U.S. patent application number 15/297384 was filed with the patent office on 2017-12-14 for calicheamicin-antibody-drug conjugates and methods of use.
The applicant listed for this patent is Genentech, Inc.. Invention is credited to Thomas Pillow.
Application Number | 20170355725 15/297384 |
Document ID | / |
Family ID | 57218959 |
Filed Date | 2017-12-14 |
United States Patent
Application |
20170355725 |
Kind Code |
A9 |
Pillow; Thomas |
December 14, 2017 |
CALICHEAMICIN-ANTIBODY-DRUG CONJUGATES AND METHODS OF USE
Abstract
The invention relates generally to a calicheamicin molecule
activated with a leaving group. The invention further relates
generally to an antibody-drug conjugate comprising an antibody
directly conjugated by a disulfide to one or more calicheamicin
molecules.
Inventors: |
Pillow; Thomas; (South San
Francisco, CA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
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Prior
Publication: |
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Document Identifier |
Publication Date |
|
US 20170107243 A1 |
April 20, 2017 |
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Family ID: |
57218959 |
Appl. No.: |
15/297384 |
Filed: |
October 19, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62243967 |
Oct 20, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6807 20170801;
A61P 35/00 20180101; C07H 15/207 20130101; A61K 47/545 20170801;
A61K 39/39558 20130101; A61K 47/6809 20170801 |
International
Class: |
C07H 15/207 20060101
C07H015/207; A61K 39/395 20060101 A61K039/395 |
Claims
1. A drug intermediate composition of Formula I or Formula II:
##STR00009## wherein R is selected from H, --C(O)R.sup.1,
--C(O)NR.sup.1R.sup.2, --S(O).sub.2R.sup.1, and
--S(O)2NR.sup.2R.sup.1; R.sup.1 and R.sup.2 are independently
selected from C.sub.1-C.sub.6 alkyl and C.sub.6-C.sub.20 aryl;
R.sup.3 is selected from NO.sub.2, Cl, F, CN, CO.sub.2H, and Br;
and q is 0, 1, or 2.
2. The drug intermediate composition of claim 1 wherein R is
--C(O)CH.sub.3.
3. The drug intermediate composition of claim 1 wherein R.sup.3 is
NO.sub.2 and q is 1.
4. The drug intermediate composition of claim 3 having Formula Ia:
##STR00010##
5. The drug intermediate composition of claim 1 having Formula IIa:
##STR00011##
6. An antibody-drug conjugate compound having Formula III:
##STR00012## or a pharmaceutically acceptable salt thereof, wherein
R is selected from H, --C(O)R.sup.1, --C(O)NR.sup.1R.sup.2,
--S(O).sub.2R.sup.1, and --S(O).sub.2NR.sup.2R.sup.1; R.sup.1 and
R.sup.2 are independently selected from C.sub.1-C.sub.6 alkyl and
C.sub.6-C.sub.20 aryl; p is an integer from 1 to 8; and Ab is an
antibody which binds to one or more tumor-associated antigens or
cell-surface receptors selected from (1)-(53): (1) BMPR1B (bone
morphogenetic protein receptor-type IB); (2) E16 (LAT1, SLC7A5);
(3) STEAP1 (six transmembrane epithelial antigen of prostate); (4)
MUC16 (0772P, CA125); (5) MPF (MPF, MSLN, SMR, megakaryocyte
potentiating factor, mesothelin); (6) Napi2b (NAPI-3B, NPTIIb,
SLC34A2, solute carrier family 34 (sodium phosphate), member 2,
type II sodium-dependent phosphate transporter 3b); (7) Sema 5b
(FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog,
sema domain, seven thrombospondin repeats (type 1 and type 1-like),
transmembrane domain (TM) and short cytoplasmic domain,
(semaphorin) 5B); (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN
cDNA 2700050C12, RIKEN cDNA 2700050C12 gene); (9) ETBR (Endothelin
type B receptor); (10) MSG783 (RNF124, hypothetical protein
FLJ20315); (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2,
STMP, prostate cancer associated gene 1, prostate cancer associated
protein 1, six transmembrane epithelial antigen of prostate 2, six
transmembrane prostate protein); (12) TrpM4 (BR22450, FLJ20041,
TRPM4, TRPM4B, transient receptor potential cation channel,
subfamily M, member 4); (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,
teratocarcinoma-derived growth factor); (14) CD21 (CR2 (Complement
receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs 73792);
(15) CD79b (CD79B, CD79.beta., IGb (immunoglobulin-associated
beta), B29); (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain
containing phosphatase anchor protein 1a), SPAP1B, SPAP1C); (17)
HER2; (18) NCA; (19) MDP; (20) IL20R.alpha.; (21) Brevican; (22)
EphB2R; (23) ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R (B
cell-activating factor receptor, BLyS receptor 3, BR3); (27) CD22
(B-cell receptor CD22-B isoform); (28) CD79a (CD79A, CD79.alpha.,
immunoglobulin-associated alpha); (29) CXCR5 (Burkitt's lymphoma
receptor 1); (30) HLA-DOB (Beta subunit of MHC class II molecule
(Ia antigen)); (31) P2X5 (Purinergic receptor P2X ligand-gated ion
channel 5); (32) CD72 (B-cell differentiation antigen CD72, Lyb-2);
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein
of the leucine rich repeat (LRR) family); (34) FcRH1 (Fc
receptor-like protein 1); (35) FcRH5 (IRTA2, Immunoglobulin
superfamily receptor translocation associated 2); (36) TENB2
(putative transmembrane proteoglycan); (37) PMEL17 (silver homolog;
SILV; D12S53E; PMEL17; SI; SIL); (38) TMEFF1 (transmembrane protein
with EGF-like and two follistatin-like domains 1; Tomoregulin-1);
(39) GDNF-Ra1 (GDNF family receptor alpha 1, GFRA1; GDNFR; GDNFRA;
RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); (40) Ly6E
(lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1);
(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); (42) Ly6G6D
(lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1); (43) LGR5
(leucine-rich repeat-containing G protein-coupled receptor 5;
GPR49, GPR67); (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B;
MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE1); (45) LY6K
(lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226);
(46) GPR19 (G protein-coupled receptor 19; Mm.4787); (47) GPR54
(KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12); (48) ASPHD1
(aspartate beta-hydroxylase domain containing 1; LOC253982); (49)
Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3); (50) TMEM118
(ring finger protein, transmembrane 2; RNFT2; FLJ14627); (51)
GPR172A (G protein-coupled receptor 172A, GPCR41; FLJ11856;
D15Ertd747e); (52) CD33; and (53) CLL-1.
7. The antibody-drug conjugate compound according to claim 6,
wherein Ab is a cysteine-engineered antibody.
8. The antibody-drug conjugate compound according to claim 7,
wherein the cysteine-engineered antibody is a mutant selected from
LC K149C, HC A140, HC A118C, and HC L177C.
9. The antibody-drug conjugate compound according to claim 6,
wherein Ab is selected from anti-HER2 4D5, anti-CD22, anti-CD33,
anti-Ly6E, anti-Napi3b, anti-HER2 7C2, and anti-CLL-1.
10. The antibody-drug conjugate compound according to claim 6,
wherein p is 1, 2, 3, or 4.
11. The antibody-drug conjugate compound according to claim 6,
comprising a mixture of the antibody-drug conjugate compounds,
wherein the average drug loading per antibody in the mixture of
antibody-drug conjugate compounds is about 2 to about 5.
12. A pharmaceutical composition comprising the antibody-drug
conjugate compound according to claim 6 and a pharmaceutically
acceptable diluent, carrier or excipient.
13. The pharmaceutical composition of claim 12, further comprising
a therapeutically effective amount of a chemotherapeutic agent.
14. Use of an antibody-drug conjugate compound according to claim 6
in the manufacture of a medicament for the treatment of cancer in a
mammal.
15. A method of treating cancer comprising administering to a
patient the pharmaceutical composition of claim 14.
16. The method of claim 15 wherein the patient is administered a
chemotherapeutic agent, in combination with the antibody-drug
conjugate.
17. An antibody-drug conjugate compound according to claim 6 for
use in a method for treating cancer.
18. A method of making an antibody-drug conjugate compound of claim
6, the method comprising (a) reacting an antibody which binds to
one or more tumor-associated antigens or cell-surface receptors
selected from (1)-(53): (1) BMPR1B (bone morphogenetic protein
receptor-type IB); (2) E16 (LAT1, SLC7A5); (3) STEAP1 (six
transmembrane epithelial antigen of prostate); (4) MUC16 (0772P,
CA125); (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,
mesothelin); (6) Napi2b (NAPI-3B, NPTIIb, SLC34A2, solute carrier
family 34 (sodium phosphate), member 2, type II sodium-dependent
phosphate transporter 3b); (7) Sema 5b (FLJ10372, KIAA1445,
Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven
thrombospondin repeats (type 1 and type 1-like), transmembrane
domain (TM) and short cytoplasmic domain, (semaphorin) 5B); (8)
PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12,
RIKEN cDNA 2700050C12 gene); (9) ETBR (Endothelin type B receptor);
(10) MSG783 (RNF124, hypothetical protein FLJ20315); (11) STEAP2
(HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer
associated gene 1, prostate cancer associated protein 1, six
transmembrane epithelial antigen of prostate 2, six transmembrane
prostate protein); (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B,
transient receptor potential cation channel, subfamily M, member
4); (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,
teratocarcinoma-derived growth factor); (14) CD21 (CR2 (Complement
receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs 73792);
(15) CD79b (CD79B, CD79.beta., IGb (immunoglobulin-associated
beta), B29); (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain
containing phosphatase anchor protein 1a), SPAP1B, SPAP1C); (17)
HER2; (18) NCA; (19) MDP; (20) IL20R.alpha.; (21) Brevican; (22)
EphB2R; (23) ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R (B
cell-activating factor receptor, BLyS receptor 3, BR3); (27) CD22
(B-cell receptor CD22-B isoform); (28) CD79a (CD79A, CD79.alpha.,
immunoglobulin-associated alpha); (29) CXCR5 (Burkitt's lymphoma
receptor 1); (30) HLA-DOB (Beta subunit of MHC class II molecule
(Ia antigen)); (31) P2X5 (Purinergic receptor P2X ligand-gated ion
channel 5); (32) CD72 (B-cell differentiation antigen CD72, Lyb-2);
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein
of the leucine rich repeat (LRR) family); (34) FcRH1 (Fc
receptor-like protein 1); (35) FcRH5 (IRTA2, Immunoglobulin
superfamily receptor translocation associated 2); (36) TENB2
(putative transmembrane proteoglycan); (37) PMEL17 (silver homolog;
SILV; D12S53E; PMEL17; SI; SIL); (38) TMEFF1 (transmembrane protein
with EGF-like and two follistatin-like domains 1; Tomoregulin-1);
(39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA;
RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); (40) Ly6E
(lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1);
(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); (42) Ly6G6D
(lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1); (43) LGR5
(leucine-rich repeat-containing G protein-coupled receptor 5;
GPR49, GPR67); (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B;
MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE1); (45) LY6K
(lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226);
(46) GPR19 (G protein-coupled receptor 19; Mm.4787); (47) GPR54
(KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12); (48) ASPHD1
(aspartate beta-hydroxylase domain containing 1; LOC253982); (49)
Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3); (50) TMEM118
(ring finger protein, transmembrane 2; RNFT2; FLJ14627); (51)
GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856;
D15Ertd747e); (52) CD33; and (53) CLL-1 (b) with a drug
intermediate of Formula I or Formula II ##STR00013## wherein: R is
selected from H, --C(O)R.sup.1, --C(O)NR.sup.1R.sup.2,
--S(O).sub.2R.sup.1, and --S(O)2NR.sup.2R.sup.1; R.sup.1 and
R.sup.2 are independently selected from C.sub.1-C.sub.6 alkyl and
C.sub.6-C.sub.20 aryl; R.sup.3 is selected from NO.sub.2, Cl, F,
CN, CO.sub.2H, and Br; and q is 0, 1, or 2.
19. The method according to claim 18, wherein Ab is a
cysteine-engineered antibody.
20. The method according to claim 18, wherein the
cysteine-engineered antibody is a mutant selected from LC K149C, HC
A140, HC A118C, and HC L177C.
21. The method according to claim 18, wherein Ab is selected from
anti-HER2 4D5, anti-CD22, anti-CD33, anti-Ly6E, anti-Napi3b,
anti-HER2 7C2, and anti-CLL-1.
22. The method according to claim 18, wherein p is 1, 2, 3, or
4.
23. The method according to claim 18, comprising a mixture of the
antibody-drug conjugate compounds, wherein the average drug loading
per antibody in the mixture of antibody-drug conjugate compounds is
about 2 to about 5.24.
24. An article of manufacture comprising a pharmaceutical
composition of claim 12, a container, and a package insert or label
indicating that the pharmaceutical composition can be used to treat
cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority benefit of U.S. provisional
application Ser. No. 62/243,967 filed on Oct. 20, 2015, which is
incorporated herein in its entirety.
FIELD OF THE INVENTION
[0002] The field of the invention relates generally to an
antibody-drug conjugate comprising an antibody directly conjugated
to one or more calicheamicin molecules.
BACKGROUND
[0003] Calicheamicin and calicheamicin derivatives refer to a
family of antibacterial and antitumor agents, as described, for
instance, in U.S. Pat. No. 4,970,198 (which is incorporated herein
in its entirety). Calicheamicin derivatives within the scope of the
disclosure include, without limitation, dihydro derivatives as
described in U.S. Pat. No. 5,037,651 and N-acylated derivatives as
described in U.S. Pat. No. 5,079,233 (both of which are
incorporated in their entirety herein). As used herein, a
calicheamicin derivative refers to calicheamicin that has been
substituted at one or more positions to obtain a different
compound.
[0004] The calicheamicin family of antibiotics, and derivatives and
analogs thereof, are capable of producing double-stranded DNA
breaks at sub-picomolar concentrations (Hinman et al., (1993)
Cancer Research 53:3336-3342; Angew Chem. Intl. Ed. Engl. (1994)
33:183-186; Lode et al., (1998) Cancer Research 58:2925-2928).
Calicheamicin comprises a warhead comprising an enediyne ring
structure (a ring comprising a double bond flanked by triple bonds)
and a methyl trisulfide (i.e., --S--S--S--CH.sub.3) group. It is
believed that the warhead is activated by reduction of a disulfide
bond, and that the activated warhead functions by causing breaks in
double-stranded DNA. A mechanism of action was proposed by
Bouchard, H., et al., Ab-drug conjugates-A new wave of cancer
drugs, Bioorganic & Medicinal Chemistry Letters 24 (2014)
5357-5363 where the enediyne ring is activated by reductive
cleavage of the disulfide bond by the steps: (i) formation of a
calicheamicin=CHCH.sub.2SH moiety by nucleophilic attack of the
methyl trisulfide moiety and cleavage of CH.sub.3--S--S--, (ii)
formation of a fused 2,5-dihydrothiophene ring from
calicheamicin=CHCH.sub.2SH, and (iii) formation of a fused benzene
free di-radical from the enediyne. Activated calicheamicin then
cleaves double stranded DNA.
[0005] Calicheamicin has intracellular sites of action, but, in
some instances, does not effectively cross the plasma membrane.
Therefore, cellular uptake of these agents through
antibody-mediated internalization may, in some embodiments, greatly
enhance cytotoxic effect. It is known that
calicheamicin-linker-antibody conjugates provide for the
specificity and effective plasma membrane permeability
(internalization) of the antibody in combination with the cytotoxic
potency of calicheamicin. Therefore, cellular uptake of
calicheamicin may, in some aspects, greatly enhance its cytotoxic
effect. Methods of forming calicheamicin-linker-antibody drug
conjugates are known and described, for example, in U.S. Pat. Nos.
5,877,296, 5,773,001, 5,712,374, 5,714,586, 5,739,116 and 5,767,285
(each of which is incorporated by reference herein).
[0006] Antibody-drug conjugates, comprising an antibody-linker-drug
conjugate, are attractive targeted chemo-therapeutic molecules, as
they combine ideal properties of both antibodies and cytotoxic
drugs by targeting potent cytotoxic drugs to the antigen-expressing
tumor cells, thereby enhancing their anti-tumor activity.
Successful antibody-drug conjugate development for a given target
antigen depends on optimization of antibody selection, linker
stability, cytotoxic drug potency and mode of linker-drug
conjugation to the antibody. More particularly, effective
antibody-drug conjugates are characterized by at least one or more
of the following: (i) an antibody-drug conjugate formation method
wherein the antibody retains sufficient specificity to target
antigens and wherein the drug efficacy is maintained; (ii)
antibody-drug conjugate stability sufficient to limit drug release
in the blood and concomitant damage to non-targeted cells; (iii)
sufficient cell membrane transport efficiency (endocytosis) to
achieve a therapeutic intracellular antibody-drug conjugate
concentration; (iv) sufficient intracellular drug release from the
antibody-drug conjugate sufficient to achieve a therapeutic drug
concentration; and (v) drug cytotoxicity in nanomolar or
sub-nanomolar amounts.
[0007] Conventional means of attaching, i.e., covalent bonding of a
drug moiety to an antibody via a linker, generally leads to a
heterogeneous mixture of molecules where the drug moieties are
attached at a number of sites on the antibody. For example,
cytotoxic drugs have typically been conjugated to antibodies
through the often-numerous lysine residues of an antibody,
generating a heterogeneous antibody-drug conjugate mixture.
Depending on reaction conditions, the heterogeneous mixture
typically contains a distribution of antibodies with from 0 to
about 8, or more, attached drug moieties. In addition, within each
subgroup of conjugates with a particular integer ratio of drug
moieties to a single antibody, there is a potentially heterogeneous
mixture where the drug moiety is attached at various sites on the
antibody. Analytical and preparative methods are inadequate to
separate and characterize the antibody-drug conjugate species
molecules within the heterogeneous mixture resulting from a
conjugation reaction. Antibodies are large, complex and
structurally diverse biomolecules, often with many reactive
functional groups. Antibody reactivity with linker reagents and
drug-linker intermediates are dependent on factors such as pH,
concentration, salt concentration, and co-solvents. Furthermore,
the multistep conjugation process may be nonreproducible due to
difficulties in controlling the reaction conditions and
characterizing reactants and intermediates.
