations in complex structure allow the formation of dimers,
trimers, tetramers, pentamers, hexamers and other multimers. In
some embodiments, the DNL.TM. complex may comprise two or more
antibodies, antibody fragments or fusion proteins which bind to the
same antigenic determinant or to two or more different antigens.
The DNL.TM. complex may also comprise one or more other effectors,
such as proteins, peptides, immunomodulators, cytokines,
interleukins, interferons, binding proteins, peptide ligands,
carrier proteins, toxins, ribonucleases such as onconase,
inhibitory oligonucleotides such as siRNA, antigens or
xenoantigens, polymers such as PEG, enzymes, therapeutic agents,
hormones, cytotoxic agents, anti-angiogenic agents, pro-apoptotic
agents or any other molecule or aggregate.
[0137] 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 RII), and each type has
.alpha. and .beta. isoforms (Scott, Pharmacol. Ther. 1991; 50:123).
Thus, the four isoforms of PKA regulatory subunits are RI.alpha.,
RI.beta., RII.alpha. and RII.beta.. 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 of
RII.alpha. (Newlon et al., Nat. Struct. Biol. 1999; 6:222). As
discussed below, similar portions of the amino acid sequences of
other regulatory subunits are involved in dimerization and docking,
each located near the N-terminal end of the regulatory subunit.
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)
[0138] 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). 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.
[0139] We have developed a platform technology to utilize the DDD
of human PKA regulatory subunits and the AD of AKAP 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 DNL.TM. complex 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 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.2 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 stabilized 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. Using various combinations of
linkers, adaptor modules and precursors, a wide variety of DNL.TM.
constructs of different stoichiometry may be produced and used
(see, e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866;
7,527,787 and 7,666,400.)
[0140] By attaching the DDD and AD away from the functional groups
of the two precursors, such site-specific ligations are also
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, including peptides, proteins, antibodies, antibody
fragments, and other effector moieties with a wide range of
activities. Utilizing the fusion protein method of constructing AD
and DDD conjugated effectors described in the Examples below,
virtually any protein or peptide may be incorporated into a DNL.TM.
construct. However, the technique is not limiting and other methods
of conjugation may be utilized.
[0141] 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, 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. Site-specific attachment of a variety
of effector moieties may be performed using techniques known in the
art, such as the use of bivalent cross-linking reagents and/or
other chemical conjugation techniques.
[0142] Structure-Function Relationships in AD and DDD Moieties
[0143] For different types of DNL.TM. constructs, different AD or
DDD sequences may be utilized. Exemplary DDD and AD sequences are
provided below.
TABLE-US-00003 DDD1 (SEQ ID NO: 1)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2 (SEQ ID NO: 2)
CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1 (SEQ ID NO: 3)
QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4) CGQIEYLAKQIVDNAIQQAGC
[0144] The skilled artisan will realize that DDD1 and DDD2 are
based on the DDD sequence of the human RII.alpha. isoform of
protein kinase A. However, in alternative embodiments, the DDD and
AD moieties may be based on the DDD sequence of the human RI.alpha.
form of protein kinase A and a corresponding AKAP sequence, as
exemplified in DDD3, DDD3C and AD3 below.
TABLE-US-00004 DDD3 (SEQ ID NO: 5)
SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK DDD3C (SEQ ID
NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLE KEEAK AD3
(SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC
[0145] In other alternative embodiments, other sequence variants of
AD and/or DDD moieties may be utilized in construction of the
DNL.TM. complexes. For example, there are only four variants of
human PKA DDD sequences, corresponding to the DDD moieties of PKA
RI.alpha., RII.alpha., RI.beta. and RII.beta.. The RII.alpha. DDD
sequence is the basis of DDD1 and DDD2 disclosed above. The four
human PKA DDD sequences are shown below. The DDD sequence
represents residues 1-44 of RII.alpha., 1-44 of RII.beta., 12-61 of
RI.alpha. and 13-66 of RI.beta.. (Note that the sequence of DDD1 is
modified slightly from the human PKA RII.alpha. DDD moiety.)
TABLE-US-00005 PKA RI.alpha. (SEQ ID NO: 8)
SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEA K PKA RI.beta.
(SEQ ID NO: 9) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR
QILA PKA RII.alpha. (SEQ ID NO: 10)
SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RII.beta. (SEQ ID
NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER
[0146] 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, the entire text of each of which is incorporated herein
by reference.)
[0147] For example, Kinderman et al. (2006, Mol Cell 24:397-408)
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:1 below. (See
FIG. 1 of Kinderman et al., 2006, incorporated herein by
reference.) 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.
TABLE-US-00006 (SEQ ID NO: 1)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
[0148] As discussed in more detail below, conservative amino acid
substitutions have been characterized for each of the twenty common
L-amino acids. Thus, based on the data of Kinderman (2006) and
conservative amino acid substitutions, potential alternative DDD
sequences based on SEQ ID NO:1 are shown in Table 2. In devising
Table 2, only highly conservative amino acid substitutions were
considered. For example, charged residues were only substituted for
residues of the same charge, residues with small side chains were
substituted with residues of similar size, hydroxyl side chains
were only substituted with other hydroxyls, etc. Because of the
unique effect of proline on amino acid secondary structure, no
other residues were substituted for proline. A limited number of
such potential alternative DDD moiety sequences are shown in SEQ ID
NO:12 to SEQ ID NO:31 below. The skilled artisan will realize that
an almost unlimited number of alternative species within the genus
of DDD moieties can be constructed by standard techniques, for
example using a commercial peptide synthesizer or well known
site-directed mutagenesis techniques. The effect of the amino acid
substitutions on AD moiety binding may also be readily determined
by standard binding assays, for example as disclosed in Alto et al.
(2003, Proc Natl Acad Sci USA 100:4445-50).
TABLE-US-00007 TABLE 2 Conservative Amino Acid Substitutions in
DDD1 (SEQ ID NO: 1). Consensus sequence disclosed as SEQ ID NO: 87.
S H I Q I P P G L T E L L Q G Y T V E V L R T K N A S D N A S D K R
Q Q P P D L V E F A V E Y F T R L R E A R A N N E D L D S K K D L K
L I I I V V V
TABLE-US-00008 (SEQ ID NO: 12)
THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 13)
SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 14)
SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 15)
SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 16)
SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 17)
SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 18)
SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 19)
SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 20)
SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 21)
SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 22)
SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 23)
SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 24)
SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25)
SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 26)
SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 27)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 28)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 29)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 30)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 31)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA
[0149] Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50)
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:3), 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:3 below. The skilled artisan will realize that in
designing sequence variants of the AD 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 DDD binding. Table 3 shows potential conservative
amino acid substitutions in the sequence of AKAP-IS (AD1, SEQ ID
NO:3), similar to that shown for DDD1 (SEQ ID NO:1) in Table 2
above.
[0150] A limited number of such potential alternative AD moiety
sequences are shown in SEQ ID NO:32 to SEQ ID NO:49 below. Again, a
very large number of species within the genus of possible AD moiety
sequences could be made, tested and used by the skilled artisan,
based on the data of Alto et al. (2003). It is noted that FIG. 2 of
Alto (2003) shows an even large number of potential amino acid
substitutions that may be made, while retaining binding activity to
DDD moieties, based on actual binding experiments.
TABLE-US-00009 AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 3)
TABLE-US-00010 TABLE 3 Conservative Amino Acid Substitutions in AD1
(SEQ ID NO: 3). Consensus sequence disclosed as SEQ ID NO: 88. Q I
E Y L A K Q I V D N A I Q Q A N L D F I R N E Q N N L V T V I S
V
TABLE-US-00011 NIEYLAKQIVDNAIQQA (SEQ ID NO: 32) QLEYLAKQIVDNAIQQA
(SEQ ID NO: 33) QVEYLAKQIVDNAIQQA (SEQ ID NO: 34) QIDYLAKQIVDNAIQQA
(SEQ ID NO: 35) QIEFLAKQIVDNAIQQA (SEQ ID NO: 36) QIETLAKQIVDNAIQQA
(SEQ ID NO: 37) QIESLAKQIVDNAIQQA (SEQ ID NO: 38) QIEYIAKQIVDNAIQQA
(SEQ ID NO: 39) QIEYVAKQIVDNAIQQA (SEQ ID NO: 40) QIEYLARQIVDNAIQQA
(SEQ ID NO: 41) QIEYLAKNIVDNAIQQA (SEQ ID NO: 42) QIEYLAKQIVENAIQQA
(SEQ ID NO: 43) QIEYLAKQIVDQAIQQA (SEQ ID NO: 44) QIEYLAKQIVDNAINQA
(SEQ ID NO: 45) QIEYLAKQIVDNAIQNA (SEQ ID NO: 46) QIEYLAKQIVDNAIQQL
(SEQ ID NO: 47) QIEYLAKQIVDNAIQQI (SEQ ID NO: 48) QIEYLAKQIVDNAIQQV
(SEQ ID NO: 49)
[0151] Gold et al. (2006, Mol Cell 24:383-95) utilized
crystallography and peptide screening to develop a SuperAKAP-IS
sequence (SEQ ID NO:50), 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, which 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 DNL.TM. constructs. Other alternative sequences
that might be substituted for the AKAP-IS AD sequence are shown in
SEQ ID NO:51-53. Substitutions relative to the AKAP-IS sequence are
underlined. It is anticipated that, as with the AD2 sequence shown
in SEQ ID NO:4, the AD moiety may also include the additional
N-terminal residues cysteine and glycine and C-terminal residues
glycine and cysteine.
TABLE-US-00012 SuperAKAP-IS QIEYVAKQIVDYAIHQA (SEQ ID NO: 50)
Alternative AKAP sequences QIEYKAKQIVDHAIHQA (SEQ ID NO: 51)
QIEYHAKQIVDHAIHQA (SEQ ID NO: 52) QIEYVAKQIVDHAIHQA (SEQ ID NO:
53)
[0152] FIG. 2 of Gold et al. disclosed additional DDD-binding
sequences from a variety of AKAP proteins, shown below.
TABLE-US-00013 RII-Specific AKAPs AKAP-KL PLEYQAGLLVQNAIQQAI (SEQ
ID NO: 54) AKAP79 LLIETASSLVKNAIQLSI (SEQ ID NO: 55) AKAP-Lbc
LIEEAASRIVDAVIEQVK (SEQ ID NO: 56) RI-Specific AKAPs AKAPce
ALYQFADRFSELVISEAL (SEQ ID NO: 57) RIAD LEQVANQLADQIIKEAT (SEQ ID
NO: 58) PV38 FEELAWKIAKMIWSDVF (SEQ ID NO: 59) Dual-Specificity
AKAPs AKAP7 ELVRLSKRLVENAVLKAV (SEQ ID NO: 60) MAP2D
TAEEVSARIVQVVTAEAV (SEQ ID NO: 61) DAKAP1 QIKQAAFQLISQVILEAT (SEQ
ID NO: 62) DAKAP2 LAWKIAKMIVSDVMQQ (SEQ ID NO: 63)
[0153] Stokka et al. (2006, Biochem J 400:493-99) also developed
peptide competitors of AKAP binding to PKA, shown in SEQ ID
NO:64-66. The peptide antagonists were designated as Ht31 (SEQ ID
NO:64), RIAD (SEQ ID NO:65) and PV-38 (SEQ ID NO:66). 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-00014 Ht31 DLIEEAASRIVDAVIEQVKAAGAY (SEQ ID NO: 64) RIAD
LEQYANQLADQIIKEATE (SEQ ID NO: 65) PV-38 FEELAWKIAKMIWSDVFQQC (SEQ
ID NO: 66)
[0154] Hundsrucker et al. (2006, Biochem J 396:297-306) 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 are
provided in Table 1 of Hundsrucker et al., reproduced in Table 4
below. AKAPIS represents a synthetic RII subunit-binding peptide.
All other peptides are derived from the RII-binding domains of the
indicated AKAPs.
TABLE-US-00015 TABLE 4 AKAP Peptide sequences Peptide Sequence
AKAPIS QIEYLAKQIVDNAIQQA (SEQ ID NO: 3) AKAPIS-P QIEYLAKQIPDNAIQQA
(SEQ ID NO: 67) Ht31 KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68)
Ht31-P KGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69)
AKAP7.delta.-wt-pep PEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70)
AKAP7.delta.-L304T-pep PEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71)
AKAP7.delta.-L308D-pep PEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72)
AKAP7.delta.-P-pep PEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73)
AKAP7.delta.-PP-pep PEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74)
AKAP7.delta.-L314E-pep PEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75)
AKAP1-pep EEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pep
LVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pep
QYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pep
LEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pep
NTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pep
VNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pep
NGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pep
TQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) Rab32-pep
ETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)
[0155] 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:3). 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 were those of AKAP-IS, AKAP7.delta.-wt-pep,
AKAP7.delta.-L304T-pep and AKAP7.delta.-L308D-pep.
TABLE-US-00016 AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 3)
[0156] Can et al. (2001, J Biol Chem 276:17332-38) 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:1. 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. The skilled artisan will
realize that in designing sequence variants of DDD, it would be
most preferred to avoid changing the most conserved residues
(italicized), and it would be preferred to also avoid changing the
conserved residues (underlined), while conservative amino acid
substitutions may be considered for residues that are neither
underlined nor italicized.
TABLE-US-00017 (SEQ ID NO: 1)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
[0157] A modified set of conservative amino acid substitutions for
the DDD1 (SEQ ID NO:1) sequence, based on the data of Carr et al.
(2001) is shown in Table 5. Even with this reduced set of
substituted sequences, there are over 65,000 possible alternative
DDD moiety sequences that may be produced, tested and used by the
skilled artisan without undue experimentation. The skilled artisan
could readily derive such alternative DDD amino acid sequences as
disclosed above for Table 2 and Table 3.
TABLE-US-00018 TABLE 5 Conservative Amino Acid Substitutions in
DDD1 (SEQ ID NO: 1). Consensus sequence disclosed as SEQ ID NO: 89.
S H I Q P T E Q V T N S I L A Q P V E V E T R R E A A N I D S K K L
L L I I A V V
[0158] The skilled artisan will realize that these and other amino
acid substitutions in the DDD or AD amino acid sequences may be
utilized to produce alternative species within the genus of AD or
DDD moieties, using techniques that are standard in the field and
only routine experimentation.
[0159] Amino Acid Substitutions
[0160] In alternative 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, the
DDD and/or AD sequences used to make DNL.TM. constructs may be
modified as discussed above.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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).
[0165] 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.
[0166] 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. (Id.) 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 (Idem.)
[0167] 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.
[0168] 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.
[0169] Therapeutic Agents
[0170] In alternative 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, either
conjugated to the subject immunotoxins or separately administered
before, simultaneously with, or after the immunotoxin. 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
[0171] 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, epipodophyllotoxin, 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, nitrosourea,
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.
[0172] 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.
[0173] Chemokines of use may include RANTES, MCAF, MIP1-alpha,
MIP1-Beta and IP-10.
[0174] In certain embodiments, anti-angiogenic agents, such as
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-B, 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.
[0175] Immunomodulators of use may be selected from 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". 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); 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-B;
platelet-growth factor; transforming growth factors (TGFs) such as
TGF-.alpha. and TGF-.beta..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 and LT.
[0176] Radionuclides of use include, but are not limited
to--.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. The therapeutic radionuclide preferably
has a decay-energy in the range of 20 to 6,000 keV, preferably in
the ranges 60 to 200 keV for an Auger emitter, 100-2,500 keV for a
beta emitter, and 4,000-6,000 keV for an alpha emitter. Maximum
decay energies of useful beta-particle-emitting nuclides are
preferably 20-5,000 keV, more preferably 100-4,000 keV, and most
preferably 500-2,500 keV. 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. Some useful
diagnostic nuclides may include .sup.18F, .sup.52Fe, .sup.62Cu,
.sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.86Y, .sup.89Zr,
.sup.94Tc, .sup.94mTc, .sup.99mTc, or .sup.111In.
[0177] Therapeutic agents may include a photoactive agent or dye.
Fluorescent compositions, such as fluorochrome, and other
chromogens, or dyes, such as porphyrins sensitive to visible light,
have been used to detect and to treat lesions by directing the
suitable light to the lesion. In therapy, this has been termed
photoradiation, phototherapy, or photodynamic therapy. See Joni et
al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES
(Libreria Progetto 1985); van den Bergh, Chem. Britain (1986),
22:430. Moreover, monoclonal antibodies have been coupled with
photoactivated dyes for achieving phototherapy. See Mew et al., J.
Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;
Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,
Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin.
Biol. Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med.
(1989), 9:422; Pelegrin et al., Cancer (1991), 67:2529.
[0178] 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. The skilled artisan will realize that any
siRNA or interference RNA species may be attached to an antibody or
fragment thereof for delivery to a targeted tissue. Many siRNA
species against a wide variety of targets are known in the art, and
any such known siRNA may be utilized in the claimed methods and
compositions.
[0179] Known siRNA species of potential use include those specific
for IKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR
(U.S. Pat. No. 7,148,342); Bcl2 and EGFR (U.S. Pat. No. 7,541,453);
CDC20 (U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S.
Pat. No. 7,576,196); KRAS (U.S. Pat. No. 7,576,197); carbonic
anhydrase II (U.S. Pat. No. 7,579,457); complement component 3
(U.S. Pat. No. 7,582,746); interleukin-1 receptor-associated kinase
4 (IRAK4) (U.S. Pat. No. 7,592,443); survivin (U.S. Pat. No.
7,608,7070); superoxide dismutase 1 (U.S. Pat. No. 7,632,938); MET
proto-oncogene (U.S. Pat. No. 7,632,939); amyloid beta precursor
protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R (U.S. Pat. No.
7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complement factor B
(U.S. Pat. No. 7,696,344); p53 (U.S. Pat. No. 7,781,575), and
apolipoprotein B (U.S. Pat. No. 7,795,421), the Examples section of
each referenced patent incorporated herein by reference.
[0180] Additional siRNA species are available from known commercial
sources, such as Sigma-Aldrich (St Louis, Mo.), Invitrogen
(Carlsbad, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.),
Ambion (Austin, Tex.), Dharmacon (Thermo Scientific, Lafayette,
Colo.), Promega (Madison, Wis.), Minis Bio (Madison, Wis.) and
Qiagen (Valencia, Calif.), among many others. Other publicly
available sources of siRNA species include the siRNAdb database at
the Stockholm Bioinformatics Centre, the MIT/ICBP siRNA Database,
the RNAi Consortium shRNA Library at the Broad Institute, and the
Probe database at NCBI. For example, there are 30,852 siRNA species
in the NCBI Probe database. The skilled artisan will realize that
for any gene of interest, either a siRNA species has already been
designed, or one may readily be designed using publicly available
software tools. Any such siRNA species may be delivered using the
subject DNL complexes.
[0181] Exemplary siRNA species known in the art are listed in Table
6. Although siRNA is delivered as a double-stranded molecule, for
simplicity only the sense strand sequences are shown in Table
6.
TABLE-US-00019 TABLE 6 Exemplary siRNA Sequences SEQ ID Target
Sequence NO VEGF R2 AATGCGGCGGTGGTGACAGTA SEQ ID NO: 90 VEGF R2
AAGCTCAGCACACAGAAAGAC SEQ ID NO: 91 CXCR4 UAAAAUCUUCCUGCCCACCdTdT
SEQ ID NO: 92 CXCR4 GGAAGCUGUUGGCUGAAAAdTdT SEQ ID NO: 93 PPARC1
AAGACCAGCCUCUUUGCCCAG SEQ ID NO: 94 Dynamin 2 GGACCAGGCAGAAAACGAG
SEQ ID NO: 95 Catenin CUAUCAGGAUGACGCGG SEQ ID NO: 96 E1A binding
UGACACAGGCAGGCUUGACUU SEQ ID protein NO: 97 Plasminogen
GGTGAAGAAGGGCGTCCAA SEQ ID activator NO: 98 K-ras
GATCCGTTGGAGCTGTTGGCGTAGTT SEQ ID CAAGAGACTCGCCAACAGCTCCAACT NO: 99
TTTGGAAA Sortilin 1 AGGTGGTGTTAACAGCAGAG SEQ ID NO: 100 Apolipopro-
AAGGTGGAGCAAGCGGTGGAG SEQ ID tein E NO: 101 Apolipopro-
AAGGAGTTGAAGGCCGACAAA SEQ ID tein E NO: 102 Bcl-X
UAUGGAGCUGCAGAGGAUGdTdT SEQ ID NO: 103 Raf-1
TTTGAATATCTGTGCTGAGAACACA SEQ ID GTTCTCAGCACAGATATTCTTTTT NO: 104
Heat shock AATGAGAAAAGCAAAAGGTGCCCTGTCTC SEQ ID transcrip- NO: 105
tion factor 2 IGFBP3 AAUCAUCAUCAAGAAAGGGCA SEQ ID NO: 106
Thioredoxin AUGACUGUCAGGAUGUUGCdTdT SEQ ID NO: 107 CD44
GAACGAAUCCUGAAGACAUCU SEQ ID NO: 108 MMP14
AAGCCTGGCTACAGCAATATGCCTGTCTC SEQ ID NO: 109 MAPKAPK2
UGACCAUCACCGAGUUUAUdTdT SEQ ID NO: 110 FGFR1 AAGTCGGACGCAACAGAGAAA
SEQ ID NO: 111 ERBB2 CUACCUUUCUACGGACGUGdTdT SEQ ID NO: 112 BCL2L1
CTGCCTAAGGCGGATTTGAAT SEQ ID NO: 113 ABL1 TTAUUCCUUCUUCGGGAAGUC SEQ
ID NO: 114 CEACAM1 AACCTTCTGGAACCCGCCCAC SEQ ID NO: 115 CD9
GAGCATCTTCGAGCAAGAA SEQ ID NO: 116 CD151 CATGTGGCACCGTTTGCCT SEQ ID
NO: 117 Caspase 8 AACTACCAGAAAGGTATACCT SEQ ID NO: 118 BRCA1
UCACAGUGUCCUUUAUGUAdTdT SEQ ID NO: 119 p53 GCAUGAACCGGAGGCCCAUTT
SEQ ID NO: 120 CEACAM6 CCGGACAGTTCCATGTATA SEQ ID NO: 121
[0182] The skilled artisan will realize that Table 6 represents a
very small sampling of the total number of siRNA species known in
the art, and that any such known siRNA may be utilized in the
claimed methods and compositions.
