U.S. patent application number 12/939117 was filed with the patent office on 2011-05-12 for multivalent antibodies and uses therefor.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Kathy L. Miller, Leonard G. Presta.
Application Number | 20110110852 12/939117 |
Document ID | / |
Family ID | 22722944 |
Filed Date | 2011-05-12 |
United States Patent
Application |
20110110852 |
Kind Code |
A1 |
Miller; Kathy L. ; et
al. |
May 12, 2011 |
Multivalent Antibodies and Uses Therefor
Abstract
The present application describes engineered antibodies, with
three or more functional antigen binding sites, and uses, such as
therapeutic applications, for such engineered antibodies.
Inventors: |
Miller; Kathy L.; (San
Francisco, CA) ; Presta; Leonard G.; (San Francisco,
CA) |
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
22722944 |
Appl. No.: |
12/939117 |
Filed: |
November 3, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11535031 |
Sep 25, 2006 |
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12939117 |
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11218821 |
Sep 2, 2005 |
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11535031 |
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09813341 |
Mar 20, 2001 |
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11218821 |
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60195819 |
Apr 11, 2000 |
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Current U.S.
Class: |
424/1.49 ;
424/130.1; 424/178.1; 424/183.1; 424/9.1; 424/9.6; 530/387.1;
530/391.3; 530/391.7 |
Current CPC
Class: |
C07K 16/2896 20130101;
C07K 2317/77 20130101; C07K 16/00 20130101; C07K 16/32 20130101;
C07K 2317/73 20130101; C07K 2317/64 20130101; A61K 2039/505
20130101; C07K 16/2878 20130101; A61P 31/00 20180101; C07K 16/468
20130101; A61P 35/00 20180101; C07K 2319/00 20130101; A61K 39/3955
20130101; A61K 47/6879 20170801; C07K 2317/55 20130101; A61K 45/06
20130101 |
Class at
Publication: |
424/1.49 ;
424/9.1; 424/9.6; 424/130.1; 424/178.1; 424/183.1; 530/387.1;
530/391.3; 530/391.7 |
International
Class: |
A61K 51/10 20060101
A61K051/10; A61K 49/00 20060101 A61K049/00; A61K 39/395 20060101
A61K039/395; C07K 16/00 20060101 C07K016/00 |
Claims
1.-93. (canceled)
94. A binding protein comprising four polypeptide chains, wherein
two polypeptide chains comprise VD1-(X1)n-VD2-C-(X2)n, wherein VD1
is a first heavy chain variable domain, VD2 is a second heavy chain
variable domain, C is a heavy chain constant domain, X1 is a linker
with the proviso that it is not CH1, and X2 is an Fc region; and
two polypeptide chains comprise VD1-(X1)n-VD2-C-(X2)n, wherein VD1
is a first light chain variable domain, VD2 is a second light chain
variable domain, C is a light chain constant domain, X1 is a linker
with the proviso that it is not CH1, and X2 does not comprise an Fc
region; and n is 0 or 1; wherein said four polypeptide chains of
said binding protein form four functional antigen binding
sites.
95. The binding protein according to claim 94, wherein said binding
protein is capable of binding one or more targets.
96. The binding protein according to claim 95, wherein said one or
more targets is selected from the group consisting of ABCF1; ACVR1;
ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA;
AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3;
ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1;
B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15);
BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B;
BMPR2; BPAG1 (plectin); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1;
CANT1; CASP1; CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (1-309); CCL11
(eotaxin); CCL13 (MCP-4); CCL15 (MT-1d); CCL16 (HCC-4); CCL17
(TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20
(MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC/STC-1); CCL23
(MPIF-1); CCL24 (MPIF-2/eotaxin-2); CCL25 (TECK); CCL26
(eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3 (MT-1a); CCL4 (MIP-1b);
CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1;
CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-1RB/RA); CCR3
(CKR3/CMKBR3); CCR4; CCR5 (CMKBRS/ChemR13); CCR6
(CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBI1); CCR8
(CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR);
CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38;
CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72;
CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDH1 (E-cadherin);
CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9;
CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wap1/Cip1);
CDKN1B (p27Kip1); CDKN1C; CDKN2A (p161NK4a); CDKN2B; CDKN2C; CDKN3;
CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3;
CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7
(claudin-7); CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1); CNR1;
COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2
(GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsin
B); CX3CL1 (SCYD1); CX3CR1 (V28); CXCL1 (GRO1); CXCL10(IP-10);
CXCL11 (1-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2
(GRO2); CXCL3 (GRO3); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9
(MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo);
CYB5; CYC1; CYSLTR1; DAB21P; DES; DKFZp451J0118; DNCL1; DPP4; E2F1;
ECGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1;
ENO2; ENO3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3
(TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (aFGF);
FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18;
FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4
(HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF
(VEGFD); FIL1 (EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1
(fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa);
GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GM-CSF;
GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10
(C10); GRP; GSN (Gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A;
HDAC9; HGF; HIF1A; HIP1; histamine and histamine receptors; HLA-A;
HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNA1;
IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; IFNW1; IGBP1;
IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA;
IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13;
IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C;
IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; ILIA; IL1B; IL1F10;
IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; EMU; IL1R2; IL1RAP;
IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2; IL1RN; IL2; 1L20; IL20RA;
IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25; IL26; IL27; IL28A;
IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5;
IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8; IL8RA;
IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1;
IRAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3;
ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR;
KITLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15;
KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A;
KRTHB6 (hair-specific type II keratin); LAMAS; LEP (leptin);
Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16);
LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1;
midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3
(metallothionectin-III); MTS S1; MUC1 (mucin); MYC; MYD88; NCK2;
neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66
(Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NROB1; NROB2;
NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NRII2; NRII3; NR2C1; NR2C2;
NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2;
NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1;
P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1;
PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG;
PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2;
PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB;
RGS1; RGS13; RGS3; RNF110 (ZNF144); ROBO2; SI00A2; SCGB1D2
(lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1);
SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPINA1;
SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1); SERPINF1; SHBG;
SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Sprl);
ST6GAL1; STAB1; STATE; STEAP; STEAP2; TB4R2; TBX21; TCP10; TDGF1;
TEK; TGFA; TGFB1; TGFB111; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2;
TGFBR3; TH1L; THBS1 (thrombospondin-1); THBS2; THBS4; THPO; TIE
(Tie-1); TIMP3; tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6;
TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF11A;
TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7;
TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12
(APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15
(VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6
(FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB
ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase Iia);
TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6;
TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL
C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5/CCXCR1);
YY1; and ZFPM2.
97. The binding protein according to claim 94, wherein said binding
protein is capable of binding a two targets, wherein the two
targets are selected from the group consisting of CD138 and CD20;
CD138 and CD40; CD20 and CD3; CD38 & CD138; CD38 and CD20; CD38
and CD40; CD40 and CD20; CD19 and CD20; CD-8 and IL-6; PDL-1 and
CTLA-4; CTLA-4 and BTNO2; CSPGs and RGM A; IGF1 and IGF2; IGF1/2
and Erb2B; IL-12 and IL-18; IL-12 and TWEAK; IL-13 and ADAMS; IL-13
and CL25; IL-13 and IL-1beta; IL-13 and IL-25; IL-13 and IL-4;
IL-13 and IL-5; IL-13 and IL-9; IL-13 and LHR agonist; IL-13 and
MDC; IL-13 and MIF; IL-13 and PED2; IL-13 and SPRR2a; IL-13 and
SPRR2b; IL-13 and TARC; IL-13 and TGF-.beta.; IL-1.alpha. and
IL-1.beta.; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and
RGM A; RGM A and RGM B; Te38 and TNF.alpha.; TNF.alpha. and IL-12;
TNF.alpha. and IL-12p40; TNF.alpha. and IL-13; TNF.alpha. and
IL-15; TNF.alpha. and IL-17; TNF.alpha. and IL-18; TNF.alpha. and
IL-1beta; TNF.alpha. and IL-23; TNF.alpha. and MIF; TNF.alpha. and
PEG2; TNF.alpha. and PGE4; TNF.alpha. and VEGF; and VEGFR and EGFR;
TNF.alpha. and RANK ligand; TNF.alpha. and Blys; TNF.alpha. and
GP130; TNF.alpha. and CD-22; and TNF.alpha. and CTLA-4.
98. The binding protein according to claim 95, wherein the binding
protein is capable of modulating a biological function of one or
more targets.
99. The binding protein according to claim 95, wherein the binding
protein is capable of neutralizing one or more targets.
100. The binding protein according to claim 95, wherein said one or
more targets is selected from the group consisting of cytokine,
chemokine, cell surface protein, enzyme and receptor.
101. The binding protein according to claim 100, wherein the
cytokine is selected from the group consisting of lymphokines,
monokines, and polypeptide hormones.
102. The binding protein according to claim 101, wherein said
cytokines are IL-1.alpha. and IL-1.beta..
103. The binding protein according to claim 101, wherein said
cytokines are TNF-.alpha. and IL-13.
104. The binding protein according to claim 101, wherein said
cytokines are IL-12 and IL-18.
105. The binding protein according to claim 100, wherein the cell
surface protein is an integrin.
106. The binding protein according to claim 100, wherein the cell
surface proteins are CD-20 and CD3.
107. The binding protein according to claim 100, wherein the enzyme
is selected from the group consisting of kinases and proteases.
108. The binding protein according to claim 100, wherein the
receptor is selected from the group consisting of lymphokine
receptor, monokine receptor, and polypeptide hormone receptor.
109. A binding protein conjugate comprising a binding protein
described in any one of claims 94-101 and 104, said binding protein
conjugate further comprising an agent selected from the group
consisting of; an immunoadhesion molecule, an imaging agent, a
therapeutic agent, and a cytotoxic agent.
110. The binding protein conjugate according to claim 109, wherein
said agent is an imaging agent selected from the group consisting
of a radiolabel, an enzyme, a fluorescent label, a luminescent
label, a bioluminescent label, a magnetic label, and biotin.
111. The binding protein conjugate according to claim 110, wherein
said imaging agent is a radiolabel selected from the group
consisting of: .sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc,
.sup.111In, .sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, and
.sup.153Sm.
112. The binding protein conjugate according to claim 109, wherein
said agent is a therapeutic or cytotoxic agent selected from the
group consisting of; an anti-metabolite, an alkylating agent, an
antibiotic, a growth factor, a cytokine, an anti-angiogenic agent,
an anti-mitotic agent, an anthracycline, toxin, and an apoptotic
agent.
113. A binding protein described in claim 94 produced according to
a method comprising culturing a host cell in culture medium under
conditions sufficient to produce said binding protein, wherein said
host cell comprises a vector, said vector comprising a nucleic acid
encoding said binding protein.
114. A pharmaceutical composition comprising a binding protein of
any one of claims 94-101, 104 and 113, and a pharmaceutically
acceptable carrier.
115. The pharmaceutical composition of claim 114, further
comprising at least one additional therapeutic agent.
116. The pharmaceutical composition according to claim 115, wherein
said additional agent is a therapeutic or imaging agent.
117. The pharmaceutical composition of claim 116, wherein said
additional agent is selected from the group consisting of:
Therapeutic agent, imaging agent, cytotoxic agent, angiogenesis
inhibitors; kinase inhibitors; co-stimulation molecule blockers;
adhesion molecule blockers; anti-cytokine antibody or functional
fragment thereof; methotrexate; cyclosporin; rapamycin; FK506;
detectable label or reporter; a TNF antagonist; an antirheumatic; a
muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug
(NSAID), an analgesic, an anesthetic, a sedative, a local
anesthetic, a neuromuscular blocker, an antimicrobial, an
antipsoriatic, a corticosteriod, an anabolic steroid, an
erythropoietin, an immunization, an immunoglobulin, an
immunosuppressive, a growth hormone, a hormone replacement drug, a
radiopharmaceutical, an antidepressant, an antipsychotic, a
stimulant, an asthma medication, a beta agonist, an inhaled
steroid, an epinephrine or analog, a cytokine, and a cytokine
antagonist.
118. A pharmaceutical composition comprising a binding protein
conjugate according to claim 109 and a pharmaceutically acceptable
carrier.
119. The pharmaceutical composition according to claim 118, wherein
said binding protein conjugate comprises an imaging agent selected
from the group consisting of a radiolabel, an enzyme, a fluorescent
label, a luminescent label, a bioluminescent label, a magnetic
label, and biotin.
120. The pharmaceutical composition according to claim 119, wherein
said imaging agent is a radiolabel selected from the group
consisting of: .sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc,
.sup.111In, .sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, and
.sup.153Sm.
121. The pharmaceutical composition according to claim 118, wherein
said binding protein conjugate comprises a therapeutic or cytotoxic
agent selected from the group consisting of an anti-metabolite, an
alkylating agent, an antibiotic, a growth factor, a cytokine, an
anti-angiogenic agent, an anti-mitotic agent, an anthracycline, a
toxin, and an apoptotic agent.
122. The pharmaceutical composition of claim 118 further comprising
a second agent.
123. The pharmaceutical composition of claim 122, wherein said
second agent is a therapeutic or imaging agent.
124. The pharmaceutical composition of claim 123, wherein said
therapeutic or imaging agent is selected from the group: cytotoxic
agent, angiogenesis inhibitors, kinase inhibitors; co-stimulation
molecule blockers; adhesion molecule blockers; anti-cytokine
antibody or functional fragment thereof; methotrexate; cyclosporin;
rapamycin; FK506; detectable label or reporter; a TNF antagonist;
an antiheumatic; a muscle relaxant, a narcotic, anon-steroid
anti-inflammatory dug (NSAID), an analgesic, an anesthetic, a
sedative, a local anesthetic, a neuromuscular blocker, an
antimicrobial, an antipsoriatic, a corticosteriod, an anabolic
steroid, an erythropoietin, an immunization, an immunoglobulin, an
immunosuppressive, a growth hormone, a hormone replacement drug, a
radiopharmaceutical, .sup.3H, .sup.14C, .sup.35S, .sup.90Y,
.sup.99Tc, .sup.111In, .sup.125I, .sup.131I, .sup.177Lu,
.sup.166Ho, .sup.153Sm, a fluorescent label, a luminescent label, a
bioluminescent label, a magnetic label, biotin, an antidepressant,
an antipsychotic, a stimulant, an asthma medication, a beta
agonist, an inhaled steroid, an epinephrine or analog, a cytokine,
and a cytokine antagonist.
125. An isolated antibody comprising four polypeptide chains,
wherein two polypeptide chains comprise VD1-(X1)n-VD2-(X2)n-Fc,
wherein VD1 is a first heavy chain variable domain, VD2 is a second
heavy chain variable domain, X1 is a linker with the proviso that
it is not CH1, and X2 is a heavy chain constant domain; and two
polypeptide chains comprise VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a
first light chain variable domain, VD2 is a second light chain
variable domain, C is a light chain constant domain, X1 is a linker
with the proviso that it is not CH1, and X2 does not comprise an Fc
region; and n is 0 or 1; wherein said four polypeptide chains of
said binding protein form four functional antigen binding
sites.
126. The binding protein according to claim 125, wherein said
binding protein is capable of binding one or more targets.
127. The isolated antibody according to claim 126, wherein said one
or more targets is selected from the group consisting of BDNF; CCL3
(MIP-1a); CCL5 (RANTES); CD19; CD1c; CD20; CD22; CD24; CD28; CD3;
CD37; CD38; CD4; CD40; CD44; CD52; CD69; CD72; CD74; CD79A; CD79B;
C D8; CD80; CD81; CD83; CD86; CSF1 (M-CSF); CSF2 (MG-CSF); CSF3
(G-CSF); CTLA-4; EGF; EGFR; EPO; ErbB2 (Her-2); FGFI (aFGF); FGF2
(bFGF); GM-CSF; IFN-a; IFN-gamma; IGF-I; IGF-H; IL-1; IL-2; IL-3;
IL-4; IL-5; IL6; IL-7; IL-8; IL-9; IL-10; NGFB; TGF-alpha;
TGF-beta1; TGF-beta2; TGF-beta3; TGF-beta4; TNF; TNF-alpha; and
VEGF.