[0008] Antibody-drug conjugates are typically formed by conjugating
one or more antibody cysteine thiol groups to one or more linker
moieties bound to a drug thereby forming an antibody-linker-drug
complex. Cysteine thiols are reactive at neutral pH, unlike most
amines which are protonated and less nucleophilic near pH 7. Since
free thiol (RSH, sulfhydryl) groups are relatively reactive,
proteins with cysteine residues often exist in their oxidized form
as disulfide-linked oligomers or have internally bridged disulfide
groups. Antibody cysteine thiol groups are generally more reactive,
i.e. more nucleophilic, towards electrophilic conjugation reagents
than antibody amine or hydroxyl groups. Engineering in cysteine
thiol groups by the mutation of various amino acid residues of a
protein to cysteine amino acids is potentially problematic,
particularly in the case of unpaired (free Cys) residues or those
which are relatively accessible for reaction or oxidation. In
concentrated solutions of the protein, whether in the periplasm of
E. coli, culture supernatants, or partially or completely purified
protein, unpaired Cys residues on the surface of the protein can
pair and oxidize to form intermolecular disulfides, and hence
protein dimers or multimers. Disulfide dimer formation renders the
new Cys unreactive for conjugation to a drug, ligand, or other
label. Furthermore, if the protein oxidatively forms an
intramolecular disulfide bond between the newly engineered Cys and
an existing Cys residue, both Cys groups are unavailable for active
site participation and interactions. Furthermore, the protein may
be rendered inactive or non-specific, by misfolding or loss of
tertiary structure (Zhang et al. (2002) Anal. Biochem.
311:1-9).
[0009] Improved antibody-drug conjugates, THIOMAB.TM., have been
developed that provide for site-specific conjugation of a drug to
an antibody through cysteine substitutions at sites where the
engineered cysteines are available for conjugation but do not
perturb immunoglobulin folding and assembly or alter antigen
binding and effector functions (Junutula, et al., 2008b Nature
Biotech., 26(8):925-932; Dornan et al. (2009) Blood
114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No.
7,723,485; WO2009/052249). These THIOMAB.TM. antibodies can then be
conjugated to cytotoxic drugs through the engineered cysteine thiol
groups to obtain THIOMAB.TM. drug conjugates (TDC) with uniform
stoichiometry (e.g., up to 2 drugs per antibody in an antibody that
has a single engineered cysteine site). Studies with multiple
antibodies against different antigens have shown that TDCs are as
efficacious as conventional antibody-drug conjugate in xenograft
models and are tolerated at higher doses in relevant preclinical
models. THIOMAB.TM. antibodies have been engineered for drug
attachment at different locations of the antibody (e.g., specific
amino acid positions (i.e., sites) within the light chain-Fab,
heavy chain-Fab and heavy chain-Fc). The in vitro and in vivo
stability, efficacy and PK properties of THIOMAB.TM. antibodies
provide a unique advantage over conventional antibody-drug
conjugates due to their homogeneity and site-specific conjugation
to cytotoxic drugs.
[0010] There are still other limitations or challenges to the
preparation and use of antibody-drug conjugates, and in particular
antibody-calicheamicin derivative conjugates. For example, some
linkers may be labile in the blood stream, thereby releasing
unacceptable amounts of the drug prior to internalization in a
target cell. Other linkers may provide stability in the
bloodstream, but intracellular release effectiveness may be
negatively impacted. Linkers that provide for desired intracellular
release typically have poor stability in the bloodstream.
Alternatively stated, bloodstream stability and intracellular
release are typically inversely related. Second, in standard
conjugation processes, the amount of calicheamicin loaded on the
carrier protein (the drug loading), the amount of aggregate that is
formed in the conjugation reaction, and the yield of final purified
conjugate that can be obtained are interrelated. For example,
aggregate formation is generally positively correlated to the
number of equivalents of calicheamicin and derivatives thereof
conjugated to the carrier-antibody. Because drug potency and
efficacy increases with calicheamicin content, it is desirable to
maximize calicheamicin loading on an antibody carrier while
retaining the affinity of the antibody. However, under high drug
loading, formed aggregates must be removed for therapeutic
applications. As a result, drug loading-mediated aggregate
formation decreases antibody-drug conjugate yield and can renders
process scale-up difficult. For example, prior art conjugation
methods using linkers have been found to require a compromise
between higher drug loading and antibody-drug conjugate yield, by
limiting the amount of calicheamicin that is added to the
conjugation reaction.
[0011] Accordingly, there is a continuing need for improved
efficacious calicheamicin-antibody conjugates that provide for
optimized safety and efficacy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows a plot of in vivo tumor volume change over time
in a HCC1569X2 xenograph model in SCID Beige mice after IV dosing
with Thio Hu anti-Ly6E LC K149C-p-nitro-PDS-Calicheamicin at doses
of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg and 10 mg/kg and with a
Thio Hu anti-CD22 LC K149C-p-nitro-PDS-Calicheamicin control dosed
IV at 3 mg/kg
[0013] FIG. 2 shows a plot of in vivo tumor volume change over time
in a WSU-DLCL2 xenograph model in CB-17 Fox Chase SCID mice after
IV dosing with Thio Hu anti-CD22 10F4v3 LC
K149C-p-nitro-PDS-Calicheamicin at doses of 0.3 mg/kg, 1 mg/kg, 3
mg/kg, 6 mg/kg and 10 mg/kg and with a Thio Hu anti-Ly6E 9B12v12 LC
K149C-p-nitro-PDS-Calicheamicin control dosed IV at 3 mg/kg
[0014] FIG. 3A shows a plot of IC.sub.50 potency versus log
concentration against CD22 positive Burkitt's human lymphoma cells
(BJAB) for targeted Thio Hu Anti-CD22 10F4v3 LC K149C-Calicheamicin
IC50 as compared to non-targeted control Thio Hu Anti-Ly6E 9B12.v12
LC K149C-Calicheamicin. The targeted potency is >1500-fold
greater than non-targeted potency.
[0015] FIG. 3B shows a plot of IC.sub.50 potency versus log
concentration against CD22 positive WSU-DLCL2 human diffuse large
B-cell lymphoma-derived cell line for targeted Thio Hu Anti-Ly6E
10F4v3 LC K149C-Calicheamicin IC.sub.50 potency as compared to
non-targeted control Thio Hu Anti-Ly6E 9B12.v12 LC
K149C-Calicheamicin. The targeted potency is >2000-fold greater
than non-targeted potency.
[0016] FIG. 3C shows a plot of IC.sub.50 potency versus log
concentration against Jurkat cells for Thio Hu Anti-Ly6E 10F4v3 LC
K149C-Calicheamicin and Thio Hu Anti-Ly6E 9B12.v12 LC
K149C-Calicheamicin IC.sub.50 potency versus log concentration.
SUMMARY
[0017] In one aspect of the present disclosure drug intermediates
of Formula I and of Formula II are provided:
##STR00001##
[0018] In such aspects, R is selected from H, --C(O)R.sup.1,
--C(O)NR.sup.1R.sup.2, --S(O).sub.2R.sup.1, and
--S(O)2NR.sup.2R.sup.1; R.sup.1 and R.sup.2 are independently
selected from C.sub.1-C.sub.6 alkyl and C.sub.6-C.sub.20 aryl;
R.sup.3 is selected from NO.sub.2, Cl, F, CN, CO.sub.2H, and Br;
and q is 0, 1, or 2.
[0019] In another aspect of the present disclosure, an
antibody-drug conjugate of Formula III, or a pharmaceutically
acceptable salt thereof, is provided:
##STR00002##
In such aspects, R is selected from H, --C(O)R.sup.1,
--C(O)NR.sup.1R.sup.2, --S(O).sub.2R.sup.1, and
--S(O).sub.2NR.sup.2R.sup.1, and R.sup.1 and R.sup.2 are
independently selected from C.sub.1-C.sub.6 alkyl and
C.sub.6-C.sub.20 aryl. The designator p is an integer from 1 to 8.
Ab is an antibody which binds to one or more tumor-associated
antigens or cell-surface receptors selected from (1)-(53), as
listed herein: (1) BMPR1B (bone morphogenetic protein receptor-type
IB); (2) E16 (LAT1, SLC7A5); (3) STEAP1 (six transmembrane
epithelial antigen of prostate); (4) MUC16 (0772P, CA125); (5) MPF
(MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin);
(6) Napi2b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34
(sodium phosphate), member 2, type II sodium-dependent phosphate
transporter 3b); (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMASB,
SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin
repeats (type 1 and type 1-like), transmembrane domain (TM) and
short cytoplasmic domain, (semaphorin) 5B); (8) PSCA hlg
(2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA
2700050C12 gene); (9) ETBR (Endothelin type B receptor); (10)
MSG783 (RNF124, hypothetical protein F1120315); (11) STEAP2
(HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer
associated gene 1, prostate cancer associated protein 1, six
transmembrane epithelial antigen of prostate 2, six transmembrane
prostate protein); (12) TrpM4 (BR22450, F1120041, TRPM4, TRPM4B,
transient receptor potential cation channel, subfamily M, member
4); (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,
teratocarcinoma-derived growth factor); (14) CD21 (CR2 (Complement
receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs 73792);
(15) CD79b (CD79B, CD79.beta., IGb (immunoglobulin-associated
beta), B29); (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain
containing phosphatase anchor protein 1a), SPAP1B, SPAP1C); (17)
HER2; (18) NCA; (19) MDP; (20) IL20R.alpha.; (21) Brevican; (22)
EphB2R; (23) ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R (B
cell-activating factor receptor, BLyS receptor 3, BR3); (27) CD22
(B-cell receptor CD22-B isoform); (28) CD79a (CD79A, CD79a,
immunoglobulin-associated alpha); (29) CXCR5 (Burkitt's lymphoma
receptor 1); (30) HLA-DOB (Beta subunit of MHC class II molecule
(Ia antigen)); (31) P2X5 (Purinergic receptor P2X ligand-gated ion
channel 5); (32) CD72 (B-cell differentiation antigen CD72, Lyb-2);
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein
of the leucine rich repeat (LRR) family); (34) FcRH1 (Fc
receptor-like protein 1); (35) FcRH5 (IRTA2, Immunoglobulin
superfamily receptor translocation associated 2); (36) TENB2
(putative transmembrane proteoglycan); (37) PMEL17 (silver homolog;
SILV; D12S53E; PMEL17; SI; SIL); (38) TMEFF1 (transmembrane protein
with EGF-like and two follistatin-like domains Tomoregulin-1); (39)
GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA;
RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); (40) Ly6E
(lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1);
(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); (42) Ly6G6D
(lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1); (43) LGR5
(leucine-rich repeat-containing G protein-coupled receptor 5;
GPR49, GPR67); (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B;
MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE1); (45) LY6K
(lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226);
(46) GPR19 (G protein-coupled receptor 19; Mm.4787); (47) GPR54
(KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12); (48) ASPHD1
(aspartate beta-hydroxylase domain containing 1; LOC253982); (49)
Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3); (50) TMEM118
(ring finger protein, transmembrane 2; RNFT2; FLJ14627); (51)
GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856;
D15Ertd747e); (52) CD33; and (53) CLL-1.
[0020] In some other aspects of the disclosure, pharmaceutical
compositions comprising an antibody-drug conjugate of the present
disclosure is provided, the pharmaceutical composition further
comprising at least one of a diluent, a carrier, and an
excipient.
[0021] In other aspects, a pharmaceutical composition comprising an
antibody-drug conjugate of the present disclosure and at least one
of a diluent, a carrier, and an excipient is provided for
administration to a patient for the treatment of cancer.
[0022] In still other aspects, antibody-drug conjugates of the
present disclosure are provided for use in the manufacture of a
medicament for the treatment of cancer in an animal.
[0023] In yet other aspects, methods for treating cancer with the
antibody-drug conjugates of the present disclosure are
provided.
[0024] In other aspects, a method of making antibody-drug
conjugates of the present disclosure is provided wherein the method
comprises reacting an antibody which binds with one or more
tumor-associated antigens or cell-surface receptors selected from
(1)-(53) as described elsewhere herein with a drug-leaving group
intermediate composition of formula I or formula II as described
elsewhere herein.
[0025] In other aspects, an article of manufacture is provided, the
article comprising: an antibody-drug conjugate of the present
disclosure and at least one of a diluent, a carrier, and an
excipient; a container; and a package insert or label indicating
that the pharmaceutical composition can be used to treat
cancer.
DETAILED DESCRIPTION
[0026] Reference will now be made in detail to certain embodiments
of the invention, examples of which are illustrated in the
accompanying structures and formulas. While the invention will be
described in conjunction with the enumerated embodiments, it will
be understood that they are not intended to limit the invention to
those embodiments. On the contrary, the invention is intended to
cover all alternatives, modifications, and equivalents, which may
be included within the scope of the present invention as defined by
the claims.
[0027] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. The present
invention is in no way limited to the methods and materials
described.
[0028] The present disclosure is generally directed to
antibody-drug conjugates comprising an antibody directly conjugated
to one or more calicheamicin derivatives, in the absence of a
linker or linking moiety. The present disclosure is further
directed to calicheamicin derivative intermediate compositions
comprising a leaving group. Such intermediate compositions are
suitable substrates for formation of antibody-drug conjugates
wherein an antibody is covalently bound directly to calicheamicin
derivative, after loss of the leaving group and in the absence of a
conventional linker or linking moiety. The present disclosure is
further directed to use of such an antibody-calicheamicin conjugate
in the treatment of an illness, in particular cancer. As used
herein, unless otherwise specified, calicheamicin refers to the
calicheamicin derivative compounds encompassed by the present
disclosure.
[0029] In this regard it is to be noted that prior art methods for
forming antibody-calicheamicin and antibody-calicheamicin
derivative conjugates, such as those disclosed in U.S. 55,877,296
and 5,773,001, result in a large percentage of conjugate
aggregates, rendering scale-up impractical and presenting
purification problems (see U.S. publication no. 2007/0213511 A1,
which is incorporated herein by reference, at, for example,
paragraphs [0008] and [0009]). It is further known that the methods
disclosed in U.S. Pat. No. 5,714,586 and U.S. Pat. No. 5,712,374
produce antibody-drug conjugates having from 50% to 60% of an
undesired low conjugated fraction (see, e.g., U.S. publication no.
2007/0213511 A1 at paragraph [0010]). Furthermore, problematically,
linkers may be instable in the bloodstream, thereby resulting
significant drug release prior to internalization. Further, the use
of such calicheamicin-linker-antibody conjugates may be limited by
the capabilities of known conjugation processes, which typically
result in the formation aggregates, particularly when the drug
loading per antibody molecule is increased.
[0030] Based on experimental evidence to-date, it has been
discovered that the direct conjugation, by means of a disulfide
covalent bond between an antibody and a calicheamicin derivative,
in the absence of a linker, provides for improved
antibody-calicheamicin conjugates, characterized by reproducible
calicheamicin drug loading per antibody (DAR), reduced aggregate
formation, improved bloodstream stability and improved
intracellular release. Accordingly, it is believed that direct
conjugation of an antibody to calicheamicin by a disulfide bond
effectively decouples bloodstream stability and intracellular
release such that both improved bloodstream stability and improved
intracellular release are enabled.
DEFINITIONS
[0031] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs, and are
consistent with: Singleton et al. (1994) Dictionary of Microbiology
and Molecular Biology, 2nd Ed., J. Wiley & Sons, New York,
N.Y.; and Janeway, C., Travers, P., Walport, M., Shlomchik (2001)
Immunobiology, 5th Ed., Garland Publishing, New York.
[0032] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Specific illustrative and
exemplary embodiments for measuring binding affinity are described
in the following.
[0033] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
dimers, multimers, multispecific antibodies (e.g., bispecific
antibodies), and antibody fragments, so long as they exhibit the
desired biological activity (Miller et al. (2003) Jour. of
Immunology 170:4854-4861). Antibodies may be murine, human,
humanized, chimeric, or derived from other species. An antibody is
a protein generated by the immune system that is capable of
recognizing and binding to a specific antigen. (Janeway, C.,
Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed.,
Garland Publishing, New York). A target antigen generally has
numerous binding sites, also called epitopes, recognized by CDRs on
multiple antibodies. Each antibody that specifically binds to a
different epitope has a different structure. Thus, one antigen may
have more than one corresponding antibody. An antibody includes a
full-length immunoglobulin molecule or an immunologically active
portion of a full-length immunoglobulin molecule, i.e., a molecule
that contains an antigen binding site that immunospecifically binds
an antigen of a target of interest or part thereof, such targets
including but not limited to, cancer cell or cells that produce
autoimmune antibodies associated with an autoimmune disease. The
immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE,
IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2) or subclass of immunoglobulin molecule. The immunoglobulins
can be derived from any species. In one aspect, however, the
immunoglobulin is of human, murine, or rabbit origin.
[0034] "Antibody fragments" comprise a portion of a full length
antibody, generally the antigen binding or variable region thereof.
Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv
fragments; diabodies; linear antibodies; minibodies (Olafsen et al.
(2004) Protein Eng. Design & Sel. 17(4):315-323), fragments
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies, CDR (complementary determining region), and
epitope-binding fragments of any described herein which
immunospecifically bind to cancer cell antigens, viral antigens or
microbial antigens, single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[0035] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al.
(1975) Nature 256:495, or may be made by recombinant DNA methods
(see for example: U.S. Pat. No. 4,816,567; U.S. Pat. No.
5,807,715). The monoclonal antibodies may also be isolated from
phage antibody libraries using the techniques described in Clackson
et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol.
Biol., 222:581-597; for example.
[0036] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl.
Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest
herein include "primatized" antibodies comprising variable domain
antigen-binding sequences derived from a non-human primate (e.g.,
Old World Monkey, Ape, etc.) and human constant region
sequences.
[0037] An "intact antibody" herein is one comprising a VL and VH
domains, as well as a light chain constant domain (CL) and heavy
chain constant domains, CH1, CH2 and CH3. The constant domains may
be native sequence constant domains (e.g., human native sequence
constant domains) or amino acid sequence variant thereof. The
intact antibody may have one or more "effector functions" which
refer to those biological activities attributable to the Fc
constant region (a native sequence Fc region or amino acid sequence
variant Fc region) of an antibody. Examples of antibody effector
functions include C1q binding; complement dependent cytotoxicity;
Fc receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; and down regulation of cell surface receptors
such as B cell receptor and BCR.
[0038] Depending on the amino acid sequence of the constant domain
of their heavy chains, intact antibodies can be assigned to
different "classes." There are five major classes of intact
immunoglobulin antibodies: IgA, IgD, IgE, IgG, and IgM, and several
of these may be further divided into "subclasses" (isotypes), e.g.,
IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant
domains that correspond to the different classes of antibodies are
called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known. Ig forms include hinge-modifications or hingeless forms
(Roux et al. (1998) J. Immunol. 161:4083-4090; Lund et al. (2000)
Eur. J. Biochem. 267:7246-7256; US 2005/0048572; US
2004/0229310).