[0183] Diagnostic Agents
[0184] Diagnostic agents are preferably 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.
[0185] Methods of Therapeutic Treatment
[0186] Various embodiments concern methods of treating a cancer in
a subject, such as a mammal, including humans, domestic or
companion pets, such as dogs and cats, comprising administering to
the subject a therapeutically effective amount of a cytotoxic
immunoconjugate.
[0187] In one embodiment, immunological diseases which may be
treated with the subject immunotoxins may include, for example,
joint diseases such as ankylosing spondylitis, juvenile rheumatoid
arthritis, rheumatoid arthritis; neurological disease such as
multiple sclerosis and myasthenia gravis; pancreatic disease such
as diabetes, especially juvenile onset diabetes; gastrointestinal
tract disease such as chronic active hepatitis, celiac disease,
ulcerative colitis, Crohn's disease, pernicious anemia; skin
diseases such as psoriasis or scleroderma; allergic diseases such
as asthma and in transplantation related conditions such as graft
versus host disease and allograft rejection.
[0188] The administration of the cytotoxic immunoconjugates 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 CD4, CD5, CD8, CD14, CD15, CD16, CD19, IGF-1R, CD20,
CD21, CD22, CD23, CD25, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L,
CD45, CD46, CD52, CD54, CD70, CD74, CD79a, CD80, CD95, CD126,
CD133, CD138, CD154, CEACAM5, CEACAM6, B7, AFP, PSMA, EGP-1, EGP-2,
carbonic anhydrase IX, PAM4 antigen, MUC1, MUC2, MUC3, MUC4, MUC5,
Ia, MIF, HM1.24, HLA-DR, tenascin, Flt-3, VEGFR, P1GF, ILGF, IL-6,
IL-25, tenascin, TRAIL-R1, TRAIL-R2, complement factor C5, oncogene
product, or a combination thereof. Various antibodies of use, such
as anti-CD19, anti-CD20, and anti-CD22 antibodies, are known to
those of skill in the art. 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; 7,501,498; 7,612,180; 7,670,804; and U.S.
Patent Application Publ. Nos. 20080131363; 20070172920;
20060193865; and 20080138333, the Examples section of each
incorporated herein by reference.
[0189] The immunotoxin therapy can be further supplemented with the
administration, either concurrently or sequentially, of at least
one therapeutic agent. 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). Additional useful drugs
include phenyl butyrate, bendamustine, and bryostatin-1.
[0190] The subject immunotoxins can be formulated according to
known methods to prepare pharmaceutically useful compositions,
whereby the immunotoxin 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.
[0191] The subject immunotoxins can be formulated for intravenous
administration via, for example, bolus injection or continuous
infusion. Preferably, the immunotoxin is infused over a period of
less than about 4 hours, and more preferably, over a period of less
than about 3 hours. For example, the first 25-50 mg could be
infused within 30 minutes, preferably even 15 min, and the
remainder infused over the next 2-3 hrs. 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.
[0192] Additional pharmaceutical methods may be employed to control
the duration of action of the immunotoxins. Control release
preparations can be prepared through the use of polymers to complex
or adsorb the immunotoxins. 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
immunotoxin, the amount of immunotoxin 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.
[0193] The immunotoxin 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 immunotoxin is infused over a
period of less than about 4 hours, and more preferably, over a
period of less than about 3 hours.
[0194] More generally, the dosage of an administered immunotoxin
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 immunotoxin that is in the range of from about 1 mg/kg
to 25 mg/kg as a single intravenous infusion, 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 example, once per week for 4-10 weeks, once
per week for 8 weeks, or once per week for 4 weeks. It may also be
given less frequently, such as every other week for several months,
or monthly or quarterly for many months, as needed in a maintenance
therapy.
[0195] Alternatively, an immunotoxin may be administered as one
dosage every 2 or 3 weeks, repeated for a total of at least 3
dosages. Or, the construct may be administered twice per week for
4-6 weeks. If the dosage is lowered to approximately 200-300
mg/m.sup.2 (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for
a 70 kg patient), it may be administered once or even twice weekly
for 4 to 10 weeks. Alternatively, the dosage schedule may be
decreased, namely every 2 or 3 weeks for 2-3 months. It has been
determined, however, that even higher doses, such as 20 mg/kg once
weekly or once every 2-3 weeks can be administered by slow i.v.
infusion, for repeated dosing cycles. The dosing schedule can
optionally be repeated at other intervals and dosage may be given
through various parenteral routes, with appropriate adjustment of
the dose and schedule.
[0196] In preferred embodiments, the immunotoxins 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). Cancers
conducive to treatment methods of the present invention involves
cells which express, over-express, or abnormally express
IGF-1R.
[0197] 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.
[0198] 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)).
[0199] 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.
[0200] 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.
[0201] In preferred embodiments, the method of the invention is
used to inhibit growth, progression, and/or metastasis of cancers,
in particular those listed above.
[0202] 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.
[0203] Expression Vectors
[0204] Still other embodiments may concern DNA sequences comprising
a nucleic acid encoding an antibody, antibody fragment, toxin or
constituent fusion protein of an immunotoxin, such as a DNL.TM.
construct. Fusion proteins may comprise an antibody or fragment or
toxin attached to, for example, an AD or DDD moiety.
[0205] 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.
[0206] 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. Pat. Nos. 7,531,327 and 7,537,930, the Examples section of
each incorporated herein by reference.
[0207] Kits
[0208] Various embodiments may concern kits containing components
suitable for treating or diagnosing diseased tissue in a patient.
Exemplary kits may contain one or more immunotoxins 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.
[0209] 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.
Examples
[0210] The following examples are provided to illustrate, but not
to limit, the claims of the present invention.
Example 1
Production and Use of DNL.TM. Complexes Showing Improved Stability,
Pharmacokinetics and Efficacy by Attaching AD Moieties to the
C-Terminal End of the Antibody Light Chain
[0211] We explored the production and use of improved
Dock-and-Lock.TM. (DNL.TM.) complexes, incorporating IgG molecules
with an AD moiety fused to the C-terminal end of the kappa light
chain (hereafter denoted as "C.sub.k" complexes or fusion
proteins), instead of the C-terminal end of the Fc (hereafter
denoted as "C.sub.H"). In the Examples below, the C.sub.k DNL.TM.
complexes are also indicated by an asterisk (e.g., 20*-2b). Two
exemplary C.sub.k-derived prototypes, an anti-CD22/CD20 bispecific
hexavalent antibody, comprising epratuzumab (anti-CD22) and four
Fabs of veltuzumab (anti-CD20), and a CD20-targeting
immunocytokine, comprising veltuzumab and four molecules of
interferon-.alpha.2b, displayed enhanced Fc-effector functions in
vitro, as well as improved pharmacokinetics, stability and
anti-lymphoma activity in vivo, compared to their Fc-derived
counterparts. These unexpected superior results favor the use of
DNL.TM. conjugates with the C.sub.k-design for clinical
development.
[0212] The C.sub.k-IgG-IFN.alpha., designated 20*-2b, had a similar
molecular size and composition to its Fc-IgG-IFN.alpha.
counterpart, 20-2b, each comprising veltuzumab and 4 copies of
IFN.alpha.2b fused at the C-terminal ends of the light or heavy
chains, respectively. The C.sub.k-bsHexAb, designated
22*-(20)-(20), and its Fc-bsHexAb homologue, 22-(20)-(20), each
comprised epratuzumab and 4 veltuzumab Fabs, which were fused at
the C-terminal ends of the light and heavy chains, respectively.
Compared to the analogous Fc-based immunoconjugates, the
C.sub.k-IgG-IFN.alpha. and C.sub.k-b sHexAb were more stable in
vivo, cleared more slowly from the circulation and had improved
Fc-effector function, significantly enhancing efficacy in vivo.
[0213] Methods
[0214] Antibodies and Cell Culture--
[0215] Immunomedics provided veltuzumab (anti-CD20 IgG1),
epratuzumab (anti-CD22 IgG.sub.1), a murine anti-IFN.alpha. mAb,
hMN-14 (labetuzumab), a rat anti-idiotype mAb veltuzumab (WR2), and
a rat anti-idiotype mAb to epratuzumab (WN). HRP-conjugated second
antibodies were from Jackson Immunoresearch (Westgrove, Pa.).
Heat-inactivated fetal bovine serum (FBS) was obtained from Hyclone
(Logan, Utah). All other cell culture media and supplements were
purchased from Invitrogen Life Technologies (Carlsbad, Calif.).
SpESFX-10 cells (Rossi et al., 2011, Biotechnol. Prog. 27:766-775)
and production clones were maintained in H-SFM. Daudi cell line was
purchased from ATCC and grown in 10% FBS-RPMI (Manassas, Va.).
[0216] DNL.TM. Constructs--
[0217] Methods for production of C.sub.k-based DNL.TM. constructs
are described in further detail below. For
C.sub.H3-AD2-IgG-veltuzumab, C.sub.H3-AD2-IgG-epratuzumab,
C.sub.H1-DDD2-Fab-veltuzumab, and IFN.alpha.2b-DDD2, generation of
the mammalian expression vectors and production clones, and their
use for the DNL.TM. conjugation of 20-2b and 22-(20)-(20), have
been reported previously (Rossi et al., 2008, Cancer Res.
68:8384-8392; Chang et al., 2009, Bioconjug. Chem. 20:1899-1907;
Rossi et al., 2009, Blood 114:3864-3871; Rossi et al., 2009, Blood
113:6161-6171). C.sub.k-AD2-IgG, was generated by recombinant
engineering, whereby the AD2 peptide was fused to the C-terminal
end of the kappa light chain (FIG. 1a). Because the natural
C-terminus of C.sub.K is a cysteine residue, which forms a
disulfide bridge to C.sub.H1, a 16-amino acid residue "hinge"
linker (SEQ ID NO:122) was used to space the AD2 from the
C.sub.K-V.sub.H1 disulfide bridge. The goal of this approach was to
obtain full binding and activities of all Fabs and effector groups,
while maintaining a full Fc effector function. The ultimate goal
was to maintain a Pk that approaches that of IgG and prevent the
intracellular dissociation of the modules, which presumably occurs
by proteolysis following uptake of the complex into the cell.
[0218] The first C.sub.K-AD2-IgG module was constructed for
veltuzumab (hA20), with additional C.sub.K-AD2-IgG modules produced
subsequently for milatuzumab (hLL1), epratuzumab (hLL2) and hR1
(anti-IGF-1R). These modules have been used to generate hexavalent
antibodies and immunocytokines, which were compared to constructs
of similar composition that were made with the corresponding
C.sub.H3-AD2-IgG modules. The mammalian expression vectors for
C.sub.k-AD2-IgG-veltuzumab and C.sub.k-AD2-IgG-epratuzumab were
constructed using the pdHL2 vector, which was used previously for
expression of the homologous C.sub.H3-AD2-IgG modules. A 2208-bp
nucleotide sequence (SEQ ID NO:130) was synthesized comprising the
pdHL2 vector sequence ranging from the Bam HI restriction site
within the V.sub..kappa./C.sub.K intron to the Xho I restriction
site 3' of the C.sub.k intron, with the insertion of the coding
sequence for the hinge linker (EFPKPSTPPGSSGGAP, SEQ ID NO:122) and
AD2 in frame at the 3' end of the coding sequence for C.sub.K. This
synthetic sequence was inserted into the IgG-pdHL2 expression
vectors for veltuzumab and epratuzumab via Bam HI and Xho I
restriction sites. Generation of production clones with SpESFX-10
were performed as described for the C.sub.H3-AD2-IgG modules (Rossi
et al., 2008, Cancer Res. 68:8384-8392; Rossi et al., 2009, Blood
113:6161-6171). C.sub.k-AD2-IgG-veltuzumab and
C.sub.k-AD2-IgG-epratuzumab were produced by stably-transfected
production clones in batch roller bottle culture, and purified from
the supernatant fluid in a single step using MABSELECT.TM. (GE
Healthcare) Protein A affinity chromatography.
[0219] Following the same process described previously for
22-(20)-(20) (Rossi et al., 2009, Blood 113:6161-6171),
C.sub.k-AD2-IgG-epratuzumab was conjugated with
C.sub.H1-DDD2-Fab-veltuzumab (FIG. 1b), a Fab-based module derived
from veltuzumab, to generate the bsHexAb 22*-(20)-(20), where the
22* indicates the C.sub.k-AD2 module of epratuzumab and each (20)
symbolizes a stabilized dimer of veltuzumab Fab (FIG. 1c). The
properties of 22*-(20)-(20) were compared with those of
22-(20)-(20), the homologous Fc-bsHexAb comprising
C.sub.H3-AD2-IgG-epratuzumab (FIG. 1d), which has similar
composition and molecular size, but a different architecture.
[0220] Following the same process described previously for 20-2b
(Rossi et al., 2009, Blood 114:3864-3871),
C.sub.k-AD2-IgG-veltuzumab (FIG. 1a), was conjugated with
IFN.alpha.2b-DDD2, a module of IFN.alpha.2b with a DDD2 peptide
fused at its C-terminal end (FIG. 1e), to generate 20*-2b (FIG. 10,
which comprises veltuzumab with a dimeric IFN.alpha.2b fused to
each light chain. The properties of 20*-2b were compared with those
of 20-2b (FIG. 1g), which is the homologous Fc-IgG-IFN.alpha.. Each
of the bsHexAbs and IgG-IFN.alpha. were isolated from the reaction
mixture by MABSELECT.TM. affinity chromatography.
[0221] Production of DNA Vectors for the Expression of
C.sub.K-AD2-IgG Modules.--
[0222] A 2208 basepair DNA sequence (SEQ ID NO:130) was
synthesized, comprising the sequence of the pdHL2 expression vector
from the Bam HI restriction site (within the V.sub.K/C.sub.K
intron) to the Xho I restriction site (preceding the heavy chain
expression cassette), with the insertion of the coding sequence for
the hinge linker (SEQ ID NO:122) and AD2 (SEQ ID NO:4), in frame at
the 3' end of the coding sequence for C.sub.K. This synthetic
sequence was inserted into the Bam HI/XhoI restriction sites in the
expression vector for veltuzumab (hA20-pdHL2) in a single cloning
step, to generate C.sub.K-AD2-IgG-hA20-pdHL2 (FIG. 11). Similarly,
the 2208 basepair fragment was inserted into the pGSHL expression
vectors for epratuzumab, milatuzumab and hR1 using Bam HI/Xho I
restriction sites (FIG. 12).
[0223] The synthetic nucleic acid sequence for conversion of
IgG-pdHL2 to CK-AD2-IgG-pdHL2 vector is shown in SEQ ID NO:130. 5'
Bam HI and 3' Xho I restriction sites are underlined. The coding
sequence for the C.sub.K-hinge linker-AD2 peptide is shown in
bold.
TABLE-US-00020 (SEQ ID NO: 130)
GGATCCCGCAATTCTAAACTCTGAGGGGGTCGGATGACGTGGCCATTCTT
TGCCTAAAGCATTGAGTTTACTGCAAGGTCAGAAAAGCATGCAAAGCCCT
CAGAATGGCTGCAAAGAGCTCCAACAAAACAATTTAGAACTTTATTAAGG
AATAGGGGGAAGCTAGGAAGAAACTCAAAACATCAAGATTTTAAATACGC
TTCTTGGTCTCCTTGCTATAATTATCTGGGATAAGCATGCTGTTTTCTGT
CTGTCCCTAACATGCCCTGTGATTATCCGCAAACAACACACCCAAGGGCA
GAACTTTGTTACTTAAACACCATCCTGTTTGCTTCTTTCCTCAGGAACTG
TGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAA
TCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA
GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCC
AGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC
AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGC
CTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCA
ACAGGGGAGAGTGTGAGTTCCCTAAACCCAGCACTCCACCCGGATCTTCC
GGCGGCGCTCCCTGTGGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGA
CAACGCCATCCAGCAGGCCGGGTGCTAGAGGGAGAAGTGCCCCCACCTGC
TCCTCAGTTCCAGCCTGACCCCCTCCCATCCTTTGGCCTCTGACCCTTTT
TCCACAGGGGACCTACCCCTATTGCGGTCCTCCAGCTCATCTTTCACCTC
ACCCCCCTCCTCCTCCTTGGCTTTAATTATGCTAATGTTGGAGGAGAATG
AATAAATAAAGTGAATCTTTGCACCTGTGGTTTCTCTCTTTCCTCATTTA
ATAATTATTATCTGTTGTTTTACCAACTACTCAATTTCTCTTATAAGGGA
CTAAATATGTAGTCATCCTAAGGCGCATAACCATTTATAAAAATCATCCT
TCATTCTATTTTACCCTATCATCCTCTGCAAGACAGTCCTCCCTCAAACC
CACAAGCCTTCTGTCCTCACAGTCCCCTGGGCCATGGTAGGAGAGACTTG
CTTCCTTGTTTTCCCCTCCTCAGCAAGCCCTCATAGTCCTTTTTAAGGGT
GACAGGTCTTACAGTCATATATCCTTTGATTCAATTCCCTGAGAATCAAC
CAAAGCAAATTTTTCAAAAGAAGAAACCTGCTATAAAGAGAATCATTCAT
TGCAACATGATATAAAATAACAACACAATAAAAGCAATTAAATAAACAAA
CAATAGGGAAATGTTTAAGTTCATCATGGTACTTAGACTTAATGGAATGT
CATGCCTTATTTACATTTTTAAACAGGTACTGAGGGACTCCTGTCTGCCA
AGGGCCGTATTGAGTACTTTCCACAACCTAATTTAATCCACACTATACTG
TGAGATTAAAAACATTCATTAAAATGTTGCAAAGGTTCTATAAAGCTGAG
AGACAAATATATTCTATAACTCAGCAATTCCCACTTCTAGGGGTTCGACT
GGCAGGAAGCAGGTCATGTGGCAAGGCTATTTGGGGAAGGGAAAATAAAA
CCACTAGGTAAACTTGTAGCTGTGGTTTGAAGAAGTGGTTTTGAAACACT
CTGTCCAGCCCCACCAAACCGAAAGTCCAGGCTGAGCAAAACACCACCTG
GGTAATTTGCATTTCTAAAATAAGTTGAGGATTCAGCCGAAACTGGAGAG
GTCCTCTTTTAACTTATTGAGTTCAACCTTTTAATTTTAGCTTGAGTAGT
TCTAGTTTCCCCAAACTTAAGTTTATCGACTTCTAAAATGTATTTAGAAT
TTCGACCAATTCTCATGTTTGACAGCTTATCATCGCTGCACTCCGCCCGA
AAAGTGCGCTCGGCTCTGCCAAGGACGCGGGGCGCGTGACTATGCGTGGG
CTGGAGCAACCGCCTGCTGGGTGCAAACCCTTTGCGCCCGGACTCGTCCA
ACGACTATAAAGAGGGCAGGCTGTCCTCTAAGCGTCACCACGACTTCAAC
GTCCTGAGTACCTTCTCCTCACTTACTCCGTAGCTCCAGCTTCACCAGAT CCCTCGAG
[0224] Production and Purification of C.sub.K-AD2-IgG Modules--
[0225] The C.sub.K-AD2-IgG-hA20-pdHL2 vector was linearized by
digestion with Sal I restriction enzyme and transfected into
SpESFX-10 myeloma cells by electroporation. Following
electroporation, the cells were plated in 96-well tissue culture
plates and transfectant clones were selected with 0.05 .mu.M
methotrexate (MTX). Clones were screened for protein expression by
sandwich ELISA using wells coated with WR2 (hA20 anti-Id) and
detection with peroxidase-conjugated goat anti-human Fab.
[0226] The three C.sub.K-AD2-IgG-pGSHL expression vectors were
transfected similarly to above, but plated in glutamine-free media
for selection, instead of MTX. Clones were screened for protein
expression by sandwich ELISA using wells coated with
antibody-specific anti-Ids and detection with peroxidase-conjugated
goat anti-human Fab.
[0227] The highest producing clones were expanded and cultured in
roller bottles for protein expression. The C.sub.K-AD2-IgG modules
were purified using Protein A affinity chromatography. The
productivity of the cell lines was similar to that of IgG or
C.sub.H3-AD2-IgG. Reducing SDS-PAGE resolved a protein band for the
hA20 Kappa-AD2 polypeptide with a relative mobility consistent with
its calculated molecular weight (26,951 Da) and larger than hA20
Kappa (23,204 Da) (not shown). Expectedly, the heavy chain
polypeptides of C.sub.k-AD2-IgG-hA20 co-migrated with those of hA20
IgG.