128. The isolated antibody according to claim 126, wherein said one
or more targets is selected from the group consisting of ABCFI;
ACVRI; ACVRIB; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2;
AICDA; AIFI; AlGI; AKAPI; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2;
ANGPTL3; ANGPTL4; ANPEP; APC; APOCI; AR;AZGPI
(zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAGI; BAH; BCL2;
BCL6; BLNK; BLR1 (MDR15); BlyS; BMPI; BMP2; BMP3B (GDFIO); BMP4;
BMP6; BMP8; BMPRIA; BMPRIB; BMPR2; BPAGI (plectin); BRCAI; CI9orflO
(IL27w); C3; C4A; C5; C5R1; CANT!; CASPI; CASP4; CAVI; CCBP2
(D6/JAB61); CCL1 (1-309); CCM (eotaxin); CCL13 (MCP-4); CCL15
(MIP-Id); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP3b);
CCL2 (MCP-I); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC; exodus-2;
CCL22 (MDC/STC-I); CCL23 (MPIF-I); CCL24 (MPIF-2/eotaxin-2); CCL25
(TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL4 (MIP-Ib);
CCL7 (MCPS); CCL8 (mcp-2); CCNAI; CCNA2; CCND1; CCNEI; CCNE2; CCR1
(CKR1/HMI45); CCR2 (mcp-IRB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5
(CMKBR5/ChemR13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7
(CKR7/EB11); CCR8 (CMKBR8/TERI!CKR-L1); CCR9 (GPR-9-6); CCRL1
(VSHKI); CCRU (L-CCR); CD164; CD200; CD3E; CD3G; CD3Z; CD45RB; CDHI
(E-cadherin); CDHIO; CDHI2; CDH13; CDHI8; CDHI9; CDH20; CDH5; CDH7;
CDH8; CDH9; CDK2; CDK3;CDK4; CDK5; CDK6; CDK7; CDK9; CDKNIA
(p21WapI/Cip1); CDKNIB (p27Kip1); CDKNIC; CDKN2A (p16INK4a);
CDKN2B; CDKN2C; CDKN3; CEBPB; CERI; CHGA; CHGB; Chitinase; CHST!O;
CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8;
CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLRI; CMKORI
(RDCI); CNRI; COL18AI; COL1AI; COL4A3; COL6AI; CR2; CRP; CTNNBI
(b-catenin); CTSB (cathepsin B); CX3CL1 (SCYD1); CX3CRI (V28);
CXCL1 (GRO1); CXCL10(IP-10); CXCL1I (1-TAC/IP-9); CXCL12 (SDFI);
CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GR03); CXCL5
(ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKRL2);
CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYCI; CYSLTRI; DAB2IP;
DES; DKFZp45IJO118; DNCL1; DPP4; E2FI; ECGFI; EDGI; EFNAI; EFNA3;
EFNB2; ELAC2; ENG; ENO1; EN02; EN03; EPHB4; EREG; ERK8; ESRI; ESR2;
F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1O;
FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19;
FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6
(HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FILL
(EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin);
FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1;
GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GNAS1; GNRH1; GPR2 (CCR10);
GPR31; GPR44; GPR81 (FKSG80); GRCC10(C10); GRP; GSN (Gelsolin);
GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HIFI;
histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1;
HUMCYT2A; ICEBERG; ICOSL; ID2; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6;
IFNA7; IFNB1; IFNW1; IGBP1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6;
IL1 ORA; IL1 ORB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1;
IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17;
IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; ILIA;
IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1;
IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2; IL1R.sup.N;
IL20; IL20RA; IL21R; IL22; 1L22R; IL22RA2; IL23; IL24; IU5; IL26;
1L27; IL28A; IL28B; IL29; IURA; IURB; IL2R.sup.G; IL30; IL3RA;
IL4R; IL5RA; IL6R; IL6ST (glycoprotein 130); IL7R; ILSRA; ILSRB;
ILSRB; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1;
ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4
integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC
Box BP); KLF6; KLKIO; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5;
KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specific
type II keratin); LAMAS; LEP (leptin); Lingo-p75; Lingo-Troy; LPS;
LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or
Omgp; MAP2K7 (c-Jun); MDK; MEM; midkine; MIF; MIP-2; MKI67 (Ki-67);
MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUCI
(mucin); MYC; MYD88; NCK2; neurocan; NFKB1; NFKB2; NGFR; NgR-Lingo;
NgR-Nog066 (Nogo); NgR-p75; NgR-Troy; NMEI (NM23A); NOX5; NPPB;
NROB1; NROB2; NRID1; NRID2; NRIH2; NRIH3; NRIH4; NRII2; NRII3;
NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2;
NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4;
ODZ1; OPRD1; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PDGFA;
PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG;
PLAU (uPA); PLG; PLXDC1; PPBP(CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL;
PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2
(p21Rac2); RARB; RGS1; RGS13; RGS3; RNF11O (ZNF144); ROB02; SIOOA2;
SCGBID2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2
(mammaglobin 1); SCYEI (endothelial Monocyte-activating cytokine);
SDF2; SERPINA1; SERPINA3; SERPINB5 (maspin); SERPINEI (PA1-I);
SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRRIB
(Spr1); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCPIO;
TDGF1; TEK; TGFBR1; TGFBR2; TGFBR3; TH1L; THBSI (thrombospondin-1);
THBS2; THBS4; THPO; TIE (Tie-I); TIMP3; tissue factor; TLR10; TLR2;
TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-ALPHAIP2 (B94);
TNF-ALPHAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5;
TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF1O (TRAIL); TNFSF11
(TRANCE); TNFSF12 (AP03L); TNFSF13 (April); TNFSF13B; TNFSF14
(HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5
(CD40 ligand); TNFSF6 (FasL); TNFSF7 (C027 ligand); TNFSF8 (CmO
ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors; TOP2A
(topoisomerase Iia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3;
TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB;
VEGFC; versican; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-1b);
XCR1 (GPR5/CCXCR1); YY1; and ZFPM2.
129. The binding protein according to claim 125, wherein said
binding protein is capable of binding a two targets, wherein the
two targets are selected from the group consisting of CD20 and CD3;
CD38 and CD20; CD38 and CD40; CD40 and CD20; CD19 and CD20; CD8 and
IL-6; IGF-I and IGF-II; IGF-I/II and Erb2B; TNF-alpha and IL-1beta;
TNF-alpha and MIF; TNF-alpha and VEGF; TNF-alpha and CTLA-4.
130. The binding protein according to claim 125, wherein said
binding protein is capable of binding a two targets, wherein the
two targets are selected from the group consisting of CD138 and
CD20; CD138 and CD40; CD38 and CD138; PDL-1 and CTLA4;CTLA-4 and
BTN02; CSPGs and RGM A; IL-12 and IL-18; IL-12 and TWEAK; IL-13 and
ADAM8; IL-13 and CL25; IL-13 and IL-1beta; IL-13 and IL-25; IL-13
and IL-4; IL-13 and IL-5; IL-13 and IL-9; IL-13 and LHR agonist;
IL-13 and MDC; IL-13 and MIF; IL-13 and PED2; IL-13 and SPRR2a;
IL-13 and SPRR2b; IL-13 and TARC; IL-13 and TGF-beta; IL-1 alpha
and IL-1 beta; MAG and RGM A; NgR and RGM A; NogoA and RGMA; OMGp
and RGMA; RGMA and RGM B; Te38 and TNF-alpha; TNF-alpha and IL-12;
TNF-alpha and IL-12p40; TNF-alpha and IL-13; TNF-alpha and IL-15;
TNF-alpha and IL-17; TNF-alpha and IL-18; TNF-alpha and IL-23;
TNF-alpha and MIF; TNF-alpha and PEG2; TNF-alpha and PGE4; and
VEGFR and EGFR; TNF-alpha and RANK ligand; TNF-alpha and Blys;
TNF-alpha and GP130; and TNF-alpha and CD-22.
Description
RELATED APPLICATION
[0001] This application is a continuation application claiming
priority to application Ser. No. 11/218,821, filed Sep. 2, 2005,
which is a continuation application claiming priority to
application Ser. No. 09/813,341, filed Mar. 20, 2001, which claims
priority under 35 U.S.C. .sctn.119(e) to provisional application
No. 60/195,819, filed Apr. 11, 2000, the entire contents of which
are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention concerns engineered antibodies, with
three or more functional antigen binding sites, and uses, such as
therapeutic uses, for such engineered antibodies.
[0004] 2. Description of Related Art
Structure of Naturally Occurring Antibodies
[0005] Naturally occurring antibodies (immunoglobulins) comprise
two heavy chains linked together by disulfide bonds and two light
chains, one light chain being linked to each of the heavy chains by
disulfide bonds. Each heavy chain has at one end a variable domain
(VH) followed by a number of constant domains (three or four
constant domains, CH1, CH2, CH3 and CH4, depending on the antibody
class). Each light chain has a variable domain (VL) at one end and
a constant domain (CL) at its other end; the constant domain of the
light chain is aligned with the first constant domain of the heavy
chain, and the light chain variable domain is aligned with the
variable domain of the heavy chain. See FIG. 1 herein. Particular
amino acid residues are believed to form an interface between the
light and heavy chain variable domains, see e.g. Chothia et al., J.
Mol. Biol. 186:651-663 (1985); and Novotny and Haber, Proc. Natl.
Acad. Sci. USA 82:4592-4596 (1985).
[0006] The constant domains are not involved directly in binding
the antibody to an antigen, but are involved in various effector
functions, such as participation of the antibody in
antibody-dependent cell-mediated cytotoxicity (ADCC) and complement
dependent cytotoxicity (CDC). The variable domains of each pair of
light and heavy chains are involved directly in binding the
antibody to the antigen. The variable domains of naturally
occurring light and heavy chains have the same general structure;
each comprising four framework regions (FRs), whose sequences are
somewhat conserved, connected by three complementarity determining
regions (CDRs) (see Kabat et al., Sequences of Proteins of
Immunological Interest, National Institutes of Health, Bethesda,
Md., (1991)). The four FRs largely adopt a beta-sheet conformation
and the CDRs form loops connecting, and in some cases forming part
of, the beta-sheet structure. The CDRs in each chain are held in
close proximity by the FRs and, with the CDRs from the other chain,
contribute to the formation of the antigen binding site.
[0007] FIGS. 2A-E herein depict the structures of the five major
naturally occurring immunoglobulin isotypes. IgG, IgD and IgE
immunoglobulins possess only two antigen binding sites. IgA and
IgM, on the other hand, are capable of forming polymeric structures
with higher valencies.
[0008] IgM is secreted by plasma cells as a pentamer in which five
monomer units are held together by disulfide bonds linking their
carboxyl-terminal (CIA/CO) domains and C.mu.3/C.mu.3 domains. The
five monomer subunits are arranged with their Fc regions in the
center of the pentamer and the 10 antigen-binding sites on the
periphery of the molecule. Each pentamer contains an additional
Fc-linked polypeptide called the J (joining) chain, which is
disulfide-bonded to the carboxyl-terminal cysteine residue of 2 of
the 10.mu. chains. The J chain appears to be required for
polymerization of the monomers to form pentameric IgM; it is added
just before secretion of the pentamer. An IgM molecule can bind 10
small hapten molecules; however, because of steric hindrance, only
5 molecules of larger antigens can be bound simultaneously. The
increased valency of pentameric IgM increases its capacity to bind
such multi-dimensional antigens as viral particles and red blood
cells (RBCs).
[0009] IgA exists primarily as a monomer, although polymeric forms
such as dimers, trimers, and even tetramers are sometimes seen. The
IgA of external secretions consists of a dimer or tetramer, a
J-chain polypeptide, and a polypeptide chain called secretory
component.
Antibodies for Clinical Uses
[0010] Widespread use has been made of monoclonal antibodies,
particularly those derived from rodents including mice, however
they are frequently antigenic in human clinical use. For example, a
major limitation in the clinical use of rodent monoclonal
antibodies is an anti-globulin response during therapy (Miller at
al., Blood 62:988-995 (1983); and Schroff, R. W. et al., Cancer
Res. 45:879-885 (1985)).
[0011] The art has attempted to overcome this problem by
constructing "chimeric" antibodies in which an animal antigen
binding variable domain is coupled to a human constant domain
(Cabilly et al., U.S. Pat. No. 4,816,567; Morrison et al., Proc.
Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature
312:643-646 (1984); and Neuberger et al., Nature 314:268-270
(1985)). The isotype of the human constant domain may be selected
to tailor the chimeric antibody for participation in ADCC and CDC
(see e.g. Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987);
Riechmann et al., Nature 332:323-327 (1988); Love et al., Methods
in Enzymology 178:515-527 (1989); and Bindon et al., J. Exp. Med.
168:127-142 (1988)). In the typical embodiment, such chimeric
antibodies contain about one third rodent (or other non-human
species) sequence and thus are capable of eliciting a significant
anti-globulin response in humans. For example, in the case of the
murine anti-CD3 antibody, OKT3, much of the resulting anti-globulin
response is directed against the variable region rather than the
constant region (Jeffers et al., Transplantation 41:572-578
(1986)).
[0012] In a further effort to resolve the antigen binding functions
of antibodies and to minimize the use of heterologous sequences in
human antibodies, Winter and colleagues (Jones et al., Nature
321:522-525 (1986); Riechmann et al., i Nature 332:323-327 (1988);
and Verhoeyen et al., Science 239:1534-1536 (1988)) have
substituted rodent CDRs or CDR sequences for the corresponding
segments of a human antibody.
[0013] The therapeutic promise of this approach is supported by the
clinical efficacy of a humanized antibody specific for the
CAMPATH-1 antigen with two non-Hodgkin lymphoma patients, one of
whom had previously developed an anti-globulin response to the
parental rat antibody (Riechmann et al., Nature 332:323-327 (1988);
and Hale et al., Lancet i:1394-1399 (1988)).
[0014] In some cases, substituting CDRs from rodent antibodies for
the human CDRs in human frameworks is sufficient to transfer high
antigen binding affinity (Jones et al., Nature 321:522-525 (1986);
Verhoeyen et al., Science 239:1534-1536 (1988)), whereas in other
cases it has been necessary to additionally replace one (Riechmann
et al., Nature 332:323-327 (1988)) or several (Queen et al., Proc.
Natl. Acad. Sci. USA 86:10029-10033 (1989)) framework residues. See
also Co et al., Proc. Natl. Acad. Sci. USA 88:2869-2873 (1991);
U.S. Pat. No. 5,821,337 (Carter et al.); and U.S. Pat. No.
5,530,101 (Queen et al.). Additional references relating to
humanization of antibodies include Gorman et al., Proc. Natl. Acad.
Sci. USA 88:4181-4185 (1991); Daugherty et al., Nucleic Acids
Research 19(9):2471-2476 (1991); Brown et al., Proc. Natl. Acad.
Sci. USA 88:2663-2667 (1991); and Junghans et al., Cancer Research
50:1495-1502 (1990).
[0015] Instead of a chimeric/humanized antibody, one may treat a
patient with a human antibody in order to avoid human antibodies
raised against a murine antibody (known as the "HAMA response").
Several technologies are available for generating human
antibodies.
[0016] Human antibodies may be selected using phage display
technology. Phage display has been adapted to select human
antibodies from an unimmunized donor (Marks et al. J. Mol. Biol.
222:581-597 (1991)). According to this approach, PCR is used to
amplify variable domain genes from mRNA prepared from human
peripheral blood lymphocytes (PBLs). Primers are used such that DNA
from both IgG and IgM heavy chains and both .kappa. and .lamda.
chains is amplified. These genes are then randomly combined and
expressed as single chain Fv (scFv) fused to the gene III coat
protein of M13 phage. Human antibodies against an antigen of
interest may then be identified by rounds of growth and selection
by binding to that antigen (e.g. to the immobilized antigen). See
Griffiths et al. EMBO J. 12:725-734 (1993).
[0017] "Synthetic" phage-antibody repertoires have also been built
from cloned human VH-gene segments. A repertoire (2.times.10.sup.7
clones) was first constructed using a short H3 loop of five or
eight random residues with each of 49 segments, and combined with a
fixed light chain (Hoogenboom et al. J. Mol. Biol. 227:381-388
(1992)). By adding a range of H3 loops of different lengths, up to
12 residues, a single library was created from which a range of
more than 20 binding specificities could be selected (Winter et al.
Ann. Rev. Immuno. 12:433-55 (1994)). Other synthetic libraries have
been built from the framework of a single antibody by randomizing
CDRs of the human antibody (Garrard and Henner Gene 128:103-109
(1993)). Antibodies derived from such synthetic phage-antibody
repertoires are also considered to be "human" antibodies
herein.
[0018] The affinity of low affinity "primary" phage-antibodies may
be improved by using phage display technology. One approach is to
use a chain-shuffling strategy in which the VH domain is held
constant and then recombined with the original library of VL genes
and tighter binders selected by binding to immobilized antigen.
This cycle is repeated by fixing the new VL domain and recombining
with the original VH library (Marks et al. Bio/Technology
10:779-783 (1992)). Alternatively, point mutations in the primary
antibody may be introduced using error-prone PCR and higher
affinity binders selected by using phage display. Gram et al. PNAS
(USA) 89: 3576-3580 (1992).
[0019] One may also produce human antibodies by immunizing mice
which have been genetically engineered to express human antibodies.
Severe combined immune deficient (SCID) mice lack the ability to
produce their own immunoglobulins due to a defect in the
recombinase gene. Several groups have reconstituted a functional
humoral immune system in these mice by transfer of human peripheral
blood lymphocytes (PBLs). These hu-PBL-SCID mice can be used to
raise human antibodies upon immunization with antigen. Duchosal et
al. Nature 355:258-262 (1992). Using another approach, the heavy-
and light-chain genes within mice are turned off and then yeast
artificial chromosomes (YACs) engineered with large DNA sequences
containing human heavy- and light-chain genes are introduced into
the mice. Such "XenoMice" are able to produce human antibodies upon
immunization with an antigen of interest. See U.S. Pat. No.
5,434,340; U.S. Pat. No. 5,591,699; U.S. Pat. No. 5,569,825; U.S.
Pat. No. 5,545,806; and U.S. Pat. No. 5,545,807.