[0039] A "cysteine engineered antibody" or "cysteine engineered
antibody variant" is an antibody in which one or more residues of
an antibody are substituted with cysteine residues. In accordance
with the present disclosure, the thiol group(s) of the cysteine
engineered antibodies can be conjugated to calicheamicin to form a
THIOMAB.TM. antibody (i.e., a THIOMAB.TM. drug conjugate (TDC),
wherein in accordance with the present disclosure the drug is a
calicheamicin derivative). In particular embodiments, the
substituted residues occur at accessible sites of the antibody. By
substituting those residues with cysteine, reactive thiol groups
are thereby positioned at accessible sites of the antibody and may
be used to conjugate the antibody to the drug moiety to create an
immunoconjugate, as described further herein. For example, a
THIOMAB.TM. antibody may be an antibody with a single mutation of a
non-cysteine native residue to a cysteine in the light chain (e.g.,
G64C, K149C or R142C according to Kabat numbering) or in the heavy
chain (e.g., D101C or V184C or T205C according to Kabat numbering).
In specific examples, a THIOMAB.TM. antibody has a single cysteine
mutation in either the heavy or light chain such that each
full-length antibody (i.e., an antibody with two heavy chains and
two light chains) has two engineered cysteine residues. Cysteine
engineered antibodies and preparatory methods are disclosed by US
2012/0121615 A1 (incorporated by reference herein in its
entirety).
[0040] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth/proliferation. Examples of cancer
include, but are not limited to, carcinoma, lymphoma (e.g.,
Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and
leukemia. More particular examples of such cancers include acute
myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic
myelogenous leukemia (CML), chronic myelomonocytic leukemia, acute
promyelocytic leukemia (APL), chronic myeloproliferative disorder,
thrombocytic leukemia, precursor B-cell acute lymphoblastic
leukemia (pre-B-ALL), precursor T-cell acute lymphoblastic leukemia
(preT-ALL), multiple myeloma (MM), mast cell disease, mast cell
leukemia, mast cell sarcoma, myeloid sarcomas, lymphoid leukemia,
and undifferentiated leukemia. In some embodiments, the cancer is
myeloid leukemia. In some embodiments, the cancer is acute myeloid
leukemia (AML).
[0041] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0042] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0043] "Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g. B cell receptor); and B cell activation.
[0044] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0045] The term "epitope" refers to the particular site on an
antigen molecule to which an antibody binds. In some embodiments,
the particular site on an antigen molecule to which an antibody
binds is determined by hydroxyl radical footprinting.
[0046] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc regions and variant Fc regions. In one embodiment, a human IgG
heavy chain Fc region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991.
[0047] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0048] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0049] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0050] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0051] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0052] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0053] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887
(1993); Clarkson et al., Nature 352:624-628 (1991).
[0054] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors."
[0055] The term "hypervariable region" or "HVR," as used herein,
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops ("hypervariable loops"). Generally, native four-chain
antibodies comprise six HVRs; three in the VH (H1, H2, H3), and
three in the VL (L1, L2, L3). HVRs generally comprise amino acid
residues from the hypervariable loops and/or from the
"complementarity determining regions" (CDRs), the latter being of
highest sequence variability and/or involved in antigen
recognition. Exemplary hypervariable loops occur at amino acid
residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55
(H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2,
and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2,
89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991).) With the exception of CDR1 in VH, CDRs generally comprise
the amino acid residues that form the hypervariable loops. CDRs
also comprise "specificity determining residues," or "SDRs," which
are residues that contact antigen. SDRs are contained within
regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary
a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and
a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2,
89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See
Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless
otherwise indicated, HVR residues and other residues in the
variable domain (e.g., FR residues) are numbered herein according
to Kabat et al., supra.
[0056] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0057] An "isolated antibody" is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman et al., J. Chromatogr. B 848:79-87 (2007).
[0058] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
[0059] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0060] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0061] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y where X is
the number of amino acid residues scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of A and B, and where Y is the total number of amino acid residues
in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the
% amino acid sequence identity of A to B will not equal the % amino
acid sequence identity of B to A. Unless specifically stated
otherwise, all % amino acid sequence identity values used herein
are obtained as described in the immediately preceding paragraph
using the ALIGN-2 computer program.
[0062] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0063] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0064] The terms "treat" and "treatment" refer to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down (lessen) an undesired
physiological change or disorder, such as the development or spread
of cancer. For purposes of this invention, beneficial or desired
clinical results include, but are not limited to, alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. Those in
need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder
or those in which the condition or disorder is to be prevented.
[0065] The term "therapeutically effective amount" refers to an
amount of a drug effective to treat a disease or disorder in a
mammal. In the case of cancer, the therapeutically effective amount
of the drug may reduce the number of cancer cells; reduce the tumor
size; inhibit (i.e., slow to some extent and preferably stop)
cancer cell infiltration into peripheral organs; inhibit (i.e.,
slow to some extent and preferably stop) tumor metastasis; inhibit,
to some extent, tumor growth; and/or relieve to some extent one or
more of the symptoms associated with the cancer. To the extent the
drug may prevent growth and/or kill existing cancer cells, it may
be cytostatic and/or cytotoxic. For cancer therapy, efficacy can,
for example, be measured by assessing the time to disease
progression (TTP) and/or determining the response rate (RR).
[0066] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. A "tumor" comprises one or more
cancerous cells. Examples of cancer include, but are not limited
to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g., epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer ("NSCLC"), 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, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, rectal cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney or renal
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, anal carcinoma, penile carcinoma, as well as head and
neck cancer.
[0067] The term "leaving group," as used herein, refers to a
sulfhydryl moiety that leaves in the course of a chemical reaction
involving the groups as described herein.
[0068] The term "hydrocarbyl" as used herein describes organic
compounds or radicals consisting exclusively of the elements carbon
and hydrogen. These moieties include, without limitation, alkyl,
alkenyl, alkynyl, and aryl moieties. These moieties also include
alkyl, alkenyl, alkynyl, and aryl moieties substituted with other
aliphatic or cyclic hydrocarbon groups, such as alkaryl, alkenaryl
and alkynaryl. Unless otherwise indicated, these moieties
preferably comprise 1 to 20 carbon atoms, 1 to 10 carbon atoms or 1
to 6 carbon atoms.
[0069] Unless otherwise indicated, the alkyl groups described
herein are preferably lower alkyl containing from one to eight
carbon atoms in the principal chain. They may be straight or
branched chain or cyclic including, but not limited to, methyl,
ethyl, propyl, isopropyl, allyl, benzyl, hexyl and the like.
[0070] Unless otherwise indicated, the alkynyl groups described
herein are preferably lower alkynyl containing from two to eight
carbon atoms in the principal chain and up to 20 carbon atoms. They
may be straight or branched chain including, but not limited to,
ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like.
[0071] The term "aryl" as used herein alone or as part of another
group denotes optionally substituted homocyclic aromatic groups,
preferably monocyclic or bicyclic groups containing from 5 to 20
carbons, from 5 to 10 carbons, or from 5 to 6 carbons in the ring
portion, including, but not limited to, phenyl, biphenyl, naphthyl,
substituted phenyl, substituted biphenyl or substituted naphthyl.
The aryl moieties may optionally comprise one or more hetero atoms
selected from O, S and N. Such heteroaromatics may comprise 1 or 2
nitrogen atoms, 1 or 2 sulfur atoms, 1 or 2 oxygen atoms, and
combinations thereof, in the ring, wherein the each hetero atom is
bonded to the remainder of the molecule through a carbon. Non
limiting exemplary groups include pyridine, pyrazine, pyrimidine,
pyrazole, pyrrole, imidazole, thiopene, thiopyrrilium,
parathiazine, indole, purine, benzimidazole, quinolone,
phenothiazine. Non-limiting exemplary substituents include one or
more of the following groups: hydrocarbyl, substituted hydrocarbyl,
alkyl, alkoxy, acyl, acyloxy, alkenyl, alkenoxy, aryl, aryloxy,
amino, amido, acetal, carbamyl, carbocyclo, cyano, ester, ether,
halogen, heterocyclo, hydroxy, keto, ketal, phospho, nitro, and
thio.
[0072] The "substituted" moieties described herein are moieties
such as hydrocarbyl, alkyl and aryl which are substituted with at
least one atom other than carbon, including moieties in which a
carbon chain atom is substituted with a hetero atom such as
nitrogen, oxygen, silicon, phosphorous, boron, sulfur, or a halogen
atom. These substituents include, but are not limited to, halogen,
heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy, hydroxy, keto,
acyl, acyloxy, nitro, tertiary amino, amido, nitro, cyano, thio,
sulfinate, sulfonamide, ketals, acetals, esters and ethers.
[0073] The terms "halogen" as used herein alone or as part of
another group refer to chlorine, bromine, fluorine, and iodine.
[0074] Antibodies
[0075] In any of the embodiments of the disclosure, an antibody is
humanized. In one embodiment, an antibody comprises HVRs as in any
of the embodiments of the disclosure, and further comprises a human
acceptor framework, e.g. a human immunoglobulin framework or a
human consensus framework. In certain embodiments, the human
acceptor framework is the human VL kappa I consensus (VLKI)
framework and/or the VH framework VH1. In certain embodiments, the
human acceptor framework is the human VL kappa I consensus (VLKI)
framework and/or the VH framework VH1 comprising any one of the
following mutations.
[0076] In another aspect, the antibody comprises a VH as in any of
the embodiments provided herein, and a VL as in any of the
embodiments provided herein.
[0077] In a further aspect of the invention, an antibody according
to any of the embodiments herein is a monoclonal antibody,
including a human antibody. In one embodiment, an antibody is an
antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab')2
fragment. In another embodiment, the antibody is a substantially
full length antibody, e.g., an IgG1 antibody, IgG2a antibody or
other antibody class or isotype as defined herein.
[0078] In a further aspect, an antibody according to any of the
embodiments herein may incorporate any of the features, singly or
in combination, as described herein.
[0079] 1. Antibody Affinity
[0080] In certain embodiments, an antibody provided herein has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.50 nM, .ltoreq.10 nM, .ltoreq.5 nM, .ltoreq.1 nM,
.ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001 nM, and
optionally is .gtoreq.10.sup.-13 M. (e.g. 10.sup.-8M or less, e.g.
from 10.sup.-8M to 10.sup.-13 M, e.g., from 10.sup.-9M to
10.sup.-13 M).
[0081] In one embodiment, Kd is measured by a radiolabeled antigen
binding assay (RIA) performed with the Fab version of an antibody
of interest and its antigen as described by the following assay.
Solution binding affinity of Fabs for antigen is measured by
equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled antigen in the presence of a titration series
of unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.
293:865-881(1999)). To establish conditions for the assay,
MICROTITER.RTM. multi-well plates (Thermo Scientific) are coated
overnight with 5 .mu.g/ml of a capturing anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked
with 2% (w/v) bovine serum albumin in PBS for two to five hours at
room temperature (approximately 23.degree. C.). In a non-adsorbent
plate (Nunc #269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed
with serial dilutions of a Fab of interest (e.g., consistent with
assessment of the anti-VEGF antibody, Fab-12, in Presta et al.,
Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., about 65 hours) to ensure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
polysorbate 20 (TWEEN-20.RTM.) in PBS. When the plates have dried,
150 .mu.l/well of scintillant (MICROSCINT-20.TM.; Packard) is
added, and the plates are counted on a TOPCOUNT.TM. gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.
[0082] According to another embodiment, Kd is measured using
surface plasmon resonance assays using a BIACORE.RTM.-2000,
BAICORE.RTM.-T200 or a BIACORE.RTM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) at 25.degree. C. with immobilized antigen CMS
chips at .about.10 response units (RU). Briefly, carboxymethylated
dextran biosensor chips (CMS, BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) and/or HBS-P (0.01 M Hepes pH7.4,
0.15M NaCl, 0.005% Surfactant P20) before injection at a flow rate
of 5 .mu.l/minute and/or 30 .mu.l/minute to achieve approximately
10 response units (RU) of coupled protein. Following the injection
of antigen, 1 M ethanolamine is injected to block unreacted groups.
For kinetics measurements, two-fold serial dilutions of Fab (0.78
nM to 500 nM) are injected in PBS with 0.05% polysorbate 20
(TWEEN-20.TM.2) surfactant (PBST) at 25.degree. C. at a flow rate
of approximately 25 .mu.l/min. Association rates (k.sub.on) and
dissociation rates (k.sub.off) are calculated using a simple
one-to-one Langmuir binding model (BIACORE.RTM. Evaluation Software
version 3.2) by simultaneously fitting the association and
dissociation sensorgrams. The equilibrium dissociation constant
(Kd) is calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen
et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds
10.sup.6 M.sup.-1 s.sup.-1 by the surface plasmon resonance assay
describe herein, then the on-rate can be determined by using a
fluorescent quenching technique that measures the increase or
decrease in fluorescence emission intensity (excitation=295 nm;
emission=340 nm, 16 nm band-pass) at 25.degree. C. of a 20 nM
anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of
increasing concentrations of antigen as measured in a spectrometer,
such as a stop-flow equipped spectrophometer (Aviv Instruments) or
a 8000-series SLM-AMINCO spectrophotometer (ThermoSpectronic) with
a stirred cuvette.
[0083] 2. Antibody Fragments
[0084] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other
fragments described herein. For a review of certain antibody
fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a
review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology
of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York), pp. 269-315 (1994); see also WO
93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab')2 fragments comprising salvage receptor
binding epitope residues and having increased in vivo half-life,
see U.S. Pat. No. 5,869,046.
[0085] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP
404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003);
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448
(1993). Triabodies and tetrabodies are also described in Hudson et
al., Nat. Med. 9:129-134 (2003).
[0086] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516 B1).
[0087] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0088] 3. Chimeric and Humanized Antibodies
[0089] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody
comprises a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) and a human constant region. In a further
example, a chimeric antibody is a "class switched" antibody in
which the class or subclass has been changed from that of the
parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0090] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0091] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol.
Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua
et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and
Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J.
Cancer, 83:252-260 (2000) (describing the "guided selection"
approach to FR shuffling).
[0092] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.
USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623
(1993)); human mature (somatically mutated) framework regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions derived
from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0093] 4. Human Antibodies
[0094] In certain embodiments, an antibody provided herein is a
human antibody. Human antibodies can be produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459
(2008).
[0095] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HUMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELoCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0096] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boemer et al., J.
Immunol., 147: 86 (1991).) Human antibodies generated via human
B-cell hybridoma technology are also described in Li et al., Proc.
Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826
(describing production of monoclonal human IgM antibodies from
hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3): 185-91 (2005).
[0097] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described herein.
[0098] 5. Library-Derived Antibodies
[0099] Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom et al. in Methods in
Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,
Totowa, N.J., 2001) and further described, e.g., in the McCafferty
et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628
(1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and
Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed.,
Human Press, Totowa, N.J., 2003); Sidhu et al., J Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472
(2004); and Lee et al., J. Immunol. Methods 284(1-2):
119-132(2004).
[0100] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Patent publications describing human antibody phage libraries
include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0101] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0102] 6. Multispecific Antibodies
[0103] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. The term
"multispecific antibody" is used in the broadest sense and
specifically covers an antibody comprising an antigen-binding
domain that has polyepitopic specificity (i.e., is capable of
specifically binding to two, or more, different epitopes on one
biological molecule or is capable of specifically binding to
epitopes on two, or more, different biological molecules). In some
embodiments, multispecific antibodies are monoclonal antibodies
that have binding specificities for at least two different sites.
In some embodiments, an antigen-binding domain of a multispecific
antibody (such as a bispecific antibody) comprises two VH/VL units,
wherein a first VH/VL unit specifically binds to a first epitope
and a second VH/VL unit specifically binds to a second epitope,
wherein each VH/VL unit comprises a heavy chain variable domain
(VH) and a light chain variable domain (VL). Such multispecific
antibodies include, but are not limited to, full length antibodies,
antibodies having two or more VL and VH domains, antibody fragments
such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and
triabodies, antibody fragments that have been linked covalently or
non-covalently. A VH/VL unit that further comprises at least a
portion of a heavy chain variable region and/or at least a portion
of a light chain variable region may also be referred to as an
"arm" or "hemimer" or "half antibody." In some embodiments, a
hemimer comprises a sufficient portion of a heavy chain variable
region to allow intramolecular disulfide bonds to be formed with a
second hemimer. In some embodiments, a hemimer comprises a knob
mutation or a hole mutation, for example, to allow
heterodimerization with a second hemimer or half antibody that
comprises a complementary hole mutation or knob mutation. Knob
mutations and hole mutations are discussed further herein.
[0104] In certain embodiments, a multispecific antibody provided
herein may be a bispecific antibody. The term "bispecific antibody"
is used in the broadest sense and covers a multispecific antibody
comprising an antigen-binding domain that is capable of
specifically binding to two different epitopes on one biological
molecule or is capable of specifically binding to epitopes on two
different biological molecules. A bispecific antibody may also be
referred to herein as having "dual specificity" or as being "dual
specific." Bispecific antibodies can be prepared as full length
antibodies or antibody fragments. The term "biparatopic antibody"
as used herein, refers to a bispecific antibody where a first
antigen-binding domain and a second antigen-binding domain bind to
two different epitopes on the same antigen molecule or it may bind
to epitopes on two different antigen molecules.
[0105] In some embodiments, the first antigen-binding domain and
the second antigen-binding domain of the biparatopic antibody may
bind the two epitopes within one and the same antigen molecule
(intramolecular binding). For example, the first antigen-binding
domain and the second antigen-binding domain of the biparatopic
antibody may bind to two different epitopes on the same antibody
molecule. In certain embodiments, the two different epitopes that a
biparatopic antibody binds are epitopes that are not normally bound
at the same time by one monospecific antibody, such as e.g. a
conventional antibody or one immunoglobulin single variable
domain.
[0106] In some embodiments, the first antigen-binding domain and
the second antigen-binding domain of the biparatopic antibody may
bind epitopes located within two distinct antigen molecules.
[0107] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and
Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168), WO2009/089004,
US2009/0182127, US2011/0287009, Marvin and Zhu, Acta Pharmacol.
Sin. (2005) 26(6):649-658, and Kontermann (2005) Acta Pharmacol.
Sin., 26:1-9). The term "knob-into-hole" or "KnH" technology as
used herein refers to the technology directing the pairing of two
polypeptides together in vitro or in vivo by introducing a
protuberance (knob) into one polypeptide and a cavity (hole) into
the other polypeptide at an interface in which they interact. For
example, KnHs have been introduced in the Fc:Fc binding interfaces,
CL:CH1 interfaces or VH/VL interfaces of antibodies (see, e.g., US
2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, and Zhu
et al., 1997, Protein Science 6:781-788). In some embodiments, KnHs
drive the pairing of two different heavy chains together during the
manufacture of multispecific antibodies. For example, multispecific
antibodies having KnH in their Fc regions can further comprise
single variable domains linked to each Fc region, or further
comprise different heavy chain variable domains that pair with
similar or different light chain variable domains. KnH technology
can be also be used to pair two different receptor extracellular
domains together or any other polypeptide sequences that comprises
different target recognition sequences (e.g., including affibodies,
peptibodies and other Fc fusions).