[0228] Bispecific Hexavalent Antibodies Made by DNL.TM. with
C.sub.K-AD2-IgG--
[0229] Bispecific hexavalent antibodies (bsHexAbs) were generated
by combining C.sub.k-AD2-IgG modules with C.sub.H3-DDD2-Fab modules
of a different specificity and performing DNL.TM. conjugation under
mild redox conditions. Six bsHexAbs and one monospecific HexAb were
produced and characterized, as exemplified by the construct named
20C.sub.k-(74)-(74) (alternatively, 20*-(74)-(74)), where the first
code (20C.sub.k or 20*) indicates the Ck-AD2-IgG module and codes
in parentheses indicate stabilized dimeric Fab-DDD2 modules. Thus,
20C.sub.k-(74)-(74) (or 20*-(74)-(74)) comprises veltuzumab
(anti-CD20) fused with four anti-CD74 Fabs derived from
milatuzumab. The component parts and valencies of the 7 HexAbs are
given in Table 7.
TABLE-US-00021 TABLE 7 Bispecific hexavalent antibodies targeting
B-cell malignancies. Valency HexAb IgG-AD2 module (parent mAb)
Fab-DDD2 module(parent mAb) CD20 CD22 CD74 20Ck-(22)-(22)
C.sub.k-AD2-IgG-hA20 (veltuzumab) C.sub.H3-DDD2-Fab-hLL2
(epratuzumab) 2 4 20Ck-(74)-(74) C.sub.k-AD2-IgG-hA20 (veltuzumab)
C.sub.H3-DDD2-Fab-hLL1 (milatuzumab) 2 4 20Ck-(20)-(20)
C.sub.k-AD2-IgG-hA20 (veltuzumab) C.sub.H3-DDD2-Fab-hA20
(veltuzumab) 6 22Ck-(20)-(20)* C.sub.k-AD2-IgG-hLL2 (epratuzumab)
C.sub.H3-DDD2-Fab-hA20 (veltuzumab) 4 2 22Ck-(74)-(74)
C.sub.k-AD2-IgG-hLL2 (epratuzumab) C.sub.H3-DDD2-Fab-hLL1
(milatuzumab) 2 4 74Ck-(20)-(20) C.sub.k-AD2-IgG-hLL1 (milatuzumab)
C.sub.H3-DDD2-Fab-hA20 (veltuzumab) 4 2 74Ck-(22)-(22)
C.sub.k-AD2-IgG-hLL1 (milatuzumab) C.sub.H3-DDD2-Fab-hLL2
(epratuzumab) 4 2 *monospecific hexavalent
[0230] Each of the HexAbs was produced and purified in a similar
fashion. A detailed description of one preparation of 22*-(20)-(20)
is provided as an example. A molar excess of C.sub.H3-DD2-Fab-hA20
(42 mg) was mixed with 25 mg of C.sub.K-AD2-IgG-hLL2 in
Tris-Citrate buffer (pH 7.5.+-.0.2). Reduced glutathione and EDTA
were added at 2 mM and 1 mM, respectively, and the reaction was
held overnight at room temperature, prior to addition of 4 mM
oxidized glutathione and an additional 4-hour incubation at room
temperature. The reaction mixture was applied to a 5-ml
MABSELECT.TM. (Protein A) chromatography column, which was washed
with PBS prior to elution of the bsHexAb with 0.1M Citrate, pH 3.5.
The 22*-(20)-(20) construct was dialyzed into 0.04M PBS, pH 7.4. A
total of 56 mg of 22*-(20)-(20) was recovered, representing 96%
yield. Size exclusion HPLC (SE-HPLC) resolved a single homogeneous
protein peak with a retention time consistent with a protein of
.about.368 kDa molecular weight (not shown). The SE-HPLC peak for
the C.sub.k-AD2-based bsHexAbs resolve with a slightly longer
retention time compared to the corresponding C.sub.H3-AD2-based
bsHexAbs (not shown), which have a similar composition and
molecular weight, indicating that the former have a smaller Stokes
radius and are more compact molecules, compared to the latter.
[0231] 20 (C.sub.k)-2b, an IgG-IFN.alpha. Immunocytokine Based on
C.sub.k-AD2-IgG-hA20--
[0232] An immunocytokine comprising veltuzumab fused with four
IFN.alpha.2b groups was prepared using the DNL.TM. method by
combining C.sub.k-AD2-IgG-hA20 with IFN.alpha.2b-DDD2 (FIG. 10.
C.sub.K-AD2-IgG-hA20 (54 mg) was combined with 81.1 mg of
IFN.alpha.-DDD2. EDTA (1 mM) and reduced glutathione (2 mM) were
added and the solution was held for 5 hours at room temperature.
Oxidized glutathione (4 mM) was added to the mixture, which was
held overnight at room temperature. The 20*-2b was purified to near
homogeneity using two sequential affinity chromatography steps.
First, the reaction mixture was applied to a 4-ml MABSELECT.TM.
(Protein A) column. Protein was eluted with 4 column volumes (16
ml) of 0.02% Polysorbate-80, 50 mM citrate, pH 3.5 directly into 16
ml of 0.02% P-80, 80 mM imidazole, 1 M NaCl, 100 mM
Na.sub.2HPO.sub.4 and the solution was adjusted to pH 7.3 with 50
mM Na.sub.2HPO.sub.4, 40 mM imidazole, 500 mM NaCl. The adjusted
eluent was applied to an 8-ml Ni-SEPHAROSE.RTM. 6 FF column
equilibrated with 0.02% P-80, 40 mM imidazole, 0.5 M NaCl, 50 mM
NaPO.sub.4, pH 7.5. A total of 85 mg of 20(C.sub.k)-2b was eluted
with 5 column volumes of 500 mM imidazole, 0.02% P-80, 50 mM NaCl,
20 mM NaH.sub.2PO.sub.4, pH 7.5.
[0233] SE-HPLC resolved a major protein peak for 20*-2b with a
retention time consistent with a protein of -250 kDa (not shown).
The 20*-2b peak resolved with a longer retention time than that of
20-2b, which comprises the same components (veltuzumab and four
IFN.alpha.2b) and has a similar molecular weight, indicating that
the former has a smaller Stokes radius and is more compact than the
latter, similar to what was observed for the HexAbs.
[0234] Analytical Methods--
[0235] Size-exclusion high performance liquid chromatography
(SE-HPLC) was performed using a 4 .mu.m UHR SEC column (Waters
Corp., Milford Mass.). SDS-PAGE was performed using 4-20% gradient
Tris-glycine gels (Invitrogen, Gaithersburg, Md.). IEF was
performed at 1000 V, 20 mM and 25 watts for 1 h, using pH 6-10.5
ISOGEL.RTM. Agarose IEF plates (Lonza, Basel, Switzerland) on a
BIO-PHORESIS.RTM. horizontal electrophoresis cell (Bio-Rad,
Hercules, Calif.). All colorimetric (ELISA and MTS) and
fluorometric (CDC and ADCC) assays were quantified with an
ENVISION.RTM. 2100 Multilabel Plate Reader (PerkinElmer, Waltham,
Mass.).
[0236] Cell Binding--
[0237] Binding to cells was measured by flow cytometry on a
GUAVA.RTM. PCA using GUAVA.RTM. Express software (Millipore Corp.,
Billerica, Mass.). Veltuzumab and 20*-2b were labeled with
phycoerythrin (PE) using a ZENON.RTM. R-Phycoerythrin human IgG
labeling kit following the manufacturer's protocol (Invitrogen,
Molecular Probes). Daudi cells were incubated with the
PE-veltuzumab and PE-20*-2b (0.1-15 nM) for 30 min at room
temperature and washed with 1% BSA-PBS prior to analysis. Plots of
concentration vs. mean fluorescence intensity (MFI) were analyzed
by linear regression.
[0238] In Vitro Cytotoxicity--
[0239] Daudi cells were plated at 10,000 cells/well in 96-well
plates and incubated at 37.degree. C. for 3 days in the presence of
increasing concentrations of 20*-2b or 20-2b. Viable cell densities
were determined using the MTS-based CELLTITER 96.RTM. Cell
Proliferation Assay (Promega, Madison, Wis.).
[0240] FcRn Binding Measurements--
[0241] FcRn binding was evaluated by surface plasmon resonance on a
BIACORE.RTM. X instrument (GE Healthcare) following the methods of
Wang et al. (2011, Drug Metab Dispos. 39:1469-1477). Soluble
single-chain FcRn was generated following the methods of Feng et
al. (2011, Protein Expr. Purif. 79:66-71). The extracellular domain
of the human FcRn heavy chain was fused with .beta.2-microglobulin
via a flexible peptide linker. The fusion protein was expressed
using a modified pdHL2 vector in transfectant SpESFX-10 cells, and
purified using Ni-Sepharose. Purified scFcRn was immobilized onto a
CM5 biosensor chip using an amine coupling kit (GE Healthcare) to a
density of .about.600 response units (RU). The test articles were
diluted with pH 6.0 running buffer [50 mM NaPO.sub.4, 150 mM NaCl,
and 0.05% (v/v) Surfactant 20] to 400, 200, 100, 50, and 25 nM and
bound to the immobilized scFcRn for 3 min to reach equilibrium,
followed by 2 min of dissociation with the flow rate at 30
.mu.L/min. The sensorchip was regenerated with pH 7.5 running
buffer between runs. To determine FcRn binding affinity (K.sub.D)
at pH 6.0, the data from all five concentrations were used
simultaneously to fit a two-state reaction model (BIAevaluation
software; GE Healthcare). Goodness of fit was indicated by
.chi..sup.2 values.
[0242] Pk Analyses--
[0243] The pharmacokinetics (Pk) and in vivo stability were
compared between 20*-2b and 20-2b following intravenous (i.v.) or
subcutaneous (s.c) injection in mice. Groups of 18 Swiss-Webster
mice were administered 1-mg doses of 20*-2b or 20-2b by either i.v.
or s.c. injection. Using 3 mice per time point, animals were
sacrificed and bled at 6, 16, 24, 48, 72 and 96 hours. Therefore,
each serum sample represented an independent animal/time point. For
measurement of intact and total (intact plus dissociated)
IgG-IFN.alpha., microtiter wells were adsorbed with WR2, a rat
anti-Id for veltuzumab, at 5 .mu.g/mL in 0.5 M Na.sub.2CO.sub.3, pH
9.5. Following blocking with 2% BSA-PBS, serum dilutions in
antibody buffer (0.1% gelatin, 0.05% proclin, 0.05% Tween-20, 0.1 M
NaCl, 0.1 M NaPO.sub.4, pH 7.4) were incubated in the coated wells
for 2 h. For measurement of intact IgG-IFN.alpha., wells were
probed with a mouse anti-IFN.alpha. mAb (5 .mu.g/mL in antibody
buffer) for 1 h, followed by detection with HRP-conjugated goat
anti-mouse IgG-Fc. For measurement of total veltuzumab IgG, wells
were probed with HRP-conjugated goat anti-human IgG-Fc for 1 h.
[0244] For measurement of intact and total bsHexAbs, microtiter
wells were adsorbed with WN, a rat anti-idiotype for epratuzumab.
Serum dilutions were incubated in the coated wells for 2 h. For
detection of intact bsHexAb, wells were probed with HRP-conjugated
WR2 (1 .mu.g/mL in antibody buffer) for 1 h. For detection of total
epratuzumab IgG, wells were probed with HRP-conjugated goat
anti-human IgG-Fc for 1 h.
[0245] Signal was developed with o-phenylenediamine dihydrochloride
substrate solution and OD was measured at 490 nM. The
concentrations of intact and total species were extrapolated from
construct-specific standard curves. Pk was analyzed using the
WINNONLIN.RTM. Pk software package (v5.1; Pharsight Corp.; Mountain
View, Calif.).
[0246] In Vivo and Ex Vivo Methods--
[0247] Injection and collection of sera from rabbits was performed
by Lampire Biological Laboraories (Pipersville, Pa.). For Pk
studies, 10-week old male Swiss-Webster mice (Taconic, Germantown,
N.Y.) and New Zealand White rabbits were injected subcutaneously
(SC), and also intravenously (IV) for mice, with test agents
diluted in PBS. Blood samples were obtained by cardiac puncture and
from the ear vein for mice and rabbits, respectively. Serum was
isolated from clotted blood by centrifugation, and diluted in
antibody buffer, prior to analysis by ELISA.
[0248] Human blood specimens were collected from healthy donors.
In-vitro ADCC and CDC activity were assayed as described previously
(Rossi et al., 2008, Cancer Res. 68, 8384-8392). For ADCC, Daudi
cells were incubated for 4 h at 37.degree. C. with PBMCs, which
were isolated from the blood of healthy donors, at a 50:1
effector:target ratio using test agents at 33 nM.
[0249] In Vivo Efficacy in Mice--
[0250] Female 8-12-week old C.B.17 homozygous SCID mice (Taconic)
were inoculated intravenously with 1.5.times.10.sup.7 Daudi cells
on day 0. For comparison of the bsHexAbs, treatment was
administered by SC injection on days 1 and 5. For comparison of the
IgG-IFN.alpha., treatments were administered as a single SC
injection on day 7. Saline was used as a control treatment.
Animals, monitored daily, were humanely euthanized when hind-limb
paralysis developed or if they became otherwise moribund.
Additionally, mice were euthanized if they lost more than 20% of
initial body weight. Survival curves were analyzed using
Kaplan-Meier plots, using the Prism (v4.03) software package
(GraphPad Software, Inc., San Diego, Calif.). Some outliers
determined by critical Z test were censored from analyses.
[0251] Statistical Analyses--
[0252] Statistical significance (P<0.05) was determined using
student's T-tests for all results except for the in vivo survival
curves, which were evaluated by log-rank analysis.
[0253] Results
[0254] Synthesis of C.sub.k-Based Immunoconjugates--
[0255] The DNL.TM. synthesis produced homogeneous preparations of
22*-(20)-(20), 22-(20)-(20), 20*-2b and 20-2b. By SDS-PAGE
(non-reducing), each conjugate was resolved into a tight cluster of
bands with relative mobility conforming to their expected size
(data not shown), and under reducing conditions, only bands
representing the constituent polypeptides for each conjugate were
evident, demonstrating a high degree of purity (not shown). For
each conjugate, SE-HPLC resolved a major peak having a retention
time consistent with their molecular size (not shown). The longer
retention times observed for 22*-(20)-(20) and 20*-2b are likely
due to their more compact structure, as compared to 22-(20)-(20)
and 20-2b, respectively. Isoelectric focusing showed that 20*-2b
and 20-2b have a similar pI (calculated pI=pH 7.22), with no
evidence of unreacted IgG-AD2 (pI=pH 7.86) or IFN.alpha.2b-DDD2
(pI=pH 6.87) modules (not shown).
[0256] Both conjugates retain full binding of the parental mAbs, as
shown for 20*-2b, which exhibited identical binding as veltuzumab
to live Daudi cells (FIG. 6). Cytotoxicity also was similar between
the C.sub.k and Fc versions in Daudi cells (EC.sub.50=0.2 pM),
demonstrating equivalent CD20 binding and IFN.alpha. specific
activity (FIG. 7).
[0257] Pharmacokinetics--
[0258] We reported previously that the T.sub.1/2 for Fc-bsHexAbs
were approximately half as long as their parental mAbs in mice
(Rossi et al., 2009, Blood 113:6161-6171). In the initial study,
which measured the serum concentrations of 22*-(20)-(20),
22-(20)-(20) and epratuzumab in mice over a period of 72 h after
subcutaneous (SC) injection (FIG. 2a), 22-(20)-(20) reached maximal
concentration at 16 h and was cleared with a T.sub.1/2 about 1 day,
similar to the findings before. In comparison, both epratuzumab and
22*-(20)-(20) reached peak levels between 24 and 48 h, while
clearing similarly, but slower than 22-(20)-(20). A subsequent
study monitoring clearance over 5 days again found 22*-(20)-(20)
with superior Pk, showing .about.2-fold higher maximum
concentration in serum, with longer T.sub.1/2 and mean residence
time (MRT), culminated in a 3.8-fold greater area under the curve
(AUC). (FIG. 2b; Table 8).
[0259] As in mice, the Pk parameters determined in rabbits were
.about.2-fold greater for 22*-(20)-(20), resulting in a 3.3-fold
greater AUC, compared to 22-(20)-(20) (FIGS. 2c and d; Table 8).
Importantly, the concentrations of the 22*-(20)-(20) following SC
administration in both mice and rabbits were sustained for longer
periods.
TABLE-US-00022 TABLE 8 Summary of pharmacokinetic parameters Dose
T.sub.1/2 T.sub.max C.sub.max AUC(0-.infin.) MRT Species Route (mg)
Construct (h) (h) (.mu.g/mL) (h * .mu.g/ml) (h) Mouse IV 1.0 20*-2b
36.2 6.0 649.0 32516.5 55.2 20-2b 17.1 6.0 629.8 15514.0 19.1 Mouse
SC 1.0 20*-2b 37.9 16.0 312.1 18318.2 62.1 20-2b 16.0 16.0 146.0
6498.6 30.9 Mouse SC 0.5 22*-(20)-(20) 106.5 24.0 50.6 6704.7 153.1
22-(20)-(20) 54.5 16.0 26.5 1752.9 85.2 Rabbit SC 18 22*-(20)-(20)
117.9 53.3 31.6 6079.1 179.6 22-(20)-(20) 51.1 37.3 17.8 1838.4
89.2 T.sub.1/2, elimination half-life; T.sub.max, time of maximal
concentration; C.sub.max, maximal concentration; AUC, area under
the curve; MRT, mean residence time.
[0260] Binding affinity (K.sub.D) of the bsHexAbs to the neonatal
Fc receptor (FcRn) was assessed by surface plasmon resonance and
found to be 166 and 310 nM for 22*-(20)-(20) and 22-(20)-(20),
respectively (P=0.01). The affinity of epratuzumab (16 nM) was
approximately 10-fold stronger than 22*-(20)-(20) (P=0.007) (Table
9).
TABLE-US-00023 TABLE 9 Summary of Biacore analysis for neonatal Fc
receptor binding affinity Epratuzumab 22*-(20)-(20) 22-(20)-(20)
k.sub.d k.sub.a K.sub.D k.sub.d k.sub.a K.sub.D k.sub.d k.sub.a
K.sub.D Run 1 0.0242 1.64 .times. 10.sup.6 15.0 0.0395 2.80 .times.
10.sup.5 141.1 0.0458 1.48 .times. 10.sup.5 309.5 Run 2 0.0218 1.56
.times. 10.sup.6 17.9 0.0404 2.26 .times. 10.sup.5 178.8 0.0411
1.54 .times. 10.sup.5 266.9 Run 3 0.0239 1.56 .times. 10.sup.6 15.8
0.0441 2.48 .times. 10.sup.5 177.8 0.0419 1.19 .times. 10.sup.5
352.1 Mean .+-. 0.0233 .+-. 1.59 .times. 10.sup.6 .+-. 16.3 .+-.
0.0413 .+-. 2.51 .times. 10.sup.5 .+-. 165.9 .+-. 0.0429 .+-. 1.40
.times. 10.sup.5 .+-. 309.5 .+-. S.D 0.0013 4.62 .times. 10.sup.4
1.5 0.0024 2.72 .times. 10.sup.4 21.5 0.0025 1.87 .times. 10.sup.4
42.6 k.sub.d = 1/s; k.sub.a = 1/Ms; K.sub.D = k.sub.d/k.sub.a given
as nM concentration
[0261] Fc-IgG-IFN.alpha. constructs, such as 20-2b, also were
cleared from circulation faster than their parental mAb (Rossi et
al., 2009, Blood 114:3864-3871). However, when the Pk parameters of
20*-2b and 20-2b following either SC or intravenous (IV) injection
were compared (FIG. 3), the T.sub.1/2, C.sub.max, and MRT were each
again about 2-fold higher for 20*-2b, resulting in a 2.8-fold
greater AUC, compared to 20-2b (Table 8). For IV administration,
20*-2b had a 2- and 2.8-fold longer T.sub.1/2 and MRT,
respectively, and a 2-fold greater AUC.
[0262] In Vivo Stability--
[0263] The Fc-bsHexAbs and Fc-IgG-IFN.alpha. are stable ex vivo in
serum (Rossi et al., 2009, Blood 114:3864-3871; Rossi et al., 2009,
Blood 113:6161-6171). However, analysis of serum samples from
earlier Pk studies suggested these constructs dissociate in vivo
over time, presumably by intracellular processing. We compared the
in vivo stability of 20*-2b and 20-2b by measuring the
concentrations of the intact IgG-IFN.alpha. and the total
veltuzumab, which allowed for differentiating the intact from the
dissociated species (FIG. 3c). The % intact IgG-IFN.alpha. was
plotted versus time (FIG. 3d), and in vivo dissociation rates for
20-2b and 20*-2b were calculated by linear regression to 0.97%/h
and 0.18%/h, respectively. A similar analysis was performed on
serum samples following SC injection of the bsHexAbs in mice, with
in vivo dissociation rates for 22-(20)-(20) and 22*-(20)-(20)
calculated to 0.55%/h and 0.19%/h, respectively (FIG. 8).
Interestingly, both 22-(20)-(20) and 22*-(20)-(20) were completely
stable in vivo following SC injections in rabbits (FIG. 9). The
reason for the difference in in vivo stabilities between mice and
rabbits is not known.
[0264] Effector Function--
[0265] We reported that Fc-IgG-IFN.alpha. and Fc-bsHexAbs did not
induce measurable CDC in vitro, even when their parental mAb had
potent activity (Rossi et al., 2009, Blood 114:3864-3871; Rossi et
al., 2009, Blood 113:6161-6171). Consistent with the prior results,
veltuzumab exhibited strong CDC, yet no activity was evident for
20-2b (FIG. 4a). However, 20*-2b induced strong CDC, which
approached the potency of veltuzumab (FIG. 4a). Under these in
vitro conditions, epratuzumab lacked CDC, whereas 22-(20)-(20)
achieved a modest increase, and 22*-(20)-(20) induced even greater
activity, which was .about.10-fold less potent than veltuzumab
(FIG. 4b).