[0020] Human monoclonal antibodies may also be generated by
immortalizing a human B lymphocyte producing an antibody of
interest. The ethical issues surrounding immunizing humans in order
to generate activated human B lymphocytes can be avoided by
immunizing human lymphocytes in vitro. Both human PBLs (Borrebaeck
et al. Proc. Natl. Acad. ScL USA 85:3995-4000 (1988)) and human
splenocytes (Boerner et al. J. Immunol. 147, 86-95 (1991)) have
been successfully immunized in vitro. Improvements in human
hybridoma technology have been achieved by using a mouse-human
heterohybrid as the fusion partner (Boerner et al.).
Antibody Variants
[0021] Antibodies have been modified in order to increase their
antigen-binding valency. For instance, Ghetie et al. homodimerized
tumor-reactive monoclonal antibodies (anti-CD19, anti-CD20,
anti-CD21, anti-CD22 and anti-HER2 antibodies) by chemically
introducing a thioether bond between a pair of IgGs using two
heterobifunctional crosslinkers . Ghetie et al. PNAS (USA)
94:7509-7514 (1997); and WO 99/02567. Wolff et al. Cancer Research
53: 2560-2565 (1993) also chemically linked an IgG monoclonal
antibody (CHiBR96) using heterobifunctional cross-linkers to
generate a monoclonal antibody homodimer with enhanced anti-tumor
activity in nude mice.
[0022] Shopes et al., replaced a serine residue near the carboxyl
terminus of a human IgG1 heavy chain (Ser.sup.444) with a cysteine.
The introduced intermolecular disulfide bonds between Cys.sup.444
residues linked pairs of immunoglobulins "tail-to-tail" to form
covalent dimers (H.sub.2L2).sub.2. The anti-dansyl dimers were said
to be more efficient than monomeric human IgG1 at
antibody-dependent complement-mediated cytolysis of hapten-bearing
erythrocytes. Shopes, B. J. Immunol. 148(9): 2918-2922 (1992); and
WO 91/19515. This approach, involving introduction of cysteine
residues, has also been used to generate a homodimeric form of the
CAMPATH-1H antibody. The homodimeric CAMPATH-1H antibody exhibited
improved lysis using target cells expressing antigen at low
density, but no improvement in lysis was observed using cells
expressing antigen at high density. Greenwood et al. Ther. Immunol.
1:247-255 (1994). See, also, Caron et al. J. Exp. Med.
176:1191-1195 (1992), concerning an engineered anti-CD33 antibody
with a serine to cysteine substitution at position 444 of the heavy
chain allowing interchain disulfide bond formation at the COOH
terminus of the IgG. The homodimeric IgG was said to have similar
avidity to the parent IgG, but apparently showed an improved
ability to internalize and retain radioisotope in target leukemia
cells, and was more potent at complement-mediated leukemia cell
killing and antibody-dependent cellular cytotoxicity using human
effectors.
[0023] Coloma and Morrison Nature Biotech. 15: 159-163 (1997)
describe a tetravalent bispecific antibody which was engineered by
fusing DNA encoding a single chain anti-dansyl antibody Fv (scFv)
after the C terminus (CH3-scFv) or after the hinge (Hinge-scFv) of
an IgG3 anti-dansyl antibody. See, also, WO95/09917. Smith and
Morrison engineered three versions of mu-like IgG3 by engineering
either (1) Cys414 of an IgM heavy chain or (2) Cys575 of an IgM
heavy chain, or both (1) and (2), into the IgG3 heavy chain gene.
All three mutant constructs were expressed by Sp2/0 cells and
assembled into polymers containing up to six H.sub.2L2 subunits.
The thus-produced `IgM-like` polymers of IgG were considered to
possess both the Fc gamma receptor binding properties of IgG and
the more potent complement activity of IgM. See, Smith and Morrison
Bio/Technology 12:683-688 (1994).
[0024] Shuford and collegues isolated a human IgG1 anti-group B
streptococci antibody oligomer from a transfected myeloma cell
line. Shuford et al. Science 252:724-727 (1991). Immunochemical
analysis and DNA sequencing indicated that the cell line produced
both a normal kappa light chain and a 37 kD V-V-C variant light
chain (L37). Contransfection of vectors encoding the heavy chain
and L37 resulted in the production of oligomeric IgG.
[0025] U.S. Pat. No. 5,641,870 (Rinderknecht et al.) describes a
bivalent, linear F(ab').sub.2 fragment comprising tandem repeats of
a heavy chain fragment (VH-CH1-VH-CH1) cosecreted with a light
chain. The C-terminus of CH1 was joined directed to the N-terminus
of VH without any extraneous linking protein sequences.
[0026] Other publications on antibody variants include WO 00/06605;
U.S. Pat. No. 5,591,828; U.S. Pat. No. 5,959,083; U.S. Pat. No.
6,027,725; WO98/58965; WO94/13804; Tutt et al. J. Immunol.
147:60-69 (1991); WO99/37791; U.S. Pat. No. 5,989,830; WO94/15642;
EP 628,078B1; WO97/14719; Stevenson et al. Anti-Cancer Drug Design
3:219-230 (1989).
ErbB Receptor Tyrosine Kinases
[0027] The ErbB receptor tyrosine kinases are important mediators
of cell growth, differentiation and survival. The receptor family
includes at least four distinct members including Epidermal Growth
Factor Receptor (EGFR or ErbB1), HER2 (ErbB2 or p185.sup.neu), HER3
(ErbB3) and HER4 (ErbB4 or tyro2).
[0028] EGFR, encoded by the erbB1 gene, has been causally
implicated in human malignancy. In particular, increased expression
of EGFR has been observed in breast, bladder, lung, head, neck and
stomach cancer, as well as glioblastomas. Increased EGFR receptor
expression is often associated with increased production of the
EGFR ligand, Transforming Growth Factor alpha (TGF-alpha), by the
same tumor cells resulting in receptor activation by an autocrine
stimulatory pathway. Baselga and Mendelsohn Pharmac. Ther.
64:127-154 (1994). Monoclonal antibodies directed against the EGFR
or its ligands, TGF-alpha and EGF, have been evaluated as
therapeutic agents in the treatment of such malignancies. See,
e.g., Baselga and Mendelsohn., supra; Masui et al. Cancer Research
44:1002-1007 (1984); and Wu et al. J. Clin. Invest. 95:1897-1905
(1995).
[0029] The second member of the ErbB family, p185.sup.neu, was
originally identified as the product of the transforming gene from
neuroblastomas of chemically treated rats. The activated form of
the neu proto-oncogene results from a point mutation (valine to
glutamic acid) in the transmembrane region of the encoded protein.
Amplification of the human homolog of neu is observed in breast and
ovarian cancers and correlates with a poor prognosis (Slamon et
al., Science, 235:177-182 (1987); Slamon et al., Science,
244:707-712 (1989); and U.S. Pat. No. 4,968,603). To date, no point
mutation analogous to that in the neu proto-oncogene has been
reported for human tumors. Overexpression of HER2 (frequently but
not uniformly due to gene amplification) has also been observed in
other carcinomas including carcinomas of the stomach, endometrium,
salivary gland, lung, kidney, colon, thyroid, pancreas and
bladder.
[0030] Antibodies directed against the rat p185.sup.neu and human
HER2 protein products have been described. Drebin and colleagues
have raised antibodies against the rat neu gene product,
p185.sup.neu. See, for example, Drebin et al., Cell 41:695-706
(1985); Myers et al., Meth. Enzym. 198:277-290 (1991); and
WO94/22478. Drebin et al. Oncogene 2:273-277 (1988) report that
mixtures of antibodies reactive with two distinct regions of
p185.sup.neu result in synergistic anti-tumor effects on
neu-transformed NIH-3T3 cells implanted into nude mice. See also
U.S. Pat. No. 5,824,311 issued Oct. 20, 1998.
[0031] Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989)
describe the generation of a panel of anti-HER2 antibodies which
were characterized using the human breast tumor cell line SKBR3.
Relative cell proliferation of the SKBR3 cells following exposure
to the antibodies was determined by crystal violet staining of the
monolayers after 72 hours. Using this assay, maximum inhibition was
obtained with the antibody called 4D5 which inhibited cellular
proliferation by 56%. Other antibodies in the panel reduced
cellular proliferation to a lesser extent in this assay. The
antibody 4D5 was further found to sensitize HER2-overexpressing
breast tumor cell lines to the cytotoxic effects of TNF-alpha. See,
also, U.S. Pat. No. 5,677,171, issued Oct. 14, 1997. The anti-HER2
antibodies discussed in Hudziak et al. were further characterized
in Fendly et al. Cancer Research 50:1550-1558 (1990); Kotts et al.
In Vitro 26(3):59A (1990); Sarup et al. Growth Regulation 1:72-82
(1991); Shepard et al. J. Clin. Immunol. 11(3):117-127 (1991);
Kumar et al. Mol. Cell. Biol. 11(2):979-986 (1991); Lewis et al.
Cancer Immunol. Immunother. 37:255-263 (1993); Pietras et al.
Oncogene 9:1829-1838 (1994); Vitetta et al. Cancer Research
54:5301-5309 (1994); Sliwkowski et al. J. Biol. Chem.
269(20):14661-14665 (1994); Scott et al. J. Biol. Chem. 266:14300-5
(1991); D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994);
Lewis et al. Cancer Research 56:1457-1465 (1996); and Schaefer et
al. Oncogene 15:1385-1394 (1997).
[0032] A recombinant humanized IgG1 version of the murine anti-HER2
antibody 4D5 (rhuMAb HER2 or HERCEPTIN.RTM.; commercially available
from Genentech, Inc., South San Francisco) is clinically active in
patients with HER2-overexpressing metastatic breast cancers that
have received extensive prior anti-cancer therapy (Baselga et al.,
J. Clin. Oncol. 14:737-744 (1996)). HERCEPTIN.RTM. received
marketing approval from the Food and Drug Administration Sep. 25,
1998 for the treatment of patients with metastatic breast cancer
whose tumors overexpress the HER2 protein.
[0033] Other anti-HER2 antibodies with various properties have been
described in Tagliabue et al. Int. J. Cancer 47:933-937 (1991);
McKenzie et al. Oncogene 4:543-548 (1989); Maier et al. Cancer Res.
51:5361-5369 (1991); Bacus et al. Molecular Carcinogenesis
3:350-362 (1990); Stancovski et al. PNAS (USA) 88:8691-8695 (1991);
Bacus et al. Cancer Research 52:2580-2589 (1992); Xu et al. Int. J.
Cancer 53:401-408 (1993); WO94/00136; Kasprzyk et al. Cancer
Research 52:2771-2776 (1992); Hancock et al. Cancer Res.
51:4575-4580 (1991); Shawver et al. Cancer Res. 54:1367-1373
(1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994); Harwerth et
al. J. Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No. 5,783,186;
Klapper et al. Oncogene 14:2099-2109 (1997); WO 98/77797; and U.S.
Pat. No. 5,783,186. Homology screening has resulted in the
identification of two other ErbB receptor family members; HER3
(U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus et al.
PNAS (USA) 86:9193-9197 (1989)) and HER4 (EP Pat Appln No 599,274;
Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993);
and Plowman et al., Nature, 366:473-475 (1993)). Both of these
receptors display increased expression on at least some breast
cancer cell lines.
[0034] The ErbB receptors are generally found in various
combinations in cells and heterodimerization is thought to increase
the diversity of cellular responses to a variety of ErbB ligands
(Earp et al. Breast Cancer Research and Treatment 35: 115-132
(1995)). EGFR is bound by six different ligands; Epidermal Growth
Factor (EGF), Transforming Growth Factor alpha (TGF-alpha),
amphiregulin, Heparin Binding Epidermal Growth Factor (HB-EGF),
betacellulin and epiregulin (Groenen et al. Growth Factors
11:235-257 (1994)). A family of heregulin proteins resulting from
alternative splicing of a single gene are ligands for HER3 and
HER4. The heregulin family includes alpha, beta and gamma
heregulins (Holmes et al., Science, 256:1205-1210 (1992); U.S. Pat.
No. 5,641,869; and Schaefer et al. Oncogene 15:1385-1394 (1997));
neu differentiation factors (NDFs), glial growth factors (GGFs);
acetylcholine receptor inducing activity (ARIA); and sensory and
motor neuron derived factor (SMDF). For a review, see Groenen et
al. Growth Factors 11:235-257 (1994); Lemke, G. Molec. & Cell.
Neurosci. 7:247-262 (1996) and Lee et al. Pharm. Rev. 47:51-85
(1995). Recently, two additional ErbB ligands were identified;
neuregulin-2 (NRG-2) which is reported to bind either HER3 or HER4
(Chang et al. Nature 387 509-512 (1997); and Carraway et al Nature
387:512-516 (1997)) and neuregulin-3 which binds HER4 (Zhang et al.
PNAS (USA) 94(18):9562-7 (1997)). HB-EGF, betacellulin and
epiregulin also bind to HER4.
[0035] While EGF and TGF-alpha do not bind HER2, EGF stimulates
EGFR and HER2 to form a heterodimer, which activates EGFR and
results in transphosphorylation of HER2 in the heterodimer.
Dimerization and/or transphosphorylation appears to activate the
HER2 tyrosine kinase. See Earp et al., supra. Likewise, when HER3
is co-expressed with HER2, an active signaling complex is formed
and antibodies directed against HER2 are capable of disrupting this
complex (Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665
(1994)). Additionally, the affinity of HER3 for heregulin (HRG) is
increased to a higher affinity state when co-expressed with HER2.
See also, Levi et al., Journal of Neuroscience 15: 1329-1340
(1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92: 1431-1435
(1995); and Lewis et al., Cancer Res., 56:1457-1465 (1996) with
respect to the HER2-HER3 protein complex. HER4, like HER3, forms an
active signaling complex with HER2 (Carraway and Cantley, Cell
78:5-8 (1994)).
TNF Receptor Superfamily
[0036] Various molecules, such as Tumor Necrosis Factor-alpha
("TNF-alpha"), Tumor Necrosis Factor-beta ("TNF-beta"),
Lymphotoxin-alpha ("LT-alpha"), CD30 ligand, CD27 ligand, CD40
ligand, OX-40 ligand, 4-1 BB ligand, Apo-1 ligand (also referred to
as Fas ligand or CD95 ligand), Apo-2 ligand (also referred to as
TRAIL), Apo-3 ligand (also referred to as TWEAK), osteoprotegerin
(OPG), APRIL, RANK ligand (also referred to as TRANCE), and TALL-1
(also referred to as BlyS, BAFF or THANK) have been identified as
members of the Tumor Necrosis Factor ("TNF") family of cytokines
(See, e.g., Gruss and Dower, Blood, 85:3378-3404 (1995); Pitti et
al., J. Biol. Chem., 271:12687-12690 (1996); Wiley et al.,
Immunity, 3:673-682 (1995); Browning et al., Cell, 72:847-856
(1993); Armitage et al. Nature, 357:80-82 (1992); WO 97/01633
published Jan. 16, 1997; WO 97/25428 published Jul. 17, 1997;
Marsters et al., Curr. Biol., 8:525-528 (1998); Simonet et al.,
Cell, 89:309-319 (1997); Chicheportiche et al., Biol. Chem.,
272:32401-32410 (1997); Hahne et al., J. Exp. Med., 188:1185-1190
(1998); WO98/28426 published Jul. 2, 1998; WO98/46751 published
Oct. 22, 1998; WO/98/18921 published May 7, 1998; Moore et al.,
Science, 285:260-263 (1999); Shu et al., J. Leukocyte Biol., 65:680
(1999); Schneider et al., J. Exp. Med., 189:1747-1756 (1999); and
Mukhopadhyay et al., J. Biol. Chem., 274:15978-15981 (1999)). Among
these molecules, TNF-alpha, TNF-beta, CD30 ligand, 4-1BB ligand,
Apo-1 ligand, Apo-2 ligand (Apo2L/TRAIL) and Apo-3 ligand (TWEAK)
have been reported to be involved in apoptotic cell death. Both
TNF-alpha and TNF-beta have been reported to induce apoptotic death
in susceptible tumor cells (Schmid et al., Proc. Natl. Acad. Sci.,
83:1881 (1986); Dealtry et al., Eur. J. Immunol., 17:689
(1987)).
[0037] Various molecules in the TNF family also have purported
role(s) in the function or development of the immune system (Gruss
et al., Blood, 85:3378 (1995)). Zheng et al. have reported that
TNF-alpha is involved in post-stimulation apoptosis of CD8-positive
T cells (Zheng et al., Nature, 377:348-351 (1995)). Other
investigators have reported that CD30 ligand may be involved in
deletion of self-reactive T cells in the thymus (Amakawa et al.,
Cold Spring Harbor Laboratory Symposium on Programmed Cell Death,
Abstr. No. 10, (1995)). CD40 ligand activates many functions of B
cells, including proliferation, immunoglobulin secretion, and
survival (Renshaw et al., J. Exp. Med., 180:1889 (1994)). Another
recently identified TNF family cytokine, TALL-1 (BlyS), has been
reported, under certain conditions, to induce B cell proliferation
and immunoglobulin secretion. (Moore et al., supra; Schneider et
al., supra; Mackay et al., J. Exp. Med., 190:1697 (1999)).