[0108] The term "knob mutation" as used herein refers to a mutation
that introduces a protuberance (knob) into a polypeptide at an
interface in which the polypeptide interacts with another
polypeptide. In some embodiments, the other polypeptide has a hole
mutation.
[0109] A "protuberance" refers to at least one amino acid side
chain which projects from the interface of a first polypeptide and
is therefore positionable in a compensatory cavity in the adjacent
interface (i.e. the interface of a second polypeptide) so as to
stabilize the heteromultimer, and thereby favor heteromultimer
formation over homomultimer formation, for example. The
protuberance may exist in the original interface or may be
introduced synthetically (e.g. by altering nucleic acid encoding
the interface). In some embodiments, nucleic acid encoding the
interface of the first polypeptide is altered to encode the
protuberance. To achieve this, the nucleic acid encoding at least
one "original" amino acid residue in the interface of the first
polypeptide is replaced with nucleic acid encoding at least one
"import" amino acid residue which has a larger side chain volume
than the original amino acid residue. It will be appreciated that
there can be more than one original and corresponding import
residue. The side chain volumes of the various amino residues are
shown, for example, in Table 1 of US2011/0287009. A mutation to
introduce a "protuberance" may be referred to as a "knob
mutation."
[0110] In some embodiments, import residues for the formation of a
protuberance are naturally occurring amino acid residues selected
from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan
(W). In some embodiments, an import residue is tryptophan or
tyrosine. In some embodiment, the original residue for the
formation of the protuberance has a small side chain volume, such
as alanine, asparagine, aspartic acid, glycine, serine, threonine
or valine.
[0111] A "cavity" refers to at least one amino acid side chain
which is recessed from the interface of a second polypeptide and
therefore accommodates a corresponding protuberance on the adjacent
interface of a first polypeptide. The cavity may exist in the
original interface or may be introduced synthetically (e.g. by
altering nucleic acid encoding the interface). In some embodiments,
nucleic acid encoding the interface of the second polypeptide is
altered to encode the cavity. To achieve this, the nucleic acid
encoding at least one "original" amino acid residue in the
interface of the second polypeptide is replaced with DNA encoding
at least one "import" amino acid residue which has a smaller side
chain volume than the original amino acid residue. It will be
appreciated that there can be more than one original and
corresponding import residue. In some embodiments, import residues
for the formation of a cavity are naturally occurring amino acid
residues selected from alanine (A), serine (S), threonine (T) and
valine (V). In some embodiments, an import residue is serine,
alanine or threonine. In some embodiments, the original residue for
the formation of the cavity has a large side chain volume, such as
tyrosine, arginine, phenylalanine or tryptophan. A mutation to
introduce a "cavity" may be referred to as a "hole mutation."
[0112] The protuberance is "positionable" in the cavity which means
that the spatial location of the protuberance and cavity on the
interface of a first polypeptide and second polypeptide
respectively and the sizes of the protuberance and cavity are such
that the protuberance can be located in the cavity without
significantly perturbing the normal association of the first and
second polypeptides at the interface. Since protuberances such as
Tyr, Phe and Trp do not typically extend perpendicularly from the
axis of the interface and have preferred conformations, the
alignment of a protuberance with a corresponding cavity may, in
some instances, rely on modeling the protuberance/cavity pair based
upon a three-dimensional structure such as that obtained by X-ray
crystallography or nuclear magnetic resonance (NMR). This can be
achieved using widely accepted techniques in the art.
[0113] In some embodiments, a knob mutation in an IgG1 constant
region is T366W. In some embodiments, a hole mutation in an IgG1
constant region comprises one or more mutations selected from
T366S, L368A and Y407V. In some embodiments, a hole mutation in an
IgG1 constant region comprises T366S, L368A and Y407V.
[0114] In some embodiments, a knob mutation in an IgG4 constant
region is T366W. In some embodiments, a hole mutation in an IgG4
constant region comprises one or more mutations selected from
T366S, L368A, and Y407V. In some embodiments, a hole mutation in an
IgG4 constant region comprises T366S, L368A, and Y407V.
[0115] Multi-specific antibodies may also be made by engineering
electrostatic steering effects for making antibody Fc-heterodimeric
molecules (WO 2009/089004A1); cross-linking two or more antibodies
or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et
al., Science, 229: 81 (1985)); using leucine zippers to produce
bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992)); using "diabody" technology for making
bispecific antibody fragments (Hollinger et al., Proc. Natl. Acad.
Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)
dimers (Gruber et al., J. Immunol., 152:5368 (1994)); and preparing
trispecific antibodies as described, e.g., in Tutt et al. J.
Immunol. 147: 60 (1991).
[0116] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies" or "dual-variable
domain immunoglobulins" (DVDs) are also included herein (US
2006/0025576A1, and Wu et al. (2007) Nature Biotechnology).
[0117] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant. The Fc region
variant may comprise a human Fc region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions.
[0118] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc(RIII only, whereas monocytes express Fc(RI,
Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is
summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays
to assess ADCC activity of a molecule of interest is described in
U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l
Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see
Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).
Alternatively, non-radioactive assays methods may be employed (see,
for example, ACTI.TM. non-radioactive cytotoxicity assay for flow
cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox
96.RTM. non-radioactive cytotoxicity assay (Promega, Madison,
Wis.). Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in an animal model such as
that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA
95:652-656 (1998). C1q binding assays may also be carried out to
confirm that the antibody is unable to bind C1q and hence lacks CDC
activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879
and WO 2005/100402. To assess complement activation, a CDC assay
may be performed (see, for example, Gazzano-Santoro et al., J.
Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood
101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood
103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the art
(Petkova, S. B. et al., Intl. Immunol. 18(12):1759-1769
(2006)).
[0119] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0120] In certain embodiments Pro329 of a wild-type human Fc region
is substituted with glycine or arginine or an amino acid residue
large enough to destroy the proline sandwich within the
Fc/Fc.gamma. receptor interface, that is formed between the
proline329 of the Fc and tryptophan residues Trp 87 and Trp 110 of
FcgRIII (Sondermann et al.: Nature 406, 267-273 (20 Jul. 2000)). In
a further embodiment, at least one further amino acid substitution
in the Fc variant is S228P, E233P, L234A, L235A, L235E, N297A,
N297D, or P331S and still in another embodiment said at least one
further amino acid substitution is L234A and L235A of the human
IgG1 Fc region or S228P and L235E of the human IgG4 Fc region (U.S.
Pat. No. 8,969,526 which is incorporated by reference in its
entirety).
[0121] In certain embodiments, a polypeptide comprises the Fc
variant of a wild-type human IgG Fc region wherein the polypeptide
has Pro329 of the human IgG Fc region substituted with glycine and
wherein the Fc variant comprises at least two further amino acid
substitutions at L234A and L235A of the human IgG1 Fc region or
S228P and L235E of the human IgG4 Fc region, and wherein the
residues are numbered according to the EU index of Kabat (U.S. Pat.
No. 8,969,526 which is incorporated by reference in its entirety).
In certain embodiments, the polypeptide comprising the P329G, L234A
and L235A substitutions exhibit a reduced affinity to the human
Fc.gamma.RIIIA and Fc.gamma.RIIA, for down-modulation of ADCC to at
least 20% of the ADCC induced by the polypeptide comprising the
wild type human IgG Fc region, and/or for down-modulation of ADCP
(U.S. Pat. No. 8,969,526 which is incorporated by reference in its
entirety).
[0122] In a specific embodiment the polypeptide comprising an Fc
variant of a wild type human Fc polypeptide comprises a triple
mutation: an amino acid substitution at position Pro329, a L234A
and a L235A mutation (P329/LALA) (U.S. Pat. No. 8,969,526 which is
incorporated by reference in its entirety). In specific
embodiments, the polypeptide comprises the following amino acid
substitutions: P329G, L234A, and L235A.
[0123] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001)).
[0124] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0125] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al., J. Immunol. 164: 4178-4184 (2000).
[0126] Antibodies with increased half-lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc
region residue 434 (U.S. Pat. No. 7,371,826).
[0127] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
[0128] 7. Cysteine Engineered Antibody Variants
[0129] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "THIOMAB.TM. antibody," in
which one or more residues of an antibody are substituted with
cysteine residues. In particular embodiments, the substituted
residues occur at accessible sites of the antibody. By substituting
those residues with cysteine, reactive thiol groups are thereby
positioned at accessible sites of the antibody and may be used to
conjugate the antibody to the drug moiety to create an
immunoconjugate, as described further herein. In certain
embodiments, any one or more of the following residues may be
substituted with cysteine: V205 (Kabat numbering) of the light
chain; K149 (Kabat numbering) of the light chain; A118 (EU
numbering) of the heavy chain; and 5400 (EU numbering) of the heavy
chain Fc region. Cysteine engineered antibodies may be generated as
described, e.g., in U.S. Pat. No. 7,521,541.
[0130] In some aspects, a THIOMAB.TM. antibody comprises one of the
heavy or light chain cysteine substitutions listed in Table 1
below.
TABLE-US-00001 TABLE 1 Screening GNE Kabat Chain Mutation Mutation
Mutation (HC/LC) Residue Site # Site # Site # LC T 22 22 22 LC K 39
39 39 LC Y 49 49 49 LC Y 55 55 55 LC T 85 85 85 LC T 97 97 97 LC I
106 106 106 LC R 108 108 108 LC R 142 142 142 LC K 149 149 149 LC V
205 205 205 HC T 117 114 110 HC A 143 140 136 HC L 177 174 170 HC L
182 179 175 HC T 190 187 183 HC T 212 209 205 HC V 265 262 258 HC G
374 371 367 HC Y 376 373 369 HC E 385 382 378 HC S 427 424 420 HC N
437 434 430 HC Q 441 438 434
[0131] In other aspects, a THIOMAB.TM. antibody comprises one of
the heavy chain cysteine substitutions listed in Table 2.
TABLE-US-00002 TABLE 2 Screening GNE Kabat Chain Mutation Mutation
Mutation (HC/LC) Residue Site # Site # Site # HC T 117 114 110 HC A
143 140 136 HC L 177 174 170 HC L 182 179 175 HC T 190 187 183 HC T
212 209 205 HC V 265 262 258 HC G 374 371 367 HC Y 376 373 369 HC E
385 382 378 HC S 427 424 420 HC N 437 434 430 HC Q 441 438 434
[0132] In some other aspects, a THIOMAB.TM. antibody comprises one
of the light chain cysteine substitutions listed in Table 3.
TABLE-US-00003 TABLE 3 Screening GNE Kabat Chain Mutation Mutation
Mutation (HC/LC) Residue Site # Site # Site # LC I 106 106 106 LC R
108 108 108 LC R 142 142 142 LC K 149 149 149
[0133] In some other aspects, a THIOMAB.TM. antibody comprises one
of the heavy or light chain cysteine substitutions listed in Table
4.
TABLE-US-00004 TABLE 4 Screening GNE Kabat Chain Mutation Mutation
Mutation (HC/LC) Residue Site # Site # Site # LC K 149 149 149 HC A
143 140 136 HC A 121 118 114
[0134] Cysteine engineered antibodies which may be useful in the
antibody-drug conjugates of the invention in the treatment of
cancer include, but are not limited to, antibodies against cell
surface receptors and tumor-associated antigens (TAA).
Tumor-associated antigens are known in the art, and can be prepared
for use in generating antibodies using methods and information
which are well known in the art. In attempts to discover effective
cellular targets for cancer diagnosis and therapy, researchers have
sought to identify transmembrane or otherwise tumor-associated
polypeptides that are specifically expressed on the surface of one
or more particular type(s) of cancer cell as compared to on one or
more normal non-cancerous cell(s). Often, such tumor-associated
polypeptides are more abundantly expressed on the surface of the
cancer cells as compared to on the surface of the non-cancerous
cells. The identification of such tumor-associated cell surface
antigen polypeptides has given rise to the ability to specifically
target cancer cells for destruction via antibody-based
therapies.
[0135] Examples of tumor-associated antigens TAA include, but are
not limited to, TAA (1)-(53) listed herein. For convenience,
information relating to these antigens, all of which are known in
the art, is listed herein and includes names, alternative names,
Genbank accession numbers and primary reference(s), following
nucleic acid and protein sequence identification conventions of the
National Center for Biotechnology Information (NCBI). Nucleic acid
and protein sequences corresponding to TAA (1)-(53) are available
in public databases such as GenBank. Tumor-associated antigens
targeted by antibodies include all amino acid sequence variants and
isoforms possessing at least about 70%, 80%, 85%, 90%, or 95%
sequence identity relative to the sequences identified in the cited
references, or which exhibit substantially the same biological
properties or characteristics as a TAA having a sequence found in
the cited references. For example, a TAA having a variant sequence
generally is able to bind specifically to an antibody that binds
specifically to the TAA with the corresponding sequence listed. The
sequences and disclosure in the reference specifically recited
herein are expressly incorporated by reference.
[0136] Tumor-Associated Antigens
[0137] (1) BMPR1B (bone morphogenetic protein receptor-type IB,
Genbank accession no. NM_001203) ten Dijke, P., et al. Science 264
(5155):101-104 (1994), Oncogene 14 (11):1377-1382 (1997));
WO2004063362 (Claim 2); WO2003042661 (Claim 12); U52003134790-A1
(Page 38-39); WO2002102235 (Claim 13; Page 296); WO2003055443 (Page
91-92); WO200299122 (Example 2; Page 528-530); WO2003029421 (Claim
6); WO2003024392 (Claim 2; FIG. 112); WO200298358 (Claim 1; Page
183); WO200254940 (Page 100-101); WO200259377(Page 349-350);
WO200230268 (Claim 27; Page 376); WO200148204 (Example; FIG. 4)
NP_001194 bone morphogenetic protein receptor, type
IB/pid=NP_001194.1--Cross-references: MIM:603248; NP_001194.1;
AY065994.
[0138] (2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486)
Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395
(6699):288-291 (1998), Gaugitsch, H. W., et al. (1992) J. Biol.
Chem. 267 (16):11267-11273); WO2004048938 (Example 2); WO2004032842
(Example IV); WO2003042661 (Claim 12); WO2003016475 (Claim 1);
WO200278524 (Example 2); WO200299074 (Claim 19; Page 127-129);
WO200286443 (Claim 27; Pages 222, 393); WO2003003906 (Claim 10;
Page 293); WO200264798 (Claim 33; Page 93-95); WO200014228 (Claim
5; Page 133-136); US2003224454 (FIG. 3); WO2003025138 (Claim 12;
Page 150); NP_003477 solute carrier family 7 (cationic amino acid
transporter, y+system), member 5/pid=NP_003477.3--Homo sapiens
Cross-references: MIM:600182; NP_003477.3; NM_015923;
NM_003486_1.
[0139] (3) STEAP1 (six transmembrane epithelial antigen of
prostate, Genbank accession no. NM_012449) Cancer Res. 61 (15),
5857-5860 (2001), Hubert, R. S., et al. (1999) Proc. Natl. Acad.
Sci. U.S.A. 96 (25):14523-14528); WO2004065577 (Claim 6);
WO2004027049 (FIG. 1L); EP1394274 (Example 11); WO2004016225 (Claim
2); WO2003042661 (Claim 12); US2003157089 (Example 5); US2003185830
(Example 5); US2003064397 (FIG. 2); WO200289747 (Example 5; Page
618-619); WO2003022995 (Example 9; FIG. 13A, Example 53; Page 173,
Example 2; FIG. 2A); NP_036581 six transmembrane epithelial antigen
of the prostate Cross-references: MIM:604415; NP_036581.1;
NM_012449_1.
[0140] (4) 0772P (CA125, MUC16, Genbank accession no. AF361486) J.
Biol. Chem. 276 (29):27371-27375 (2001)); WO2004045553 (Claim 14);
WO200292836 (Claim 6; FIG. 12); WO200283866 (Claim 15; Page
116-121); US2003124140 (Example 16); U.S. Pat. No. 798,959.
Cross-references: GI:34501467; AAK74120.3; AF361486_1.
[0141] (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,
mesothelin, Genbank accession no. NM_005823) Yamaguchi, N., et al.
Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A.
96 (20):11531-11536 (1999), Proc. Natl. Acad. Sci. U.S.A. 93
(1):136-140 (1996), J. Biol. Chem. 270 (37):21984-21990 (1995));
WO2003101283 (Claim 14); (WO2002102235 (Claim 13; Page 287-288);
WO2002101075 (Claim 4; Page 308-309); WO200271928 (Page 320-321);
WO9410312 (Page 52-57); Cross-references: MIM:601051; NP_005814.2;
NM_005823_1.
[0142] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family
34 (sodium phosphate), member 2, type II sodium-dependent phosphate
transporter 3b, Genbank accession no. NM_006424) J. Biol. Chem. 277
(22):19665-19672 (2002), Genomics 62 (2):281-284 (1999), J. A., et
al. (1999) Biochem. Biophys. Res. Commun. 258 (3):578-582);
WO2004022778 (Claim 2); EP1394274 (Example 11); WO2002102235 (Claim
13; Page 326); EP875569 (Claim 1; Page 17-19); WO200157188 (Claim
20; Page 329); WO2004032842 (Example IV); WO200175177 (Claim 24;
Page 139-140); Cross-references: MIM:604217; NP_006415.1;
NM_006424_1.
[0143] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMASB, SEMAG,
Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type
1 and type 1-like), transmembrane domain (TM) and short cytoplasmic
domain, (semaphorin) 5B, Genbank accession no. AB040878) Nagase T.,
et al. (2000) DNA Res. 7 (2):143-150); WO2004000997 (Claim 1);
WO2003003984 (Claim 1); WO200206339 (Claim 1; Page 50); WO200188133
(Claim 1; Page 41-43, 48-58); WO2003054152 (Claim 20); WO2003101400
(Claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew;
HGNC:10737.
[0144] (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA
2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no.
AY358628); Ross et al. (2002) Cancer Res. 62:2546-2553;
US2003129192 (Claim 2); US2004044180 (Claim 12); US2004044179
(Claim 11); US2003096961 (Claim 11); US2003232056 (Example 5);
WO2003105758 (Claim 12); US2003206918 (Example 5); EP1347046 (Claim
1); WO2003025148 (Claim 20); Cross-references: GI:37182378;
AAQ88991.1; AY358628_1.
[0145] (9) ETBR (Endothelin type B receptor, Genbank accession no.