[0266] Unlike CDC, the Fc-based conjugates did not have reduced
ADCC, but instead, 20-2b exhibited enhanced ADCC compared to
veltuzumab (Rossi et al., 2009, Blood 114:3864-3871). Depending on
the PBMC donor, epratuzumab induced little or no ADCC in vitro, and
not surprisingly, 22-(20)-(20) did not show a statistically
significant improvement (FIG. 4c). However, the ADCC associated
with 22*-(20)-(20) was not significantly different from veltuzumab,
when PBMCs of a high-ADCC donor were used (FIG. 4c). With a
low-ADCC PBMC donor, 22*-(20)-(20) had enhanced activity (11.4%
lysis), compared to epratuzumab (2.3%) and 22-(20)-(20) (4.3%), but
it was lower than veltuzumab (18.5%) (P=0.0326, data not
shown).
[0267] In Vivo Efficacy--
[0268] As reported previously, 20-2b is remarkably potent in
treating mice bearing human Daudi Burkitt lymphoma xenografts,
which are highly sensitive to direct killing by IFN.alpha. (Rossi
et al., 2009, Blood 114:3864-3871). Using the same model, the
C.sub.k-based conjugates demonstrated even more potent anti-tumor
activity than their Fc-based counterparts (FIG. 5a). While both
20-2b and 20*-2b at a single 1 .mu.g-dose cured the majority of the
animals, with median survival time (MST) greater than 189 days,
20*2b, but not 20-2b, at 0.25 maintained its potency, providing
evidence of significantly improved therapeutic efficacy (MST>189
days with 7/8 cures for 20*2b vs. 134.5 days with just 3/8
survivors for 20-2b; P=0.0351). A molar equivalent of veltuzumab
(0.6 .mu.s) to 1 .mu.g of 20-2b increased the MST by only 12.5 days
over saline control, The superiority of another different C.sub.k
construct over the Fc-parental construct was shown again in the
disseminated Daudi model, where animals were administered two
injections (days 1 and 5) of high (1 mg) or low (10 .mu.s) doses of
22*-(20)-(20) or 22-(20)-(20) (FIG. 5b). For the high dose, the MST
was >123 and 71 days with 100% and 10% survival for
22*-(20)-(20) and 22-(20-(20), respectively (P<0.0001). With the
low-dose treatment, the MST was 91 days for 22*-(20)-(20) with 2
mice surviving, compared to 50.5 days for 22-(20-(20) with no
survivors (P=0.0014). High doses of each bsHexAb improved survival
significantly more (P<0.0001) than either epratuzumab alone or
in combination with C.sub.H1-DDD2-Fab-veltuzumab, which were given
at a molar equivalent to the 1-mg dose of bsHexAb. At the 100-fold
lower dosing, both bsHexAbs were superior to high-dose epratuzumab
(P<0.003), and 22*-(20)-(20), but not 22-(20)-(20), was superior
to high-dose epratuzumab plus C.sub.H1-DDD2-Fab-veltuzumab
(P<0.0001).
[0269] Discussion
[0270] The various formats of antibody-based fusion proteins,
including bsAbs (Kontermann, 2010, Curr Opin Mol Ther 12:176-183)
and immunocytokines (Kontermann, 2012, Arch Biochem Biophys
526:194-205), can largely be categorized into three groups, based
on where additional moieties are fused to a whole IgG, an Fc, or an
antigen-binding fragment such as Fab, scFv or diabody. Whereas
Fc-fusion may increase T.sub.1/2, and fusion to antigen-binding
fragments should impart targeting, only fusion to IgG could expect
to achieve antibody targeting, full Fc effector function and
markedly extended Pk. Because not all IgG-fusion designs are
created equal, effector activities and Pk are known to vary widely
among the different formats and even between particular constructs
of the same design.
[0271] DNL.TM. complexes are exceptional for producing
immunoconjugates that retain full antigen-binding avidity of the
targeting antibody and biological activity of the appending
effector molecules (e.g., cytokines), and have potent efficacy both
in vitro and in vivo (Rossi et al., 2012, Bioconjug. Chem
23:309-323; Rossi et al., 2009, Blood 114:3864-3871; Rossi et al.,
2009, Blood 113:6161-6171; Rossi et al., 2010, Cancer Res.
70:7600-7609; Rossi et al., 2011, Blood 118:1877-1884). However,
Fc-bsHexAbs and Fc-IgG-IFN.alpha. were cleared from circulation at
approximately twice the rate of their parental mAbs. Sub-optimal Pk
is a common deficiency associated with immunoconjugates that is
primarily attributed to impaired dynamic binding to the FcRn (Kuo
& Aveson, 2011, MAbs. 3:422-430). To improve Pk, we engineered
a new class of IgG-AD2 module having the AD2 peptide fused at the
C-terminal end of the light chain. The new module was used to
assemble C.sub.k-bsHexAbs and C.sub.k-IgG-IFN.alpha., which not
only exhibited comparable in vitro properties to their Fc-based
homologues, including antigen binding, IFN.alpha. specific activity
and in vitro cytotoxicity, but also had superior Pk, in vivo
stability and Fc effector activity, which together resulted in
increased in vivo efficacy, compared to the already potent Fc-based
counterparts.
[0272] The superior Pk of the C.sub.k-bsHexAbs and
C.sub.k-IgG-IFN.alpha. is most likely attributed to their increased
binding affinity to the FcRn, which was twice as strong at pH 6.0
for 22*-(20)-(20), compared to 22-(20)-(20). FcRn binding is
mediated by portions of the C.sub.H2 and C.sub.H3 domains of IgG,
with critical contact sites located near the C-terminal end of the
Fc (Huber et al., 1993, J Mol Biol 230:1077-1083; Raghavan et al.,
1994, Immunity. 1:303-315). Considering that the T.sub.1/2 of
22*-(20)-(20) was in the range of epratuzumab (Rossi et al., 2009,
Blood 113:6161-6171), it was unanticipated that FcRn binding was
approximately 10-fold weaker for the former (155 nM). However,
using this same method, we measured the FcRn K.sub.D at 42 and 92
nM for other humanized mAbs, which typically have Pk similar to
epratuzumab (data not shown). T.sub.1/2 is not necessarily directly
correlated with FcRn K.sub.D at pH 6.0 (Dall'Acqua, 2002, J Immunol
169:5171-5180; Gurbaxani et al., 2006, Mol Immunol 43, 1462-1473).
It has been suggested that the rate of dissociation at pH 7.4 is
equally or perhaps more important in determining T.sub.1/2 (Wang,
2011, Drug Metab Dispos. 39:1469-1477). Although FcRn:IgG contacts
are limited to the Fc domain, the antigen-binding domain can
negatively impact FcRn binding, as evidenced by the fact that most
therapeutic antibodies share a very similar Fc (IgG.sub.1), yet
vary widely in FcRn K.sub.D and T.sub.1/2 (Suzuki, 2010, J Immunol
184:1968-1976). Additional factors include endocytosis,
ligand:antibody ratio, antibody structural stability, antibody pI,
and methionine oxidation (Kuo & Aveson, 2011, MAbs.
3:422-430).
[0273] For fusion proteins, the FcRn K.sub.D and T.sub.1/2 can be
influenced by the nature and location of the fusion partner (Suzuki
et al., 2010, J Immunol 184:1968-1976; Lee et al., 2003, Clin
Pharmacol Ther 73, 348-365). We observed that the T.sub.1/2 of each
IgG-IFN.alpha. was shorter than the corresponding bsHexAb that was
assembled using the same class of IgG-AD2 module. For example, the
T.sub.1/2 of 20*-2b (37.9 h) was markedly shorter than that of
22*-(20)-(20) (106.5 h), suggesting that, independent of their
location, the IFN.alpha. groups negatively impact FcRn binding,
perhaps by lowering the pI of the adduct.
[0274] The present Example identifies the C-terminal end of the
light chain as the most advantageous location for fusion to IgG. An
immunocytokine of single-chain IL-12 fused to the N-terminal end of
the heavy chain of an anti-HER2 IgG.sub.3 retained HER2 binding
(Peng et al., 1999, J Immunol 163:250-258).sup.17. We applied a
similar strategy using DNL.TM. by constructing an IgG-AD2 module
having the AD2 peptide fused to the N-terminal end of veltuzumab
heavy chain. However, bsHexAbs and IgG-IFN.alpha. made with this
module did not bind CD20 on cells (data not shown). This might have
been because of the large size of the additional (Fab).sub.2 or
(IFN.alpha.2b).sub.2 groups. That these conjugates bound to
anti-idiotype mAbs suggests that the nature of the antigen, which
is a small extracellular loop of CD20, might be a factor. The
C-terminal end of the heavy chain is the most common and convenient
location for fusion to IgG(Kontermann, 2012, Arch Biochem Biophys
526:194-205). However, this is also the most likely location to
impact FcRn binding and Pk negatively. For example, an
immunocytokine of GM-CSF fused at the C-terminus of the heavy chain
of an anti-HER2 IgG.sub.3 exhibited markedly reduced T.sub.1/2 (10
hours) compared to the parental mAb (110 hours) (Dela Cruz et al.,
2000, J Immunol 165:5112-5121). Fc-based bsAbs also suffer from
diminished Pk. As an example, a bsAb having an anti-IGF-1R scFv
fused to the C-terminal end of the heavy chain of an anti-EGFR IgG
cleared from circulation in mice twice as fast (T.sub.v2=9.93 h),
compared to the parental mAb (T.sub.1/2=20.36 h) (Dong et al.,
2011, MAbs. 3:273-288).
[0275] Croasdale and colleagues systematically studied the effect
of fusion location with IgG-scFv tetravalent bsAbs using an
anti-IGF-1R IgG.sub.1 fused at the N- or C-termini of the heavy or
light chains, with an anti-EGFR scFv (Croasdale et al., 2012, Arch.
Biochem Biophys 526:206-18). Fusion of scFv to the IgG at the
C-terminus of the light chain produced the highest yields, had the
longest T.sub.1/2 and was the most effective in vivo. The authors
indicated that each construct bound FcRn and Fc.gamma.RIIIa;
however, K.sub.D was not reported. Among the different formats, the
C-terminal heavy chain fusion had the shortest T.sub.1/2. Fusion at
the N- or C-terminus of the heavy chain resulted in substantially
reduced or complete loss, respectively, of ADCC. Alternatively,
fusion at the C-terminus of the light chain did not decrease
ADCC.
[0276] Our results show that fusion location can impact ADCC. For
the bsHexAbs comprising epratuzumab as the IgG, which has minimal
ADCC, strong ADCC was measured for 22*-(20)-(20), but not
22-(20)-(20), suggesting that this Fc effector function was
provided by the addition of the four anti-CD20 Fabs, and that their
fusion location is critical. Additionally, 22*-(20)-(20) showed
moderate CDC, which was not detected for epratuzumab, and only
modestly increased for 22-(20)-(20), suggesting that this effector
function also can be bestowed to a CDC-lacking mAb by the addition
of Fabs of a CDC-inducing mAb, and that, the activity is sensitive
to the location of the fusion site. This was demonstrated clearly
with the IgG-IFN.alpha., where the Fc-IgG-IFN, 20-2b, did not have
detectable CDC and 20*-2b induced potent activity, similar to
veltuzumab.
[0277] Although the Fc-bsHexAbs and Fc-IgG-IFN.alpha. are quite
stable in human or mouse sera and whole blood (Rossi et al., 2009,
Blood 114:3864-3871; Rossi et al., 2009, Blood 113:6161-6171), the
Fc-fusions, in particular, were not completely stable in vivo. The
Fc-based conjugates dissociated at a rate of 0.5-1.0%/h in mice,
compared to <0.2%/h for the C.sub.k-based constructs. Because
dissociation has never been observed ex vivo, we presume it occurs
by an intracellular process. Interestingly, there was no evidence
of in-vivo instability in rabbits, even after 5 days. The
C-terminal lysine residue of the heavy chain is often cleaved
proteolytically during antibody production. The common Fc-based
fusion proteins, where additional groups are fused to the
C-terminal lysine, potentially can be cleaved in vivo by proteases,
such as plasmin, which cleave after exposed lysine residues
(Gillies et al., 1992, Proc Natl Acad Sci U. S. A 89,
1428-1432).
[0278] In summary, this study demonstrates the superior in vivo
properties of bsAbs and immunocytokines made as DNL.TM. complexes
with fusion at the C-terminal end of the light chain, suggesting
that the C-terminus of the light chain is the preferred fusion
location for most immunoconjugates with intended clinical use.
Example 2
Production and Use of C.sub.k-Based DNL.TM. Complexes for Treatment
of Autoimmune Disease
Background
[0279] Systemic lupus erythematosus (SLE) has been classified as an
autoimmune disease that may involve many organ systems, as an
inflammatory multisystem rheumatic disorder, or as a collagen
vascular disease. Corticosteroids remain the foundation for
long-term management with most patients, even those in clinical
remission, maintained using low doses. High-dose steroids,
particularly 0.5-1.0 g pulse i.v. methylprednisolone, are standard
treatment for management of an acute flare, with immunosuppressants
(azathioprine, cyclophosphamide, methotrexate, etc.) generally used
in severe cases when other treatments are ineffective. The
cytotoxicity associated with immunosuppressants as well as the
problems of long-term systemic corticosteroid therapy provide
incentives to develop targeted and less toxic therapies,
particularly those with steroid-sparing effects. No new agent has
been approved as a therapeutic for SLE in over 50 years until the
recent approval of Benlysta (belimumab) in March of 2011.
[0280] Although the conventional view of B cells is as precursors
of immunoglobulin-producing plasma cells, they may also play other
roles in the pathogenesis of SLE, such as presenting autoantigens,
promoting the breakdown of peripheral T-cell tolerance, and
possibly by activating populations of T cells with low affinity
toward autoantigens (Looney, 2010, Drugs 70:529-540; Mok, 2010, Int
J Rheum Dis 13:3-11; Goldenberg, 2006, Expert Rev Anticancer Ther
6:1341-1353; Thaunat et al., 2010, Blood 116:515-521). Because of
the central role of B cells in the pathogenesis of autoimmunity,
targeted anti-B-cell immunotherapies are expected to offer
therapeutic value in for SLE. For example, Benlysta is a monoclonal
antibody (mAb) that inhibits activation of B cells by blocking
B-cell activating factor.
[0281] Another B-cell target is CD22, a 135-kD glycoprotein that is
a B-lymphocyte-restricted member of the immunoglobulin superfamily,
and a member of the sialoadhesin family of adhesion molecules that
regulate B cell activation and interaction with T cells (e.g.,
Carnahan et al., 2007, Mol Immunol 44:1331-1341; Haas et al., 2006,
J Immunol 177:3063-3073; Haas et al., 2010, J Immunol
184:4789-4800). CD22 has 7 extracellular domains and is rapidly
internalized when cross-linked with its natural ligand, producing a
potent costimulatory signal in primary B cells. CD22 is an
attractive molecular target for therapy because of its restricted
expression; it is not exposed on embryonic stem or pre-B cells nor
is it normally shed from the surface of antigen-bearing cells.
[0282] Epratuzumab is a humanized anti-CD22 antibody that is in
advanced clinical trials. Clinical trials with epratuzumab have
been undertaken for patients with non-Hodgkin's lymphoma,
leukemias, Waldenstrom's macroglobulinemia, Sjogren's syndrome and
SLE, and encompass an experience in more than 1000 patients. In the
initial clinical study with epratuzumab in non-Hodgkin's lymphoma
(NHL) or other B-cell malignancies, patients received 4 consecutive
weekly epratuzumab infusions at doses of ranging from 120 to 1000
mg/m.sup.2/week (Goldenberg, 2006, Expert Rev Anticancer Ther
6:1341-1353; Goldenberg et al., 2006, Arthritis Rheum 54:2344-2345;
Leonard et al., 2003, J Clin Oncol 21:3051-3059; Leonard et al.,
2008, Cancer 113:2714-2723). Treatment-related toxicity typically
involved nausea, chills/rigors, fever and other transient mild or
moderate infusion reactions occurring primarily during the first
weekly infusion. Peripheral B-cell levels decreased following
epratuzumab therapy, but otherwise no consistent changes were seen
in RBC, ANC, platelets, immunoglobulins, or T-cell levels following
treatment. Epratuzumab blood levels after the 4.sup.th weekly
infusion increased in a dose-dependent manner, and epratuzumab
remained in circulation with a half-life of 23 days. Interestingly,
enhanced anti-tumor effects in indolent and aggressive forms of NHL
were reported when epratuzumab was combined with the anti-CD20
rituximab (Goldenberg, 2006, Expert Rev Anticancer Ther
6:1341-1353; Leonard et al., 2005, J Clin Oncol 23:5044-5051;
Leonard et al., 2008, Cancer 113:2714-2723; Strauss et al., 2006, J
Clin Oncol 24:3880-3886). Epratuzumab is entering two Phase-III
registration trials for the treatment of SLE patients. Also
noteworthy is that rituximab has also shown activity in patients
with SLE (Ramos-Casals et al., 2009, Lupus 18:767-776).
[0283] Since the combination of rituximab and epratuzumab showed
improved anti-lymphoma efficacy without increased toxicity in
patients (Leonard et al., 2008, Cancer 113:2714-2723), we
engineered and evaluated bsAbs against both CD20 and CD22,
including an earlier design based on the IgG-(scFv).sub.2 format
(Qu et al., 2008, Blood 111:2211-2219) and the more recent DNL.TM.
design based on the hexavalent IgG-(Fab).sub.4 format, which
resulted in 22-(20)-(20) and 20-(22)-(22) (Rossi et al., 2009 Blood
113:6161-6171). Specifically, 22-(20)-(20) comprises epratuzumab
and 4 additional Fabs of veltuzumab, and thus binds CD22 bivalently
and CD20 tetravalently. Likewise, the other bsAb, 20-(22)-(22),
comprising veltuzumab and 4 Fabs of epratuzumab, binds CD20
bivalently and CD22 tetravalently. For the original HexAbs,
referred to henceforth as C.sub.H3-HexAbs, the two types of modules
are C.sub.H3-AD2-IgG and C.sub.H1-DDD2-Fab. Each of these modules
is produced as a fusion protein in myeloma cells and purified by
protein A (C.sub.H3-AD2-IgG) or KappaSelect (C.sub.H1-DDD2-Fab)
affinity chromatography.
[0284] A HexAb can be either monospecific or bispecific. The
C.sub.H3-HexAbs comprise a pair of Fab-DDD2 dimers linked to a full
IgG at the carboxyl termini of the two heavy chains, thus having
six Fab-arms and a common Fc domain. For example, the code of
20-(22)-(22) designates the bispecific HexAb comprising a divalent
anti-CD20 IgG of veltuzumab and a pair of dimeric anti-CD22
Fab-arms of epratuzumab, whereas 22-(20)-(20) specifies the
bispecific HexAb comprising a divalent anti-CD22 IgG of epratuzumab
and a pair of dimeric anti-CD20 Fab-arms of veltuzumab.
[0285] As discussed in Example 1 above, we have developed an
alternative HexAb format by utilizing a new IgG-AD2 module,
C.sub.k-AD2-IgG, which has the AD2 peptide fused to the carboxyl
end of the kappa light chain, instead of at the end of the Fc.
Combination with C.sub.H1-DDD2-Fab results in a C.sub.k-HexAb
structure, having a different architecture, but similar composition
(6 Fabs and an Fc), to the C.sub.H3-HexAb, having the four
additional Fabs linked at the end of the light chain. As discussed
above, the C.sub.k-HexAbs exhibit superior effector functions and
have significantly improved pharmacokinetics (Pk), compared to the
original C.sub.H3-HexAbs.
[0286] The C.sub.k-HexAbs are particularly well suited for in vivo
applications as they display favorable Pk, are stable in vivo, and
may be less immunogenic as both DDD- and AD-peptides are derived
from human proteins and the constitutive antibody components are
humanized. In addition, each of the anti-CD22/CD20 potently induce
direct cytotoxicity against various CD20/CD22-expressing lymphoma
and leukemic cell lines in vitro without the need for a secondary
antibody to effect hypercrosslinking, which is required for the
parental mAbs. In vivo studies confirmed the efficacy of the bsAbs
to inhibit growth of Burkitt lymphoma xenografts in mice, thus
indicating their larger size has not affected tumor targeting and
tissue penetration.
[0287] Preliminary Results
[0288] Clinical Experience with Epratuzumab--
[0289] Clinical studies have been conducted to examine the efficacy
of epratuzumab in indolent and aggressive forms of NHL alone in
combination with rituximab. The published data show that the
antibody can be given weekly for 4 weeks in a<1-h infusion up to
doses of 1,000 mg/m.sup.2, with the optimal dose appearing to be
360 mg/m.sup.2, and resulting in very durable objective responses
in 43% of follicular patients given this optimal dose, with
one-third comprising CRs. When combined weekly for four weeks with
rituximab, follicular, indolent NHL patients showed an overall 67%
objective response (7% PR and 60% CR/Cru), with only one patient
relapsing at 19 months follow-up. We and others have studied the
combination of rituximab and epratuzumab at their recommended full
doses weekly.times.4 in multicenter US and European trials, with
results indicating a higher CR rate than observed in historical
studies with rituximab alone in similar patient populations.