[0038] Mutations in the mouse Fas/Apo-1 receptor or ligand genes
(called lpr and gld, respectively) have been associated with some
autoimmune disorders, indicating that Apo-1 ligand may play a role
in regulating the clonal deletion of self-reactive lymphocytes in
the periphery (Krammer et al., Curr. Op. Immunol., 6:279-289
(1994); Nagata et al., Science, 267:1449-1456 (1995)). Apo-1 ligand
is also reported to induce post-stimulation apoptosis in
CD4-positive T lymphocytes and in B lymphocytes, and may be
involved in the elimination of activated lymphocytes when their
function is no longer needed (Krammer et al., supra; Nagata et al.,
supra). Agonist mouse monoclonal antibodies specifically binding to
the Apo-1 receptor have been reported to exhibit cell killing
activity that is comparable to or similar to that of TNF-alpha
(Yonehara et al., J. Exp. Med., 169:1747-1756 (1989)).
[0039] Induction of various cellular responses mediated by such TNF
family cytokines is believed to be initiated by their binding to
specific cell receptors. Previously, two distinct TNF receptors of
approximately 55-kDa (TNFR1) and 75-kDa (TNFR2) were identified
(Hohman et al., J. Biol. Chem., 264:14927-14934 (1989); Brockhaus
et al., Proc. Natl. Acad. Sci., 87:3127-3131 (1990); EP 417,563,
published Mar. 20, 1991; Loetscher et al., Cell, 61:351 (1990);
Schall et al., Cell, 61:361 (1990); Smith et al., Science,
248:1019-1023 (1990); Lewis et al., Proc. Natl. Acad. Sci.,
88:2830-2834 (1991); Goodwin et al., Mol. Cell. Biol., 11:3020-3026
(1991)). Those TNFRs were found to share the typical structure of
cell surface receptors including extracellular, transmembrane and
intracellular regions. The extracellular portions of both receptors
were found naturally also as soluble TNF-binding proteins (Nophar
et al., EMBO J., 9:3269 (1990); and Kohno et al., Proc. Natl. Acad.
Sci. U.S.A., 87:8331 (1990); Hale et al., J. Cell. Biochem.
Supplement 15F, 1991, p. 113 (P424)).
[0040] The extracellular portion of type 1 and type 2 TNFRs (TNFR1
and TNFR2) contains a repetitive amino acid sequence pattern of
four cysteine-rich domains (CRDs) designated 1 through 4, starting
from the NH.sub.2-terminus. (Schall et al., supra; Loetscher et
al., supra; Smith et al., supra; Nophar et al., supra; Kohno et
al., supra; Banner et al., Cell, 73:431-435 (1993)). A similar
repetitive pattern of CRDs exists in several other cell-surface
proteins, including the p75 nerve growth factor receptor (NGFR)
(Johnson et al., Cell, 47:545 (1986); Radeke et al., Nature,
325:593 (1987)), the B cell antigen CD40 (Stamenkovic et al., EMBO
J., 8:1403 (1989)), the T cell antigen OX40 (Mallet et al., EMBO
J., 9:1063 (1990)) and the Fas antigen (Yonehara et al., supra and
Itoh et al., Cell, 66:233-243 (1991)). CRDs are also found in the
soluble TNFR (sTNFR)-like T2 proteins of the Shope and myxoma
poxviruses (Upton et al., Virology, 160:20-29 (1987); Smith et al.,
Biochem. Biophys. Res. Commun., 176:335 (1991); Upton et al.,
Virology, 184:370 (1991)). Optimal alignment of these sequences
indicates that the positions of the cysteine residues are well
conserved. These receptors are sometimes collectively referred to
as members of the TNF/NGF receptor superfamily.
[0041] The TNF family ligands identified to date, with the
exception of Lymphotoxin-alpha, are type II transmembrane proteins,
whose C-terminus is extracellular. In contrast, most receptors in
the TNF receptor (TNFR) family identified to date are type I
transmembrane proteins. In both the TNF ligand and receptor
families, however, homology identified between family members has
been found mainly in the extracellular domain ("ECD"). Several of
the TNF family cytokines, including TNF-alpha, Apo-1 ligand and
CD40 ligand, are cleaved proteolytically at the cell surface; the
resulting protein in each case typically forms a homotrimeric
molecule that functions as a soluble cytokine. TNF receptor family
proteins are also usually cleaved proteolytically to release
soluble receptor ECDs that can function as inhibitors of the
cognate cytokines.
[0042] More recently, other members of the TNFR family have been
identified. In von Bulow et al., Science, 278:138-141 (1997),
investigators describe a plasma membrane receptor referred to as
Transmembrane Activator and CAML-Interactor or "TACI". The TACI
receptor is reported to contain a cysteine-rich motif
characteristic of the TNFR family. In an in vitro assay, cross
linking of TACI on the surface of transfected Jurkat cells with
TACI-specific antibodies led to activation of NF-KB (see also, WO
98/39361 published Sep. 18, 1998).
[0043] Laabi et al., EMBO J., 11:3897-3904 (1992) reported
identifying a new gene called "BCM" whose expression was found to
coincide with B cell terminal maturation. The open reading frame of
the BCM normal cDNA predicted a 184 amino acid long polypeptide
with a single transmembrane domain. These investigators later
termed this gene "BCMA." (Laabi et al., Nucleic Acids Res.,
22:1147-1154 (1994)). BCMA mRNA expression was reported to be
absent in human malignant B cell lines which represent the pro-B
lymphocyte stage, and thus, is believed to be linked to the stage
of differentiation of lymphocytes (Gras et al., Int. Immunology,
7:1093-1106 (1995)). In Madry et al., Int. Immunology, 10:1693-1702
(1998), the cloning of murine BCMA cDNA was described. The murine
BCMA cDNA is reported to encode a 185 amino acid long polypeptide
having 62% identity to the human BCMA polypeptide. Alignment of the
murine and human BCMA protein sequences revealed a conserved motif
of six cysteines in the N-terminal region, suggesting that the BCMA
protein belongs to the TNFR superfamily (Madry et al., supra).
[0044] In Marsters et al., Curr. Biol., 6:750 (1996), investigators
describe a full length native sequence human polypeptide, called
Apo-3, which exhibits similarity to the TNFR family in its
extracellular cysteine-rich repeats and resembles TNFR1 and CD95 in
that it contains a cytoplasmic death domain sequence (see also
Marsters et al., Curr. Biol., 6:1669 (1996)). Apo-3 has also been
referred to by other investigators as DR3, wsl-1, TRAMP, and LARD
(Chinnaiyan et al., Science, 274:990 (1996); Kitson et al., Nature,
384:372 (1996); Bodmer et al., Immunity, 6:79 (1997); Screaton et
al., Proc. Natl. Acad. Sci., 94:4615-4619 (1997)).
[0045] Pan et al. have disclosed another TNF receptor family member
referred to as "DR4" (Pan et al., Science, 276:111-113 (1997); see
also WO98/32856 published Jul. 30, 1998). The DR4 was reported to
contain a cytoplasmic death domain capable of engaging the cell
suicide apparatus. Pan et al. disclose that DR4 is believed to be a
receptor for the ligand known as Apo2L/TRAIL.
[0046] In Sheridan et al., Science, 277:818-821 (1997) and Pan et
al., Science, 277:815-818 (1997), another molecule believed to be a
receptor for Apo2L/TRAIL is described (see also, WO98/51793
published Nov. 19, 1998; and WO98/41629 published Sep. 24, 1998).
That molecule is referred to as DR5 (it has also been alternatively
referred to as Apo-2; TRAIL-R, TR6, Tango-63, hAPO8, TRICK2 or
KILLER (Screaton et al., Curr. Biol., 7:693-696 (1997); Walczak et
al., EMBO J., 16:5386-5387 (1997); Wu et al., Nature Genetics,
17:141-143 (1997); WO98/35986 published Aug. 20, 1998; EP870,827
published Oct. 14, 1998; WO98/46643 published Oct. 22, 1998;
WO99/02653 published Jan. 21, 1999; WO99/09165 published Feb. 25,
1999; and WO99/11791 published Mar. 11, 1999). Like DR4, DR5 is
reported to contain a cytoplasmic death domain and be capable of
signaling apoptosis. The crystal structure of the complex formed
between Apo2L/TRAIL and DR5 is described in Hymowitz et al.,
Molecular Cell, 4:563-571 (1999).
[0047] Yet another death domain-containing receptor, DR6, was
recently identified (Pan et al., FEBS Letters, 431:351-356 (1998)).
Aside from containing four putative extracellular cysteine rich
domains and a cytoplasmic death domain, DR6 is believed to contain
a putative leucine-zipper sequence that overlaps with a
proline-rich motif in the cytoplasmic region. The proline-rich
motif resembles sequences that bind to src-homology-3 domains,
which are found in many intracellular signal-transducing
molecules.
[0048] A further group of recently identified receptors are
referred to as "decoy receptors," which are believed to function as
inhibitors, rather than transducers of signaling. This group
includes DcR1 (also referred to as TRID, LIT or TRAIL-R3) (Pan et
al., Science, 276:111-113 (1997); Sheridan et al., Science,
277:818-821 (1997); McFarlane et al., J. Biol. Chem.,
272:25417-25420 (1997); Schneider et al., FEBS Letters, 416:329-334
(1997); Degli-Esposti et al., J. Exp. Med., 186:1165-1170 (1997);
and Mongkolsapaya et al., J. Immunol., 160:3-6 (1998)) and DcR2
(also called TRUNDD or TRAIL-R4) (Marsters et al., Curr. Biol.,
7:1003-1006 (1997); Pan et al., FEBS Letters, 424:41-45 (1998);
Degli-Esposti et al., Immunity, 7:813-820 (1997)), both cell
surface molecules, as well as OPG (Simonet et al., supra; Emery et
al., infra) and DcR3 (Pitti et al., Nature, 396:699-703 (1998)),
both of which are secreted, soluble proteins.
[0049] Additional newly identified members of the TNFR family
include CAR1, HVEM, GITR, ZTNFR-5, NTR-1, and TNFL1 (Brojatsch et
al., Cell, 87:845-855 (1996); Montgomery et al., Cell, 87:427-436
(1996); Marsters et al., J. Biol. Chem., 272:14029-14032 (1997);
Nocentini et al., Proc. Natl. Acad. Sci. USA 94:6216-6221 (1997);
Emery et al., J. Biol. Chem., 273:14363-14367 (1998); WO99/04001
published Jan. 28, 1999; WO99/07738 published Feb. 18, 1999;
WO99/33980 published Jul. 8, 1999).
[0050] As reviewed recently by Tewari et al., TNFR1, TNFR2 and CD40
modulate the expression of proinflammatory and costimulatory
cytokines, cytokine receptors, and cell adhesion molecules through
activation of the transcription factor, NF-.kappa.B (Tewari et al.,
Curr. Op. Genet. Develop., 6:39-44 (1996)). NF-.kappa.B is the
prototype of a family of dimeric transcription factors whose
subunits contain conserved Rel regions (Verma et al., Genes
Develop., 9:2723-2735 (1996); Baldwin, Ann. Rev. Immunol.,
14:649-681 (1996)). In its latent form, NF-.kappa.B is complexed
with members of the I-.kappa.B inhibitor family; upon inactivation
of the I-.kappa.B in response to certain stimuli, released
NF-.kappa.B translocates to the nucleus where it binds to specific
DNA sequences and activates gene transcription. As described above,
the TNFR members identified to date either include or lack an
intracellular death domain region. Some TNFR molecules lacking a
death domain, such as TNFR2, CD40, HVEM, and GITR, are capable of
modulating NF-.kappa.B activity. (see, e.g., Lotz et al., J.
Leukocyte Biol., 60:1-7 (1996)).
[0051] For a review of the TNF family of cytokines and their
receptors, see Ashkenazi and Dixit, Science, 281:1305-1308 (1998);
Golstein, Curr. Biol., 7:750-753 (1997); Gruss and Dower, supra,
and Nagata, Cell, 88:355-365 (1997).
B Cell Surface Antigens
[0052] Lymphocytes are one of many types of white blood cells
produced in the bone marrow during the process of hematopoiesis.
There are two major populations of lymphocytes: B lymphocytes (B
cells) and T lymphocytes (T cells). The lymphocytes of particular
interest herein are B cells.
[0053] B cells mature within the bone marrow and leave the marrow
expressing an antigen-binding antibody on their cell surface. When
a naive B cell first encounters the antigen for which its
membrane-bound antibody is specific, the cell begins to divide
rapidly and its progeny differentiate into memory B cells and
effector cells called "plasma cells". Memory B cells have a longer
life span and continue to express membrane-bound antibody with the
same specificity as the original parent cell. Plasma cells do not
produce membrane-bound antibody but instead produce the antibody in
a form that can be secreted. Secreted antibodies are the major
effector molecule of humoral immunity.
[0054] The CD20 antigen (also called human B-lymphocyte-restricted
differentiation antigen, Bp35) is a hydrophobic transmembrane
protein with a molecular weight of approximately 35 kD located on
pre-B and mature B lymphocytes (Valentine et al. J. Biol. Chem.
264(19):11282-11287 (1989); and Einfeld et al. EMBO J. 7(3):711-717
(1988)). The antigen is also expressed on greater than 90% of B
cell non-Hodgkin's lymphomas (NHL) (Anderson et al. Blood
63(6):1424-1433 (1984)), but is not found on hematopoietic stem
cells, pro-B cells, normal plasma cells or other normal tissues
(Tedder et al. J. Immunol. 135(2):973-979 (1985)). CD20 regulates
an early step(s) in the activation process for cell cycle
initiation and differentiation (Tedder et al., supra) and possibly
functions as a calcium ion channel (Tedder et al. J. Cell. Biochem.
14D:195 (1990)).
[0055] Given the expression of CD20 in B cell lymphomas, this
antigen can serve as a candidate for "targeting" of such lymphomas.
In essence, such targeting can be generalized as follows:
antibodies specific to the CD20 surface antigen of B cells are
administered to a patient. These anti-CD20 antibodies specifically
bind to the CD20 antigen of (ostensibly) both normal and malignant
B cells; the antibody bound to the CD20 surface antigen may lead to
the destruction and depletion of neoplastic B cells. Additionally,
chemical agents or radioactive labels having the potential to
destroy the tumor can be conjugated to the anti-CD20 antibody such
that the agent is specifically "delivered" to the neoplastic B
cells. Irrespective of the approach, a primary goal is to destroy
the tumor; the specific approach can be determined by the
particular anti-CD20 antibody which is utilized and, thus, the
available approaches to targeting the CD20 antigen can vary
considerably.
[0056] CD19 is another antigen that is expressed on the surface of
cells of the B lineage. Like CD20, CD19 is found on cells
throughout differentiation of the lineage from the stem cell stage
up to a point just prior to terminal differentiation into plasma
cells (Nadler, L. Lymphocyte Typing II 2: 3-37 and Appendix,
Renling et al. eds. (1986) by Springer Verlag). Unlike CD20
however, antibody binding to CD19 causes internalization of the
CD19 antigen. CD19 antigen is identified by the HD237-CD19 antibody
(also called the "B4" antibody) (Kiesel et al. Leukemia Research
II, 12: 1119 (1987)), among others. The CD19 antigen is present on
4-8% of peripheral blood mononuclear cells and on greater than 90%
of B cells isolated from peripheral blood, spleen, lymph node or
tonsil. CD19 is not detected on peripheral blood T cells, monocytes
or granulocytes. Virtually all non-T cell acute lymphoblastic
leukemias (ALL), B cell chronic lymphocytic leukemias (CLL) and B
cell lymphomas express CD19 detectable by the antibody B4 (Nadler
et al. J. Immunol. 131:244 (1983); and Nadler et al. in Progress in
Hematology Vol. XII pp. 187-206. Brown, E. ed. (1981) by Grune
& Stratton, Inc).
[0057] Additional antibodies which recognize differentiation
stage-specific antigens expressed by cells of the B cell lineage
have been identified. Among these are the B2 antibody directed
against the CD21 antigen; B3 antibody directed against the CD22
antigen; and the J5 antibody directed against the CD10 antigen
(also called CALLA). See U.S. Pat. No. 5,595,721 issued Jan. 21,
1997 (Kaminski et al.).
[0058] The rituximab (RITUXAN.RTM.) antibody is a genetically
engineered chimeric murine/human monoclonal antibody directed
against the CD20 antigen. Rituximab is the antibody called "C2B8"
in U.S. Pat. No. 5,736,137 issued Apr. 7, 1998 (Anderson et al.).
RITUXAN.RTM. is indicated for the treatment of patients with
relapsed or refractory low-grade or follicular, CD20 positive, B
cell non-Hodgkin's lymphoma. In vitro mechanism of action studies
have demonstrated that RITUXAN.RTM. binds human complement and
lyses lymphoid B cell lines through CDC (Reff et al. Blood
83(2):435-445 (1994)). Additionally, it has significant activity in
assays for ADCC. More recently, RITUXAN.RTM. has been shown to have
anti-proliferative effects in tritiated thymidine incorporation
assays and to induce apoptosis directly, while other anti-CD19 and
CD20 antibodies do not (Maloney et al. Blood 88(10):637a (1996)).