AY275463); Nakamuta M., et al. Biochem. Biophys. Res. Commun. 177,
34-39, 1991; Ogawa Y., et al. Biochem. Biophys. Res. Commun. 178,
248-255, 1991; Arai H., et al. Jpn. Circ. J. 56, 1303-1307, 1992;
Arai H., et al. J. Biol. Chem. 268, 3463-3470, 1993; Sakamoto A.,
Yanagisawa M., et al. Biochem. Biophys. Res. Commun. 178, 656-663,
1991; Elshourbagy N. A., et al. J. Biol. Chem. 268, 3873-3879,
1993; Haendler B., et al. J. Cardiovasc. Pharmacol. 20, s1-S4,
1992; Tsutsumi M., et al. Gene 228, 43-49, 1999; Strausberg R. L.,
et al. Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002;
Bourgeois C., et al. J. Clin. Endocrinol. Metab. 82, 3116-3123,
1997; Okamoto Y., et al. Biol. Chem. 272, 21589-21596, 1997;
Verheij J. B., et al. Am. J. Med. Genet. 108, 223-225, 2002;
Hofstra R. M. W., et al. Eur. J. Hum. Genet. 5, 180-185, 1997;
Puffenberger E. G., et al. Cell 79, 1257-1266, 1994; Attie T., et
al., Hum. Mol. Genet. 4, 2407-2409, 1995; Auricchio A., et al. Hum.
Mol. Genet. 5:351-354, 1996; Amiel J., et al. Hum. Mol. Genet. 5,
355-357, 1996; Hofstra R. M. W., et al. Nat. Genet. 12, 445-447,
1996; Svensson P. J., et al. Hum. Genet. 103, 145-148, 1998; Fuchs
S., et al. Mol. Med. 7, 115-124, 2001; Pingault V., et al. (2002)
Hum. Genet. 111, 198-206; WO2004045516 (Claim 1); WO2004048938
(Example 2); WO2004040000 (Claim 151); WO2003087768 (Claim 1);
WO2003016475 (Claim 1); WO2003016475 (Claim 1); WO200261087 (FIG.
1); WO2003016494 (FIG. 6); WO2003025138 (Claim 12; Page 144);
WO200198351 (Claim 1; Page 124-125); EP522868 (Claim 8; FIG. 2);
WO200177172 (Claim 1; Page 297-299); US2003109676; U.S. Pat. No.
6,518,404 (FIG. 3); U.S. Pat. No. 5,773,223 (Claim 1a; Col 31-34);
WO2004001004.
[0146] (10) MSG783 (RNF124, hypothetical protein F1120315, Genbank
accession no. NM_017763); WO2003104275 (Claim 1); WO2004046342
(Example 2); WO2003042661 (Claim 12); WO2003083074 (Claim 14; Page
61); WO2003018621 (Claim 1); WO2003024392 (Claim 2; FIG. 93);
WO200166689 (Example 6); Cross-references: LocusID:54894;
NP_060233.2; NM_017763_1.
[0147] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2,
STMP, prostate cancer associated gene 1, prostate cancer associated
protein 1, six transmembrane epithelial antigen of prostate 2, six
transmembrane prostate protein, Genbank accession no. AF455138)
Lab. Invest. 82 (11):1573-1582 (2002)); WO2003087306; US2003064397
(Claim 1; FIG. 1); WO200272596 (Claim 13; Page 54-55); WO200172962
(Claim 1; FIG. 4B); WO2003104270 (Claim 11); WO2003104270 (Claim
16); US2004005598 (Claim 22); WO2003042661 (Claim 12); US2003060612
(Claim 12; FIG. 10); WO200226822 (Claim 23; FIG. 2); WO200216429
(Claim 12; FIG. 10); Cross-references: GI:22655488; AAN04080.1;
AF455138_1.
[0148] (12) TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient
receptor potential cation channel, subfamily M, member 4, Genbank
accession no. NM_017636) Xu, X. Z., et al. Proc. Natl. Acad. Sci.
U.S.A. 98 (19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J.
Biol. Chem. 278 (33):30813-30820 (2003)); US2003143557 (Claim 4);
WO200040614 (Claim 14; Page 100-103); WO200210382 (Claim 1; FIG.
9A); WO2003042661 (Claim 12); WO200230268 (Claim 27; Page 391);
US2003219806 (Claim 4); WO200162794 (Claim 14; FIG. 1A-D);
Cross-references: MIM:606936; NP_060106.2; NM_017636_1.
[0149] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,
teratocarcinoma-derived growth factor, Genbank accession no.
NP_003203 or NM_003212) Ciccodicola, A., et al. EMBO J. 8
(7):1987-1991 (1989), Am. J. Hum. Genet. 49 (3):555-565 (1991));
US2003224411 (Claim 1); WO2003083041 (Example 1); WO2003034984
(Claim 12); WO200288170 (Claim 2; Page 52-53); WO2003024392 (Claim
2; FIG. 58); WO200216413 (Claim 1; Page 94-95, 105); WO200222808
(Claim 2; FIG. 1); U.S. Pat. No. 5,854,399 (Example 2; Col 17-18);
U.S. Pat. No. 5,792,616 (FIG. 2); Cross-references: MIM:187395;
NP_003203.1; NM_003212_1.
[0150] (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein
Barr virus receptor) or Hs.73792 Genbank accession no. M26004)
Fujisaku et al. (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J.
J., et al. J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al.
Proc. Natl. Acad. Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al.
Mol. Immunol. 35, 1025-1031, 1998; Weis J. J., et al. Proc. Natl.
Acad. Sci. U.S.A. 83, 5639-5643, 1986; Sinha S. K., et al. (1993)
J. Immunol. 150, 5311-5320; WO2004045520 (Example 4); US2004005538
(Example 1); WO2003062401 (Claim 9); WO2004045520 (Example 4);
WO9102536 (FIGS. 9.1-9.9); WO2004020595 (Claim 1); Accession:
P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.
[0151] (15) CD79b (CD79B, CD79.beta., IGb
(immunoglobulin-associated beta), B29, Genbank accession no.
NM_000626 or 11038674) Proc. Natl. Acad. Sci. U.S.A. (2003) 100
(7):4126-4131, Blood (2002) 100 (9):3068-3076, Muller et al. (1992)
Eur. J. Immunol. 22 (6):1621-1625); WO2004016225 (Claim 2, FIG.
140); WO2003087768, US2004101874 (Claim 1, page 102); WO2003062401
(Claim 9); WO200278524 (Example 2); US2002150573 (Claim 5, page
15); U.S. Pat. No. 5,644,033; WO2003048202 (Claim 1, pages 306 and
309); WO 99/558658, U.S. Pat. No. 6,534,482 (Claim 13, FIG. 17A/B);
WO200055351 (Claim 11, pages 1145-1146); Cross-references:
MIM:147245; NP_000617.1; NM_000626_1.
[0152] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing
phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession
no. NM_030764, AY358130) Genome Res. 13 (10):2265-2270 (2003),
Immunogenetics 54 (2):87-95 (2002), Blood 99 (8):2662-2669 (2002),
Proc. Natl. Acad. Sci. U.S.A. 98 (17):9772-9777 (2001), Xu, M. J.,
et al. (2001) Biochem. Biophys. Res. Commun. 280 (3):768-775;
WO2004016225 (Claim 2); WO2003077836; WO200138490 (Claim 5; FIG.
18D-1-18D-2); WO2003097803 (Claim 12); WO2003089624 (Claim 25);
Cross-references: MIM:606509; NP_110391.2; NM_030764_1.
[0153] (17) HER2 (ErbB2, Genbank accession no. M11730) Coussens L.,
et al. Science (1985) 230(4730):1132-1139); Yamamoto T., et al.
Nature 319, 230-234, 1986; Semba K., et al. Proc. Natl. Acad. Sci.
U.S.A. 82, 6497-6501, 1985; Swiercz J. M., et al. J. Cell Biol.
165, 869-880, 2004; Kuhns J. J., et al. J. Biol. Chem. 274,
36422-36427, 1999; Cho H.-S., et al. Nature 421, 756-760, 2003;
Ehsani A., et al. (1993) Genomics 15, 426-429; WO2004048938
(Example 2); WO2004027049 (FIG. 10; WO2004009622; WO2003081210;
WO2003089904 (Claim 9); WO2003016475 (Claim 1); US2003118592;
WO2003008537 (Claim 1); WO2003055439 (Claim 29; FIG. 1A-B);
WO2003025228 (Claim 37; FIG. 5C); WO200222636 (Example 13; Page
95-107); WO200212341 (Claim 68; FIG. 7); WO200213847 (Page 71-74);
WO200214503 (Page 114-117); WO200153463 (Claim 2; Page 41-46);
WO200141787 (Page 15); WO200044899 (Claim 52; FIG. 7); WO200020579
(Claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (Claim 3; Col 31-38);
WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7); WO2004043361
(Claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4);
Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761;
AAA35808.1.
[0154] (18) NCA (CEACAM6, Genbank accession no. M18728); Barnett
T., et al. Genomics 3, 59-66, 1988; Tawaragi Y., et al. Biochem.
Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R. L., et al.
Proc. Natl. Acad. Sci. U.S.A. 99:16899-16903, 2002; WO2004063709;
EP1439393 (Claim 7); WO2004044178 (Example 4); WO2004031238;
WO2003042661 (Claim 12); WO200278524 (Example 2); WO200286443
(Claim 27; Page 427); WO200260317 (Claim 2); Accession: P40199;
Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728.
[0155] (19) MDP (DPEP1, Genbank accession no. BC017023) Proc. Natl.
Acad. Sci. U.S.A. 99 (26):16899-16903 (2002)); WO2003016475 (Claim
1); WO200264798 (Claim 33; Page 85-87); JP05003790 (FIG. 6-8);
WO9946284 (FIG. 9); Cross-references: MIM:179780; AAH17023.1;
BC017023_1.
[0156] (20) IL20R.alpha. (IL20R.alpha., ZCYTOR7, Genbank accession
no. AF184971); Clark H. F., et al. Genome Res. 13, 2265-2270, 2003;
Mungall A. J., et al. Nature 425, 805-811, 2003; Blumberg H., et
al. Cell 104, 9-19, 2001; Dumoutier L., et al. J. Immunol. 167,
3545-3549, 2001; Parrish-Novak J., et al. J. Biol. Chem. 277,
47517-47523, 2002; Pletnev S., et al. (2003) Biochemistry
42:12617-12624; Sheikh F., et al. (2004) J. Immunol. 172,
2006-2010; EP1394274 (Example 11); US2004005320 (Example 5);
WO2003029262 (Page 74-75); WO2003002717 (Claim 2; Page 63);
WO200222153 (Page 45-47); US2002042366 (Page 20-21); WO200146261
(Page 57-59); WO200146232 (Page 63-65); WO9837193 (Claim 1; Page
55-59); Accession: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971;
AAF01320.1.
[0157] (21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053)
Gary S. C., et al. Gene 256, 139-147, 2000; Clark H. F., et al.
Genome Res. 13, 2265-2270, 2003; Strausberg R. L., et al. Proc.
Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002; US2003186372 (Claim
11); US2003186373 (Claim 11); US2003119131 (Claim 1; FIG. 52);
US2003119122 (Claim 1; FIG. 52); US2003119126 (Claim 1);
US2003119121 (Claim 1; FIG. 52); US2003119129 (Claim 1);
US2003119130 (Claim 1); US2003119128 (Claim 1; FIG. 52);
US2003119125 (Claim 1); WO2003016475 (Claim 1); WO200202634 (Claim
1).
[0158] (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession
no. NM_004442) Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061
(1991) Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci.
21:309-345 (1998), Int. Rev. Cytol. 196:177-244 (2000));
WO2003042661 (Claim 12); WO200053216 (Claim 1; Page 41);
WO2004065576 (Claim 1); WO2004020583 (Claim 9); WO2003004529 (Page
128-132); WO200053216 (Claim 1; Page 42); Cross-references:
MIM:600997; NP_004433.2; NM_004442_1.
[0159] (23) ASLG659 (B7h, Genbank accession no. AX092328)
US20040101899 (Claim 2); WO2003104399 (Claim 11); WO2004000221
(FIG. 3); US2003165504 (Claim 1); US2003124140 (Example 2);
US2003065143 (FIG. 60); WO2002102235 (Claim 13; Page 299);
US2003091580 (Example 2); WO200210187 (Claim 6; FIG. 10);
WO200194641 (Claim 12; FIG. 7b); WO200202624 (Claim 13; FIG.
1A-1B); US2002034749 (Claim 54; Page 45-46); WO200206317 (Example
2; Page 320-321, Claim 34; Page 321-322); WO200271928 (Page
468-469); WO200202587 (Example 1; FIG. 1); WO200140269 (Example 3;
Pages 190-192); WO200036107 (Example 2; Page 205-207); WO2004053079
(Claim 12); WO2003004989 (Claim 1); WO200271928 (Page 233-234,
452-453); WO 0116318.
[0160] (24) PSCA (Prostate stem cell antigen precursor, Genbank
accession no. AJ297436) Reiter R. E., et al. Proc. Natl. Acad. Sci.
U.S.A. 95, 1735-1740, 1998; Gu Z., et al. Oncogene 19, 1288-1296,
2000; Biochem. Biophys. Res. Commun. (2000) 275(3):783-788;
WO2004022709; EP1394274 (Example 11); US2004018553 (Claim 17);
WO2003008537 (Claim 1); WO200281646 (Claim 1; Page 164);
WO2003003906 (Claim 10; Page 288); WO200140309 (Example 1; FIG.
17); US2001055751 (Example 1; FIG. 1b); WO200032752 (Claim 18; FIG.
1); WO9851805 (Claim 17; Page 97); WO9851824 (Claim 10; Page 94);
WO9840403 (Claim 2; FIG. 1B); Accession: 043653; EMBL; AF043498;
AAC39607.1.
[0161] (25) GEDA (Genbank accession No. AY260763); AAP14954 lipoma
HMGIC fusion-partner-like protein/pid=AAP14954.1--Homo sapiens
Species: Homo sapiens (human) WO2003054152 (Claim 20); WO2003000842
(Claim 1); WO2003023013 (Example 3, Claim 20); US2003194704 (Claim
45); Cross-references: GI:30102449; AAP14954.1; AY260763_1.
[0162] (26) BAFF-R (B cell-activating factor receptor, BLyS
receptor 3, BR3, Genbank accession No. AF116456); BAFF
receptor/pid=NP_443177.1--Homo sapiens Thompson, J. S., et al.
Science 293 (5537), 2108-2111 (2001); WO2004058309; WO2004011611;
WO2003045422 (Example; Page 32-33); WO2003014294 (Claim 35; FIG.
6B); WO2003035846 (Claim 70; Page 615-616); WO200294852 (Col
136-137); WO200238766 (Claim 3; Page 133); WO200224909 (Example 3;
FIG. 3); Cross-references: MIM:606269; NP_443177.1; NM_052945_1; AF
132600.
[0163] (27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8,
Lyb8, SIGLEC-2, F1122814, Genbank accession No. AK026467); Wilson
et al. (1991) J. Exp. Med. 173:137-146; WO2003072036 (Claim 1; FIG.
1); Cross-references: MIM:107266; NP_001762.1; NM_001771_1.
[0164] (28) CD79a (CD79A, CD79.alpha., immunoglobulin-associated
alpha, a B cell-specific protein that covalently interacts with Ig
beta (CD79B) and forms a complex on the surface with Ig M
molecules, transduces a signal involved in B-cell differentiation),
pI: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19q13.2, Genbank
accession No. NP_001774.10) WO2003088808, US20030228319;
WO2003062401 (Claim 9); US2002150573 (Claim 4, pages 13-14);
WO9958658 (Claim 13, FIG. 16); WO9207574 (FIG. 1); U.S. Pat. No.
5,644,033; Ha et al. (1992) J. Immunol. 148(5):1526-1531; Mueller
et al. (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al.
(1994) Immunogenetics 40(4):287-295; Preud'homme et al. (1992)
Clin. Exp. Immunol. 90(1):141-146; Yu et al. (1992) J. Immunol.
148(2) 633-637; Sakaguchi et al. (1988) EMBO J.
7(11):3457-3464.
[0165] (29) CXCR5 (Burkitt's lymphoma receptor 1, a G
protein-coupled receptor that is activated by the CXCL13 chemokine,
functions in lymphocyte migration and humoral defense, plays a role
in HIV-2 infection and perhaps development of AIDS, lymphoma,
myeloma, and leukemia); 372 aa, pI: 8.54 MW: 41959 TM: 7 [P] Gene
Chromosome: 11q23.3, Genbank accession No. NP_001707.1)
WO2004040000; WO2004015426; US2003105292 (Example 2); U.S. Pat. No.
6,555,339 (Example 2); WO200261087 (FIG. 1); WO200157188 (Claim 20,
page 269); WO200172830 (pages 12-13); WO200022129 (Example 1, pages
152-153, Example 2, pages 254-256); WO9928468 (Claim 1, page 38);
U.S. Pat. No. 5,440,021 (Example 2, col 49-52); WO9428931 (pages
56-58); WO9217497 (Claim 7, FIG. 5); Dobner et al. (1992) Eur. J.
Immunol. 22:2795-2799; Barella et al. (1995) Biochem. J.
309:773-779.
[0166] (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia
antigen) that binds peptides and presents them to CD4+T
lymphocytes); 273 aa, pI: 6.56 MW: 30820 TM: 1 [P] Gene Chromosome:
6p21.3, Genbank accession No. NP_002111.1) Tonnelle et al. (1985)
EMBO J. 4(11):2839-2847; Jonsson et al. (1989) Immunogenetics
29(6):411-413; Beck et al. (1992) J. Mol. Biol. 228:433-441;
Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99:16899-16903;
Servenius et al. (1987) J. Biol. Chem. 262:8759-8766; Beck et al.
(1996) J. Mol. Biol. 255:1-13; Naruse et al. (2002) Tissue Antigens
59:512-519; WO9958658 (Claim 13, FIG. 15); U.S. Pat. No. 6,153,408
(Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat. No.
6,011,146 (col 145-146); Kasahara et al. (1989) Immunogenetics
30(1):66-68; Larhammar et al. (1985) J. Biol. Chem.
260(26):14111-14119.
[0167] (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel
5, an ion channel gated by extracellular ATP, may be involved in
synaptic transmission and neurogenesis, deficiency may contribute
to the pathophysiology of idiopathic detrusor instability); 422
aa), pI: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3,
Genbank accession No. NP_002552.2) Le et al. (1997) FEBS Lett.
418(1-2):195-199; WO2004047749; WO2003072035 (Claim 10); Touchman
et al. (2000) Genome Res. 10:165-173; WO200222660 (Claim 20);
WO2003093444 (Claim 1); WO2003087768 (Claim 1); WO2003029277 (page
82).
[0168] (32) CD72 (B-cell differentiation antigen CD72, Lyb-2)
PROTEIN SEQUENCE Full maeaity . . . tafrfpd (1.359; 359 aa), pI:
8.66, MW: 40225 TM: 1 [P] Gene Chromosome: 9p13.3, Genbank
accession No. NP_001773.1) WO2004042346 (Claim 65); WO2003026493
(pages 51-52, 57-58); WO200075655 (pages 105-106); Von Hoegen et
al. (1990) J. Immunol. 144(12):4870-4877; Strausberg et al. (2002)
Proc. Natl. Acad. Sci USA 99:16899-16903.