[0290] For lupus, completed studies have enrolled 331 unique
individuals who received at least one dose of epratuzumab
(Shoenfeld et al. (Eds.), Immunotherapeutic Agents for SLE. Future
Medicine Ltd; 2012). In the initial study, Dorner et al.
administered 360 mg/m.sup.2 to 14 patients with moderately active
SLE (Dorner et al., 2006, Arthritis Res Ther 8:R74). Patients
received 360 mg/m.sup.2 epratuzumab intravenously every 2 weeks for
four doses, with analgesic/antihistamine premedications (but no
steroids), and were followed for up to 32 weeks. The drug had
effects as early as 6 weeks, with 93% demonstrating improvements in
British Isles Lupus Activity Group (BILAG) Index in at least one B-
or C-level disease activity at 6 weeks, and all patients achieved
improvement in at least one BILAG body system at 10 weeks.
Epratuzumab was well tolerated and had a median infusion time of 32
min. Blood B-cell levels decreased by an average of 35% at 6 weeks
and remained decreased at 6 months post-therapy. No adverse safety
signals were detected. B-cell levels decreased post-treatment by
about 40%, which is much less than the experience with anti-CD20
mAbs. Post-treatment T-cell levels, immunoglobulins and other
standard safety laboratory tests remained unchanged from baseline.
No evidence of HAHA was found in these patients. No consistent
changes in autoantibodies and other disease-related laboratory
tests were seen.
[0291] This led to two Phase III studies known as ALLEVIATE I and
II (SL0003/SL0004; ClinicalTrials.gov registry: NCT00111306 and
NCT00383214) that were intended to be 48-week, randomized,
double-blind, placebo-controlled trials, followed by an open-label,
long-term, safety study for patients in the USA (SL0006). The
protocol included infusing patients with epratuzumab at 360 or 720
mg/m.sup.2 (in addition to background therapy, which included
corticosteroids and immunosuppressives) over four consecutive
12-week cycles: in the first cycle, four infusions were given at
weeks 0, 1, 2 and 3; for the three subsequent retreatment cycles,
two infusions were given at weeks 0 and 1. The primary efficacy end
point was a BILAG responder analysis at week 12, since too few
patients completed the originally intended 24 patient response
variable evaluation. Responders had a reduction of BILAG A or B by
one level, no new BILAG A or less than two new Bs, and no
introduction of immunosuppression or increase in steroid doses
during the treatment period. Initiated in 2005, the study was
prematurely discontinued in 2006 due to drug supply interruption.
At that point, only 90 patients had been studied long enough for
analysis and the two groups were pooled.
[0292] A total of 29 US patients were given open-label follow-up
therapy in SL0006. Subjects generally had serious lupus: the mean
BILAG score was 13.2 and 43% had at least one BILAG A. In total,
63% were on immunosuppressive agents and 43% were on 25 mg or more
of prednisone daily. A total of 91% received four infusions and 69%
reached week 24. Using an intention-to-treat analysis, a BILAG
response was achieved at week 12 in 44.1, 20 and 30.3% of the 360
mg/m.sup.2, 720 mg/m.sup.2 and placebo groups, respectively, with
responses seen within 6 weeks. Epratuzumab demonstrated significant
steroid-sparing properties and correlated with improvements in
health-related quality of life. The improvements were sustained in
those who stayed in the open label follow-up. No significant
intergroup differences were found in adverse events or serious
adverse events. B-cell depletion was approximately 20-40% among
treated patients.
[0293] EMBLEM was a Phase IIb, 12-week, double-blind study of six
different dosing regimens for patients with at least one BILAG A
and/or two BILAG B's (ClinicalTrials.gov registry: NCT00624351).
This study included 227 SLE patients with a mean total BILAG score
of 15.2 and a mean SLE disease activity index of 14.8 who were on a
mean 14 mg of prednisone daily, and the majority were also taking
immunosuppressive agents. Study participants thus had more
multisystem disease activity than has been seen in any other
published lupus clinical trial. Four weekly infusions, two
infusions every other week, or placebo, were given against a
background of prednisone and, for most, immunosuppressive therapy.
Those who received a combined dose of 2400 mg had meaningful and
statistically significant improvements, with 37.9% achieving an
`enhanced BILAG improvement`, whereby at least two levels (e.g., A
to C, B to D) of improvement were noted. Again, there were no
safety signals or significant immunosuppression. Only four out of
187 (2.1%) patients developed HAHA.
[0294] EMBODY, a pivotal 48-week trial consisting of two large
cohorts totaling nearly 2000 patients, was initiated in December
2010 (ClinicalTrials.gov registry: NCT01262365).
[0295] Clinical Experience with Veltuzumab--
[0296] We have studied veltuzumab in over 80 NHL patients
refractory/relapsed to prior therapies, including rituximab, and it
has been found to have about a 43% objective response and a 27%
complete response rate in FL patients at all doses summarized,
which appear to be durable (15-25 months) (Morchhauser et al.,
2009, J Clin Oncol 27:3346-3353). Activity was seen even at doses
of 80 mg/m.sup.2. Importantly, the infusion profile appears better
than rituximab's, with no grade 3-4 adverse reactions and infusion
times of .ltoreq.2 h (compared to 4-8 h for rituximab). Veltuzumab
has been examined also in a subcutaneous (SC) formulation in B-cell
lymphoma (Negrea et al., 2011, Haematologica 96:567-573).
[0297] Veltuzumab has also been studied in patients with immune
thrombocytopenia (ITP) (ClinicalTrials.gov registry: NCT00547066),
and has been shown to be active in this disease, even when low
doses have been administered (twice, on weeks 1 and 3)
intravenously and subcutaneously (data not shown). Forty-one
patients received 2 doses of veltuzumab 2 weeks apart. Veltuzumab
was well-tolerated (limited Grade 1-2 transient reactions, except
one Grade 3 infusion reaction), with no other safety issues. Of 38
assessable patients, 9 with newly-diagnosed or persistent disease
(ITP.ltoreq.1 year) previously treated only with steroids and/or
immunoglobulins, had 7 (78%) responses including 3 (33%) CRs and 4
(44%) PRs, while 29 with chronic disease up to 31 years and
additional prior therapies had 20 (69%) responses, including 4
(13%) CRs and 10 (35%) PRs. For the 27 responders, median time to
relapse increased with response category (MR: 2.4, PR: 5.5, CR:
14.4 months) with 10 (37%) responding >1 year (3 ongoing at
3.0-3.8 years). Eight responders were retreated, with 3 again
achieving PRs, including one retreated 4 times. Both IV and SC
routes depleted B cells after the first injection at all doses.
Eight patients developed low HAHA titers of uncertain clinical
significance. Thus, veltuzumab is a promising therapeutic on its
own, both in NHL and in an autoimmune disease.
[0298] Hexavalent bsAbs Made by DNL.TM.--
[0299] The molecular engineering, production, purification and
biochemical/biological characterization of 22-(20)-(20) and
20-(22)-(22) have been reported (Rossi et al., 2009, Blood
113:6161-6171). A detailed examination of the mechanism of action
and cell signaling induced by 22-(20)-(20) and 20-(22)-(22) has
also been published (Gupta et al., 2010, Blood 116:3258-3267). The
key findings are as follows.
[0300] Both 22-(20)-(20) and 20-(22)-(22) retained the binding
properties of their parental Fab/IgGs with all 6 Fabs capable of
binding simultaneously. Competitive ELISAs showed that each
construct possesses the functional valency as designed, and that
each Fab binds with a similar affinity to those of the parental
mAb. Flow cytometry demonstrate bispecific binding to live NHL
cells with longer retention than the parental mAbs. The
internalization rate of the bsAbs is largely influenced by both
valency and the internalizing nature of the constitutive
antibodies. Specifically, 22-(20)-(20) with four Fabs from the
slowly internalizing veltuzumab and two Fabs from the rapidly
internalizing epratuzumab behaves similar to veltuzumab, showing a
slow internalization rate. Conversely 20-(22)-(22) with four Fabs
from the rapidly internalizing epratuzumab and two Fabs from the
slowly internalizing veltuzumab exhibits rapid internalization,
similar to epratuzumab.
[0301] The two anti-CD20/CD22 bsAbs induced caspase-independent
apoptosis more potently than veltuzumab or epratuzumab, either
alone or in combination. Despite the incorporation of veltuzumab,
which alone displays potent CDC, neither bsAb is able to induce
CDC. Both bsAbs exhibit ADCC, with 20-(22)-(22) more potent than
22-(20)-(20), presumably due to the higher density of CD20 than
CD22 in normal B cells and NHL as well as the fact that veltuzumab
mediates ADCC more efficiently than epratuzumab.
[0302] The bsAbs inhibit lymphoma cells without immobilization
(required for epratuzumab) or hypercrosslinking with a secondary
antibody (required for veltuzumab). Such direct cytotoxicity is
about 50-fold more potent in Daudi Burkitt lymphoma cells than the
combination of both parental mAbs in the absence of immobilization
or hypercrosslinking. In Raji and Ramos cells, 22-(20)-(20) is 8-
to 10-fold more potent than 20-(22)-(22), which is in turn 8- to
10-fold more potent than the combination of both parental Abs.
Thus, 22-(20)-(20) can be 100-fold more potent than the parental
mAbs given in combination in vitro in the absence of other factors,
such as effect cells.
[0303] Both bsAbs induce extensive translocation of CD22 (as well
as CD20) into lipid rafts. Both bsAbs induce strong homotypic
adhesion in lymphoma cells, whereas under the same conditions the
parental mAbs are ineffective, indicating that crosslinking CD20
and CD22 leads to homotypic adhesion, which may contribute to the
enhanced in vitro cytotoxicity.
[0304] Pk analyses show that the circulating half-life of the bsAbs
in mice is 2-3-fold shorter than that of the parental mAbs.
Biodistribution studies in mice show that both bsAbs have tissue
uptakes similar to veltuzumab and epratuzumab, indicating that the
bsAbs are not cleared more rapidly than their parental mAbs because
of increased uptake in normal tissues.
[0305] In vivo studies in Daudi xenografts reveal 20-(22)-(22),
despite having a shorter serum half-life, had anti-tumor efficacy
comparable to equimolar veltuzumab. Although 22-(20)-(20) is less
potent than 20-(22)-(22), it is still more effective than
epratuzumab and the control bsAbs. The greatly enhanced direct
toxicity of the bsAbs correlates with their ability to alter the
basal expression of various intracellular proteins involved in
regulating cell growth, survival, and apoptosis, with the net
outcome leading to cell death. In an ex vivo setting, both
22-(20)-(20) and 20-(22)-(22) deplete NHL cells as well as normal B
cells from whole blood of healthy volunteers.
[0306] Because Pk analyses revealed that the circulating half-life
of the C.sub.H3-HexAbs in mice is 2-3-fold shorter than that of the
parental mAbs, we have developed the alternative C.sub.k-HexAb
format, with the goal of improving the Pk. The studies in Example 1
above indicate that the increased rate of blood clearance observed
for the C.sub.H3-based HexAbs is due to the location of the
additional Fab groups at the end of the Fc, interfering with the
binding (and/or release) of the neonatal Fc receptor (FcRn), which
is responsible for recirculation of IgG following endocytosis,
resulting in greatly extended Pk. Indeed, 22*-(20)-(20) exhibited
markedly superior Pk compared to 22-(20)-(20) (Example 1).
Following subcutaneous injection in normal mice, 22*-(20)-(20)
achieved a two-fold greater C.sub.max and three-fold longer
circulating half-life, resulting in a three-fold greater area under
the curve, compared to 22-(20)-(20). Additionally, 22*-(20)-(20)
was found to be highly stable in vivo over the entire 5-day Pk
study. This was evident because two different ELISA formats, one of
which detects any form of epratuzumab, and the other quantifying
only intact 22*-(20)-(20), generated essentially overlapping Pk
curves.
[0307] Use in SLE
[0308] The 22*-(20)-(20) DNL.TM. construct is selected for
therapeutic use in SLE. 22*-(20)-(20) is derived from veltuzumab,
the humanized anti-CD20 monoclonal antibody (mAb) and epratuzumab,
the humanized anti-CD22 mAb, to form a covalent conjugate with four
Fab fragments of veltuzumab attached to one IgG of epratuzumab (see
Example 1). Both epratuzumab and veltuzumab have shown clinical
activity in autoimmune disease and combination therapy with both
mAbs will be more effective than either as monotherapies. A more
potent therapy, using two different mechanisms of action (B-cell
depletion by anti-CD20 mAb and B-cell modulation by anti-CD22 mAb),
is accomplished by using a bispecific antibody capable of targeting
both CD20 and CD22 that is more convenient and economical than
administering two different mAbs sequentially, which presently
requires patients to be infused for many hours in each treatment
session.
[0309] Use of 22*-(20)-(20) as a therapeutic agent for SLE is
evaluated in an SCID mouse model, in which animals are engrafted
with peripheral blood lymphocytes (PBL) from SLE patients
(Mauermann et al., 2004, Clin Exp Immunol 137:513-520). The
efficacy of the bsAb is compared to epratuzumab and veltuzumab
independently and in combination by monitoring the serum level of
anti-dsDNA antibody, a hallmark of SLE.
[0310] Blood samples are collected from SLE patients. For
engraftment, 3.times.10.sup.7 PBLs obtained from individual SLE
patients are injected intraperitoneally (i.p.) into an 8-10 week
old female SCID mouse. Thus, each animal represents an individual
lupus patient. Approximately two-thirds of the mice have successful
engraftment, with evidence of human antibody production in
concentrations .gtoreq.100 .mu.g/mL within 2 weeks, with peak
production within 4 weeks. Mice having evidence of engraftment, are
used for treatment. To monitor the effect of treatment, mice are
bled on days 24, 34, 44, and 54 and the sera are tested by ELISA or
Protein-A HPLC for the presence of total hIgG, anti-dsDNA
(measurement of lupus disease state) and anti-tetanus toxoid
antibodies (to demonstrate functional human humoral immune
system).
[0311] Human anti-dsDNA antibodies in the recipient mouse sera, as
an indicator of SLE, are determined using maxisorb 96-well
microtitre plates coated with poly L-lysine (5 mg/ml, Sigma, St.
Louis, Mo., USA), followed by coating with lambda phage dsDNA (5
mg/well, Boehringer Mannheim, Germany). Plates are blocked with 10%
fetal calf serum (FCS) in PBS, and incubated with mouse sera
(diluted 1:5-1:40) for 2 h. Bound anti-dsDNA is detected with a
goat antihuman IgG antibody conjugated to horseradish peroxidase
(Jackson).
[0312] The effect of using the 22*-(20)-(20) DNL.TM. on SLE mice is
examined. Prior studies with the 22-(20)-(20) bsAb in vitro found
it was effective in killing human B-cell lymphoma cell lines at
concentrations of .about.1 nM (.about.350 .mu.g/mL), and in vivo,
three 10-.mu.g doses of 22-(20)-(20) in 1 week controlled the
outgrowth of IV implanted Daudi B-cell lymphoma cell line in SCID
mice (Rossi et al., 2009, Blood 113:6161-6171). However, the bsAbs
were less effective in killing normal B-cells ex vivo (FIG. 10).
Based on these results, SLE-engrafted SCID mice are treated
initially with 400 .mu.g of 22*-(20)-(20) i.p. twice weekly for 2
weeks (starting on day 14). If titers return, a second cycle of
treatment is initiated, continuing until study termination on day
60. If disease control at 400 .mu.g is insufficient after 2 weeks
of treatment, treatment in subsequent groups of animals given 400
.mu.g is uninterrupted for 4 sequential weeks. If disease control
is significantly improved after 2 weeks with 400 .mu.g, a lower
dose of 40 .mu.g using the same twice weekly schedule for 2 weeks
is followed by an observation period. Equal numbers of animals
receive only the excipient (buffer) dosing so that a baseline for
disease progression is established. Our goal is to establish a
treatment protocol using a minimum dose that significantly
decreases antibody production, proteinuria, and evidence of renal
damage. In addition to the buffer control, the effects are
determined of the parental mAbs of 22*-(20)-(20), epratuzumab,
veltuzumab and a combination of epratuzumab and veltuzumab IgG, as
well as veltuzumab-DDD2 (bivalent Fab), each given at equal molar
amounts and following the same dosing schedule as the 22*-(20)-(20)
test group.
[0313] The primary comparator among treatment groups is the change
in anti-dsDNA antibody serum titer following treatment. Based on
the results of Maurermann et al. using this model system, the
anti-dsDNA titer in control mice peaks at 30-40 days, before slowly
declining. Successful therapy results in a much lower C.sub.max and
more rapid decline in anti-dsDNA titer, to levels below those at
the onset of therapy. The C.sub.max and the change in anti-dsDNA
titer from day 14 to day 70 (.DELTA.C.sub.70/14) are measured for
each animal. At the end of the therapy study, animals are assessed
for proteinuria and inflammatory glomerulonephritis as additional
measurements of disease progression or control.
[0314] It is observed that SLE mice treated with a 400 .mu.g dose
of 22*-(20)-(20) twice weekly for four weeks show a significant
decrease in anti-dsDNA titer, with lower levels of proteinuria and
inflammatory glomerulonephritis, compared to the buffer control,
either epratuzumab or veltuzumab administered alone, or the
combination of epratuzumab and veltuzumab, when administered at the
same molar dosages and schedules as 22*-(20)-(20).
Example 3
General Techniques for DOCK-AND-LOCK.TM.
[0315] The general techniques discussed below were used to generate
DNL.TM. complexes with AD or DDD moieties attached to the
C-terminal end of the antibody heavy chain. Light chain appended AD
moieties were constructed as described in Example 1 above. With the
exception of superior Pk, in vivo stability and improved efficacy,
the C.sub.k DNL.TM. constructs were found to function similarly to
their C.sub.H counterparts.
[0316] Expression Vectors
[0317] 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 (V.sub.H and V.sub.L) 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.
[0318] To generate Fab-DDD expression vectors, the coding sequences
for the hinge, CH2 and CH3 domains of the heavy chain were replaced
with a sequence encoding the first 4 residues of the hinge, a 14
residue Gly-Ser linker and a DDD moiety, such as the first 44
residues of human RII.alpha. (referred to as DDD1, SEQ ID NO:1). To
generate Fab-AD expression vectors, the sequences for the hinge,
CH2 and CH3 domains of IgG were replaced with a sequence encoding
the first 4 residues of the hinge, a 15 residue Gly-Ser linker and
an AD moiety, such as a 17 residue synthetic AD called AKAP-IS
(referred to as AD1, SEQ ID NO:3), 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. 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.
[0319] Preparation of CH1
[0320] 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:123) followed by four glycines and a serine,
with the final two codons (GS) comprising a Bam HI restriction
site. The 410 by 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.
[0321] A duplex oligonucleotide was synthesized 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-00024 (SEQ ID NO: 124)
GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRL REARA
[0322] Two oligonucleotides, designated RIIA1-44 top and RIIA1-44
bottom, which overlap by 30 base pairs on their 3' ends, were
synthesized 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.
[0323] A duplex oligonucleotide was synthesized 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-00025 GSGGGGSGGGGSQIEYLAKQIVDNAIQQA (SEQ ID NO: 125)
[0324] 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.
[0325] Ligating DDD1 with CH1
[0326] 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..
[0327] Ligating AD1 with CH1
[0328] 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..
[0329] 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.RTM. shuttle vector.
[0330] C-DDD2-Fd-hMN-14-pdHL2
[0331] 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 (SEQ ID NO:2) 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.
[0332] The expression vector was engineered as follows. Two
overlapping, complimentary oligonucleotides, which comprise the
coding sequence for part of the linker peptide 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.
[0333] 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.
[0334] h679-Fd-AD2-pdHL2
[0335] h679-Fab-AD2, was designed to pair to C-DDD2-Fab-hMN-14.
h679-Fd-AD2-pdHL2 is an expression vector for the production of
h679-Fab-AD2, which possesses an anchoring domain sequence of AD2
(SEQ ID NO:4) 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.
[0336] The expression vector was engineered as follows. Two
overlapping, complimentary oligonucleotides (AD2 Top and AD2
Bottom), 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.
[0337] 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.
[0338] Generation of TF2 DNL.TM. Construct
[0339] A trimeric DNL.TM. 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).
[0340] 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).
[0341] Production of TF10 DNL.TM. Construct
[0342] A similar protocol was used to generate a trimeric TF10
DNL.TM. construct, comprising two copies of a C-DDD2-Fab-hPAM4 and
one copy of C-AD2-Fab-679. The TF10 bispecific ([hPAM4].sub.2 x
h679) antibody was produced using the method disclosed for
production of the (anti CEA).sub.2x anti HSG bsAb TF2, as described
above. The TF10 construct bears two humanized PAM4 Fabs and one
humanized 679 Fab.
[0343] The two fusion proteins (hPAM4-DDD2 and h679-AD2) were
expressed independently in stably transfected myeloma cells. The
tissue culture supernatant fluids were combined, resulting in a
two-fold molar excess of hPAM4-DDD2. The reaction mixture was
incubated at room temperature for 24 hours under mild reducing
conditions using 1 mM reduced glutathione. Following reduction, the
reaction was completed by mild oxidation using 2 mM oxidized
glutathione. TF10 was isolated by affinity chromatography using
IMP291-affigel resin, which binds with high specificity to the h679
Fab.
Example 4
Production of AD- and DDD-linked Fab and IgG Fusion Proteins From
Multiple Antibodies
[0344] Using the techniques described in the preceding Examples,
the IgG and Fab fusion proteins shown in Table 10 were constructed
and incorporated into DNL.TM. constructs. The fusion proteins
retained the antigen-binding characteristics of the parent
antibodies and the DNL.TM. constructs exhibited the antigen-binding
activities of the incorporated antibodies or antibody
fragments.