Synergy between RITUXAN.RTM. and chemotherapies and toxins has also
been observed experimentally. In particular, RITUXAN.RTM.
sensitizes drug-resistant human B cell lymphoma cell lines to the
cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria toxin and
ricin (Demidem et al. Cancer Chemotherapy &
Radiopharmaceuticals 12(3):177-186 (1997)). In vivo preclinical
studies have shown that RITUXAN.RTM. depletes B cells from the
peripheral blood, lymph nodes, and bone marrow of cynomolgus
monkeys, presumably through complement and cell-mediated processes
(Reff et al. Blood 83(2):435-445 (1994)).
SUMMARY OF THE INVENTION
[0059] The present invention provides multivalent antibodies (e.g.
tetravalent antibodies) with three or more antigen binding sites,
which can be readily produced by recombinant expression of nucleic
acid encoding the polypeptide chains of the antibody. In one
embodiment, the multivalent antibody comprises a dimerization
domain and three or more antigen binding sites. The preferred
dimerization domain comprises (or consists of) an Fc region or a
hinge region. In one embodiment, the invention provides an isolated
antibody comprising a dimerization domain and three or more antigen
binding sites amino-terminal thereto. The invention further
provides an isolated antibody comprising an Fc region and three or
more antigen binding sites amino-terminal to the Fc region. The
preferred multivalent antibody herein comprises (or consists of)
three to about eight, but preferably four, antigen binding sites
(which are generally all "functional", as hereindefined). In one
embodiment, the multivalent antibody comprises five or more (e.g.
up to about eight) antigen binding sites. The multivalent antibody
herein is preferably not a native sequence IgA or IgM, and may lack
an Fc region or have only one Fc region.
[0060] In the preferred embodiment, the multivalent antibody
comprises at least one polypeptide chain (and preferably two
polypeptide chains), wherein the polypeptide chain(s) comprise two
or more variable domains. For instance, the polypeptide chain(s)
may comprise VD1-(X1).sub.n-VD2-(X2).sub.n-Fc, wherein VD1 is a
first variable domain, VD2 is a second variable domain, Fc is one
polypeptide chain of an Fc region, X1 and X2 represent an amino
acid or polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region
chain; VH-CH1-VH-CH1-Fc region chain; VL-CL-flexible
linker-VL-CL-Fc region chain; or VL-CL-VL-CL-Fc region chain. Where
the polypeptide chain (or polypeptide chains) comprise Fd-flexible
linker-Fd, the flexible linker may comprise a peptide such as
gly-ser, gly-ser-gly-ser (SEQ ID NO:10), ala-ser, or
gly-gly-gly-ser (SEQ ID NO:11).
[0061] The multivalent antibody herein preferably further comprises
at least two (and preferably four) light chain variable domain
polypeptides. The multivalent antibody herein may, for instance,
comprise from about two to about eight light chain variable domain
polypeptides. The light chain variable domain polypeptides
contemplated here comprise a light chain variable domain and,
optionally, further comprise a CL domain.
[0062] The multivalent antibodies herein have properties which are
desirable, among other things, from a therapeutic standpoint. For
instance, the multivalent antibody may (1) be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind; (2) be an agonist
antibody; and/or (3) induce cell death and/or apoptosis of a cell
expressing an antigen which the multivalent antibody is capable of
binding to. The "parent antibody" which provides at least one
antigen binding specificity of the multivalent antibody may be one
which is internalized (and/or catabolized) by a cell expressing an
antigen to which the antibody binds; and/or may be an agonist,
cell-death-inducing, and/or apoptosis-inducing antibody, and the
multivalent form of the antibody as described herein may display
improvement(s) in one or more of these properties. Moreover, the
parent antibody may lack any one or more of these properties, but
may be endowed with them when constructed as a multivalent antibody
as hereindescribed.
[0063] The three or more antigen binding sites of the multivalent
antibodies herein may all bind the same antigen; or may bind two or
more (e.g. from two to about three) different antigens.
[0064] The multivalent antibody may bind (1) a cell surface protein
expressed (or overexpressed) by tumor cells, e.g. Epidermal Growth
Factor Receptor (EGFR), HER2 receptor, HER3 receptor, HER4
receptor, or DcR3; (2) a receptor in the Tumor Necrosis Factor
(TNF) receptor superfamily (e.g. an Apo2L receptor, such as DR4,
DR5, DcR1 or DcR2); and/or (3) a B cell surface antigen (such as
CD19, CD.sub.20, CD22 or CD40). In the preferred embodiment of the
invention, all of the functional antigen binding sites of the
multivalent antibody bind the same antigen as listed above (e.g.
all four antigen binding sites of a tetravalent antibody bind
either (1), (2) or (3)).
[0065] The invention also provides immunoconjugates comprising the
multivalent antibody conjugated with a cytotoxic agent. The
cytotoxic agent here may be one which is active in killing cells
once internalized.
[0066] The invention additionally pertains to a polypeptide chain
comprising VD1-(X1).sub.n-VD2 (X2).sub.n-Fc, wherein VD1 is a first
variable domain, VD2 is a second variable domain, Fc is one
polypeptide chain of an Fc region, X1 and X2 represent an amino
acid or polypeptide, and n is 0 or 1. For instance, the polypeptide
chain may comprise VH-CH1-flexible linker-VH-CH1-Fc region chain;
VH-CH1-VH-CH1-Fc region chain; VL-CL-flexible linker-VL-CL-Fc
region chain; or VL-CL-VL-CL-Fc region chain. In another
embodiment, the polypeptide chain comprises VH-CH1-flexible
linker-VH-CH1-dimerization domain; VH-CH1-VH-CH1-dimerization
domain; VL-CL-flexible linker-VL-CL-dimerization domain; or
VL-CL-VL-CL-dimerization domain. For instance, the polypeptide
chain may comprise VH-CH1-flexible linker-VH-CH.sub.1-hinge region;
VH-CH1-VH-CH1-hinge region. The invention additionally provides an
antibody comprising one or more (preferably two) of such
polypeptide chains. The antibody preferably further comprises at
least two (and preferably four) light chain or heavy chain variable
domain polypeptides, e.g., where the light chain variable domain
polypeptides comprise VL-CL and the heavy chain variable domain
polypeptides comprise VH-CH1.
[0067] The invention further provides a polypeptide chain
comprising three or more heavy chain or light chain variable
domains, wherein each of the variable domains is able to combine
with three or more light chain or heavy chain variable domain
polypeptides to form three or more antigen binding sites, each
directed against the same antigen. The invention also provides an
isolated antibody comprising the polypeptide chain. In the
preferred embodiment, where the polypeptide chain comprises three
or more heavy chain variable domains, the antibody preferably
further comprises three or more light chain variable domain
polypeptides which can combine with the heavy chain variable
domains to form the three or more antigen binding sites. Examples
of such antibodies are shown in FIG. 23 D (with three antigen
binding sites) and FIG. 23E (with four antigen binding sites). In
addition, the invention provides a polypeptide chain comprising the
formula: (a) VL-CL-flexible linker-VL-CL-flexible linker-VL-CL; (b)
VH-CH1-flexible linker-VH-CH1-flexible linker-VH-CH1; (c)
(VL-CL).sub.n, wherein n is three or more; or (d) (VH-CH 1).sub.n,
wherein n is three or more.
[0068] The invention further provides: isolated nucleic acid
encoding the multivalent antibody or polypeptide chain; a vector
comprising nucleic acid encoding the multimeric antibody or
polypeptide chain, optionally, operably linked to control sequences
recognized by a host cell transformed with the vector; a host cell
comprising (e.g. transformed with) nucleic acid encoding the
multimeric antibody or polypeptide chain; a method for producing
the multivalent antibody or polypeptide chain comprising culturing
the host cell so that the nucleic acid is expressed and,
optionally, recovering the multivalent antibody or polypeptide
chain from the host cell culture (e.g. from the host cell culture
medium). Nucleic acids encoding (1) the heavy chain variable
domains and (2) the light chain variable domains of the multivalent
antibody are preferrably co-expressed by a host cell transformed
with both (1) and (2). Nucleic acids (1) and (2) may be present in
the same, or different, vectors.
[0069] Diagnostic and therapeutic uses for the multivalent
antibodies disclosed herein are contemplated. In one diagnostic
application, the invention provides a method for determining the
presence of an antigen of interest comprising exposing a sample
suspected of containing the antigen to the multivalent antibody and
determining binding of the multivalent antibody to the sample. Both
in vitro and in vivo diagnostic methods are provided.
[0070] In one therapeutic application, the invention provides a
method of treating a mammal suffering from, or predisposed to, a
disease or disorder, comprising administering to the mammal a
therapeutically effective amount of a multivalent antibody as
disclosed herein, or of a composition comprising the multivalent
antibody and a pharmaceutically acceptable carrier. The disorder to
be treated herein may be cancer, in which case the method may
further comprise administering a therapeutically effective amount
of a cytotoxic agent to the mammal. The present invention further
relates to a method of inducing apoptosis of a cancer cell
comprising exposing the cell to a multivalent antibody as described
herein, wherein the multivalent antibody binds a receptor in the
Tumor Necrosis Factor (TNF) receptor superfamily. The method may
involve killing a B cell by exposing the B cell to a multivalent
antibody that binds a B cell surface antigen. Moreover, the method
may relate to killing a cell which expresses (or overexpresses) an
ErbB receptor comprising exposing the cell to an antibody that
binds the ErbB receptor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0071] FIG. 1 is a schematic representation of a native IgG and
digestion thereof with (1) papain to generate two Fab fragments and
an Fc region or (2) pepsin to generate a F(ab').sub.2 fragment and
multiple small fragments. Disulfide bonds are represented by lines
between CH1 and CL domains and the two CH2 domains. V is variable
domain; C is constant domain; L stands for light chain and H stands
for heavy chain.
[0072] FIGS. 2A-E depict the structures of the five major naturally
occurring immunoglobulin isotypes; IgG (FIG. 2A), IgD (FIG. 2B),
IgE (FIG. 2C), IgA dimer (FIG. 2D), and IgM pentamer (FIG. 2E).
[0073] FIG. 3 depicts alignments of native sequence IgG Fc regions.
Native sequence human IgG Fc region sequences, humIgG1 (non-A and A
allotypes) (SEQ ID NOs: 1 and 2, respectively), humIgG2 (SEQ ID
NO:3), humIgG3 (SEQ ID NO:4) and humIgG4 (SEQ ID NO:5), are shown.
The human IgG1 sequence is the non-A allotype, and differences
between this sequence and the A allotype (at positions 356 and 358;
EU numbering system) are shown below the human IgG1 sequence.
Native sequence murine IgG Fc region sequences, murIgG1 (SEQ ID
NO:6), murIgG2A (SEQ ID NO:7), murIgG2B (SEQ ID NO:8) and murlgG3
(SEQ ID NO:9), are also shown.
[0074] FIGS. 4A-B depict schematically tetravalent antibodies
according to the present invention. In FIG. 4A, the four antigen
binding Fabs are numbered (1 and 2 for each arm of the tetravalent
antibody) and X represents a dimerization domain. In FIG. 4B, the
dimerization domain of the tetravalent antibody is an Fc
region.
[0075] FIG. 5 shows the construct used for expression of a
tetravalent anti-HER2 antibody (OctHER2) in Example 1.
[0076] FIGS. 6A-C illustrate binding of OctHER2 (FIG. 6A); bivalent
IgG1 rhuMAb 4D5-8 expressed by 293 cells (FIG. 6B); and vialed
HERCEPTIN.RTM. (expressed by Chinese hamster ovary (CHO) cells)
(FIG. 6C) to HER2 extracellular domain (ECD) as determined using an
enzyme-linked immunosorbent assay (ELISA).
[0077] FIG. 7 depicts ultracentrifugation analysis of binding of
OctHER2 to HER2ECD. Average molecular weights (theoretical or
experimentally determined) versus molar ratio of OctHER2 to HER2ECD
are shown. Theoretical calculated average molecular weights
assuming tetravalent antibody has four fully functional binding
sites are shown in circles; theoretical calculated average
molecular weights assuming tetravalent antibody has three fully
functional binding sites are shown in squares; and triangles
represent experimentally determined molecular weights.
[0078] FIGS. 8A-D depict the growth inhibitory activity of
HERCEPTIN.RTM. compared to OctHER2 using SKBR3 (3+ HER2
overexpressing) (FIG. 8A), MDA 361 (2+ HER2 overexpressing) (FIG.
8B), BT474 (3+ HER2 overexpressing) (FIG. 8C) and MCF7 (0+ HER2
expressing) (FIG. 8D) cell lines.
[0079] FIG. 9 depicts the effect of flexible linkers on the growth
inhibitory activity of tetravalent anti-HER2 antibodies with
respect to MDA 231 cells (1+ HER2 overexpressing) or SKBR3 cells
(3+ HER2 overexpressing).
[0080] FIGS. 10A-B compare the rate of OctHER2
internalization/catabolism (FIG. 10A) to that of HERCEPTIN.RTM.
(FIG. 10B), in relation to both MDA 453 (2+ HER2 overexpressing)
and SKBR3 (3+ HER2 overexpressing) cell lines.
[0081] FIGS. 11A-I are electron microscopy photographs showing
internalization of OctHER2. FIGS. 11A-F show subcellular
localization of .sup.125I-OctHER2 in SKBR3 cells. Autoradiographic
silver grains were observed associated with the villi of the apical
cell membrane (FIG. 11A), in close proximity with a forming coated
pit (FIG. 11B, arrow), with smooth cytosolic vesicles (FIGS. 11C
and D) and endosomes (FIGS. 11E and F). Bars=0.25 .mu.M. FIGS.
11G-I show internalization at time 0 hours (FIGS. 11G) and 5 hours
(FIGS. 11H and 11I).
[0082] FIGS. 12A-E depict apoptosis induced by an anti-DR5
tetravalent antibody (16E2 Octopus), an anti-DR5 bivalent IgG
antibody (16E2 IgG), and Apo2L/TRAIL (Apo2L) on cancer cell lines:
COLO 205 (FIG. 12A), SK-MES-1 (FIG. 12B), HCT116 (FIG. 12C), and
HOP 92 (FIG. 12D), compared to a non-cancer control cell line,
HUMEC (FIG. 12E).
[0083] FIGS. 13A-D are histology slides stained to detect apoptotic
cells. Tumor tissues from mice treated with 16E2 Octopus or
Apo2L/TRAIL were fixed in 10% formalin and then embedded into
parafilm and sectioned onto slides which were then stained with
hematoxylin and eosin and visualized under a 400.times.
magnification. The effect of 16E2 Octopus at 6 and 24 hours is
shown in FIGS. 13A and B, respectively; control-treated cells are
shown in FIG. 13C; and Apo2L/TRAIL-treated cells are shown in FIG.
13D.
[0084] FIG. 14 represents the in vivo activity of Apo2L/TRAIL (60
mg/kg, 5.times./week), 3H3 bivalent IgG (5 mg/kg given days 0, 3, 5
and 9), 16E2 bivalent IgG (16E2) (5 mg/kg given days 0, 3, 5 and
9), and 16E2 Octopus (5 mg/kg given days 0, 3, 5 and 9) with
respect to COLO 205 tumors in athymic nude mice.
[0085] FIG. 15 represents an alamarBlue in vitro assay confirming
the apoptotic activity of the material used in the mouse studies
(Apo2L/TRAIL and 16E2 Octopus) as compared to an Apo2L standard
positive control. The anti-IgE antibody (E25) used as a negative
control in the mouse studies was confirmed to have no apoptotic
activity.
[0086] FIG. 16 represents the results of a crystal violet apoptosis
assay comparing anti-DR5 3H3 Octopus to various batches of the
anti-DR5 16E2 Octopus.
[0087] FIGS. 17A-B reveal the results of the alamarBlue apoptosis
assay with respect to Apo2L/TRAIL (WO97/25428), anti-DR5 3H3
Octopus antibody, anti-DR5 16E2 Octopus antibody, and Apo2L/TRAIL
with a FLAG epitope-tag cross linked by an anti-FLAG antibody
(WO97/25428), with respect to SK-MES-1 (FIG. 17A) and Jurkat (FIG.
17B) cells in the presence of 5% fetal bovine serum (FBS).
[0088] FIGS. 18A-C depict dose response curves that show the effect
of the anti-DR5 16E2 Octopus (upper graphs) compared to Apo2L/TRAIL
(lower graphs) on the growth of leukemia, non-small cell lung
cancer, colon cancer, central nervous system (CNS) cancer,
melanoma, ovarian cancer, renal cancer, prostate cancer and breast
cancer human tumor cell lines at 2 days. Results are from the
National Cancer Institute Developmental Therapeutics Program. All
samples were tested at 5 concentrations, starting at 1% of the
stock solution (16E2 Octopus stock 0.2 mg/ml) and 4.times.0.5 log
dilutions.
[0089] FIGS. 19A-C depict dose response curves that show the effect
of the anti-DR516E2 Octopus (upper graphs) compared to Apo2L/TRAIL
(lower graphs) on the growth of leukemia, non-small cell lung
cancer, colon cancer, central nervous system (CNS) cancer,
melanoma, ovarian cancer, renal cancer, prostate cancer and breast
cancer human tumor cell lines at 6 days. Results are from the
National Cancer Institute Developmental Therapeutics Program. All
samples were tested at 5 concentrations, starting at 1% of the
stock solution (16E2 Octopus stock 0.2 mg/ml) and 4.times.0.5 log
dilutions.