[0169] (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane
protein of the leucine rich repeat (LRR) family, regulates B-cell
activation and apoptosis, loss of function is associated with
increased disease activity in patients with systemic lupus
erythematosis); 661 aa, pI: 6.20, MW: 74147 TM: 1 [P] Gene
Chromosome: 5q12, Genbank accession No. NP_005573.1) US2002193567;
WO9707198 (Claim 11, pages 39-42); Miura et al. (1996) Genomics
38(3):299-304; Miura et al. (1998) Blood 92:2815-2822;
WO2003083047; WO9744452 (Claim 8, pages 57-61); WO200012130 (pages
24-26).
[0170] (34) FcRH1 (Fc receptor-like protein 1, a putative receptor
for the immunoglobulin Fc domain that contains C2 type Ig-like and
ITAM domains, may have a role in B-lymphocyte differentiation); 429
aa, pI: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: 1q21-1q22,
Genbank accession No. NP_443170.1) WO2003077836; WO200138490 (Claim
6, FIG. 18E-1-18-E-2); Davis et al. (2001) Proc. Natl. Acad. Sci
USA 98(17):9772-9777; WO2003089624 (Claim 8); EP1347046 (Claim 1);
WO2003089624 (Claim 7).
[0171] (35) IRTA2 (Immunoglobulin superfamily receptor
translocation associated 2, a putative immunoreceptor with possible
roles in B cell development and lymphomagenesis; deregulation of
the gene by translocation occurs in some B cell malignancies); 977
aa, pI: 6.88 MW: 106468 TM: 1 [P] Gene Chromosome: 1q21, Genbank
accession No. Human:AF343662, AF343663, AF343664, AF343665,
AF369794, AF397453, AK090423, AK090475, AL834187, AY358085;
Mouse:AK089756, AY158090, AY506558; NP_112571.1. WO2003024392
(Claim 2, FIG. 97); Nakayama et al. (2000) Biochem. Biophys. Res.
Commun. 277(1):124-127; WO2003077836; WO200138490 (Claim 3, FIG.
18B-1-18B-2).
[0172] (36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative
transmembrane proteoglycan, related to the EGF/heregulin family of
growth factors and follistatin); 374 aa, NCBI Accession: AAD55776,
AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM:
605734; SwissProt Q9UIK5; Genbank accession No. AF179274; AY358907,
CAF85723, CQ782436 WO2004074320 (SEQ ID NO 810); JP2004113151 (SEQ
ID NOS 2, 4, 8); WO2003042661 (SEQ ID NO 580); WO2003009814 (SEQ ID
NO 411); EP1295944 (pages 69-70); WO200230268 (page 329);
WO200190304 (SEQ ID NO 2706); US2004249130; US2004022727;
WO2004063355; US2004197325; US2003232350; US2004005563;
US2003124579; Horie et al. (2000) Genomics 67:146-152; Uchida et
al. (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang et al.
(2000) Cancer Res. 60:4907-12; Glynne-Jones et al. (2001) Int J
Cancer. October 15; 94(2):178-84.
[0173] (37) PMEL17 (silver homologs SILV; D12S53E; PMEL17; SI;
SIL); ME20; gp100) BC001414; BT007202; M32295; M77348; NM_006928;
McGlinchey, R. P. et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106
(33), 13731-13736; Kummer, M. P. et al. (2009) J. Biol. Chem. 284
(4), 2296-2306.
[0174] (38) TMEFF1 (transmembrane protein with EGF-like and two
follistatin-like domains I; Tomoregulin-1); H7365; C9orf2; C9ORF2;
U19878; X83961; NM_080655; NM_003692; Harms, P. W. (2003) Genes
Dev. 17 (21), 2624-2629; Gery, S. et al. (2003) Oncogene 22
(18):2723-2727.
[0175] (39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR;
GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); U95847;
BC014962; NM_145793 NM_005264; Kim, M. H. et al. (2009) Mol. Cell.
Biol. 29 (8), 2264-2277; Treanor, J. J. et al. (1996) Nature 382
(6586):80-83.
[0176] (40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67,
RIG-E, SCA-2, TSA-1); NP_002337.1; NM_002346.2; de Nooij-van Dalen,
A. G. et al. (2003) Int. J. Cancer 103 (6), 768-774; Zammit, D. J.
et al. (2002) Mol. Cell. Biol. 22 (3):946-952.
[0177] (41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2);
NP_001007539.1; NM_001007538.1; Furushima, K. et al. (2007) Dev.
Biol. 306 (2), 480-492; Clark, H. F. et al. (2003) Genome Res. 13
(10):2265-2270.
[0178] (42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D,
MEGT1); NP_067079.2; NM_021246.2; Mallya, M. et al. (2002) Genomics
80 (1):113-123; Ribas, G. et al. (1999) J. Immunol. 163
(1):278-287.
[0179] (43) LGR5 (leucine-rich repeat-containing G protein-coupled
receptor 5; GPR49, GPR67); NP_003658.1; NM_003667.2; Salanti, G. et
al. (2009) Am. J. Epidemiol. 170 (5):537-545; Yamamoto, Y. et al.
(2003) Hepatology 37 (3):528-533.
[0180] (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1;
PTC; CDHF12; Hs.168114; RET51; RET-ELE1); NP_066124.1; NM_020975.4;
Tsukamoto, H. et al. (2009) Cancer Sci. 100 (10):1895-1901; Narita,
N. et al. (2009) Oncogene 28 (34):3058-3068.
[0181] (45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K;
HSJ001348; FLJ35226); NP_059997.3; NM_017527.3; Ishikawa, N. et al.
(2007) Cancer Res. 67 (24):11601-11611; de Nooij-van Dalen, A. G.
et al. (2003) Int. J. Cancer 103 (6):768-774.
[0182] (46) GPR19 (G protein-coupled receptor 19; Mm.4787);
NP_006134.1; NM_006143.2; Montpetit, A. and Sinnett, D. (1999) Hum.
Genet. 105 (1-2):162-164; O'Dowd, B. F. et al. (1996) FEBS Lett.
394 (3):325-329.
[0183] (47) GPR54 (KISS1 receptor, KISS1R; GPR54; HOT7T175;
AXOR12); NP_115940.2; NM_032551.4; Navenot, J. M. et al. (2009)
Mol. Pharmacol. 75 (6):1300-1306; Hata, K. et al. (2009) Anticancer
Res. 29 (2):617-623.
[0184] (48) ASPHD1 (aspartate beta-hydroxylase domain containing 1;
LOC253982); NP_859069.2; NM_181718.3; Gerhard, D. S. et al. (2004)
Genome Res. 14 (10B):2121-2127.
[0185] (49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3);
NP_000363.1; NM_000372.4; Bishop, D. T. et al. (2009) Nat. Genet.
41 (8):920-925; Nan, H. et al. (2009) Int. J. Cancer 125
(4):909-917.
[0186] (50) TMEM118 (ring finger protein, transmembrane 2; RNFT2;
FLJ14627); NP_001103373.1; NM_001109903.1; Clark, H. F. et al.
(2003) Genome Res. 13 (10):2265-2270; Scherer, S. E. et al. (2006)
Nature 440 (7082):346-351.
[0187] (51) GPR172A (G protein-coupled receptor 172A; GPCR41;
FLJ11856; D15Ertd747e); NP_078807.1; NM_024531.3; Ericsson, T. A.
et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100 (11):6759-6764;
Takeda, S. et al. (2002) FEBS Lett. 520 (1-3):97-101.
[0188] (52) CD33, a member of the sialic acid binding,
immunoglobulin-like lectin family, is a 67-kDa glycosylated
transmembrane protein. CD33 is expressed on most myeloid and
monocytic leukemia cells in addition to committed myelomonocytic
and erythroid progenitor cells. It is not seen on the earliest
pluripotent stem cells, mature granulocytes, lymphoid cells, or
nonhematopoietic cells (Sabbath et al., (1985) J. Clin. Invest.
75:756-56; Andrews et al., (1986) Blood 68:1030-5). CD33 contains
two tyrosine residues on its cytoplasmic tail, each of which is
followed by hydrophobic residues similar to the immunoreceptor
tyrosine-based inhibitory motif (ITIM) seen in many inhibitory
receptors.
[0189] (53) CLL-1 (CLEC12A, MICL, and DCAL2), encodes a member of
the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily.
Members of this family share a common protein fold and have diverse
functions, such as cell adhesion, cell-cell signaling, glycoprotein
turnover, and roles in inflammation and immune response. The
protein encoded by this gene is a negative regulator of granulocyte
and monocyte function. Several alternatively spliced transcript
variants of this gene have been described, but the full-length
nature of some of these variants has not been determined. This gene
is closely linked to other CTL/CTLD superfamily members in the
natural killer gene complex region on chromosome 12p13 (Drickamer K
(1999) Curr. Opin. Struct. Biol. 9 (5):585-90; van Rhenen A, et
al., (2007) Blood 110 (7):2659-66; Chen C H, et al. (2006) Blood
107 (4):1459-67; Marshall A S, et al. (2006) Eur. J. Immunol. 36
(8):2159-69; Bakker A B, et al. (2005) Cancer Res. 64 (22):8443-50;
Marshall A S, et al. (2004) J. Biol. Chem. 279 (15):14792-802).
CLL-1 has been shown to be a type II transmembrane receptor
comprising a single C-type lectin-like domain (which is not
predicted to bind either calcium or sugar), a stalk region, a
transmembrane domain and a short cytoplasmic tail containing an
ITIM motif.
[0190] Antibody Derivatives
[0191] In certain embodiments, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer is attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0192] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
[0193] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. Such
nucleic acid may encode an amino acid sequence comprising the VL
and/or an amino acid sequence comprising the VH of the antibody
(e.g., the light and/or heavy chains of the antibody). In a further
embodiment, one or more vectors (e.g., expression vectors)
comprising such nucleic acid are provided. In a further embodiment,
a host cell comprising such nucleic acid is provided. In one such
embodiment, a host cell comprises (e.g., has been transformed
with): (1) a vector comprising a nucleic acid that encodes an amino
acid sequence comprising the VL of the antibody and an amino acid
sequence comprising the VH of the antibody, or (2) a first vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and a second vector comprising a
nucleic acid that encodes an amino acid sequence comprising the VH
of the antibody. In one embodiment, the host cell is eukaryotic,
e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0,
NS0, Sp20 cell).
[0194] For recombinant production of an antibody, nucleic acid
encoding an antibody, e.g., as described herein, is isolated and
inserted into one or more vectors for further cloning and/or
expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0195] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular
Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.,
2003), pp. 245-254, describing expression of antibody fragments in
E. coli.) After expression, the antibody may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified.
[0196] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0197] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0198] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0199] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham et al.,
J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse
sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African
green monkey kidney cells (VERO-76); human cervical carcinoma cells
(HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL
3A); human lung cells (W138); human liver cells (Hep G2); mouse
mammary tumor (MMT 060562); TRI cells, as described, e.g., in
Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5
cells; and FS4 cells. Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR.sup.-CHO
cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980));
and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268
(2003).
[0200] Antibody-Drug Conjugates
[0201] The invention provides antibody-drug conjugates having an
antibody herein conjugated to one or more calicheamicin derivative
compounds. More particularly, the present disclosure provides an
antibody-drug conjugate wherein the calicheamicin derivative
compound is directly conjugated to the antibody by means of a
covalent bond, rather than the more conventional approach of a
linker, a linker-spacer, a linker-reactive group, or the like.
[0202] Antibody-drug conjugates allow for the targeted delivery of
a drug moiety to a tumor, and, in some embodiments intracellular
accumulation therein, where systemic administration of unconjugated
drugs may result in unacceptable levels of toxicity to normal cells
(Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).
[0203] Antibody-drug conjugates are targeted chemotherapeutic
molecules which combine properties of both antibodies and cytotoxic
drugs by targeting potent cytotoxic drugs to antigen-expressing
tumor cells (Teicher, B. A. (2009) Current Cancer Drug Targets
9:982-1004), thereby enhancing the therapeutic index by maximizing
efficacy and minimizing off-target toxicity (Carter, P. J. and
Senter P. D. (2008) The Cancer Jour. 14(3):154-169; Chari, R. V.
(2008) Acc. Chem. Res. 41:98-107.
[0204] The antibody-drug conjugate compounds of the invention
include those with anticancer activity. In some embodiments, the
antibody-drug conjugate compounds include an antibody directly
conjugated to the calicheamicin drug moiety or derivative (i.e.,
the antibody is directly attached or bound to the calicheamicin
drug moiety or derivative, after loss of a leaving group and
without a linking group or moiety present there between). The
antibody-drug conjugates of the invention selectively deliver an
effective dose of a drug to tumor tissue, whereby greater
selectivity (i.e., a lower efficacious dose) may be achieved while
increasing the therapeutic index ("therapeutic window").
[0205] As depicted below, an exemplary embodiment of an
antibody-drug conjugate compound comprises an antibody (Ab) which
targets a tumor cell and a calicheamicin drug moiety (D) that is
directly attached thereto by a covalent bond.
##STR00003##
In such embodiments, R is suitably selected from H, --C(O)R.sup.1,
--C(O)NR.sup.1R.sup.2, --S(O).sub.2R.sup.1, and
--S(O).sub.2NR.sup.2R.sup.1. R.sup.1 and R.sup.2 may be
independently selected from hydrogen, optionally substituted
C.sub.1-6 alkyl and C.sub.6-20 aryl. In some particular aspects, R
may be --C(O)CH.sub.3. p refers to the equivalents of calicheamicin
per Ab equivalent.
[0206] In some aspects Ab is an antibody which binds to one or more
tumor-associated antigens or cell-surface receptors as described
elsewhere herein. In some other aspects, Ab is selected from
BMPR1B, E16, STEAP1, MUC16, MPF, Napi2b, Sema 5b, PSCA hlg, ETBR,
MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP,
IL20R.alpha., Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22,
CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, FcRH5, TENB2,
PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, Ly6K,
GPR19, GPR54, ASPHD1, Tyrosinase, TMEM118, GPR172A, CD33 and CLL-1.
In yet other aspects, Ab is a cysteine-engineered antibody.
Suitable cysteine-engineered antibody is a mutant selected from LC
K149C, HC A140, HC A118C, and HC L177C. In still other aspects, Ab
is selected from anti-HER2 4D5, anti-CD22, anti-CD33, anti-Ly6E,
anti-Napi3b, anti-HER2 7C2, and anti-CLL-1.
[0207] Drug loading is represented by p, the number of drug
moieties per antibody in a molecule of Formula I. Drug loading may
range from 1 to 20 drug moieties (D) per antibody. Antibody-drug
conjugates of Formula I include collections of antibodies
conjugated with a range of drug moieties, from 1 to 20. In some
embodiments, the number of drug moieties that can be conjugated to
an antibody is limited by the number of free cysteine residues. In
some embodiments, free cysteine residues are introduced into the
antibody amino acid sequence by the methods described herein. In
such aspects, p may be 1, 2, 3, 4, 5, 6, 7, or 8, and ranges
thereof, such as from 1 to 8 or from 2 to 5. In any such aspect, p
and n are equal (i.e., p=n=1, 2, 3, 4, 5, 6, 7, or 8, or some range
there between). Exemplary antibody-drug conjugates of Formula I
include, but are not limited to, antibodies that have 1, 2, 3, or 4
engineered cysteine amino acids (Lyon, R. et al. (2012) Methods in
Enzym. 502:123-138). In some embodiments, one or more free cysteine
residues are already present in an antibody, without the use of
engineering, in which case the existing free cysteine residues may
be used to conjugate the antibody to a drug. In some embodiments,
an antibody is exposed to reducing conditions prior to conjugation
of the antibody in order to generate one or more free cysteine
residues. The average number of drug moieties per antibody (DAR) in
preparations of antibody-drug conjugates from conjugation reactions
may be characterized by conventional means such as mass
spectroscopy, ELISA assay, and HPLC. The quantitative distribution
of antibody-drug conjugates in terms of p may also be determined.
In some instances, separation, purification, and characterization
of homogeneous antibody-drug conjugates where p is a certain value
from antibody-drug conjugates with other drug loadings may be
achieved by means such as reverse phase HPLC or
electrophoresis.
[0208] For some antibody-drug conjugates, p may be limited by the
number of attachment sites on the antibody. For example, where the
attachment is a cysteine thiol, as in certain exemplary embodiments
described herein, an antibody may have only one or a limited number
of cysteine thiol groups, or may have only one or a limited number
of sufficiently reactive thiol groups, to which the drug may be
attached. In certain embodiments, higher drug loading, e.g. p>5,
may cause aggregation, insolubility, toxicity, or loss of cellular
permeability of certain antibody-drug conjugates. In certain
embodiments, the average drug loading for an antibody-drug
conjugate ranges from 1 to about 8; from about 2 to about 6; or
from about 3 to about 5. Indeed, it has been shown that for certain
antibody-drug conjugates, the optimal ratio of drug moieties per
antibody may be less than 8, and may be between about 2 to about 5
(see, e.g., U.S. Pat. No. 7,498,298).
[0209] In certain embodiments, fewer than the theoretical maximum
of drug moieties are conjugated to an antibody during a conjugation
reaction. An antibody may contain, for example, lysine residues
that do not react with the drug, as discussed herein. Generally,
antibodies do not contain many free and reactive cysteine thiol
groups which may be linked to a drug moiety; indeed most cysteine
thiol residues in antibodies exist as disulfide bridges. In certain
embodiments, an antibody may be reduced with a reducing agent such
as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under
partial or total reducing conditions, to generate reactive cysteine
thiol groups. In certain embodiments, an antibody is subjected to
denaturing conditions to reveal reactive nucleophilic groups such
as lysine or cysteine.
[0210] The loading (drug/antibody ratio) of an antibody-drug
conjugate may be controlled in different ways, and for example, by:
(i) limiting the molar excess of the drug relative to antibody,
(ii) limiting the conjugation reaction time or temperature, and
(iii) partial or limiting reductive conditions for cysteine thiol
modification.
[0211] It is to be understood that where more than one nucleophilic
group reacts with a drug, then the resulting product is a mixture
of antibody-drug conjugate compounds with a distribution of one or
more drug moieties attached to an antibody. The average number of
drugs per antibody may be calculated from the mixture by a dual
ELISA antibody assay, which is specific for antibody and specific
for the drug. Individual antibody-drug conjugate molecules may be
identified in the mixture by mass spectroscopy and separated by
HPLC, e.g. hydrophobic interaction chromatography (see, e.g.,
McDonagh et al. (2006) Prot. Engr. Design & Selection
19(7):299-307; Hamblen et al. (2004) Clin. Cancer Res.