TABLE-US-00026 TABLE 10 Fusion proteins comprising IgG or Fab
Binding Fusion Protein Specificity C-AD1-Fab-h679 HSG
C-AD2-Fab-h679 HSG C-(AD).sub.2-Fab-h679 HSG C-AD2-Fab-h734
Indium-DTPA C-AD2-Fab-hA20 CD20 C-AD2-Fab-hA20L CD20
C-AD2-Fab-hL243 HLA-DR C-AD2-Fab-hLL2 CD22 N-AD2-Fab-hLL2 CD22
C-AD2-IgG-hMN-14 CEACAM5 C-AD2-IgG-hR1 IGF-1R C-AD2-IgG-hRS7 EGP-1
C-AD2-IgG-hPAM4 MUC C-AD2-IgG-hLL1 CD74 C-DDD1-Fab-hMN-14 CEACAM5
C-DDD2-Fab-hMN-14 CEACAM5 C-DDD2-Fab-h679 HSG C-DDD2-Fab-hA19 CD19
C-DDD2-Fab-hA20 CD20 C-DDD2-Fab-hAFP AFP C-DDD2-Fab-hL243 HLA-DR
C-DDD2-Fab-hLL1 CD74 C-DDD2-Fab-hLL2 CD22 C-DDD2-Fab-hMN-3 CEACAM6
C-DDD2-Fab-hMN-15 CEACAM6 C-DDD2-Fab-hPAM4 MUC C-DDD2-Fab-hR1
IGF-1R C-DDD2-Fab-hRS7 EGP-1 N-DDD2-Fab-hMN-14 CEACAM5
Example 5
Antibody-Dendrimer DNL.TM. Complex for siRNA
[0345] Cationic polymers, such as polylysine, polyethylenimine, or
polyamidoamine (PAMAM)-based dendrimers, form complexes with
nucleic acids. However, their potential applications as non-viral
vectors for delivering therapeutic genes or siRNAs remain a
challenge. One approach to improve selectivity and potency of a
dendrimeric nanoparticle may be achieved by conjugation with an
antibody that internalizes upon binding to target cells.
[0346] We synthesized and characterized a novel immunoconjugate,
designated E1-G5/2, which was made by the DNL.TM. method to
comprise half of a generation 5 (G5) PAMAM dendrimer (G5/2)
site-specifically linked to a stabilized dimer of Fab derived from
hRS7, a humanized antibody that is rapidly internalized upon
binding to the Trop-2 antigen expressed on various solid
cancers.
[0347] Methods
[0348] E1-G5/2 was prepared by combining two self-assembling
modules, AD2-G5/2 and hRS7-Fab-DDD2, under mild redox conditions,
followed by purification on a Protein L column. To make AD2-G5/2,
we derivatized the AD2 peptide with a maleimide group to react with
the single thiol generated from reducing a G5 PAMAM with a
cystamine core and used reversed-phase HPLC to isolate AD2-G5/2. We
produced hRS7-Fab-DDD2 as a fusion protein in myeloma cells, as
described in the Examples above.
[0349] The molecular size, purity and composition of E1-G5/2 were
analyzed by size-exclusion HPLC, SDS-PAGE, and Western blotting.
The biological functions of E1-G5/2 were assessed by binding to an
anti-idiotype antibody against hRS7, a gel retardation assay, and a
DNase protection assay.
[0350] Results
[0351] E1-G5/2 was shown by size-exclusion HPLC to consist of a
major peak (>90%) flanked by several minor peaks (not shown).
The three constituents of E1-G5/2 (Fd-DDD2, the light chain, and
AD2-G5/2) were detected by reducing SDS-PAGE and confirmed by
Western blotting (not shown). Anti-idiotype binding analysis
revealed E1-G5/2 contained a population of antibody-dendrimer
conjugates of different size, all of which were capable of
recognizing the anti-idiotype antibody, thus suggesting structural
variability in the size of the purchased G5 dendrimer (not shown).
Gel retardation assays showed E1-G5/2 was able to maximally
condense plasmid DNA at a charge ratio of 6:1 (+/-), with the
resulting dendriplexes completely protecting the complexed DNA from
degradation by DNase I (not shown).
[0352] Conclusion
[0353] The DNL.TM. technique can be used to build dendrimer-based
nanoparticles that are targetable with antibodies. Such agents have
improved properties as carriers of drugs, plasmids or siRNAs for
applications in vitro and in vivo. In preferred embodiments,
anti-B-cell antibodies, such as anti-CD20 and/or anti-CD22, may be
utilized to deliver cytotoxic or cytostatic siRNA species to
targeted B-cells for therapy of lymphoma, leukemia, autoimmune or
other diseases and conditions.
Example 6
Targeted Delivery of siRNA Using Protamine Linked Antibodies
[0354] Summary
[0355] RNA interference (RNAi) has been shown to down-regulate the
expression of various proteins such as HER2, VEGF, Raf-1, bcl-2,
EGFR and numerous others in preclinical studies. Despite the
potential of RNAi to silence specific genes, the full therapeutic
potential of RNAi remains to be realized due to the lack of an
effective delivery system to target cells in vivo.
[0356] To address this critical need, we developed novel DNL.TM.
constructs having multiple copies of human protamine tethered to a
tumor-targeting, internalizing hRS7 (anti-Trop-2) antibody for
targeted delivery of siRNAs in vivo. A DDD2-L-thP1 module
comprising truncated human protamine (thP1, residues 8 to 29 of
human protamine 1) was produced, in which the sequences of DDD2 and
thP1 were fused respectively to the N- and C-terminal ends of a
humanized antibody light chain (not shown). The sequence of the
truncated hP1 (thP1) is shown below. Reaction of DDD2-L-thP1 with
the antibody hRS7-IgG-AD2 under mild redox conditions, as described
in the Examples above, resulted in the formation of an E1-L-thP1
complex (not shown), comprising four copies of thP1 attached to the
carboxyl termini of the hRS7 heavy chains.
TABLE-US-00027 tHP1 RSQSRSRYYRQRQRSRRRRRRS (SEQ ID NO: 126)
[0357] The purity and molecular integrity of E1-L-thP1 following
Protein A purification were determined by size-exclusion HPLC and
SDS-PAGE (not shown). In addition, the ability of E1-L-thP1 to bind
plasmid DNA or siRNA was demonstrated by the gel shift assay (not
shown). E1-L-thP1 was effective at binding short double-stranded
oligonucleotides (not shown) and in protecting bound DNA from
digestion by nucleases added to the sample or present in serum (not
shown).
[0358] The ability of the E1-L-thP1 construct to internalize siRNAs
into Trop-2-expressing cancer cells was confirmed by fluorescence
microscopy using FITC-conjugated siRNA and the human Calu-3 lung
cancer cell line (not shown).
[0359] Methods
[0360] The DNL.TM. technique was employed to generate E1-L-thP1.
The hRS7 IgG-AD module, constructed as described in the Examples
above, was expressed in myeloma cells and purified from the culture
supernatant using Protein A affinity chromatography. The
DDD2-L-thP1 module was expressed as a fusion protein in myeloma
cells and was purified by Protein L affinity chromatography. Since
the CH3-AD2-IgG module possesses two AD2 peptides and each can bind
to a DDD2 dimer, with each DDD2 monomer attached to a protamine
moiety, the resulting E1-L-thP1 conjugate comprises four protamine
groups. E1-L-thp1 was formed in nearly quantitative yield from the
constituent modules and was purified to near homogeneity (not
shown) with Protein A.
[0361] DDD2-L-thP1 was purified using Protein L affinity
chromatography and assessed by size exclusion HPLC analysis and
SDS-PAGE under reducing and nonreducing conditions (data not
shown). A major peak was observed at 9.6 min (not shown). SDS-PAGE
showed a major band between 30 and 40 kDa in reducing gel and a
major band about 60 kDa (indicating a dimeric form of DDD2-L-thP1)
in nonreducing gel (not shown). The results of Western blotting
confirmed the presence of monomeric DDD2-L-tP1 and dimeric
DDD2-L-tP1 on probing with anti-DDD antibodies (not shown).
[0362] To prepare the E1-L-thP1, hRS7-IgG-AD2 and DDD2-L-thP1 were
combined in approximately equal amounts and reduced glutathione
(final concentration 1 mM) was added. Following an overnight
incubation at room temperature, oxidized glutathione was added
(final concentration 2 mM) and the incubation continued for another
24 h. E1-L-thP1 was purified from the reaction mixture by Protein A
column chromatography and eluted with 0.1 M sodium citrate buffer
(pH 3.5). The product peak (not shown) was neutralized,
concentrated, dialyzed with PBS, filtered, and stored in PBS
containing 5% glycerol at 2 to 8.degree. C. The composition of
E1-L-thP1 was confirmed by reducing SDS-PAGE (not shown), which
showed the presence of all three constituents (AD2-appended heavy
chain, DDD2-L-htP1, and light chain).
[0363] The ability of DDD2-L-thP1 and E1-L-thP1 to bind DNA was
evaluated by gel shift assay. DDD2-L-thP1 retarded the mobility of
500 ng of a linear form of 3-kb DNA fragment in 1% agarose at a
molar ratio of 6 or higher (not shown). E1-L-thP1 retarded the
mobility of 250 ng of a linear 200-bp DNA duplex in 2% agarose at a
molar ratio of 4 or higher (not shown), whereas no such effect was
observed for hRS7-IgG-AD2 alone (not shown). The ability of
E1-L-thP1 to protect bound DNA from degradation by exogenous DNase
and serum nucleases was also demonstrated (not shown).
[0364] The ability of E1-L-thP1 to promote internalization of bound
siRNA was examined in the Trop-2 expressing ME-180 cervical cell
line (not shown). Internalization of the E1-L-thP1 complex was
monitored using FITC conjugated goat anti-human antibodies. The
cells alone showed no fluorescence (not shown). Addition of
FITC-labeled siRNA alone resulted in minimal internalization of the
siRNA (not shown). Internalization of E1-L-thP1 alone was observed
in 60 minutes at 37.degree. C. (not shown). E1-L-thP1 was able to
effectively promote internalization of bound FITC-conjugated siRNA
(not shown). E1-L-thP1 (10 .mu.g) was mixed with FITC-siRNA (300
nM) and allowed to form E1-L-thP1-siRNA complexes which were then
added to Trop-2-expressing Calu-3 cells. After incubation for 4 h
at 37.degree. C. the cells were checked for internalization of
siRNA by fluorescence microscopy (not shown).
[0365] The ability of E1-L-thP1 to induce apoptosis by
internalization of siRNA was examined. E1-L-thP1 (10 .mu.g) was
mixed with varying amounts of siRNA (AllStars Cell Death siRNA,
Qiagen, Valencia, Calif.). The E1-L-thP1-siRNA complex was added to
ME-180 cells. After 72 h of incubation, cells were trypsinized and
annexin V staining was performed to evaluate apoptosis. The Cell
Death siRNA alone or E1-L-thP1 alone had no effect on apoptosis
(not shown). Addition of increasing amounts of E1-L-thP1-siRNA
produced a dose-dependent increase in apoptosis (not shown). These
results show that E1-L-thP1 could effectively deliver siRNA
molecules into the cells and induce apoptosis of target cells.
[0366] Conclusions
[0367] The DNL.TM. technology provides a modular approach to
efficiently tether multiple protamine molecules to the anti-Trop-2
hRS7 antibody resulting in the novel molecule E1-L-thP1. SDS-PAGE
demonstrated the homogeneity and purity of E1-L-thP1. DNase
protection and gel shift assays showed the DNA binding activity of
E1-L-thP1. E1-L-thP1 internalized in the cells like the parental
hRS7 antibody and was able to effectively internalize siRNA
molecules into Trop-2-expressing cells, such as ME-180 and
Calu-3.
[0368] The skilled artisan will realize that the DNL.TM. technique
is not limited to any specific antibody or siRNA species. Rather,
the same methods and compositions demonstrated herein can be used
to make targeted delivery complexes comprising any antibody (e.g.,
anti-CD20 or anti-CD22), any siRNA carrier and any siRNA species.
The use of a bivalent IgG in targeted delivery complexes would
result in prolonged circulating half-life and higher binding
avidity to target cells, resulting in increased uptake and improved
efficacy.
Example 7
Ribonuclease Based DNL.TM. Immunotoxins Comprising Quadruple
Ranpirnase (Rap) Conjugated to B-Cell Targeting Antibodies
[0369] We applied the DNL.TM. method to generate a novel class of
immunotoxins, each of which comprises four copies of Rap
site-specifically linked to a bivalent IgG. We combined a
recombinant Rap-DDD module, produced in E. coli, with recombinant,
humanized IgG-AD modules, which were produced in myeloma cells and
targeted B-cell lymphomas and leukemias via binding to CD20 (hA20,
veltuzumab), CD22 (hLL2, epratuzumab) or HLA-DR (hL243, IMMU-114),
to generate 20-Rap, 22-Rap and C2-Rap, respectively. For each
construct, a dimer of Rap was covalently tethered to the C-terminus
of each heavy chain of the respective IgG. A control construct,
14-Rap, was made similarly, using labetuzumab (hMN-14), that binds
to an antigen (CEACAM5) not expressed on B-cell
lymphomas/leukemias.
TABLE-US-00028 Rap-DDD2 (SEQ ID NO: 127)
pQDWLTFQKKHITNTRDVDCDNIMSTNLFHCKDKNTFIYSRPEPVKAICK
GIIASKNVLTTSEFYLSDCNVTSRPCKYKLKKSTNKFCVTCENQAPVHFV
GVGSCGGGGSLECGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFT
RLREARAVEHHHHHH
[0370] The deduced amino acid sequence of secreted Rap-DDD2 is
shown above (SEQ ID NO:127). Rap, underlined; linker, italics;
DDD2, bold; pQ, amino-terminal glutamine converted to
pyroglutamate. Rap-DDD2 was produced in E. coli as inclusion
bodies, which were purified by IMAC under denaturing conditions,
refolded and then dialyzed into PBS before purification by
Q-Sepharose anion exchange chromatography. SDS-PAGE under reducing
conditions resolved a protein band with a Mr appropriate for
Rap-DDD2 (18.6 kDa) (not shown). The final yield of purified
Rap-DDD2 was 10 mg/L of culture.
[0371] The DNL.TM. method was employed to rapidly generate a panel
of IgG-Rap conjugates. The IgG-AD modules were expressed in myeloma
cells and purified from the culture supernatant using Protein A
affinity chromatography. The Rap-DDD2 module was produced and mixed
with IgG-AD2 to form a DNL.TM. complex. Since the CH3-AD2-IgG
modules possess two AD2 peptides and each can tether a Rap dimer,
the resulting IgG-Rap DNL.TM. construct comprises four Rap groups
and one IgG. IgG-Rap is formed nearly quantitatively from the
constituent modules and purified to near homogeneity with Protein
A.
[0372] Prior to the DNL.TM. reaction, the CH3-AD2-IgG exists as
both a monomer, and a disulfide-linked dimer (not shown). Under
non-reducing conditions, the IgG-Rap resolves as a cluster of high
molecular weight bands of the expected size between those for
monomeric and dimeric CH3-AD2-IgG (not shown). Reducing conditions,
which reduces the conjugates to their constituent polypeptides,
shows the purity of the IgG-Rap and the consistency of the DNL.TM.
method, as only bands representing heavy-chain-AD2 (HC-AD2), kappa
light chain and Rap-DDD2 were visualized (not shown).
[0373] Reversed phase HPLC analysis of 22-Rap (not shown) resolved
a single protein peak at 9.10 min eluting between the two peaks of
CH3-AD2-IgG-hLL2, representing the monomeric (7.55 min) and the
dimeric (8.00 min) forms. The Rap-DDD2 module was isolated as a
mixture of dimer and tetramer (reduced to dimer during DNL.TM.),
which were eluted at 9.30 and 9.55 min, respectively (not
shown).
[0374] LC/MS analysis of 22-Rap was accomplished by coupling
reversed phase HPLC using a C8 column with ESI-TOF mass
spectrometry (not shown). The spectrum of unmodified 22-Rap
identifies two major species, having either two GOF (GOF/GOF) or
one GOF plus one G1F (GOF/G1F) N-linked glycans, in addition to
some minor glycoforms (not shown). Enzymatic deglycosylation
resulted in a single deconvoluted mass consistent with the
calculated mass of 22-Rap (not shown). The resulting spectrum
following reduction with TCEP identified the heavy chain-AD2
polypeptide modified with an N-linked glycan of the GOF or G1F
structure as well as additional minor forms (not shown). Each of
the three subunit polypeptides comprising 22-Rap were identified in
the deconvoluted spectrum of the reduced and deglycosylated sample
(not shown). The results confirm that both the Rap-DDD2 and HC-AD2
polypeptides have an amino terminal glutamine that is converted to
pyroglutamate (pQ); therefore, 22-Rap has 6 of its 8 constituent
polypeptides modified by pQ.
[0375] In vitro cytotoxicity was evaluated in three NHL cell lines.
Each cell line expresses CD20 at a considerably higher surface
density compared to CD22; however, the internalization rate for
hLL2 (anti-CD22) is much faster than hA20 (anti-CD20). 14-Rap
shares the same structure as 22-Rap and 20-Rap, but its antigen
(CEACAM5) is not expressed by the NHL cells. Cells were treated
continuously with IgG-Rap as single agents or with combinations of
the parental MAbs plus rRap. Both 20-Rap and 22-Rap killed each
cell line at concentrations above 1 nM, indicating that their
action is cytotoxic as opposed to merely cytostatic (not shown).
20-Rap was the most potent IgG-Rap, suggesting that antigen density
may be more important than internalization rate. Similar results
were obtained for Daudi and Ramos, where 20-Rap (EC50.about.0.1 nM)
was 3-6-fold more potent than 22-Rap (not shown). The
rituximab-resistant mantle cell lymphoma line, Jeko-1, exhibits
increased CD20 but decreased CD22, compared to Daudi and Ramos.
Importantly, 20-Rap exhibited very potent cytotoxicity
(EC.sub.50.about.20 pM) in Jeko-1, which was 25-fold more potent
than 22-Rap (not shown).
[0376] The DNL.TM. method provides a modular approach to
efficiently tether multiple cytotoxins onto a targeting antibody,
resulting in novel immunotoxins that are expected to show higher in
vivo potency due to improved pharmacokinetics and targeting
specificity. LC/MS, RP-HPLC and SDS-PAGE demonstrated the
homogeneity and purity of IgG-Rap. Targeting Rap with a MAb to a
cell surface antigen enhanced its tumor-specific cytotoxicity.
Antigen density and internalization rate are both critical factors
for the observed in vitro potency of IgG-Rap. In vitro results show
that CD20-, CD22-, or HLA-DR-targeted IgG-Rap have potent biologic
activity for therapy of B-cell lymphomas and leukemias.
Example 8
Production and Use of a DNL.TM. Construct Comprising Two Different
Antibody Moieties and a Cytokine
[0377] In certain embodiments, trimeric DNL.TM. constructs may
comprise three different effector moieties, for example two
different antibody moieties and a cytokine moiety. We report here
the generation and characterization of the first bispecific
MAb-IFN.alpha., designated 20-C2-2b, which comprises two copies of
IFN-.alpha.2b and a stabilized F(ab).sub.2 of hL243 (humanized
anti-HLA-DR; IMMU-114) site-specifically linked to veltuzumab
(humanized anti-CD20). In vitro, 20-C2-2b inhibited each of four
lymphoma and eight myeloma cell lines, and was more effective than
monospecific CD20-targeted MAb-IFN.alpha. or a mixture comprising
the parental antibodies and IFN.alpha. in all but one
(HLA-DR.sup.-/CD20.sup.-) myeloma line (not shown), suggesting that
20-C2-2b should be useful in the treatment of various hematopoietic
disorders. The 20-C2-2b displayed greater cytotoxicity against
KMS12-BM (CD20.sup.+/HLA-DR.sup.+ myeloma) than monospecific
MAb-IFN.alpha. that targets only HLA-DR or CD20 (not shown),
indicating that all three components in 20-C2-2b can contribute to
toxicity. Our findings indicate that a given cell's responsiveness
to MAb-IFN.alpha. depends on its sensitivity to IFN.alpha. and the
specific antibodies, as well as the expression and density of the
targeted antigens.
[0378] Because 20-C2-2b has antibody-dependent cellular
cytotoxicity (ADCC), but not CDC, and can target both CD20 and
HLA-DR, it is useful for therapy of a broad range of hematopoietic
disorders that express either or both antigens.
[0379] Antibodies
[0380] The abbreviations used in the following discussion are: 20
(C.sub.H3-AD2-IgG-v-mab, anti-CD20 IgG DNL.TM. module); C2
(C.sub.H1-DDD2-Fab-hL243, anti-HLA-DR Fab.sub.2 DNL.TM. module); 2b
(dimeric IFN.alpha.2B-DDD2 DNL.TM. module); 734 (anti-in-DTPA IgG
DNL.TM. module used as non-targeting control). The following MAbs
were provided by Immunomedics, Inc.: veltuzumab or v-mab (anti-CD20
IgG.sub.1), hL243.gamma.4p (Immu-114, anti-HLA-DR IgG.sub.4), a
murine anti-IFN.alpha.MAb, and rat anti-idiotype MAbs to v-mab
(WR2) and hL243 (WT).
[0381] DNL.TM. Constructs
[0382] Monospecific MAb-IFN.alpha. (20-2b-2b, 734-2b-2b and
C2-2b-2b) and the bispecific HexAb (20-C2-C2) were generated by
combination of an IgG-AD2-module with DDD2-modules using the
DNL.TM. method, as described in the preceding Examples. The
734-2b-2b, which comprises tetrameric IFN.alpha.2b and MAb h734
[anti-Indium-DTPA IgG.sub.1], was used as a non-targeting control
MAb-IFN.alpha..