[0090] FIGS. 20A-B present a quantitative summary of the 2 day in
vitro results from the National Cancer Institute Developmental
Therapeutics Program comparing the anti-DR5 16E2 Octopus (FIG. 20A)
to Apo2L/TRAIL (FIG. 20B) analyzing growth inhibition (GI50),
stasis (TGI), and toxicity (LC50).
[0091] FIGS. 21A-B present a quantitative summary of the 6 day in
vitro results from the National Cancer Institute Developmental
Therapeutics Program comparing the anti-DR5 16E2 Octopus (FIG. 21A)
to Apo2L/TRAIL (FIG. 21B) analyzing growth inhibition (G150),
stasis (TGI), and toxicity (LC50).
[0092] FIG. 22 depicts apoptosis of Wil-2 cells by the anti-CD20
antibody RITUXAN.RTM., RITUXAN.RTM. cross-linked with anti-human
IgG (RITUXAN.RTM.-IgG) and a tetravalent anti-CD20 antibody
(OctCD20).
[0093] FIGS. 23A-E are cartoons depicting the full-length
Octopus/tetravalent antibody (FIG. 23B), the Octopus F(ab)'.sub.2
(FIG. 23C), POPoct-3 Fab (FIG. 23D) and POPoct-4 Fab (FIG. 23E) in
comparison to the native IgG (FIG. 23A). A representative coomassie
stained Tris-Glycine gel of anti-CD20 (C2B8) Octopus proteins
compares the sizes of the intact antibodies in non-reducing
conditions (FIG. 23F), and of the heavy chains in reducing
conditions, under which disulfide bonds are disrupted resulting in
separation of the heavy and light chains (FIG. 23G).
[0094] FIG. 24 depicts the construction of the Octopus F(ab').sub.2
backbone. Any VH/CH1 region can be substituted into the
F(ab').sub.2 backbone via the BamHI, NheI and BspEI restriction
enzyme sites.
[0095] FIG. 25 depicts the construction of the POPoct-3 heavy
chain.
[0096] FIG. 26 depicts the construction of the POPoct-4 heavy
chain.
[0097] FIG. 27 depicts the activity of multivalent anti-HER2
antibodies in cytostasis assays using BT474 cells.
[0098] FIGS. 28A-B depict the activity of multivalent anti-HER2
antibodies in cytostasis assays using SKBR3 cells. The figures are
representative plots of n=4 cytostasis assays.
[0099] FIGS. 29A-B show internalization capability of multivalent
anti-HER2 antibodies in SKBR3 cells (FIG. 29A) and BT474 cells
(FIG. 29B).
[0100] FIGS. 30A-B reveal apoptosis of COLO205 cells by multivalent
anti-DR5 antibodies
[0101] FIGS. 31A-B demonstrate signalling of multivalent anti-DR5
antibodies through the caspase pathway.
[0102] FIG. 32 compares apoptosis induced by IgG cross-linked
RITUXAN.RTM. (RITUXAN-IgG) and IgG cross-linked OctCD20
(OctCD20-IgG).
[0103] FIG. 33 shows apoptosis of WIL2 cells by multivalent
anti-CD20 antibodies, the IF5 anti-CD20 antibody (Clark et al. PNAS
(USA) 82: 1766-1770 (1985)) and IgG cross-linked IFS antibody
(IF5+IgG-X).
[0104] FIG. 34 depicts homotypic cell adhesion in WIL2S cells
induced by IF5 anti-CD20 antibody, IgG cross-linked IF5 antibody
and POPoct-3 CD20.
[0105] FIG. 35 reflects RITUXAN.RTM. or OctCD20
internalization/catabolism on DB, WIL2 and Ramos B-cell lymphoma
lines.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0106] Throughout the present specification and claims, the
numbering of the residues in an immunoglobulin heavy chain is that
of the EU index as in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991), expressly incorporated
herein by reference. The "EU index as in Kabat" refers to the
residue numbering of the human IgG1 EU antibody.
[0107] An "ErbB receptor" is a receptor protein tyrosine kinase
which belongs to the ErbB receptor family and includes EGFR, HER2,
ErbB3 and ErbB4 receptors as well as TEGFR (U.S. Pat. No.
5,708,156) and other members of this family to be identified in the
future. The ErbB receptor will generally comprise an extracellular
domain, which may bind an ErbB ligand; a lipophilic transmembrane
domain; a conserved intracellular tyrosine kinase domain; and a
carboxyl-terminal signaling domain harboring several tyrosine
residues which can be phosphorylated. The ErbB receptor may be a
native sequence ErbB receptor or an amino acid sequence variant
thereof. Preferably the ErbB receptor is native sequence human ErbB
receptor.
[0108] By "ErbB ligand" is meant a polypeptide which binds to
and/or activates an ErbB receptor. The ErbB ligand of particular
interest herein is a native sequence human ErbB ligand such as
Epidermal Growth Factor (EGF) (Savage et al., J. Biol. Chem.
247:7612-7621 (1972)); Tansforming Growth Factor alpha (TGF-alpha)
(Marquardt et al., Science 223:1079-1082 (1984)); amphiregulin also
known as schwanoma or keratinocyte autocrine growth factor (Shoyab
et al. Science 243:1074-1076 (1989); Kimura et al. Nature
348:257-260 (1990); and Cook et al. Mol. Cell. Biol. 11:2547-2557
(1991)); betacellulin (Shing et al., Science 259:1604-1607 (1993);
and Sasada et al. Biochem. Biophys. Res. Commun. 190:1173 (1993));
heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et
al., Science 251:936-939 (1991)); epiregulin (Toyoda et al., J.
Biol. Chem. 270:7495-7500 (1995); and Komurasaki et al. Oncogene
15:2841-2848 (1997)), a heregulin (see below); neuregulin-2 (NRG-2)
(Carraway et al., Nature 387:512-516 (1997)); neuregulin-3 (NRG-3)
(Zhang et al., Proc. Natl. Acad. Sci. 94:9562-9567 (1997)); or
cripto (CR-1) (Kannan et al. J. Biol. Chem. 272(6):3330-3335
(1997)). ErbB ligands which bind EGFR include EGF, TGF-alpha,
amphiregulin, betacellulin, HB-EGF and epiregulin. ErbB ligands
which bind HER3 include heregulins. ErbB ligands capable of binding
HER4 include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3 and
heregulins.
[0109] "Heregulin" (HRG) when used herein refers to a polypeptide
comprising an amino acid sequence encoded by the heregulin gene
product as disclosed in U.S. Pat. No. 5,641,869 or Marchionni et
al., Nature, 362:312-318 (1993), and biologically active variants
of such polypeptides. Examples of heregulins include
heregulin-alpha heregulin-beta1, heregulin-beta2 and
heregulin-beta3 (Holmes et al., Science, 256:1205-1210 (1992); and
U.S. Pat. No. 5,641,869); neu differentiation factor (NDF) (Peles
et al. Cell 69: 205-216 (1992)); acetylcholine receptor-inducing
activity (ARIA) (Falls et al. Cell 72:801-815 (1993)); glial growth
factors (GGFs) (Marchionni et al., Nature, 362:312-318 (1993));
sensory and motor neuron derived factor (SMDF) (Ho et al. J. Biol.
Chem. 270:14523-14532 (1995)); gamma-heregulin (Schaefer et al.
Oncogene 15:1385-1394 (1997)). An example of a biologically active
fragment/amino acid sequence variant of a native sequence HRG
polypeptide, is an EGF-like domain fragment (e.g.
HRGbeta1.sub.177-244).
[0110] An "ErbB hetero-oligomer" herein is a noncovalently
associated oligomer comprising at least two different ErbB
receptors. Such complexes may form when a cell expressing two or
more ErbB receptors is exposed to an ErbB ligand and can be
isolated by immunoprecipitation and analyzed by SDS-PAGE as
described in Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665
(1994), for example. Examples of such ErbB hetero-oligomers include
EGFR-HER2, HER2-HER3 and HER3-HER4 complexes. Moreover, the ErbB
hetero-oligomer may comprise two or more HER2 receptors combined
with a different ErbB receptor, such as HER3, HER4 or EGFR. Other
proteins, such as a cytokine receptor subunit (e.g. gp130), may be
included in the hetero-oligomer.
[0111] The terms "ErbB1", "epidermal growth factor receptor" and
"EGFR" are used interchangeably herein and refer to native sequence
EGFR as disclosed, for example, in Carpenter et al. Ann. Rev.
Biochem. 56:881-914 (1987), including variants thereof (e.g. a
deletion mutant EGFR as in Humphrey et PNAS (USA) 87:4207-4211
(1990)). erbB1 refers to the gene encoding the EGFR protein
product. Examples of antibodies which bind to EGFR include MAb 579
(ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL
8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533,
Mendelsohn et al.) and variants thereof, such as chimerized 225
(C225) and reshaped human 225 (H225) (see, WO 96/40210, Imclone
Systems Inc.).
[0112] The expressions "ErbB2" and "HER2" are used interchangeably
herein and refer to native sequence human HER2 protein described,
for example, in Semba et al., PNAS (USA) 82:6497-6501 (1985) and
Yamamoto et al. Nature 319:230-234 (1986) (Genebank accession
number X03363), and variants thereof. The term erbB2 refers to the
gene encoding human HER2 and neu refers to the gene encoding rat
p185.sup.neu. Preferred HER2 is native sequence human HER2.
Examples of antibodies which bind HER2 include MAbs 4D5 (ATCC CRL
10463), 2C4 (ATCC HB-12697), 7F3 (ATCC HB-12216), and 7C2 (ATCC HB
12215) (see, U.S. Pat. No. 5,772,997; WO98/77797; and U.S. Pat. No.
5,840,525, expressly incorporated herein by reference). Humanized
anti-HER2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3,
huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8
(HERCEPTIN.RTM.) as described in Table 3 of U.S. Pat. No. 5,821,337
expressly incorporated herein by reference; humanized 520C9
(WO93/21319). Human anti-HER2 antibodies are described in U.S. Pat.
No. 5,772,997 issued Jun. 30, 1998 and WO 97/00271 published Jan.
3, 1997.
[0113] "ErbB3" and "HER3" refer to the receptor polypeptide as
disclosed, for example, in U.S. Pat. Nos. 5,183,884 and 5,480,968
as well as Kraus et al. PNAS (USA) 86:9193-9197 (1989), including
variants thereof. Examples of antibodies which bind HER3 are
described in U.S. Pat. No. 5,968,511 (Akita and Sliwkowski), e.g.
the 8B8 antibody (ATCC HB 12070) or a humanized variant
thereof.
[0114] The terms "ErbB4" and "HER4" herein refer to the receptor
polypeptide as disclosed, for example, in EP Pat Appin No 599,274;
Plowman et al., Proc. Natl. Acad. ScL USA, 90:1746-1750 (1993); and
Plowman et al., Nature, 366:473-475 (1993), including variants
thereof such as the HER4 isoforms disclosed in WO 99/19488.
[0115] A "B cell surface marker" herein is an antigen expressed on
the surface of a B cell which can be targeted with an antibody
which binds thereto. Exemplary B cell surface markers include the
CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72,
CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81,
CD82, CD83, CDw84, CD85 and CD86 leukocyte surface markers. The B
cell surface marker of particular interest is preferentially
expressed on B cells compared to other non-B cell tissues of a
mammal and may be expressed on both precursor B cells and mature B
cells. In one embodiment, the marker is one, like CD20 or CD19,
which is found on B cells throughout differentiation of the lineage
from the stem cell stage up to a point just prior to terminal
differentiation into plasma cells. The preferred B cell surface
markers herein are CD19, CD20, CD22 and CD40.
[0116] The "CD20" antigen is an about 35 kDa, non-glycosylated
phosphoprotein found on the surface of greater than 90% of B cells
from peripheral blood or lymphoid organs. CD20 is expressed during
early pre-B cell development and remains until plasma cell
differentiation. CD20 is present on both normal B cells as well as
malignant B cells. Other names for CD20 in the literature include
"B-lymphocyte-restricted antigen" and "Bp35". The CD20 antigen is
described in Clark et al. PNAS (USA) 82:1766 (1985), for example.
Examples of antibodies which bind the CD20 antigen include: "C2B8"
which is now called "rituximab" ("RITUXAN.RTM.") (U.S. Pat. No.
5,736,137, expressly incorporated herein by reference); the
yttrium-[90]-labeled 2B8 murine antibody designated "Y2B8" (U.S.
Pat. No. 5,736,137, expressly incorporated herein by reference);
murine IgG2a "B1" optionally labeled with .sup.131I to generate the
".sup.131I-B1" antibody (BEXXAR.TM.) (U.S. Pat. No. 5,595,721,
expressly incorporated herein by reference); murine monoclonal
antibody "1F5" (Press et al. Blood 69(2):584-591 (1987)); "chimeric
2H7" antibody (U.S. Pat. No. 5,677,180, expressly incorporated
herein by reference); and monoclonal antibodies L27, G28-2, 93-1B3,
B-C1 or NU-B2 available from the International Leukocyte Typing
Workshop (Valentine et al., In: Leukocyte Typing III (McMichael,
Ed., p. 440, Oxford University Press (1987)).
[0117] The "CD19" antigen refers to the about 90 kDa antigen
identified, for example, by the HD237-CD19 or B4 antibody (Kiesel
et al. Leukemia Research II, 12: 1119 (1987)). Like CD.sub.20, CD19
is found on cells throughout differentiation of the lineage from
the stem cell stage up to a point just prior to terminal
differentiation into plasma cells. Binding of an antibody to CD19
may cause internalization of the CD19 antigen. Examples of
antibodies which bind the CD19 antigen include the anti-CD19
antibodies in Hekman et al. Cancer Immunol. Immunother. 32:364-372
(1991) and Vlasveld et al. Cancer Immunol. Immunother. 40:37-47
(1995); and the B4 antibody in Kiesel et al. Leukemia Research II,
12: 1119 (1987).
[0118] The "CD22" antigen has a molecular weight of about 140,000
kD. CD22 is expressed in the cytoplasm of early pre-B and
progenitor cells, appears on the surface of only mature B cells and
on the majority of non-Hodgkin's lymphoma (NHL) cells, and is then
lost during terminal differentiation prior to the plasma cell stage
from both the surface and cytoplasm. An example of an anti-CD22
antibody is the LL2 antibody described in Juweid et al. Cancer
Research 55:5899-5907 (1995), including chimeric/humanized variants
thereof.
[0119] The "CD40" antigen is a cell surface phosphorylated
glycoprotein that is expressed on a variety of cell types,
including B cells, B cell malignancies, follicular dendritic cells,
basal epithelial cells and carcinomas. CD40 binds CD40 ligand
(CD40L). Aside from being a B cell surface antigen, CD40 is also a
member of the TNF receptor superfamily. Examples of antibodies that
bind CD40 include those which (1) block CD40/CD40L interaction and
have anti-neoplastic properties (Armitage et al., U.S. Pat. No.
5,674,492); (2) antagonize signaling through CD40 (deBoer et al.,
U.S. Pat. No. 5,677,165); (3) deliver a stimulatory signal through
CD40 but do not increase the interaction between CD40 and CD40L,
e.g., G28-5 (Ledbetter et al., U.S. Pat. No. 5,182,368); (4)
increase the interaction between CD40 and CD40L, e.g., CD40.4 (5C3)
(PharMingen, San Diego, Calif.) and S2C6 (deposited with the
American Type Culture Collection (ATCC), Manassass, Va. on May 25,
1999 under accession number PTA-110).
[0120] The "tumor necrosis factor receptor superfamily" or "TNF
receptor superfamily" herein refers to receptor polypeptides bound
by cytokines in the TNF family. Generally, these receptors are Type
I transmembrane receptors with one or more cysteine rich repeat
sequences in their extracellular domain. The TNF receptor
superfamily may be further subdivided into (1) death receptors; (2)
decoy receptors; and (3) signaling receptors that lack death
domains. The "death receptors" contain in their cytoplasmic or
intracellular region a "death domain", i.e., a region or sequence
which acts to transduce signals in the cell which can result in
apoptosis or in induction of certain genes. The "decoy receptors"
lack a functional death domain and are incapable of transducing
signals which result in apoptosis. Examples of cytokines in the TNF
gene family include Tumor Necrosis Factor-alpha (TNF-alpha), Tumor
Necrosis Factor-beta (TNF-beta or lymphotoxin), CD30 ligand, CD27
ligand, CD40 ligand, OX-40 ligand, 4-1 BB ligand, Apo-1 ligand
(also referred to as Fas ligand or CD95 ligand), Apo-2 ligand (also
referred to as TRAIL), Apo-3 ligand (also referred to as TWEAK),
osteoprotegerin (OPG), APRIL, RANK ligand (also referred to as
TRANCE), and TALL-1 (also referred to as BlyS, BAFF or THANK).