10:7063-7070; Hamblen, K. J., et al. "Effect of drug loading on the
pharmacology, pharmacokinetics, and toxicity of an anti-CD30
antibody-drug conjugate," Abstract No. 624, American Association
for Cancer Research, 2004 Annual Meeting, Mar. 27-31, 2004,
Proceedings of the AACR, Volume 45, March 2004; Alley, S. C., et
al. "Controlling the location of drug attachment in antibody-drug
conjugates," Abstract No. 627, American Association for Cancer
Research, 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings of the
AACR, Volume 45, March 2004). In certain embodiments, a homogeneous
antibody-drug conjugate with a single loading value may be isolated
from the conjugation mixture by electrophoresis or
chromatography.
[0212] In some other aspects of the present disclosure,
calicheamicin derivative compositions comprising a thiopyridyl
leaving group or a benzimidazole leaving group are provided. Such
compositions are termed "activated calicheamicin." Activated
calicheamicin may then be conjugated with an antibody as described
elsewhere herein. Exemplary calicheamicin derivative-leaving group
compositions are depicted below as Formulae I and
##STR00004##
In such aspects, R is selected from H, --C(O)R.sup.1,
--C(O)NR.sup.1R.sup.2, --S(O).sub.2R.sup.1, and
--S(O).sub.2NR.sup.2R.sup.1; R.sup.1 and R.sup.2 are independently
selected from C.sub.1-C.sub.6 alkyl and C.sub.6-C.sub.20 aryl;
R.sup.3 is selected from NO.sub.2, Cl, F, CN, CO.sub.2H, and Br;
and q is 0, 1, or 2.
[0213] In an exemplary embodiment, R is --C(O)CH.sub.3.
[0214] In an exemplary embodiment, R.sup.3 is NO.sub.2 and q is
1.
[0215] In one exemplary embodiment, the drug intermediate has the
Formula Ia:
##STR00005##
[0216] In another exemplary embodiment, the drug intermediate has
the formula IIa:
##STR00006##
[0217] Formation of activated calicheamicin is represented by the
following scheme where X is a leaving group as defined elsewhere
herein:
##STR00007##
In the reaction, the methyltrisulfide moiety of calicheamicin is
reacted with the leaving group thiol moiety to form a disulfide
where S.sub.c refers to a calicheamicin sulfur atom and S, refers
to a leaving group sulfur atom. Examples of this reaction are
given, for instance, in U.S. Pat. Nos. 5,053,394, 5,712,374,
5,714,586, 5,739,116 and 5,767,285. Non limiting examples of
suitable solvents for forming reaction mixtures include polar
aprotic solvents such as acetonitrile, tetrahydrofuran, ethyl
acetate, acetone, N,N-dimethylformamide, dimethylsulfoxide and
dichloromethane. Calicheamicin concentration in the reaction
mixture is not narrowly critical and may suitably vary from about
0.0005 to about 0.01 mmol/mL, such as about 0.0005, about 0.001,
about 0.005, or about 0.01 mmol/mL. The leaving group is present in
stoichiometric excess, such as about 1.1:1 mole/mole, about 1.5:1
mole/mole, about 2:1 mole/mole, about 2.5:1 mole/mole or about 3:1
mole/mole. The reaction temperature is suitably about -30.degree.
C., about -20.degree. C., about -10.degree. C., about 0.degree. C.,
or about 10.degree. C., and ranges thereof, such as from about
-30.degree. C. to about 10.degree. C., from about -30.degree. C. to
about 0.degree. C., or from about -30.degree. C. to about
-10.degree. C. The reaction time to completion may suitably vary
from about 4 hours to about 4 days, such as from about 8 hours to
about 36 hours or from about 18 hours to about 36 hours.
[0218] In some aspects of the disclosure, activated calicheamicin
may be purified and isolated as a solid. Purification and isolation
methods are known in the art and include precipitation,
crystallization, filtration, centrifugation, ultrafiltration, and
various chromatographic techniques. Chromatography can involve any
number of methods including, e.g.: reverse-phase and normal phase;
size exclusion; ion exchange; high, medium and low pressure liquid
chromatography methods and apparatus; small scale analytical;
simulated moving bed (SMB) and preparative thin or thick layer
chromatography, as well as techniques of small scale thin layer and
flash chromatography. In some such aspects, the completed reaction
mixture may be evaporated to dryness followed by re-dissolution in
a polar aprotic solvent. The solution may be filtered and then
precipitated by combining the solution with a nonpolar antisolvent
such as, for instance, hexane or cyclohexane. The precipitate may
then be collected by filtration, optionally washed, and then
dried.
[0219] Formation of calicheamicin-antibody conjugates is
represented by the following scheme:
##STR00008##
In the scheme, p refers to the number of activated calicheamicin
equivalents, S.sub.c is a calicheamicin sulfur atom, X is a leaving
group as defined elsewhere herein, S.sub.x is a leaving group
sulfur atom, Ab is an antibody as described elsewhere herein,
S.sub.aH is an Ab free sulfhydryl moiety suitable for conjugation
as described elsewhere herein, n is the number of equivalents of Ab
free sulfhydryl moieties per Ab equivalent. Each p and n are
defined elsewhere herein. In some aspects, Ab-(S.sub.aH).sub.n may
be an cysteine engineered antibody as described elsewhere herein
and/or may be treated with a reducing agent for reactivity in the
conjugation reaction. The Ab is dissolved in a physiological buffer
system known in the art that will not adversely impact the
stability or antigen-binding specificity of the antibody. In some
aspects, phosphate buffered saline is used. Activated calicheamicin
is dissolved in a solvent system comprising at least one polar
aprotic solvent as described elsewhere herein. In some such
aspects, activated calicheamicin is dissolved to a concentration of
about 5 mM, 10 mM, about 20 mM, about 30 mM, about 40 mM or about
50 mM, and ranges thereof such as from about 50 mM to about 50 mM
or from about 10 mM to about 30 mM in pH 8 Tris buffer (e.g., 50 mM
Tris). In some aspects, activated calicheamicin is dissolved in
DMSO or acetonitrile, or in DMSO. In the conjugation reaction, an
equivalent excess of activated calicheamicin solution is diluted
and combined with chilled antibody solution (e.g. from about
1.degree. C. to about 10.degree. C.). The activated calicheamicin
solution may suitably be diluted with at least one polar aprotic
solvent and at least one polar protic solvent, examples of which
include water, methanol, ethanol, n-propanol, and acetic acid. In
some particular aspects the activated calicheamicin is dissolved in
DMSO and diluted with acetonitrile and water prior to admixture
with the antibody solution. The equivalents of calicheamicin to
antibody may suitably be about 1.5:1, about 3:1, about 5:1, about
10:1 about 15:1 or about 20:1, and ranges thereof, such as from
about 1.5:1 to about 20:1 from about 1.5:1 to about 15:1, from
about 1.5:1 to about 10:1, from about 3:1 to about 15:1, from about
3:1 to about 10:1, from about 5:1 to about 15:1 or from about 5:1
to about 10:1. The reaction may suitably be monitored for
completion by methods known in the art, such as LC-MS (as described
elsewhere herein), and the reaction is typically complete in from
about 1 hour to about 24 hours. After the reaction is complete, a
reagent is added to the reaction mixture to quench the reaction and
cap unreacted antibody thiol groups. An example of a suitable
reagent is maleimide.
[0220] Following conjugation, the antibody-calicheamicin conjugates
may be purified and separated from unconjugated reactants and/or
conjugate aggregates by purification methods known in the art such
as, for example and not limited to, size exclusion chromatography,
hydrophobic interaction chromatography, ion exchange
chromatography, chromatofocusing, ultrafiltration, centrifugal
ultrafiltration, and combinations thereof. For instance,
purification may be preceded by diluting the antibody-calicheamicin
conjugate, such in 20 mM sodium succinate, pH 5. The diluted
solution is applied to a cation exchange column followed by washing
with, e.g., at least 10 column volumes of 20 mM sodium succinate,
pH 5. The conjugate may be suitably eluted with PBS.
[0221] Pharmaceutical Formulations
[0222] Pharmaceutical formulations of therapeutic antibody-drug
conjugates of the invention are typically prepared for parenteral
administration, i.e. bolus, intravenous, intratumor injection in a
unit dosage injectable form with the desired degree of purity and
with one or more optional pharmaceutically acceptable carriers,
excipient, and/or vehicles (Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous solutions. Pharmaceutically acceptable
carriers are generally nontoxic to recipients at the dosages and
concentrations employed, and include, but are not limited to:
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride; benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene glycol (PEG). Exemplary pharmaceutically acceptable
carriers herein further include insterstitial drug dispersion
agents such as soluble neutral-active hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase
glycoproteins, such as rHuPH20 (HYLENEX.RTM., Baxter International,
Inc.). Certain exemplary sHASEGPs and methods of use, including
rHuPH20, are described in US Patent Publication Nos. 2005/0260186
and 2006/0104968. In one aspect, a sHASEGP is combined with one or
more additional glycosaminoglycanases, such as chondroitinases.
[0223] Exemplary lyophilized antibody or immunoconjugate
formulations are described in U.S. Pat. No. 6,267,958. Aqueous
antibody or immunoconjugate formulations include those described in
U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations
including a histidine-acetate buffer.
[0224] The formulation herein may also contain more than one active
ingredient as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other.
[0225] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0226] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody or
immunoconjugate, which matrices are in the form of shaped articles,
e.g. films, or microcapsules.
[0227] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0228] Antibody-Drug Conjugate Methods of Treatment
[0229] It is contemplated that the antibody-drug conjugates of the
present invention may be used to treat various diseases or
disorders, e.g. characterized by the overexpression of a tumor
antigen. Exemplary conditions or hyperproliferative disorders
include benign or malignant solid tumors and hematological
disorders such as leukemia and lymphoid malignancies. Others
include neuronal, glial, astrocytal, hypothalamic, glandular,
macrophagal, epithelial, stromal, blastocoelic, inflammatory,
angiogenic and immunologic, including autoimmune, disorders.
[0230] In one aspect, an antibody-drug conjugate provided herein is
used in a method of inhibiting proliferation of a cancer cell, the
method comprising exposing the cell to the antibody-drug conjugate
under conditions permissive for binding of the antibody or
antibody-drug conjugates to a tumor-associated antigen on the
surface of the cell, thereby inhibiting the proliferation of the
cell. In certain embodiments, the method is an in vitro or an in
vivo method. In further embodiments, the cell is a lymphocyte,
lymphoblast, monocyte, or myelomonocyte cell.
[0231] Inhibition of cell proliferation in vitro may be assayed
using the CellTiter-Glo.TM. Luminescent Cell Viability Assay, which
is commercially available from Promega (Madison, Wis.). That assay
determines the number of viable cells in culture based on
quantitation of ATP present, which is an indication of
metabolically active cells. See Crouch et al. (1993) J. Immunol.
Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may be
conducted in 96- or 384-well format, making it amenable to
automated high-throughput screening (HTS). See Cree et al. (1995)
AntiCancer Drugs 6:398-404. The assay procedure involves adding a
single reagent (CellTiter-Glo.RTM. Reagent) directly to cultured
cells. This results in cell lysis and generation of a luminescent
signal produced by a luciferase reaction. The luminescent signal is
proportional to the amount of ATP present, which is directly
proportional to the number of viable cells present in culture. Data
can be recorded by luminometer or CCD camera imaging device. The
luminescence output is expressed as relative light units (RLU).
[0232] In another aspect, an antibody-drug conjugate for use as a
medicament is provided. In further aspects, an antibody-drug
conjugate for use in a method of treatment is provided. In certain
embodiments, an antibody-drug conjugate for use in treating cancer
is provided. In certain embodiments, the invention provides an
antibody-drug conjugate for use in a method of treating an
individual comprising administering to the individual an effective
amount of the antibody-drug conjugate. In one such embodiment, the
method further comprises administering to the individual an
effective amount of at least one additional therapeutic agent,
e.g., as described herein.
[0233] In a further aspect, the invention provides for the use of
an antibody-drug conjugate in the manufacture or preparation of a
medicament. In one embodiment, the medicament is for treatment of
CLL-1-positive cancer. In a further embodiment, the medicament is
for use in a method of treating CLL-1-positive cancer, the method
comprising administering to an individual having CLL-1-positive
cancer an effective amount of the medicament. In one such
embodiment, the method further comprises administering to the
individual an effective amount of at least one additional
therapeutic agent, e.g., as described herein.
[0234] In a further aspect, the invention provides a method for
treating cancer. In one embodiment, the method comprises
administering to an individual having such cancer, characterized by
detection of a tumor-associated expressing antigen, an effective
amount of an antibody-drug conjugate of the invention. In one such
embodiment, the method further comprises administering to the
individual an effective amount of at least one additional
therapeutic agent, as described herein.
[0235] Antibody-drug conjugates of the invention can be used either
alone or in combination with other agents in a therapy. For
instance, an antibody or immunoconjugate of the invention may be
co-administered with at least one additional therapeutic agent. In
some embodiments, the additional therapeutic agent is an
anthracycline. In some embodiments, the anthracycline is
daunorubicin or idarubicin. In some embodiments, the additional
therapeutic agent is cytarabine. In some embodiments, the
additional therapeutic agent is cladribine. In some embodiments,
the additional therapeutic agent is fludarabine or topotecan. In
some embodiments, the additional therapeutic agent is 5-azacytidine
or decitabine.
[0236] Such combination therapies noted herein encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the antibody or immunoconjugate of
the invention can occur prior to, simultaneously, and/or following,
administration of the additional therapeutic agent and/or adjuvant.
Antibodies or immunoconjugates of the invention can also be used in
combination with radiation therapy.
[0237] An antibody or immunoconjugate of the invention (and any
additional therapeutic agent) can be administered by any suitable
means, including parenteral, intrapulmonary, and intranasal, and,
if desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration.
Dosing can be by any suitable route, e.g. by injections, such as
intravenous or subcutaneous injections, depending in part on
whether the administration is brief or chronic. Various dosing
schedules including but not limited to single or multiple
administrations over various time-points, bolus administration, and
pulse infusion are contemplated herein.
[0238] Antibodies or immunoconjugates of the invention would be
formulated, dosed, and administered in a fashion consistent with
good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular
mammal being treated, the clinical condition of the individual
patient, the cause of the disorder, the site of delivery of the
agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners.
The antibody or immunoconjugate need not be, but is optionally
formulated with one or more agents currently used to prevent or
treat the disorder in question. The effective amount of such other
agents depends on the amount of antibody or immunoconjugate present
in the formulation, the type of disorder or treatment, and other
factors discussed herein. These are generally used in the same
dosages and with administration routes as described herein, or
about from 1 to 99% of the dosages described herein, or in any
dosage and by any route that is empirically/clinically determined
to be appropriate.
[0239] For the prevention or treatment of disease, the appropriate
dosage of an antibody or immunoconjugate of the invention (when
used alone or in combination with one or more other additional
therapeutic agents) will depend on the type of disease to be
treated, the type of antibody or immunoconjugate, the severity and
course of the disease, whether the antibody or immunoconjugate is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the
antibody or immunoconjugate, and the discretion of the attending
physician. The antibody or immunoconjugate is suitably administered
to the patient at one time or over a series of treatments.
Depending on the type and severity of the disease, about 1 .mu.g/kg
to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody or
immunoconjugate can be an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. One typical
daily dosage might range from about 1 .mu.g/kg to 100 mg/kg or
more, depending on the factors mentioned herein. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the antibody or immunoconjugate would be in the range
from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of
about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any
combination thereof) may be administered to the patient. Such doses
may be administered intermittently, e.g. every week or every three
weeks (e.g. such that the patient receives from about two to about
twenty, or e.g. about six doses of the antibody). An initial higher
loading dose, followed by one or more lower doses may be
administered. However, other dosage regimens may be useful. The
progress of this therapy is easily monitored by conventional
techniques and assays.
[0240] Intracellular release of calicheamicin from the
antibody-calicheamicin conjugate in a target cell is believed to
result from reductive cleavage of the disulfide bond by
glutathione. Glutathione-mediated release provides for advantages
as compared to certain linkers known in the prior art, such as
acid-labile hydrazine linkers. More particularly, blood
concentration of glutathione is known to be very low, such as in
the micromolar range, whereas intracellular glutathione
concentration is typically up to three orders of magnitude greater,
such as in the millimolar range. It is further believed that
glutathione concentration in cancer cells is even greater due to
increased activity of reductive enzymes. Therefore, it is believed
that the calicheamicin-antibody conjugates of the present
disclosure provide for improved stability in the bloodstream and
for improved intracellular release rates.
[0241] Articles of Manufacture
[0242] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described herein is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the disorder
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antibody or immunoconjugate of the
invention. The label or package insert indicates that the
composition is used for treating the condition of choice. Moreover,
the article of manufacture may comprise (a) a first container with
a composition contained therein, wherein the composition comprises
an antibody or immunoconjugate of the invention; and (b) a second
container with a composition contained therein, wherein the
composition comprises a further cytotoxic or otherwise therapeutic
agent. The article of manufacture in this embodiment of the
invention may further comprise a package insert indicating that the
compositions can be used to treat a particular condition.
Alternatively, or additionally, the article of manufacture may
further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
or dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
Examples
[0243] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided herein.
Example 1--Preparation of Cysteine Engineered Antibodies
[0244] For large scale antibody production, antibodies were
produced in CHO cells. Vectors coding for VL and VH were
transfected into CHO cells and IgG was purified from cell culture
media by protein A affinity chromatography.
[0245] As initially isolated, the engineered cysteine residues in
antibodies exist as mixed disulfides with cellular thiols (e.g.,
glutathione) and are thus unavailable for conjugation. Partial
reduction of these antibodies (e.g., with DTT), purification, and
reoxidation with dehydroascorbic acid (DHAA) gives antibodies with
free cysteine sulfhydryl groups available for conjugation, as
previously described, e.g., in Junutula et al. (2008) Nat.
Biotechnol. 26:925-932 and US 2011/0301334. Briefly, the antibodies
were combined with the activated calicheamicin drug moiety to allow
conjugation to the free cysteine residues of the antibody. After
several hours, the antibody-drug conjugates were purified.
[0246] Under certain conditions, the cysteine engineered antibodies
were made reactive for conjugation with drugs by treatment with a
reducing agent such as DTT (Cleland's reagent, dithiothreitol) or
TCEP (tris(2-carboxyethyl)phosphine hydrochloride; Getz et al.
(1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly,
Mass.) in 50 mM Tris pH 7.5 with 2 mM EDTA for 3 hrs at 37.degree.
C. or overnight at room temperature. Full length, cysteine
engineered monoclonal antibodies (THIOMAB.TM.) expressed in CHO
cells (Gomez et al. (2010) Biotechnology and Bioeng.