[0383] The construction of the mammalian expression vector as well
as the subsequent generation of the production clones and the
purification of C.sub.H3-AD2-IgG-v-mab are disclosed in the
preceding Examples. The expressed recombinant fusion protein has
the AD2 peptide linked to the carboxyl terminus of the C.sub.H3
domain of v-mab via a 15 amino acid long flexible linker peptide.
Co-expression of the heavy chain-AD2 and light chain polypeptides
results in the formation of an IgG structure equipped with two AD2
peptides. The expression vector was transfected into Sp/ESF cells
(an engineered cell line of Sp2/0) by electroporation. The pdHL2
vector contains the gene for dihydrofolate reductase, thus allowing
clonal selection, as well as gene amplification with methotrexate
(MTX). Stable clones were isolated from 96-well plates selected
with media containing 0.2 .mu.M MTX. Clones were screened for
C.sub.H3-AD2-IgG-vmab productivity via a sandwich ELISA. The module
was produced in roller bottle culture with serum-free media.
[0384] The DDD-module, IFN.alpha.2b-DDD2, was generated as
discussed above by recombinant fusion of the DDD2 peptide to the
carboxyl terminus of human IFN.alpha.2b via an 18 amino acid long
flexible linker peptide. As is the case for all DDD-modules, the
expressed fusion protein spontaneously forms a stable
homodimer.
[0385] The C.sub.H1-DDD2-Fab-hL243 expression vector was generated
from hL243-IgG-pdHL2 vector by excising the sequence for the
C.sub.H1-Hinge-C.sub.H2-C.sub.H3 domains with SacII and EagI
restriction enzymes and replacing it with a 507 bp sequence
encoding C.sub.H1-DDD2, which was excised from the
C-DDD2-hMN-14-pdHL2 expression vector with the same enzymes.
Following transfection of C.sub.H1-DDD2-Fab-hL243-pdHL2 into Sp/ESF
cells by electroporation, stable, MTX-resistant clones were
screened for productivity via a sandwich ELISA using 96-well
microtiter plates coated with mouse anti-human kappa chain to
capture the fusion protein, which was detected with horseradish
peroxidase-conjugated goat anti-human Fab. The module was produced
in roller bottle culture.
[0386] Roller bottle cultures in serum-free H-SFM media and
fed-batch bioreactor production resulted in yields comparable to
other IgG-AD2 modules and cytokine-DDD2 modules generated to date.
C.sub.H3-AD2-IgG-v-mab and IFN.alpha.2b-DDD2 were purified from the
culture broths by affinity chromatography using MABSELECT.TM. (GE
Healthcare) and HIS-SELECT.RTM. HF Nickel Affinity Gel (Sigma),
respectively, as described previously (Rossi et al., Blood 2009,
114:3864-71). The culture broth containing the
C.sub.H1-DDD2-Fab-hL243 module was applied directly to
KAPPASELECT.RTM. affinity gel (GE-Healthcare), which was washed to
baseline with PBS and eluted with 0.1 M Glycine, pH 2.5.
[0387] Generation of 20-C2-2b by DNL.TM.
[0388] Three DNL.TM. modules (C.sub.H3-AD2-IgG-v-mab,
C.sub.H1-DDD2-Fab-hL243, and IFN-.alpha.2b-DDD2) were combined in
equimolar quantities to generate the bsMAb-IFN.alpha., 20-C2-2b.
Following an overnight docking step under mild reducing conditions
(1 mM reduced glutathione) at room temperature, oxidized
glutathione was added (2 mM) to facilitate disulfide bond formation
(locking). The 20-C2-2b was purified to near homogeneity using
three sequential affinity chromatography steps. Initially, the
DNL.TM. mixture was purified with Protein A (MABSELECT.TM.), which
binds the C.sub.H3-AD2-IgG-v-MAb group and eliminates un-reacted
IFN.alpha.2b-DDD2 or C.sub.H1-DDD2-Fab-hL243. The Protein A-bound
material was further purified by IMAC using HIS-SELECT.RTM. HF
Nickel Affinity Gel, which binds specifically to the
IFN.alpha.2b-DDD2 moiety and eliminates any constructs lacking this
group. The final process step, using an hL243-anti-idiotype
affinity gel removed any molecules lacking
C.sub.H1-DDD2-Fab-hL243.
[0389] The skilled artisan will realize that affinity
chromatography may be used to purify DNL.TM. complexes comprising
any combination of effector moieties, so long as ligands for each
of the three effector moieties can be obtained and attached to the
column material. The selected DNL.TM. construct is the one that
binds to each of three columns containing the ligand for each of
the three effector moieties and can be eluted after washing to
remove unbound complexes.
[0390] The following Example is representative of several similar
preparations of 20-C2-2b. Equimolar amounts of
C.sub.H3-AD2-IgG-v-mab (15 mg), C.sub.H1-DDD2-Fab-hL243 (12 mg),
and IFN-a2b-DDD2 (5 mg) were combined in 30-mL reaction volume and
1 mM reduced glutathione was added to the solution. Following 16 h
at room temperature, 2 mM oxidized glutathione was added to the
mixture, which was held at room temperature for an additional 6 h.
The reaction mixture was applied to a 5-mL Protein A affinity
column, which was washed to baseline with PBS and eluted with 0.1 M
Glycine, pH 2.5. The eluate, which contained .about.20 mg protein,
was neutralized with 3 M Tris-HCl, pH 8.6 and dialyzed into
HIS-SELECT.RTM. binding buffer (10 mM imidazole, 300 mM NaCl, 50 mM
NaH.sub.2PO.sub.4, pH 8.0) prior to application to a 5-mL
HIS-SELECT.RTM. IMAC column. The column was washed to baseline with
binding buffer and eluted with 250 mM imidazole, 150 mM NaCl, 50 mM
NaH.sub.2PO.sub.4, pH 8.0.
[0391] The IMAC eluate, which contained .about.11.5 mg of protein,
was applied directly to a WP (anti-hL243) affinity column, which
was washed to baseline with PBS and eluted with 0.1 M glycine, pH
2.5. The process resulted in 7 mg of highly purified 20-C2-2b. This
was approximately 44% of the theoretical yield of 20-C2-2b, which
is 50% of the total starting material (16 mg in this example) with
25% each of 20-2b-2b and 20-C2-C2 produced as side products.
[0392] Generation and Characterization of 20-C2-2b
[0393] The bispecific MAb-IFN.alpha. was generated by combining the
IgG-AD2 module, C.sub.H3-AD2-IgG-v-mab, with two different dimeric
DDD-modules, C.sub.H1-DDD2-Fab-hL243 and IFN.alpha.2b-DDD2. Due to
the random association of either DDD-module with the two AD2
groups, two side-products, 20-C2-C2 and 20-2b-2b are expected to
form, in addition to 20-C2-2b.
[0394] Non-reducing SDS-PAGE (not shown) resolved 20-C2-2b
(.about.305 kDa) as a cluster of bands positioned between those of
20-C2-C2 (.about.365 kDa) and 20-2b-2b (255 kDa). Reducing SDS-PAGE
resolved the five polypeptides (v-mab HC-AD2, hL243 Fd-DDD2,
IFN.alpha.2b-DDD2 and co-migrating v-mab and hL243 kappa light
chains) comprising 20-C2-2b (not shown). IFN.alpha.2b-DDD2 and
hL243 Fd-DDD2 are absent in 20-C2-C2 and 20-2b-2b. MABSELECT.TM.
binds to all three of the major species produced in the DNL.TM.
reaction, but removes any excess IFN.alpha.2b-DDD2 and
C.sub.H1-DDD2-Fab-hL243. The HIS-SELECT.RTM. unbound fraction
contained mostly 20-C2-C2 (not shown). The unbound fraction from WT
affinity chromatography comprised 20-2b-2b (not shown). Each of the
samples was subjected to SE-HPLC and immunoreactivity analyses,
which corroborated the results and conclusions of the SDS-PAGE
analysis.
[0395] Following reduction of 20-C2-2b, its five component
polypeptides were resolved by RP-HPLC and individual ESI-TOF
deconvoluted mass spectra were generated for each peak (not shown).
Native, but not bacterially-expressed recombinant IFN.alpha.2, is
O-glycosylated at Thr-106 (Adolf et al., Biochem J 1991; 276 (Pt
2):511-8). We determined that .about.15% of the polypeptides
comprising the IFN.alpha.2b-DDD2 module are 0-glycosylated and can
be resolved from the non-glycosylated polypeptides by RP-HPLC and
SDS-PAGE (not shown). LC/MS analysis of 20-C2-2b identified both
the 0-glycosylated and non-glycosylated species of
IFN.alpha.2b-DDD2 with mass accuracies of 15 ppm and 2 ppm,
respectively (not shown). The observed mass of the 0-glycosylated
form indicates an O-linked glycan having the structure
NeuGc-NeuGc-Gal-GalNAc, which was also predicted (<1 ppm) for
20-2b-2b (not shown). LC/MS identified both v-mab and hL243 kappa
chains as well as hL243-Fd-DDD2 (not shown) as single, unmodified
species, with observed masses matching the calculated ones (<35
ppm). Two major glycoforms of v-mab HC-AD2 were identified as
having masses of 53,714.73 (70%) and 53,877.33 (30%), indicating
GOF and G1F N-glycans, respectively, which are typically associated
with IgG (not shown). The analysis also confirmed that the amino
terminus of the HC-AD2 is modified to pyroglutamate, as predicted
for polypeptides having an amino terminal glutamine.
[0396] SE-HPLC analysis of 20-C2-2b resolved a predominant protein
peak with a retention time (6.7 min) consistent with its calculated
mass and between those of the larger 20-C2-C2 (6.6 min) and smaller
20-2b-2b (6.85 min), as well as some higher molecular weight peaks
that likely represent non-covalent dimers formed via
self-association of IFN.alpha.2b (not shown).
[0397] Immunoreactivity assays demonstrated the homogeneity of
20-C2-2b with each molecule containing the three functional groups
(not shown). Incubation of 20-C2-2b with an excess of antibodies to
any of the three constituent modules resulted in quantitative
formation of high molecular weight immune complexes and the
disappearance of the 20-C2-2b peak (not shown). The HIS-SELECT.RTM.
and WT affinity unbound fractions were not immunoreactive with WT
and anti-IFN.alpha., respectively (not shown). The MAb-IFN.alpha.
showed similar binding avidity to their parental MAbs (not
shown).
[0398] IFN.alpha. Biological Activity
[0399] The specific activities for various MAb-IFN.alpha. were
measured using a cell-based reporter gene assay and compared to
peginterferon alfa-2b (not shown). Expectedly, the specific
activity of 20-C2-2b (2454 IU/pmol), which has two IFN.alpha.2b
groups, was significantly lower than those of 20-2b-2b (4447
IU/pmol) or 734-2b-2b (3764 IU/pmol), yet greater than
peginterferon alfa-2b (P<0.001) (not shown). The difference
between 20-2b-2b and 734-2b-2b was not significant. The specific
activity among all agents varies minimally when normalized to
IU/pmol of total IFN.alpha.. Based on these data, the specific
activity of each IFN.alpha.2b group of the MAb-IFN.alpha. is
approximately 30% of recombinant IFN.alpha.2b (.about.4000
IU/pmol).
[0400] In the ex-vivo setting, the 20-C2-2b DNL.TM. construct
depleted lymphoma cells more effectively than normal B cells and
had no effect on T cells (not shown). However, it did efficiently
eliminate monocytes (not shown). Where v-mab had no effect on
monocytes, depletion was observed following treatment with hL243a4p
and MAb-IFN.alpha., with 20-2b-2b and 734-2b-2b exhibiting similar
toxicity (not shown). Therefore, the predictably higher potency of
20-C2-2b is attributed to the combined actions of anti-HLA-DR and
IFN.alpha., which may be augmented by HLA-DR targeting. These data
suggest that monocyte depletion may be a pharmacodynamic effect
associated anti-HLA-DR as well as IFN.alpha. therapy; however, this
side affect would likely be transient because the monocyte
population should be repopulated from hematopoietic stem cells.
[0401] The skilled artisan will realize that the approach described
here to produce and use bispecific immunocytokine, or other DNL.TM.
constructs comprising three different effector moieties, may be
utilized with any combinations of antibodies, antibody fragments,
cytokines or other effectors that may be incorporated into a
DNL.TM. construct, for example the combination of anti-CD20 and
anti-CD22 with IFN.alpha.2b.
Example 9
Anti-HIV DNL.TM. Complex
[0402] Among the various antibodies that neutralize HIV-1, the
murine anti-gp120 antibody, P4/D10, is distinguished by its ability
to induce antibody-dependent cellular cytotoxicity (ADCC) to
eliminate infected T cells that express the antigenic gp120 epitope
bound by P4/D10 (Broliden et al., 1990, J Virol 64:936-40).
Enhanced potency was also shown for doxorubicin-conjugated P4/D10
to neutralize and inhibit intercellular spread of HIV infection in
vitro, as well as to protect against HIV-1/MuLV infection in vivo
(Johansson et al., 2006, AIDS 20:1911-15).
[0403] DNL.TM. complexes comprising P4/D10 IgG, or other anti-HIV
antibodies or fragments thereof, along with one or more anti-HIV
agents are prepared. In a preferred embodiment illustrated herein,
the anti-HIV agent is the T20 HIV fusion inhibitor (enfuvirtide,
FUZEON.RTM.) (Asboe, 2004, HIV Clin Trials 5:1-6).
[0404] In a preferred embodiment a novel class of anti-HIV agents
that comprise multiple copies of enfuvirtide (T20) linked to a
chimeric, human or humanized antibody with specificity for HIV-1,
such as P4/D10 are prepared as DNL.TM. complexes. Such conjugates
allow less frequent dosing than with unconjugated T20 to block
entry of HIV-1 into T cells, neutralize free HIV-1 and eliminate
HIV-infected cells. In an exemplary DNL.TM. construct, The
C-terminal end of each light chain of an IgG antibody is attached
via a short linker to an AD2 moiety (SEQ ID NO:4) and expressed as
a fusion protein as described in the Examples above. The T20 HIV
fusion inhibitor is attached to a DDD2 moiety (SEQ ID NO:2) and
also expressed as a fusion protein. Two copies of the DDD2 moiety
spontaneously form a dimer that binds to the AD2 moiety, forming a
DNL.TM. complex comprising one IgG antibody and four copies of
T20.
[0405] The DDD2-T20 amino acid sequence is shown below in SEQ ID
NO:128. The sequence of DDD2 is underlined. This is followed by a
short linker and hinge region and a polyhistidine tag for affinity
purification. The sequence of T20 at the C-terminal end is in bold.
DDD2-T20 has been produced and used to make DNL.TM. complexes, as
described below.
TABLE-US-00029 DDD2-T20 (SEQ ID NO: 128)
MCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARAEFPK
PSTPPGSSGHHHHHHGSYTSLIHSLIEESQNQQEKNEQELLELDKWASLW NWF
[0406] P4/D10 is a murine antibody that may induce human anti-mouse
antibodies (HAMA) when administered to human subjects. Chimeric or
humanized forms of P4/D10 would be more suitable for human
therapeutic use. A chimeric P4/D10 (cP4/D10) was constructed by
substituting human antibody constant region sequences for the
murine constant region sequences of P4/D10. cP4/D10 was prepared
and its binding affinity for gp160 (comprising both gp120 and gp41)
was compared to the murine P4/D10 and a control non-targeting T4-2
(anti-CD4) antibody. The affinity (not shown) and in vitro activity
(not shown) of cP4/D10 were determined to be about two-fold lower
than the parental P4/D10, which is acceptable and may be further
improved by additional subcloning and humanization. Control
non-targeting antibody did not bind to gp160.
[0407] The primary target HIV patient population is individuals
failing HAART therapy, where several doses of the DNL.TM.
conjugates may effectively reduce the number of infected cells and
circulating virions. A secondary patient population is individuals
on effective HAART, with the goal to reach and delete the few
persisting, virus-producing cells.
[0408] The DDD2-T20 is combined with cP4/D10-AD2 to produce a
DNL.TM. construct that is specific for HIV. When the results are
compared on a molar concentration basis for T20, the T20-DDD2
construct is more efficacious than unconjugated T20 and the
*cP4/D1O-T20 DNL.TM. construct is more than an order of magnitude
more efficacious than unconjugated T20. The DNL.TM. construct also
exhibits a significantly greater half-life in vivo, of about 24
hours compared to 2.8 hours for T20. These results show that a
DNL.TM. complex comprising T20 exhibits a substantially longer
half-life in circulation and a substantially higher efficacy than
unconjugated T20.
Example 10
Generation of a DNL Conjugate Comprising Four IFN-.alpha.2b-DDD2
Moieties Linked to C.sub.H3-AD2-IgG
[0409] A DNL complex comprising four IFN-.alpha.2b-DDD2 moieties
linked to C.sub.k-AD2-IgG is made as follows. Briefly, a select
C.sub.k-AD2-IgG is combined with approximately two mole-equivalents
of IFN-.alpha.2b-DDD2 and the mixture is reduced under mild
conditions overnight at room temperature after adding 1 mM EDTA and
2 mM reduced glutathione (GSH). Oxidized glutathione is added to 2
mM and the mixture is held at room temperature for an additional
12-24 hours. The DNL conjugate is purified over a Protein A
affinity column. A DNL conjugate designated *20-2b, comprising four
copies of IFN-.alpha.2b anchored on C.sub.k-AD2-IgG-hA20 (with
specificity for CD20) is prepared. SE-HPLC analyses of *20-2b
generated from mammalian (m) or E. coli (e)-produced
IFN-.alpha.2b-DDD2 each show a major peak having a retention time
consistent with a covalent complex composed of an IgG and 4
IFN-.alpha.2b groups. Similar SE-HPLC profiles are observed for the
other three IFN-IgG conjugates.
[0410] The in vitro IFN.alpha. biological activity of *20-2b is
compared to that of commercial PEGylated IFN.alpha.2 agents,
PEGASYS.RTM. and PEG-INTRON.RTM., using cell-based reporter, viral
protection, and lymphoma proliferation assays. Specific activities
are determined using a cell-based kit, which utilizes a transgenic
human pro-monocyte cell line carrying a reporter gene fused to an
interferon-stimulated response element. The specific activity of
*20-2b is greater than both PEGASYS.RTM. and PEG-INTRON.RTM..
Having four IFN.alpha.2b groups contributes to the enhanced potency
of MAb-IFN.alpha.. When normalized to IFN.alpha. equivalents, the
specific activity/IFN.alpha. is about 10-fold greater than
PEGASYS.RTM. and only about 2-fold less than PEG-INTRON.RTM..
[0411] Comparison of *20-2b, PEGASYS.RTM. and PEG-INTRON.RTM. in an
in vitro viral protection assay demonstrates that *20-2b retains
IFN.alpha.2b antiviral activity with specific activities similar to
PEG-INTRON.RTM. and 10-fold greater than PEGASYS.RTM..
[0412] IFN.alpha.2b can have a direct antiproliferative or
cytotoxic effect on some tumor lines. The activity of *20-2b is
measured in an in vitro proliferation assay with a Burkitt lymphoma
cell line (Daudi) that is highly sensitive to IFN.alpha.. Each of
the IFN.alpha.2 agents efficiently inhibits (>90%) Daudi in
vitro with high potency. However, *20-2b is about 30-fold more
potent than the non-targeting MAb-IFN.alpha. constructs.
[0413] The pharmacokinetic (PK) properties of *20-2b are evaluated
in male Swiss-Webster mice and compared to those of PEGASYS.RTM.,
PEG-INTRON and .alpha.2b-413 (Pegylated IFN made by DNL.TM.).
Concentrations of IFN-.alpha. in the serum samples at various times
are determined by ELISA following the manufacturer's instructions.
Briefly, the serum samples are diluted appropriately according to
the human IFN-.alpha. standard provided in the kit. An antibody
bound to the microtiter plate wells captures interferon. A second
antibody is then used to reveal the bound interferon, which is
quantified by anti-secondary antibody conjugated to horseradish
peroxidase (HRP) following the addition of Tetramethyl benzidine
(TMB). The plates are read at 450 nm. The results of the PK
analysis, which show significantly slower elimination and longer
serum residence of *20-2b compared to the other agents.
Example 11
Generation of DDD Module Based on CD20 Xenoantigen
[0414] The cDNA sequence for murine CD20 is amplified by PCR,
resulting in a sequence in which CD20 xenoantigen is fused at its
C-terminus to a polypeptide consisting of SEQ ID NO:129.
TABLE-US-00030 (SEQ ID NO: 129)
KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEF
AVEYFTRLREARA
[0415] PCR amplification is accomplished using a full length murine
CD20 cDNA clone as a template and selected PCR primers. The PCR
amplimer is cloned into the PGEMT.RTM. vector (PROMEGA.RTM.). A
DDD2-pdHL2 mammalian expression vector is prepared for ligation
with CD20 by digestion with XbaI and Bam HI restriction
endonucleases. The CD20 amplimer is excised from PGEMT.RTM. with
XbaI and Bam HI and ligated into the DDD2-pdHL2 vector to generate
the expression vector CD2O-DDD2-pdHL2.
[0416] CD2O-DDD2-pdHL2 is linearized by digestion with SalI enzyme
and stably transfected into Sp/EEE myeloma cells by electroporation
(see, e.g., U.S. Pat. No. 7,537,930, the Examples section of which
is incorporated herein by reference). Two clones are found to have
detectable levels of CD20 by ELISA. One of the two clones is
adapted to growth in serum-free media without substantial decrease
in productivity. The clone is subsequently amplified with
increasing methotrexate (MTX) concentrations from 0.1 to 0.8 .mu.M
over five weeks. At this stage, it is subcloned by limiting
dilution and the highest producing sub-clone is expanded.