Examples of receptors in the TNF receptor superfamily include: type
1 Tumor Necrosis Factor Receptor (TNFR1), type 2 Tumor Necrosis
Factor Receptor (TNFR2), p75 Nerve Growth Factor receptor (NGFR),
the B cell surface antigen CD40, the T cell antigen OX-40, Apo-1
receptor (also called Fas or CD95), Apo-3 receptor (also called
DR3, swl-1, TRAMP and LARD), the receptor called "Transmembrane
Activator and CAML-Interactor" or "TACI", BCMA protein, DR4, DR5
(alternatively referred to as Apo-2; TRAIL-R2, TR6, Tango-63,
hAPO8, TRICK2 or KILLER), DR6, DcR1 (also referred to as TRID, LIT
or TRAIL-R3), DcR2 (also called TRAIL-R4 or TRUNDD), OPG, DcR3
(also called TR6 or M68), CAR1, HVEM (also called ATAR or TR2),
GITR, ZTNFR-5, NTR-1, TNFL1, CD30, Lymphotoxin beta receptor
(LTBr), 4-1BB receptor and TR9 (EP988, 371 A1).
[0121] The terms "Apo-2 ligand" or "Apo2L" refer to the Apo2L
polypeptides disclosed in WO97/25428, published 17 Jul. 1997 and
expressly incorporated herein by reference. For purposes of the
present application, these terms also refer to the polypeptides
referred to as TRAIL disclosed in WO97/01633, published 16 Jan.,
1997 and U.S. Pat. No. 5,763,223, issued Jun. 9, 1998 and expressly
incorporated herein by reference.
[0122] An "Apo2L receptor" is a polypeptide to which Apo2L can
specifically bind. The term "Apo2L receptor" when used herein
encompasses native sequence Apo2L receptors and variants thereof.
These terms encompass Apo2L receptor from a variety of mammals,
including humans. The Apo2L receptor may be isolated from a variety
of sources, such as from human tissue types or from another source,
or prepared by recombinant or synthetic methods. Examples of
"native sequence" Apo2L receptors include Apo-2 polypeptide or DR5
(WO98/51793, expressly incorporated herein by reference), native
sequence DR4 as described in Pan et al. Science 276:111-113 (1997);
native sequence decoy receptor 1 or DcR1 as in Sheridan et al.,
Science 277:818-821 (1997); and native sequence decoy receptor 2 or
DcR2 as in Marsters et al. Curr. Biol. 7:1003-1006 (1997); native
sequence osteoprotegerin (see Simonet et al. Cell 89:309-319
(1997); and Emery et al. J. Interferon and Cytokine Research 18(5):
A47 Abstract 2.17 (1998)). Examples of anti-DR5 antibodies include
3F11.39.7 (ATCC HB-12456), 3H3.14.5 (ATCC HB-12534), 3D5.1.10
(HB-12536) and 3H1.18.10 (HB-12535), 16E2 and 20E6 (see WO
98/51793, expressly incorporated herein by reference). Examples of
anti-DR4 antibodies include 4E7.24.3 (ATCC HB-12454) and 4H6.17.8
(ATCC HB-12455) (see, WO 99/37684, expressly incorporated herein by
reference).
[0123] Native sequence "DcR3" is described in WO99/14330, expressly
incorporated herein by reference. That patent publication describes
the following mAbs directed against DcR3: 4C4.1.4 (ATCC HB-12573);
5C4.14.7 (ATCC HB-12574); 11C5.2.8 (ATCC HB-12572); 8D3.1.5 (ATCC
HB-12571); and 4B7.1.1 (ATCC HB-12575).
[0124] A "native sequence" polypeptide comprises a polypeptide
having the same amino acid sequence as a polypeptide derived from
nature. Thus, a native sequence polypeptide can have the amino acid
sequence of naturally-occurring polypeptide from any mammal. Such
native sequence polypeptide can be isolated from nature or can be
produced by recombinant or synthetic means. The term "native
sequence" polypeptide specifically encompasses naturally-occurring
truncated or secreted forms of the polypeptide (e.g., an
extracellular domain sequence), naturally-occurring variant forms
(e.g., alternatively spliced forms) and naturally-occurring allelic
variants of the polypeptide.
[0125] A polypeptide "variant" means a biologically active
polypeptide having at least about 80% amino acid sequence identity
with the native sequence polypeptide. Such variants include, for
instance, polypeptides wherein one or more amino acid residues are
added, or deleted, at the N- or C-terminus of the polypeptide.
Ordinarily, a variant will have at least about 80% amino acid
sequence identity, more preferably at least about 90% amino acid
sequence identity, and even more preferably at least about 95%
amino acid sequence identity with the native sequence
polypeptide.
[0126] "Apoptosis" refers to programmed cell death. Physiological
events often indicative of the occurrence of apoptosis include:
fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane
vesicles (called apoptotic bodies). Various methods are available
for evaluating the cellular events associated with apoptosis. For
example, phosphatidyl serine (PS) translocation can be measured by
annexin V binding; DNA fragmentation can be evaluated through DNA
laddering or propidium-iodine staining; and nuclear/chromatin
condensation along with DNA fragmentation can be evaluated by any
increase in hypodiploid cells.
[0127] The term "antibody" is used in the broadest sense and
includes monoclonal antibodies (including full length or intact
monoclonal antibodies), polyclonal antibodies, multivalent
antibodies, multispecific antibodies (e.g., bispecific antibodies),
and antibody fragments (see below) so long as they exhibit the
desired biological activity.
[0128] Unless indicated otherwise, the expression "multivalent
antibody" is used throughout this specification to denote an
antibody comprising three or more antigen binding sites. The
multivalent antibody is preferably engineered to have the three or
more antigen binding sites and is generally not a native sequence
IgM or IgA antibody.
[0129] "Antibody fragments" comprise only a portion of an intact
antibody, generally including an antigen binding site of the intact
antibody and thus retaining the ability to bind antigen. Examples
of antibody fragments encompassed by the present definition
include: (i) the Fab fragment, having VL, CL, VH and CH1 domains;
(ii) the Fab' fragment, which is a Fab fragment having one or more
cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd
fragment having VH and CH1 domains; (iv) the Fd' fragment having VH
and CH1 domains and one or more cysteine residues at the C-terminus
of the CH1 domain; (v) the Fv fragment having the VL and VH domains
of a single arm of an antibody; (vi) the dAb fragment (Ward et al.,
Nature 341, 544-546 (1989)) which consists of a VH domain; (vii)
isolated CDR regions; (viii) F(ab').sub.2 fragments, a bivalent
fragment including two Fab' fragments linked by a disulphide bridge
at the hinge region; (ix) single chain antibody molecules (e.g.
single chain Fv; scFv) (Bird et al., Science 242:423-426 (1988);
and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies"
with two antigen binding sites, comprising a heavy chain variable
domain (VH) connected to a light chain variable domain (VL) in the
same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993));
(xi) "linear antibodies" comprising a pair of tandem Fd segments
(VH-CH1-VH-CH1) which, together with complementary light chain
polypeptides, form a pair of antigen binding regions (Zapata et al.
Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No.
5,641,870).
[0130] 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
antigen. Furthermore, in contrast to polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. The
modifier "monoclonal" 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., Nature 256:495 (1975), or may be made by recombinant
DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal
antibodies" may also be isolated from phage antibody libraries
using the techniques described in Clackson et al., Nature
352:624-628 (1991) or Marks et al., J. Mol. Biol. 222:581-597
(1991), for example.
[0131] 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., Proc. Natl. Acad.
ScL USA 81:6851-6855 (1984)).
[0132] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally will also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0133] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues. Human antibodies can be
produced using various techniques known in the art. In one
embodiment, the human antibody is selected from a phage library,
where that phage library expresses human antibodies (Vaughan et al.
Nature Biotechnology 14:309-314 (1996): Sheets et al. PNAS (USA)
95:6157-6162 (1998)); Hoogenboom and Winter, J. Mol. Biol., 227:381
(1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Human
antibodies can also be made by introducing human immunoglobulin
loci into transgenic animals, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which
closely resembles that seen in humans in all respects, including
gene rearrangement, assembly, and antibody repertoire. This
approach is described, for example, in U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the
following scientific publications: Marks et aL, Bio/Technology 10:
779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994);
Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature
Biotechnology 14: 845-51 (1996); Neuberger, Nature Biotechnology
14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93
(1995). Alternatively, the human antibody may be prepared via
immortalization of human B lymphocytes producing an antibody
directed against a target antigen (such B lymphocytes may be
recovered from an individual or may have been immunized in vitro).
See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147
(1):86-95 (1991); and U.S. Pat. No. 5,750,373.
[0134] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a beta-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the beta-sheet structure. The hypervariable
regions in each chain are held together in close proximity by the
FRs and, with the hypervariable regions from the other chain,
contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody-dependent cell-mediated cytotoxicity
(ADCC).
[0135] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in
the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (e.g. residues 26-32 (L1),
50-52 (L2) and 91-96 (L3) in the light chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable
domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
"Framework Region" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0136] 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
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into "subclasses" (isotypes), e.g., IgG1
(including non-A and A allotypes), 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.
[0137] The light chains of antibodies from any vertebrate species
can be assigned to one of two clearly distinct types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequences
of their constant domains.
[0138] The term "Fc region" is used to define the C-terminal region
of an immunoglobulin heavy chain which may be generated by papain
digestion of an intact antibody. The Fc region may be a native
sequence Fc region or a variant Fc region. Although the boundaries
of the Fc region of an immunoglobulin heavy chain might vary, the
human IgG heavy chain Fc region is usually defined to stretch from
an amino acid residue at about position Cys226, or from about
position Pro230, to the carboxyl-terminus of the Fc region. The Fc
region of an immunoglobulin generally comprises two constant
domains, a CH2 domain and a CH3 domain, and optionally comprises a
CH4 domain.
[0139] By "Fc region chain" herein is meant one of the two
polypeptide chains of an Fc region.
[0140] The "CH2 domain" of a human IgG Fc region (also referred to
as "C.gamma.2" domain) usually extends from an amino acid residue
at about position 231 to an amino acid residue at about position
340. The CH2 domain is unique in that it is not closely paired with
another domain. Rather, two N-linked branched carbohydrate chains
are interposed between the two CH2 domains of an intact native IgG
molecule. It has been speculated that the carbohydrate may provide
a substitute for the domain-domain pairing and help stabilize the
CH2 domain. Burton, Molec. Immunol. 22:161-206 (1985). The CH2
domain herein may be a native sequence CH2 domain or variant CH2
domain.
[0141] The "CH3 domain" comprises the stretch of residues
C-terminal to a CH2 domain in an Fc region (i.e. from an amino acid
residue at about position 341 to an amino acid residue at about
position 447 of an IgG). The CH3 region herein may be a native
sequence CH3 domain or a variant CH3 domain (e.g. a CH3 domain with
an introduced "protroberance" in one chain thereof and a
corresponding introduced "cavity" in the other chain thereof; see
U.S. Pat. No. 5,821,333, expressly incorporated herein by
reference). Such variant CH3 domains may be used to make
multispecific (e.g. bispecific) antibodies as herein described.
[0142] "Hinge region" is generally defined as stretching from about
Glu216, or about Cys226, to about Pro230 of human IgG1 (Burton,
Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG
isotypes may be aligned with the IgG1 sequence by placing the first
and last cysteine residues forming inter-heavy chain S--S bonds in
the same positions. The hinge region herein may be a native
sequence hinge region or a variant hinge region. The two
polypeptide chains of a variant hinge region generally retain at
least one cysteine residue per polypeptide chain, so that the two
polypeptide chains of the variant hinge region can form a disulfide
bond between the two chains. The preferred hinge region herein is a
native sequence human hinge region, e.g. a native sequence human
IgG1 hinge region.
[0143] A "functional Fc region" possesses at least one "effector
function" of a native sequence Fc region. Exemplary "effector
functions" include C1 q binding; 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; BCR), etc. Such effector functions
generally require the Fc region to be combined with a binding
domain (e.g. an antibody variable domain) and can be assessed using
various assays known in the art for evaluating such antibody
effector functions.
[0144] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. FIG. 3 provides amino acid sequences of native sequence
human and murine IgG Fc regions.
[0145] A "variant Fc region" comprises an amino acid sequence which
differs from that of a native sequence Fc region by virtue of at
least one amino acid modification. Preferably, the variant Fc
region has at least one amino acid substitution compared to a
native sequence Fc region or to the Fc region of a parent
polypeptide, e.g. from about one to about ten amino acid
substitutions, and preferably from about one to about five amino
acid substitutions in a native sequence Fc region or in the Fc
region of the parent polypeptide. The variant Fc region herein will
preferably possess at least about 80% sequence identity with a
native sequence Fc region and/or with an Fc region of a parent
polypeptide, and most preferably at least about 90% sequence
identity therewith, more preferably at least about 95% sequence
identity therewith.
[0146] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on a
target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells is summarized
in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991). To assess ADCC activity of a molecule of interest,
an in vitro ADCC assay, such as that described in U.S. Pat. No.
5,500,362 or 5,821,337 may be performed. 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 a animal model such as that disclosed in Clynes et al. PNAS
(USA) 95:652-656 (1998).
[0147] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source thereof, e.g. from blood or PBMCs as described
herein.
[0148] The terms "Fc receptor" and "FcR" are used to describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor
tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain
(reviewed in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs
are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92
(1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et
al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including
those to be identified in the future, are encompassed by the term
"FcR" herein. The term also includes the neonatal 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)).
[0149] "Complement dependent cytotoxicity" and "CDC" refer to the
lysing of a target in the presence of complement. The complement
activation pathway is initiated by the binding of the first
component of the complement system (C1q) to a molecule (e.g. an
antibody) complexed with a cognate antigen. To assess complement
activation, a CDC assay, e.g. as described in Gazzano-Santoro et
al., J. Immunol. Methods 202:163 (1996), may be performed.
[0150] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result an improvement
in the affinity of the antibody for antigen, compared to a parent
antibody which does not possess those alteration(s). Preferred
affinity matured antibodies will have nanomolar or even picomolar
affinities for the target antigen. Affinity matured antibodies are
produced by procedures known in the art. Marks et al.
Bio/Technology 10:779-783 (1992) describes affinity maturation by
VH and VL domain shuffling. Random mutagenesis of CDR and/or
framework residues is described by: Barbas et al. Proc Nat. Acad.
Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155
(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et
al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol.
Biol. 226:889-896 (1992).
[0151] "Percent (%) amino acid sequence identity" herein is defined
as the percentage of amino acid residues in a candidate sequence
that are identical with the amino acid residues in a selected
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, ALIGN-2
or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, 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 obtained as
described below by using the sequence comparison computer program
ALIGN-2. The ALIGN-2 sequence comparison computer program was
authored by Genentech, Inc. 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, and is
publicly available through Genentech, Inc., South San Francisco,
Calif. The ALIGN-2 program should be compiled for use on a UNIX
operating system, preferably digital UNIX V4.0D. All sequence
comparison parameters are set by the ALIGN-2 program and do not
vary.
[0152] For purposes herein, 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.
[0153] A "polypeptide chain" is a polypeptide wherein each of the
domains thereof is joined to other domain(s) by peptide bond(s), as
opposed to non-covalent interactions or disulfide bonds.
[0154] A "flexible linker" herein refers to a peptide comprising
two or more amino acid residues joined by peptide bond(s), and
provides more rotational freedom for two polypeptides (such as two
Fd regions) linked thereby. Such rotational freedom allows two or
more antigen binding sites joined by the flexible linker to each
access target antigen(s) more efficiently. Examples of suitable
flexible linker peptide sequences include gly-ser, gly-ser-gly-ser
(SEQ ID NO:10), ala-ser, and gly-gly-gly-ser (SEQ ID NO:11).
Preferably the flexible linker comprises 2 to about 10 amino acid
residues, and most preferably four or less residues.
[0155] A "dimerization domain" is formed by the association of at
least two amino acid residues (generally cysteine residues) or of
at least two peptides or polypeptides (which may have the same, or
different, amino acid sequences). The peptides or polypeptides may
interact with each other through covalent and/or non-covalent
association(s). Examples of dimerization domains herein include an
Fc region; a hinge region; a CH3 domain; a CH4 domain; a CH1-CL
pair; an "interface" with an engineered "knob" and/or
"protruberance" as described in U.S. Pat. No. 5,821,333, expressly
incorporated herein by reference; a leucine zipper (e.g. a jun/fos
leucine zipper, see Kostelney et al., J. Immunol., 148: 1547-1553
(1992); or a yeast GCN4 leucine zipper); an isoleucine zipper; a
receptor dimer pair (e.g., interleukin-8 receptor (IL-8R); and
integrin heterodimers such as LFA-1 and GPIIIb/IIIa), or the
dimerization region(s) thereof; dimeric ligand polypeptides (e.g.
nerve growth factor (NGF), neurotrophin-3 (NT-3), interleukin-8
(IL-8), vascular endothelial growth factor (VEGF), and
brain-derived neurotrophic factor (BDNF); see Arakawa et al. J.
Biol. Chem. 269(45): 27833-27839 (1994) and Radziejewski et al.
Biochem. 32(48): 1350 (1993)), or the dimerization region(s)
thereof; a pair of cysteine residues able to form a disulfide bond;
a pair of peptides or polypeptides, each comprising at least one
cysteine residue (e.g. from about one, two or three to about ten
cysteine residues) such that disulfide bond(s) can form between the
peptides or polypeptides (hereinafter "a synthetic hinge"); and
antibody variable domains. The most preferred dimerization domain
herein is an Fc region or a hinge region.