105(4):748-760; Gomez et al. (2010) Biotechnol. Prog. 26:1438-1445)
were reduced, for example with about a 50 fold excess of DTT
overnight at room temperature to reduce disulfide bonds which may
form between the newly introduced cysteine residues and the
cysteine present in the culture media. The reduced THIOMAB.TM. was
diluted and loaded onto a HiTrap S column in 10 mM sodium acetate,
pH 5, and eluted with PBS containing 0.3M sodium chloride.
Alternatively, the antibody was acidified by addition of
1/20.sup.th volume of 10% acetic acid, diluted with 10 mM succinate
pH 5, loaded onto the column and then washed with 10 column volumes
of succinate buffer. The column was eluted with 50 mM Tris pH7.5, 2
mM EDTA.
[0247] Light chain amino acids are numbered according to Kabat
(Kabat et al., Sequences of proteins of immunological interest,
(1991) 5th Ed., US Dept of Health and Human Service, National
Institutes of Health, Bethesda, Md.). Heavy chain amino acids are
numbered according to the EU numbering system (Edelman et al.
(1969) Proc. Natl. Acad. of Sci. 63(1):78-85), except where noted
as the Kabat system. Single letter amino acid abbreviations are
used.
[0248] Full length, cysteine engineered monoclonal antibodies
(THIOMAB.TM.) expressed in CHO cells bear cysteine adducts
(cystines) or glutathionylated on the engineered cysteines due to
cell culture conditions. To liberate the reactive thiol groups of
the engineered cysteines, the THIOMAB.TM. was dissolved in 500 mM
sodium borate and 500 mM sodium chloride at about pH 8.0 and
reduced with about a 50-100 fold excess of 1 mM TCEP
(tris(2-carboxyethyl)phosphine hydrochloride (Getz et al. (1999)
Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.) for
about 1-2 hrs at 37.degree. C. Alternatively, DTT was used as
reducing agent. The formation of inter-chain disulfide bonds was
monitored either by non-reducing SDS-PAGE or by denaturing reverse
phase HPLC PLRP column chromatography. The reduced THIOMAB.TM. was
diluted and loaded onto a HiTrap SP FF column in 10 mM sodium
acetate, pH 5, and eluted with PBS containing 0.3M sodium chloride,
or 50 mM Tris-Cl, pH 7.5 containing 150 mM sodium chloride.
[0249] Disulfide bonds were reestablished between cysteine residues
present in the parent Mab by carrying out reoxidation. The eluted
reduced THIOMAB.TM. was treated with 15.times. or 2 mM
dehydroascorbic acid (dhAA) at pH 7 for about 3 hours or for about
3 hrs in 50 mM Tris-Cl, pH 7.5, or with 200 nM to 2 mM aqueous
copper sulfate (CuSO.sub.4) at room temperature overnight. Other
oxidants, i.e. oxidizing agents, and oxidizing conditions, which
are known in the art may be used. Ambient air oxidation may also be
effective. This mild, partial reoxidation step formed intrachain
disulfides efficiently with high fidelity. The buffer was exchanged
by elution over Sephadex G25 resin and eluted with PBS with 1 mM
DTPA. The thiol/antibody value was checked by determining the
reduced antibody concentration from the absorbance at 280 nm of the
solution and the thiol concentration by reaction with DTNB
(Aldrich, Milwaukee, Wis.) and determination of the absorbance at
412 nm.
[0250] Liquid chromatography/Mass Spectrometric Analysis was
performed on a TSQ Quantum Triple Quadrupole.TM. mass spectrometer
with extended mass range (Thermo Electron, San Jose Calif.).
Samples were chromatographed on a PRLP-S.RTM., 1000 A, microbore
column (50 mm.times.2.1 mm, Polymer Laboratories, Shropshire, UK)
heated to 75.degree. C. A linear gradient from 30-40% B (solvent A:
0.05% TFA in water, solvent B: 0.04% TFA in acetonitrile) was used
and the eluent was directly ionized using the electrospray source.
Data was collected by the Xcalibur.RTM. data system and
deconvolution was performed using ProMass.RTM. (Novatia, LLC, New
Jersey). Prior to LC/MS analysis, antibodies or drug conjugates (50
micrograms) were treated with PNGase F (2 units/ml; PROzyme, San
Leandro, Calif.) for 2 hours at 37.degree. C. to remove N-linked
carbohydrates.
[0251] Hydrophobic Interaction Chromatography (HIC) samples were
injected onto a Butyl HIC NPR column (2.5 micron particle size, 4.6
mm.times.3.5 cm) (Tosoh Bioscience) and eluted with a linear
gradient from 0 to 70% B at 0.8 ml/min (A: 1.5 M ammonium sulfate
in 50 mM potassium phosphate, pH 7, B: 50 mM potassium phosphate pH
7, 20% isopropanol). An Agilent 1100 series HPLC system equipped
with a multi wavelength detector and Chemstation software was used
to resolve and quantitate antibody species with different ratios of
drugs per antibody.
Example 2--Conjugation of Calicheamicin to Antibodies
[0252] After the reduction and reoxidation procedures of Example 1,
the cysteine-engineered antibody (THIOMAB.TM.) is dissolved in PBS
(phosphate buffered saline) buffer and chilled on ice. An excess,
from about 1.5 molar to 20 equivalents of a calicheamicin,
activated with a thiol-reactive pyridyl disulfide group, is
dissolved in DMSO, diluted in acetonitrile and water, and added to
the chilled reduced, reoxidized antibody in PBS. Typically the drug
is added from a DMSO stock at a concentration of about 20 mM in 50
mM Tris, pH 8, to the antibody and monitored until the reaction is
complete from about 1 to about 24 hours as determined by LC-MS
analysis of the reaction mixture. When the reaction is complete, an
excess of maleimide is added to quench the reaction and cap any
unreacted antibody thiol groups. The conjugation mixture may be
loaded and eluted through a HiTrap SP FF column to remove excess
drug and other impurities. The reaction mixture is concentrated by
centrifugal ultrafiltration and the cysteine engineered
antibody-drug conjugate is purified and desalted by elution through
G25 resin in PBS, filtered through 0.2 .mu.m filters under sterile
conditions, and frozen for storage.
[0253] For example, the crude antibody-drug conjugate is applied to
a cation exchange column after dilution with 20 mM sodium
succinate, pH 5. The column was washed with at least 10 column
volumes of 20 mM sodium succinate, pH 5, and the antibody was
eluted with PBS. The antibody-drug conjugates were formulated into
20 mM His/acetate, pH 5, with 240 mM sucrose using gel filtration
columns. The antibody-drug conjugates were characterized by UV
spectroscopy to determine protein concentration, analytical SEC
(size-exclusion chromatography) for aggregation analysis and LC-MS
before and after treatment with Lysine C endopeptidase.
[0254] Size exclusion chromatography is performed using a Shodex
KW802.5 column in 0.2M potassium phosphate pH 6.2 with 0.25 mM
potassium chloride and 15% IPA at a flow rate of 0.75 ml/min.
Aggregation state of the conjugate was determined by integration of
eluted peak area absorbance at 280 nm.
[0255] LC-MS analysis may be performed using an Agilent QTOF 6520
ESI instrument. As an example, the antibody-drug conjugate is
treated with 1:500 w/w Endoproteinase Lys C (Promega) in Tris, pH
7.5, for 30 min at 37.degree. C. The resulting cleavage fragments
are loaded onto a 1000 .ANG. (Angstrom), 8 .mu.m (micron) PLRP-S
(highly cross-linked polystyrene) column heated to 80.degree. C.
and eluted with a gradient of 30% B to 40% B in 5 minutes. Mobile
phase A was H.sub.2O with 0.05% TFA and mobile phase B was
acetonitrile with 0.04% TFA. The flow rate was 0.5 ml/min. Protein
elution was monitored by UV absorbance detection at 280 nm prior to
electrospray ionization and MS analysis. Chromatographic resolution
of the unconjugated Fc fragment, residual unconjugated Fab and
drugged Fab was usually achieved. The obtained m/z spectra were
deconvoluted using Mass Hunter.TM. software (Agilent Technologies)
to calculate the mass of the antibody fragments.
Example 3--In Vitro Cell Proliferation Assay
[0256] Efficacy of the antibody-drug conjugates Thio Hu Anti-CD22
10F4v3 LC K149C calicheamicin and Thio Hu Anti-Ly6E 9B12.v12 LC
K149C calicheamicin was measured by a cell proliferation assay
employing the following protocol (CELLTITER GLO.TM. Luminescent
Cell Viability Assay, Promega Corp. Technical Bulletin TB288;
Mendoza et al. (2002) Cancer Res. 62:5485-5488):
[0257] 1. An aliquot of 100 .mu.l of cell culture containing about
10.sup.4 cells (CD22-positive BJAB, CD22-positive WSU-DLCL2 or
Jurkat) in medium was deposited in each well of a 96-well,
opaque-walled plate.
[0258] 2. Control wells were prepared containing medium and without
cells.
[0259] 3. Antibody-drug conjugate was added to the experimental
wells and incubated for 3-5 days.
[0260] 4. The plates were equilibrated to room temperature for
approximately 30 minutes.
[0261] 5. A volume of CELLTITER GLO.TM. Reagent equal to the volume
of cell culture medium present in each well was added.
[0262] 6. The contents were mixed for 2 minutes on an orbital
shaker to induce cell lysis.
[0263] 7. The plate was incubated at room temperature for 10
minutes to stabilize the luminescence signal.
[0264] 8. Luminescence was recorded and reported in graphs as
RLU=relative luminescence units.
[0265] Data was plotted and illustrated in FIGS. 3A to 3C as the
mean of luminescence for each set of replicates, with standard
deviation error bars. The protocol is a modification of the
CELLTITER GLO.TM. Luminescent Cell.
Example 4--Tumor Growth Inhibition, In Vivo Efficacy in High
Expressing HER2 Transgenic Explant Mice
[0266] Tumors were established and allowed to grow to 150-200
mm.sup.3 in volume (as measured using calipers) before a single
treatment on day 0. Tumor volume was measured using calipers
according to the formula: V (mm.sup.3)=0.5A.times.B.sup.2, where A
and B are the long and short diameters, respectively. Mice were
euthanized before tumor volume reached 3000 mm.sup.3 or when tumors
showed signs of impending ulceration. Data collected from each
experimental group (10 mice per group) was expressed as
mean.+-.SE.
[0267] The Fo5 mouse mammary tumor model was employed to evaluate
the in vivo efficacy of antibody-drug conjugates of the invention
after single dose intravenous injections, and as described
previously (Phillips GDL, Li G M, Dugger D L, et al. Targeting
HER2-Positive Breast Cancer with Trastuzumab-DM1, an
Antibody-Cytotoxic Drug Conjugate. (2008) Cancer Res. 68:9280-90),
incorporated by reference herein. Anti-Her2 antibody-drug
conjugates were tested with the Fo5 model, a transgenic mouse model
in which the human HER2 gene is over-expressed in mammary
epithelium under transcriptional regulation of the murine mammary
tumor virus promoter (MMTV-HER2). The HER2 over-expression causes
spontaneous development of a mammary tumor. The mammary tumor of
one of these founder animals (founder #5 [Fo5]) was propagated in
subsequent generations of FVB mice by serial transplantation of
tumor fragments (.about.2.times.2 mm in size). All studies were
conducted in accordance with the Guide for the Care and Use of
Laboratory Animals. Each antibody-drug conjugate (single dose) was
dosed in nine animals intravenously at the start of the study, and
14 days post-transplant. Initial tumor size was about 200 mm.sup.3
volume.
[0268] Another mammary fat pad transplant efficacy model may be
employed as described (Chen et al. (2007) Cancer Res 67:4924-4932),
evaluating tumor volume after a single intravenous dose and using
tumors excised from a mouse bearing an intraperitoneal tumor, then
serially passaged into the mammary fat pads of recipient mice.
[0269] Cell lines that could be tested in this way include AU565,
HCC1954, HCC1008, HCC2157, HCC202, HCC1419, HCC2218 and
HCC1569.
Example 5--Efficacy of Thio Hu Anti-Ly6E LC
K149C-p-Nitro-PDS-Calicheamicin Antibody-Drug Conjugates
[0270] Breast cancer cell line HCC1569 (CRL-2330) was obtained from
American Type Culture Collection (ATCC, Manassas, Va.). The
HCC1569.times.2 cell line is a derivative of the parental HCC1569
cell line (ATCC, CRL-2330) optimized for growth in vivo. Parental
HCC1569 cells were injected subcutaneously in the right flank of
female NCR nude mice, one tumor was harvested, minced and grown in
vitro resulting in a HCC1569 XI cell line. The HCC1569 XI line was
injected again subcutaneously in the right flank of female NCR nude
mice in an effort to improve the growth of the cell line. A tumor
from this study was collected and again adapted for in vitro growth
to generate the HCC1569.times.2 cell line. This cell line and
tumors derived from this line express Ly6E.
[0271] SCID Beige mice were inoculated in the right 2/3 mammary fat
pad with 5 million cells suspended in Hank's Balanced Salt Solution
(HBSS) and matrigel. When tumor volumes reached approximately
163-282 mm.sup.3 (day 0), the animals were randomized into groups
of 5 mice each, and administered a single intravenous (IV)
injection of either vehicle control or the antibody-drug conjugates
at the following doses: 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg and 10
mg/kg. One group of animals was administered 3 mg/kg of Thio Hu
anti-CD22 LC K149C-p-nitro-PDS-Calicheamicin. Tumor volumes were
measured twice per week until study end at 21 days. Tumor volume
was measured and calculated based on two dimensions, measured using
calipers, and was expressed in mm.sup.3 according to the formula:
V=0.5a.times.b.sup.2, wherein a and b are the long and the short
diameters of the tumor, respectively. To analyze the repeated
measurement of tumor volumes from the same animals over time, a
mixed modeling approach was used (see, e.g., Pinheiro J, et al.
nlme: linear and nonlinear mixed effects models. 2009; R package,
version 3.1-96). This approach can address both repeated
measurements and modest dropout rates due to non-treatment related
removal of animals before the study end. Cubic regression splines
were used to fit a non-linear profile to the time courses of log 2
tumor volume at each dose level. These non-linear profiles were
then related to dose within the mixed mode. All animal protocols
were approved by an Institutional Animal Care and Use Committee
(IACUC).
[0272] The experimental results are depicted in FIG. 1 and indicate
that doses of 1, 3, 6 and 10 mg/kg of Thio Hu anti-Ly6E LC
K149C-p-nitro-PDS-Calicheamicin antibody-drug conjugates reduced
tumor volume over the course of the study.
Example 6--Efficacy of Thio Hu Anti-CD22 10F4v3 LC
K149C-p-Nitro-PDS-Calicheamicin
[0273] The antitumor efficacy effect of Thio Hu anti-CD22 10F4v3 LC
K149C-p-nitro-PDS-Calicheamicin conjugates in a mouse xenograft
model of WSU-DLCL2 tumors (diffuse large B-cell lymphoma cell line)
was examined.
[0274] Female CB17 Fox Chase SCID mice were each inoculated in the
right flank with 20 million WSU-DLCL2 cells (DSMZ, German
Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany) suspended in Hank's Balanced Salt Solution (HBSS). When
the xenograft tumors reached an average tumor volume of 175-237
mm.sup.3 (day 0), the animals were randomized into groups of 5 mice
each and administered a single intravenous (IV) injection of either
vehicle control or the antibody-drug conjugate at the following
doses: 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg and 10 mg/kg. One group
of animals was administered 3 mg/kg of Thio Hu anti-LY6E 9B12.v12
LC K149-p-nitro-PDS-Calicheamicin. Tumor volumes were measured
twice per week, as described elsewhere herein, until study end at
21 days. All animal protocols were approved by an Institutional
Animal Care and Use Committee (IACUC).
[0275] The experimental results are depicted in FIG. 2 and indicate
that doses of 1, 3, 6 and 10 mg/kg of Thio Hu anti-CD22 10F4v3 LC
K149C-p-nitro-PDS-Calicheamicin reduced volume over the course of
the study.
Example 7--Additional Efficacy Studies
[0276] Example 7 evaluated the efficacy of targeted control with
Thio Hu Anti-CD22 10F4v3 LC K149C calicheamicin conjugate against
CD22 positive Burkitt's lymphoma cells ("BJAB") and against CD22
positive human diffuse large B-cell lymphoma-derived cell line
(WSU-DLCL2) versus non-targeted control with Thio Hu Anti-Ly6E
9B12.v12 LC K149C calicheamicin conjugate. Each conjugate had an
average drug to antibody ratio ("DAR") of 1.7. The efficacy of
non-targeted control for each conjugate was also evaluated against
Jurkat.
[0277] The IC.sub.50 efficacy results for the BJAB, WSU-DLCL2 and
Jurkat cells are depicted in FIGS. 3A to 3C, respectively. The
results show that treatment of the CD22-positive BJAB and WSU-DLCL2
cell lines with Thio Hu Anti-CD22 10F4v3 LC K149C-Calicheamicin
provided double-digit potency that is >1500-fold greater and
>2000-fold greater than non-targeted control with Thio Hu
Anti-Ly6E 9B12.v12 LC K149C-Calicheamicin on BJAB and WSU-DLCL2,
respectively. The results further show that Thio Hu Anti-CD22
10F4v3 LC K149C-Calicheamicin and Thio Hu Anti-Ly6E 9B12.v12 LC
K149C-Calicheamicin were each essentially non-efficacious on Jurkat
cells. The IC.sub.50 results are reported in the Table below where
"ADC" refers to antibody-drug conjugate, "Thio Hu Anti-CD22" refers
to Thio Hu Anti-CD22 10F4v3 LC K149C calicheamicin, and "Thio Hu
Anti-Ly6E" refers to Thio Hu Anti-Ly6E 9B12.v12 LC K149C
calicheamicin.
TABLE-US-00005 WSU-DLDL2 BJAB IC.sub.50 IC.sub.50 Jurkat IC.sub.50
ADC nM Ng/mL nM Ng/mL nM Ng/mL Thio Hu Anti- 0.07 10.8 0.03 4.8
121.6 18229.5 CD22 Thio Hu Anti- 125.2 18777.4 82.7 12395.0 121.0
18138.9 Ly6E
[0278] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
[0279] When introducing elements of the present disclosure or the
preferred embodiments(s) thereof, the articles "a", "an", "the" and
"said" are intended to mean that there are one or more of the
elements. The terms "comprising", "including" and "having" are
intended to be inclusive and mean that there may be additional
elements other than the listed elements.
[0280] This written description uses examples to disclose the
invention, including the best mode, and also to enable any person
skilled in the art to practice the invention, including making and
using any devices or systems and performing any incorporated
methods. The patentable scope of the invention is defined by the
claims, and may include other examples that occur to those skilled
in the art. Such other examples are intended to be within the scope
of the claims if they have structural elements that do not differ
from the literal language of the claims, or if they include
equivalent structural elements with insubstantial differences from
the literal language of the claims.
* * * * *