[0417] 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. Approximately 6 mg of
CD20-DDD2 is purified. The purity of CD2O-DDD2 is assessed by
SDS-PAGE under reducing conditions. CD2O-DDD2 is the most heavily
stained band and accounts for approximately 50% of the total
protein.
Example 12
Generation of hLL1 Fab-(CD20).sub.2 by DNL
[0418] A C.sub.k-AD2-IgG-hLL1 (anti-CD74) moiety is produced as
described in Example 1. A DDD2-mCD20 moiety is produced as
described in Example 11. A DNL reaction is performed by the
addition of reduced and lyophilized hLL1 IgG-AD2 to CD2O-DDD2 in
250 mM imidazole, 0.02% Tween 20, 150 mM NaCl, 1 mM EDTA, 50 mM
NaH.sub.2PO.sub.4, pH 7.5. After 6 h at room temperature in the
dark, the reaction mixture is dialyzed against CM Loading Buffer
(150 mM NaCl, 20 mM NaAc, pH 4.5) at 4.degree. C. in the dark. The
solution is loaded onto a 1-mL Hi-Trap CM-FF column
(AMERSHAM.RTM.), which is pre-equilibrated with CM Loading buffer.
After sample loading, the column is washed with CM loading buffer
to baseline, followed by washing with 15 mL of 0.25 M NaCl, 20 mM
NaAc, pH 4.5. The product is eluted with 12.5 mL of 0.5 M NaCl, 20
mM NaAc, pH 4.5. The DNL reaction results in the site-specific and
covalent conjugation of hLL1 IgG with a dimer of mCD20. Both the
IgG and CD20 moieties retain their respective physiological
activities in the DNL construct by cell culture assay.
[0419] The hLL1-mCD20 DNL construct is administered to subjects
with multiple myeloma (MM) and found to induce an immune response
against CD138.sup.negCD20.sup.+ putative MM stem cells. The immune
response is effective to reduce or eliminate MM disease cells in
the subjects.
[0420] 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
130144PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Ser 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 245PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 2Cys 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
317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 421PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 4Cys 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 550PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Ser Leu Arg Glu Cys Glu Leu Tyr Val Gln Lys
His Asn Ile Gln Ala 1 5 10 15 Leu Leu Lys Asp Ser Ile Val Gln Leu
Cys Thr Ala Arg Pro Glu Arg 20 25 30 Pro Met Ala Phe Leu Arg Glu
Tyr Phe Glu Arg Leu Glu Lys Glu Glu 35 40 45 Ala Lys 50
655PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 6Met Ser Cys Gly Gly Ser Leu Arg Glu Cys Glu
Leu Tyr Val Gln Lys 1 5 10 15 His Asn Ile Gln Ala Leu Leu Lys Asp
Ser Ile Val Gln Leu Cys Thr 20 25 30 Ala Arg Pro Glu Arg Pro Met
Ala Phe Leu Arg Glu Tyr Phe Glu Arg 35 40 45 Leu Glu Lys Glu Glu
Ala Lys 50 55 723PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 7Cys Gly Phe Glu Glu Leu Ala Trp Lys Ile
Ala Lys Met Ile Trp Ser 1 5 10 15 Asp Val Phe Gln Gln Gly Cys 20
851PRTHomo sapiens 8Ser Leu Arg Glu Cys Glu Leu Tyr Val Gln Lys His
Asn Ile Gln Ala 1 5 10 15 Leu Leu Lys Asp Val Ser Ile Val Gln Leu
Cys Thr Ala Arg Pro Glu 20 25 30 Arg Pro Met Ala Phe Leu Arg Glu
Tyr Phe Glu Lys Leu Glu Lys Glu 35 40 45 Glu Ala Lys 50 954PRTHomo
sapiens 9Ser Leu Lys Gly Cys Glu Leu Tyr Val Gln Leu His Gly Ile
Gln Gln 1 5 10 15 Val Leu Lys Asp Cys Ile Val His Leu Cys Ile Ser
Lys Pro Glu Arg 20 25 30 Pro Met Lys Phe Leu Arg Glu His Phe Glu
Lys Leu Glu Lys Glu Glu 35 40 45 Asn Arg Gln Ile Leu Ala 50
1044PRTHomo 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 Gly Gln Gln Pro Pro
Asp Leu Val Asp Phe Ala Val 20 25 30 Glu Tyr Phe Thr Arg Leu Arg
Glu Ala Arg Arg Gln 35 40 1144PRTHomo sapiens 11Ser Ile Glu Ile Pro
Ala Gly Leu Thr Glu Leu Leu Gln Gly Phe Thr 1 5 10 15 Val Glu Val
Leu Arg His Gln Pro Ala Asp Leu Leu Glu Phe Ala Leu 20 25 30 Gln
His Phe Thr Arg Leu Gln Gln Glu Asn Glu Arg 35 40 1244PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
12Thr 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
1344PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Ser Lys 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 1444PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 14Ser Arg 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 1544PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Ser His Ile Asn 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
1644PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 16Ser His Ile Gln Ile Pro Pro Ala 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 1744PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 17Ser His Ile Gln Ile Pro
Pro Gly Leu Ser 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 1844PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
18Ser His Ile Gln Ile Pro Pro Gly Leu Thr Asp 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
1944PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 19Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Asn 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 2044PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 20Ser His Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Ala 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 2144PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
21Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1
5 10 15 Ser 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
2244PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 22Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Asp 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 2344PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 23Ser His Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val
Leu Lys 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 2444PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
24Ser 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 Asn 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
2544PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 25Ser 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 Asn 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 2644PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 26Ser 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 Glu Leu Val Glu Phe Ala 20 25 30 Val Glu
Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40 2744PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
27Ser 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 Asp Phe
Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40
2844PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 28Ser 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 Leu 20 25 30 Val Glu Tyr Phe Thr Arg Leu
Arg Glu Ala Arg Ala 35 40 2944PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 29Ser 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 Ile 20 25 30 Val Glu
Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40 3044PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
30Ser 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
Val 20 25 30 Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40
3144PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 31Ser 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 Asp Tyr Phe Thr Arg Leu
Arg Glu Ala Arg Ala 35 40 3217PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 32Asn Ile Glu Tyr Leu Ala Lys
Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala 3317PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 33Gln
Leu Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10
15 Ala 3417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Gln Val Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 3517PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 35Gln Ile Asp Tyr Leu Ala
Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala
3617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Gln Ile Glu Phe Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 3717PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 37Gln Ile Glu Thr Leu Ala
Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala
3817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 38Gln Ile Glu Ser Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 3917PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 39Gln Ile Glu Tyr Ile Ala
Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala
4017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Gln Ile Glu Tyr Val Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 4117PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 41Gln Ile Glu Tyr Leu Ala
Arg Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala
4217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Gln Ile Glu Tyr Leu Ala Lys Asn Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 4317PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 43Gln Ile Glu Tyr Leu Ala
Lys Gln Ile Val Glu Asn Ala Ile Gln Gln 1 5 10 15 Ala
4417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Gln
Ala Ile Gln Gln 1 5 10 15 Ala 4517PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 45Gln Ile Glu Tyr Leu Ala
Lys Gln Ile Val Asp Asn Ala Ile Asn Gln 1 5 10 15 Ala
4617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Asn 1 5 10 15 Ala 4717PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 47Gln Ile Glu Tyr Leu Ala
Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Leu
4817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ile 4917PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 49Gln Ile Glu Tyr Leu Ala
Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Val
5017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Gln Ile Glu Tyr Val Ala Lys Gln Ile Val Asp Tyr
Ala Ile His Gln 1 5 10 15 Ala 5117PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 51Gln Ile Glu Tyr Lys Ala
Lys Gln Ile Val Asp His Ala Ile His Gln 1 5 10 15 Ala
5217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Gln Ile Glu Tyr His Ala Lys Gln Ile Val Asp His
Ala Ile His Gln 1 5 10 15 Ala 5317PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 53Gln Ile Glu Tyr Val Ala
Lys Gln Ile Val Asp His Ala Ile His Gln 1 5 10 15 Ala
5418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Pro Leu Glu Tyr Gln Ala Gly Leu Leu Val Gln Asn
Ala Ile Gln Gln 1 5 10 15 Ala Ile 5518PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 55Leu
Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn Ala Ile Gln Leu 1 5
10
15 Ser Ile 5618PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 56Leu Ile Glu Glu Ala Ala Ser Arg Ile
Val Asp Ala Val Ile Glu Gln 1 5 10 15 Val Lys 5718PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 57Ala
Leu Tyr Gln Phe Ala Asp Arg Phe Ser Glu Leu Val Ile Ser Glu 1 5 10
15 Ala Leu 5817PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 58Leu Glu Gln Val Ala Asn Gln Leu Ala
Asp Gln Ile Ile Lys Glu Ala 1 5 10 15 Thr 5917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 59Phe
Glu Glu Leu Ala Trp Lys Ile Ala Lys Met Ile Trp Ser Asp Val 1 5 10
15 Phe 6018PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn
Ala Val Leu Lys 1 5 10 15 Ala Val 6118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 61Thr
Ala Glu Glu Val Ser Ala Arg Ile Val Gln Val Val Thr Ala Glu 1 5 10
15 Ala Val 6218PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 62Gln Ile Lys Gln Ala Ala Phe Gln Leu
Ile Ser Gln Val Ile Leu Glu 1 5 10 15 Ala Thr 6316PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 63Leu
Ala Trp Lys Ile Ala Lys Met Ile Val Ser Asp Val Met Gln Gln 1 5 10
15 6424PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 64Asp 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
6518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Leu Glu Gln Tyr Ala Asn Gln Leu Ala Asp Gln Ile
Ile Lys Glu Ala 1 5 10 15 Thr Glu 6620PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 66Phe
Glu Glu Leu Ala Trp Lys Ile Ala Lys Met Ile Trp Ser Asp Val 1 5 10
15 Phe Gln Gln Cys 20 6717PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 67Gln Ile Glu Tyr Leu Ala Lys
Gln Ile Pro Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala 6825PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 68Lys
Gly Ala Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp Ala 1 5 10
15 Val Ile Glu Gln Val Lys Ala Ala Gly 20 25 6925PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 69Lys
Gly Ala Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Pro Asp Ala 1 5 10
15 Pro Ile Glu Gln Val Lys Ala Ala Gly 20 25 7025PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 70Pro
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 7125PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 71Pro
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 7225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 72Pro
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 7325PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 73Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Pro Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 7425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 74Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Pro Glu Asn 1 5 10
15 Ala Pro Leu Lys Ala Val Gln Gln Tyr 20 25 7525PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 75Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn 1 5 10
15 Ala Val Glu Lys Ala Val Gln Gln Tyr 20 25 7625PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 76Glu
Glu Gly Leu Asp Arg Asn Glu Glu Ile Lys Arg Ala Ala Phe Gln 1 5 10
15 Ile Ile Ser Gln Val Ile Ser Glu Ala 20 25 7725PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 77Leu
Val Asp Asp Pro Leu Glu Tyr Gln Ala Gly Leu Leu Val Gln Asn 1 5 10
15 Ala Ile Gln Gln Ala Ile Ala Glu Gln 20 25 7825PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 78Gln
Tyr Glu Thr Leu Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn 1 5 10
15 Ala Ile Gln Leu Ser Ile Glu Gln Leu 20 25 7925PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 79Leu
Glu Lys Gln Tyr Gln Glu Gln Leu Glu Glu Glu Val Ala Lys Val 1 5 10
15 Ile Val Ser Met Ser Ile Ala Phe Ala 20 25 8025PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 80Asn
Thr Asp Glu Ala Gln Glu Glu Leu Ala Trp Lys Ile Ala Lys Met 1 5 10
15 Ile Val Ser Asp Ile Met Gln Gln Ala 20 25 8125PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 81Val
Asn Leu Asp Lys Lys Ala Val Leu Ala Glu Lys Ile Val Ala Glu 1 5 10
15 Ala Ile Glu Lys Ala Glu Arg Glu Leu 20 25 8225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 82Asn
Gly Ile Leu Glu Leu Glu Thr Lys Ser Ser Lys Leu Val Gln Asn 1 5 10
15 Ile Ile Gln Thr Ala Val Asp Gln Phe 20 25 8325PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 83Thr
Gln Asp Lys Asn Tyr Glu Asp Glu Leu Thr Gln Val Ala Leu Ala 1 5 10
15 Leu Val Glu Asp Val Ile Asn Tyr Ala 20 25 8425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 84Glu
Thr Ser Ala Lys Asp Asn Ile Asn Ile Glu Glu Ala Ala Arg Phe 1 5 10
15 Leu Val Glu Lys Ile Leu Val Asn His 20 25 85330PRTHomo sapiens
85Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1
5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Ala Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 86330PRTHomo sapiens 86Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
8744PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 87Xaa Xaa Ile Xaa Ile Pro Pro Xaa Leu Xaa Xaa
Leu Leu Xaa Xaa Tyr 1 5 10 15 Xaa Val Xaa Val Leu Xaa Xaa Xaa Pro
Pro Xaa Leu Val Xaa Phe Xaa 20 25 30 Val Xaa Tyr Phe Xaa Xaa Leu
Xaa Xaa Xaa Xaa Xaa 35 40 8817PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 88Xaa Xaa Xaa Xaa Xaa Ala Xaa
Xaa Ile Val Xaa Xaa Ala Ile Xaa Xaa 1 5 10 15 Xaa 8944PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
89Xaa His Ile Xaa Ile Pro Pro Gly Leu Xaa Glu Leu Leu Gln Gly Tyr 1
5 10 15 Thr Xaa Glu Val Leu Arg Xaa Gln Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30 Xaa Xaa Tyr Phe Xaa Xaa Leu Xaa Glu Xaa Arg Xaa 35 40
9021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 90aatgcggcgg tggtgacagt a
219121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 91aagctcagca cacagaaaga c
219221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 92uaaaaucuuc cugcccacct t
219321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 93ggaagcuguu ggcugaaaat t
219421RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 94aagaccagcc ucuuugccca g
219519RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 95ggaccaggca gaaaacgag
199617RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 96cuaucaggau gacgcgg 179721RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 97ugacacaggc aggcuugacu u 219819DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 98ggtgaagaag ggcgtccaa 199960DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 99gatccgttgg agctgttggc gtagttcaag agactcgcca
acagctccaa cttttggaaa 6010020DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 100aggtggtgtt
aacagcagag 2010121DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 101aaggtggagc aagcggtgga g
2110221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 102aaggagttga aggccgacaa a
2110321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 103uauggagcug cagaggaugt t
2110449DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 104tttgaatatc tgtgctgaga acacagttct
cagcacagat attcttttt 4910529DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 105aatgagaaaa
gcaaaaggtg ccctgtctc 2910621RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 106aaucaucauc
aagaaagggc a 2110721DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 107augacuguca ggauguugct t
2110821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 108gaacgaaucc ugaagacauc u
2110929DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 109aagcctggct acagcaatat gcctgtctc
2911021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 110ugaccaucac cgaguuuaut t
2111121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 111aagtcggacg caacagagaa a
2111221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 112cuaccuuucu acggacgugt t
2111321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 113ctgcctaagg
cggatttgaa t 2111421DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 114ttauuccuuc uucgggaagu c
2111521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 115aaccttctgg aacccgccca c
2111619DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 116gagcatcttc gagcaagaa
1911719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 117catgtggcac cgtttgcct
1911821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 118aactaccaga aaggtatacc t
2111921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 119ucacaguguc cuuuauguat t
2112021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 120gcaugaaccg gaggcccaut t
2112119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 121ccggacagtt ccatgtata
1912216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 122Glu Phe Pro Lys Pro Ser Thr Pro Pro Gly Ser
Ser Gly Gly Ala Pro 1 5 10 15 1234PRTHomo sapiens 123Pro Lys Ser
Cys 1 12455PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 124Gly 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 12529PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 125Gly 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 12622PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 126Arg
Ser Gln Ser Arg Ser Arg Tyr Tyr Arg Gln Arg Gln Arg Ser Arg 1 5 10
15 Arg Arg Arg Arg Arg Ser 20 127165PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
127Pro Gln Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg
1 5 10 15 Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His
Cys Lys 20 25 30 Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro
Val Lys Ala Ile 35 40 45 Cys Lys Gly Ile Ile Ala Ser Lys Asn Val
Leu Thr Thr Ser Glu Phe 50 55 60 Tyr Leu Ser Asp Cys Asn Val Thr
Ser Arg Pro Cys Lys Tyr Lys Leu 65 70 75 80 Lys Lys Ser Thr Asn Lys
Phe Cys Val Thr Cys Glu Asn Gln Ala Pro 85 90 95 Val His Phe Val
Gly Val Gly Ser Cys Gly Gly Gly Gly Ser Leu Glu 100 105 110 Cys Gly
His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly 115 120 125
Tyr Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe 130
135 140 Ala Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala Val Glu
His 145 150 155 160 His His His His His 165 128103PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
128Met Cys Gly His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln
1 5 10 15 Gly Tyr Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu
Val Glu 20 25 30 Phe Ala Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala
Arg Ala Glu Phe 35 40 45 Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser
Gly His His His His His 50 55 60 His Gly Ser Tyr Thr Ser Leu Ile
His Ser Leu Ile Glu Glu Ser Gln 65 70 75 80 Asn Gln Gln Glu Lys Asn
Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp 85 90 95 Ala Ser Leu Trp
Asn Trp Phe 100 12963PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 129Lys Ser His His His
His His His Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Cys
Gly His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu 20 25 30 Gln
Gly Tyr Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val 35 40
45 Glu Phe Ala Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 50
55 60 1302208DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 130ggatcccgca attctaaact
ctgagggggt cggatgacgt ggccattctt tgcctaaagc 60attgagttta ctgcaaggtc
agaaaagcat gcaaagccct cagaatggct gcaaagagct 120ccaacaaaac
aatttagaac tttattaagg aataggggga agctaggaag aaactcaaaa
180catcaagatt ttaaatacgc ttcttggtct ccttgctata attatctggg
ataagcatgc 240tgttttctgt ctgtccctaa catgccctgt gattatccgc
aaacaacaca cccaagggca 300gaactttgtt acttaaacac catcctgttt
gcttctttcc tcaggaactg tggctgcacc 360atctgtcttc atcttcccgc
catctgatga gcagttgaaa tctggaactg cctctgttgt 420gtgcctgctg
aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc
480cctccaatcg ggtaactccc aggagagtgt cacagagcag gacagcaagg
acagcaccta 540cagcctcagc agcaccctga cgctgagcaa agcagactac
gagaaacaca aagtctacgc 600ctgcgaagtc acccatcagg gcctgagctc
gcccgtcaca aagagcttca acaggggaga 660gtgtgagttc cctaaaccca
gcactccacc cggatcttcc ggcggcgctc cctgtggcca 720gatcgagtac
ctggccaagc agatcgtgga caacgccatc cagcaggccg ggtgctagag
780ggagaagtgc ccccacctgc tcctcagttc cagcctgacc ccctcccatc
ctttggcctc 840tgaccctttt tccacagggg acctacccct attgcggtcc
tccagctcat ctttcacctc 900acccccctcc tcctccttgg ctttaattat
gctaatgttg gaggagaatg aataaataaa 960gtgaatcttt gcacctgtgg
tttctctctt tcctcattta ataattatta tctgttgttt 1020taccaactac
tcaatttctc ttataaggga ctaaatatgt agtcatccta aggcgcataa
1080ccatttataa aaatcatcct tcattctatt ttaccctatc atcctctgca
agacagtcct 1140ccctcaaacc cacaagcctt ctgtcctcac agtcccctgg
gccatggtag gagagacttg 1200cttccttgtt ttcccctcct cagcaagccc
tcatagtcct ttttaagggt gacaggtctt 1260acagtcatat atcctttgat
tcaattccct gagaatcaac caaagcaaat ttttcaaaag 1320aagaaacctg
ctataaagag aatcattcat tgcaacatga tataaaataa caacacaata
1380aaagcaatta aataaacaaa caatagggaa atgtttaagt tcatcatggt
acttagactt 1440aatggaatgt catgccttat ttacattttt aaacaggtac
tgagggactc ctgtctgcca 1500agggccgtat tgagtacttt ccacaaccta
atttaatcca cactatactg tgagattaaa 1560aacattcatt aaaatgttgc
aaaggttcta taaagctgag agacaaatat attctataac 1620tcagcaattc
ccacttctag gggttcgact ggcaggaagc aggtcatgtg gcaaggctat
1680ttggggaagg gaaaataaaa ccactaggta aacttgtagc tgtggtttga
agaagtggtt 1740ttgaaacact ctgtccagcc ccaccaaacc gaaagtccag
gctgagcaaa acaccacctg 1800ggtaatttgc atttctaaaa taagttgagg
attcagccga aactggagag gtcctctttt 1860aacttattga gttcaacctt
ttaattttag cttgagtagt tctagtttcc ccaaacttaa 1920gtttatcgac
ttctaaaatg tatttagaat ttcgaccaat tctcatgttt gacagcttat
1980catcgctgca ctccgcccga aaagtgcgct cggctctgcc aaggacgcgg
ggcgcgtgac 2040tatgcgtggg ctggagcaac cgcctgctgg gtgcaaaccc
tttgcgcccg gactcgtcca 2100acgactataa agagggcagg ctgtcctcta
agcgtcacca cgacttcaac gtcctgagta 2160ccttctcctc acttactccg
tagctccagc ttcaccagat ccctcgag 2208
* * * * *