[0156] "Naturally occurring amino acid residues" (i.e. amino acid
residues encoded by the genetic code) may be selected from the
group consisting of: alanine (Ala); arginine (Arg); asparagine
(Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gin);
glutamic acid (Glu); glycine (Gly); histidine (H is); isoleucine
(Ile): leucine (Leu); lysine (Lys); methionine (Met); phenylalanine
(Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan
(Trp); tyrosine (Tyr); and valine (Val). A "non-naturally occurring
amino acid residue" refers to a residue, other than those naturally
occurring amino acid residues listed above, which is able to
covalently bind adjacent amino acid residues(s) in a polypeptide
chain. Examples of non-naturally occurring amino acid residues
include norleucine, ornithine, norvaline, homoserine and other
amino acid residue analogues such as those described in Ellman et
al. Meth. Enzym. 202:301-336 (1991). To generate such non-naturally
occurring amino acid residues, the procedures of Noren et al.
Science 244:182 (1989) and Ellman et al., supra, can be used.
Briefly, these procedures involve chemically activating a
suppressor tRNA with a non-naturally occurring amino acid residue
followed by in vitro transcription and translation of the RNA.
[0157] An "isolated" polypeptide is one that has been identified
and separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the polypeptide, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the polypeptide will be purified (1) to greater than
95% by weight of polypeptide as determined by the Lowry method, and
most preferably more than 99% by weight, (2) to a degree sufficient
to obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated polypeptide
includes the polypeptide in situ within recombinant cells since at
least one component of the polypeptide's natural environment will
not be present. Ordinarily, however, isolated polypeptide will be
prepared by at least one purification step.
[0158] A "functional antigen binding site" of an antibody is one
which is capable of binding a target antigen. The antigen binding
affinity of the antigen binding site is not necessarily as strong
as the parent antibody from which the antigen binding site is
derived, but the ability to bind antigen must be measurable using
any one of a variety of methods known for evaluating antibody
binding to an antigen. Moreover, the antigen binding affinity of
each of the antigen binding sites of a multivalent antibody herein
need not be quantitatively the same. For the multimeric antibodies
herein, the number of functional antigen binding sites can be
evaluated using ultracentrifugation analysis as described in
Example 2 below. According to this method of analysis, different
ratios of target antigen to multimeric antibody are combined and
the average molecular weight of the complexes is calculated
assuming differing numbers of functional binding sites. These
theoretical values are compared to the actual experimental values
obtained in order to evaluate the number of functional binding
sites.
[0159] By "ligand activation of a receptor" is meant signal
transduction (e.g. for a tyrosine kinase receptor, that caused by
an intracellular kinase domain of a tyrosine kinase receptor
phosphorylating tyrosine residues in the receptor or a substrate
polypeptide) mediated by ligand binding to the receptor (or a
receptor complex comprising the receptor of interest). In the case
of an ErbB receptor, generally, this will involve binding of an
ErbB ligand to an ErbB hetero-oligomer which activates a kinase
domain of one or more of the ErbB receptors in the hetero-oligomer
and thereby results in phosphorylation of tyrosine residues in one
or more of the ErbB receptors and/or phosphorylation of tyrosine
residues in additional substrate polypeptides(s).
[0160] An antibody which "blocks" ligand activation of an receptor
is one which reduces or prevents such activation as hereinabove
defined. Such blocking can occur by any means, e.g. by interfering
with: ligand binding to the receptor, receptor complex formation,
tyrosine kinase activity of a tyrosine kinase receptor in a
receptor complex and/or phosphorylation of tyrosine kinase
residue(s) in or by the receptor. Examples of antibodies which
block ligand activation of an ErbB receptor include monoclonal
antibodies 2C4 and 7F3 (which block HRG activation of HER2/HER3 and
HER2/HER4 hetero-oligomers; and EGF, TGF-beta or amphiregulin
activation of an EGFR/HER2 hetero-oligomer); and L26, L96 and L288
antibodies (Klapper et al. Oncogene 14:2099-2109 (1997)), which
block EGF and NDF binding to T47D cells which express EGFR, HER2,
HER3 and HER4.
[0161] An antibody having a "biological characteristic" of a
designated antibody is one which possesses one or more of the
biological characteristics of that antibody which distinguish it
from other antibodies that bind to the same antigen.
[0162] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell in vitro
and/or in vivo. Thus, the growth inhibitory agent may be one which
significantly reduces the percentage of cells in S phase. Examples
of growth inhibitory agents include agents that block cell cycle
progression (at a place other than S phase), such as agents that
induce G1 arrest and M-phase arrest. Classical M-phase blockers
include the vincas (vincristine and vinblastine), TAXOL.RTM., and
topo II inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. (WB Saunders:
Philadelphia, 1995), especially p. 13.
[0163] Examples of "growth inhibitory" anti-HER2 antibodies are
those which bind to HER2 and inhibit the growth of cancer cells
overexpressing HER2. Preferred growth inhibitory anti-HER2
antibodies inhibit growth of SKBR3 breast tumor cells in cell
culture by greater than 20%, and preferably greater than 50% (e.g.
from about 50% to about 100%) at an antibody concentration of about
0.5 to 30 .mu.g/ml, where the growth inhibition is determined six
days after exposure of the SKBR3 cells to the antibody (see U.S.
Pat. No. 5,677,171 issued Oct. 14, 1997).
[0164] An antibody which "induces cell death" is one which causes a
viable cell to become nonviable. The cell is generally one which
expresses the antigen to which the antibody binds, especially where
the cell overexpresses the antigen. Preferably, the cell is a
cancer cell, e.g. a breast, ovarian, stomach, endometrial, salivary
gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. In
vitro, the cell may be a SKBR3, BT474, Calu 3, MDA-MB-453,
MDA-MB-361 or SKOV3 cell. Cell death in vitro may be determined in
the absence of complement and immune effector cells to distinguish
cell death induced by antibody dependent cell-mediated cytotoxicity
(ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay
for cell death may be performed using heat inactivated serum (i.e.
in the absence of complement) and in the absence of immune effector
cells. To determine whether the antibody is able to induce cell
death, loss of membrane integrity as evaluated by uptake of
propidium iodide (P1), trypan blue (see Moore et al. Cytotechnology
17:1-11 (1995)) or 7AAD can be assessed relative to untreated
cells.
[0165] An antibody which "induces apoptosis" is one which induces
programmed cell death as determined by binding of annexin V,
fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane
vesicles (called apoptotic bodies). The cell is one which expresses
the antigen to which the antibody binds and may be one which
overexpresses the antigen. The cell may be a tumor cell, e.g. a
breast, ovarian, stomach, endometrial, salivary gland, lung,
kidney, colon, thyroid, pancreatic or bladder cell. In vitro, the
cell may be a SKBR3, BT474, Calu 3 cell, MDA-MB-453, MDA-MB-361 or
SKOV3 cell. Various methods are available for evaluating the
cellular events associated with apoptosis. For example,
phosphatidyl serine (PS) translocation can be measured by annexin
binding; DNA fragmentation can be evaluated through DNA laddering
as disclosed in the example herein; and nuclear/chromatin
condensation along with DNA fragmentation can be evaluated by any
increase in hypodiploid cells. Preferably, the antibody which
induces apoptosis is one which results in about 2 to 50 fold,
preferably about 5 to 50 fold, and most preferably about 10 to 50
fold, induction of annexin binding relative to untreated cell in an
annexin binding assay using cells expressing the antigen to which
the antibody binds.
[0166] Examples of antibodies which induce apoptosis include the
anti-HER2 monoclonal antibodies 7F3 (ATCC HB-12216), and 7C2 (ATCC
HB 12215), including humanized and/or affinity matured variants
thereof; the anti-DR5 antibodies 3F11.39.7 (ATCC HB-12456);
3H3.14.5 (ATCC HB-12534); 3D5.1.10 (ATCC HB-12536); and 3H3.14.5
(ATCC HB-12534), including humanized and/or affinity matured
variants thereof; the human anti-DR5 receptor antibodies 16E2 and
20E6, including affinity matured variants thereof (WO98/51793,
expressly incorporated herein by reference); the anti-DR4
antibodies 4E7.24.3 (ATCC HB-12454); 4H6.17.8 (ATCC HB-12455);
1H5.25.9 (ATCC HB-12695); 4G7.18.8 (ATCC PTA-99); and 5G11.17.1
(ATCC HB-12694), including humanized and/or affinity matured
variants thereof.
[0167] In order to screen for antibodies which bind to an epitope
on an antigen bound by an antibody of interest, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed.
[0168] An "agonist antibody" is an antibody which binds to and
activates a receptor. Generally, the receptor activation capability
of the agonist antibody will be at least qualitatively similar (and
may be essentially quantitatively similar) to a native agonist
ligand of the receptor. An example of an agonist antibody is one
which binds to a receptor in the TNF receptor superfamily and
induces apoptosis of cells expressing the TNF receptor. Assays for
determining induction of apoptosis are described in WO98/51793 and
WO99/37684, both of which are expressly incorporated herein by
reference.
[0169] A "disorder" is any condition that would benefit from
treatment with the antibody. This includes chronic and acute
disorders or diseases including those pathological conditions which
predispose the mammal to the disorder in question. Non-limiting
examples of disorders to be treated herein include benign and
malignant tumors; leukemias and lymphoid malignancies; neuronal,
glial, astrocytal, hypothalamic and other glandular, macrophagal,
epithelial, stromal and blastocoelic disorders; and inflammatory,
angiogenic and immunologic disorders.
[0170] 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 disorder. 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 in
vivo can, for example, be measured by assessing the time to disease
progression (TTP) and/or determining the response rates (RR).
[0171] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented.
[0172] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include squamous cell
cancer, small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung, squamous carcinoma of the lung, cancer
of the peritoneum, hepatocellular cancer, gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney cancer, liver cancer, prostate
cancer, vulval cancer, thyroid cancer, hepatic carcinoma and
various types of head and neck cancer.
[0173] An "autoimmune disease" herein is a non-malignant disease or
disorder arising from and directed against an individual's own
tissues. Examples of autoimmune diseases or disorders include, but
are not limited to, inflammatory responses such as inflammatory
skin diseases including psoriasis and dermatitis (e.g. atopic
dermatitis); systemic scleroderma and sclerosis; responses
associated with inflammatory bowel disease (such as Crohn's disease
and ulcerative colitis); respiratory distress syndrome (including
adult respiratory distress syndrome; ARDS); dermatitis; meningitis;
encephalitis; uveitis; colitis; glomerulonephritis; allergic
conditions such as eczema and asthma and other conditions involving
infiltration of T cells and chronic inflammatory responses;
atherosclerosis; leukocyte adhesion deficiency; rheumatoid
arthritis; systemic lupus erythematosus (SLE); diabetes mellitus
(e.g. Type I diabetes mellitus or insulin dependent diabetes
mellitis); multiple sclerosis; Reynaud's syndrome; autoimmune
thyroiditis; allergic encephalomyelitis; Sjorgen's syndrome;
juvenile onset diabetes; and immune responses associated with acute
and delayed hypersensitivity mediated by cytokines and
T-lymphocytes typically found in tuberculosis, sarcoidosis,
polymyositis, granulomatosis and vasculitis; pernicious anemia
(Addison's disease); diseases involving leukocyte diapedesis;
central nervous system (CNS) inflammatory disorder; multiple organ
injury syndrome; hemolytic anemia (including, but not limited to
cryoglobinemia or Coombs positive anemia); myasthenia gravis;
antigen-antibody complex mediated diseases; anti-glomerular
basement membrane disease; antiphospholipid syndrome; allergic
neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome;
pemphigoid bullous; pemphigus; autoimmune polyendocrinopathies;
Reiter's disease; stiff-man syndrome; Behcet disease; giant cell
arteritis; immune complex nephritis; IgA nephropathy; IgM
polyneuropathies; immune thrombocytopenic purpura (ITP) or
autoimmune thrombocytopenia etc.
[0174] By "foreign antigen" is meant a molecule or molecules which
is/are not endogenous or native to a mammal which is exposed to it.
The foreign antigen may elicit an immune response, e.g. a humoral
and/or T cell mediated response in the mammal. Generally, the
foreign antigen will provoke the production of antibodies
thereagainst. Examples of foreign antigens contemplated herein
include immunogenic therapeutic agents, e.g. proteins such as
antibodies, particularly antibodies comprising non-human amino acid
residues (e.g. rodent, chimeric/humanized, and primatized
antibodies); toxins (optionally conjugated to a targeting molecule
such as an antibody, wherein the targeting molecule may also be
immunogenic); gene therapy viral vectors, such as retroviruses and
adenoviruses; grafts; infectious agents (e.g. bacteria and virus);
alloantigens (i.e. an antigen that occurs in some, but not in other
members of the same species) such as differences in blood types,
human lymphocyte antigens (HLA), platelet antigens, antigens
expressed on transplanted organs, blood components, pregnancy (Rh),
and hemophilic factors (e.g. Factor VIII and Factor IX).
[0175] By "blocking an immune response" to a foreign antigen is
meant reducing or preventing at least one immune-mediated response
resulting from exposure to a foreign antigen. For example, one may
dampen a humoral response to the foreign antigen, i.e., by
preventing or reducing the production of antibodies directed
against the antigen in the mammal. Alternatively, or additionally,
one may suppress idiotype; "pacify" the removal of cells coated
with alloantibody; and/or affect alloantigen presentation through
depletion of antigen-presenting cells.
[0176] The term "graft" as used herein refers to biological
material derived from a donor for transplantation into a recipient.
Grafts include such diverse material as, for example, isolated
cells such as islet cells; tissue such as the amniotic membrane of
a newborn, bone marrow, hematopoietic precursor cells, and ocular
tissue, such as corneal tissue; and organs such as skin, heart,
liver, spleen, pancreas, thyroid lobe, lung, kidney, tubular organs
(e.g., intestine, blood vessels, or esophagus), etc. The tubular
organs can be used to replace damaged portions of esophagus, blood
vessels, or bile duct. The skin grafts can be used not only for
burns, but also as a dressing to damaged intestine or to close
certain defects such as diaphragmatic hernia. The graft is derived
from any mammalian source, including human, whether from cadavers
or living donors. Preferably the graft is bone marrow or an organ
such as heart and the donor of the graft and the host are matched
for HLA class II antigens.
[0177] The term "mammalian host" as used herein refers to any
compatible transplant recipient. By "compatible" is meant a
mammalian host that will accept the donated graft. Preferably, the
host is human. If both the donor of the graft and the host are
human, they are preferably matched for HLA class II antigens so as
to improve histocompatibility.
[0178] The term "donor" as used herein refers to the mammalian
species, dead or alive, from which the graft is derived.
Preferably, the donor is human. Human donors are preferably
volunteer blood-related donors that are normal on physical
examination and of the same major ABO blood group, because crossing
major blood group barriers possibly prejudices survival of the
allograft. It is, however, possible to transplant, for example, a
kidney of a type O donor into an A, B or AB recipient.
[0179] The term "transplant" and variations thereof refers to the
insertion of a graft into a host, whether the transplantation is
syngeneic (where the donor and recipient are genetically
identical), allogeneic (where the donor and recipient are of
different genetic origins but of the same species), or xenogeneic
(where the donor and recipient are from different species). Thus,
in a typical scenario, the host is human and the graft is an
isograft, derived from a human of the same or different genetic
origins. In another scenario, the graft is derived from a species
different from that into which it is transplanted, such as a baboon
heart transplanted into a human recipient host, and including
animals from phylogenically widely separated species, for example,
a pig heart valve, or animal beta islet cells or neuronal cells
transplanted into a human host.
[0180] The expression "desensitizing a mammal awaiting
transplantation" refers to reducing or abolishing allergic
sensitivity or reactivity to a transplant, prior to administration
of the transplant to the mammal. This may be achieved by any
mechanism, such as a reduction in anti-donor antibodies in the
desensitized mammal, e.g. where such anti-donor antibodies are
directed against human lymphocyte antigen (HLA).
[0181] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof.
[0182] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclosphosphamide
(CYTOXAN.TM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; acetogenins
(especially bullatacin and bullatacinone); a camptothecin
(including the synthetic analogue topotecan); bryostatin;
callystatin; CC-1065 (including its adozelesin, carzelesin and
bizelesin synthetic analogues); cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CBI-TMI);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, ranimustine; antibiotics such as the enediyne
antibiotics (e.g. calicheamicin, especially calicheamicin
.gamma..sub.1.sup.I and calicheamicin .theta..sup.I.sub.1, see,
e.g., Agnew Chem. Intl. Ed. Engl. 33:183-186 (1994); dynemicin,
including dynemicin A; an esperamicin; as well as neocarzinostatin
chromophore and related chromoprotein enediyne antiobiotic
chromomophores), aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, carabicin, caminomycin,
carzinophilin, chromomycins, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic
acid, nogalamycin, olivomycins, peplomycin, potfiromycin,
puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,
tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such
as methotrexate and 5-fluorouracil (5-FU); folic acid analogues
such as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine, 5-FU; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate; an
epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;
lonidamine; maytansinoids such as maytansine and ansamitocins;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK.RTM.; razoxane; rhizoxin; sizofuran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g. paclitaxel (TAXOL.RTM., Bristol-Myers Squibb
Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE.RTM.,
Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine;
6-thioguanine; mercaptopurine; methotrexate; platinum analogs such
as cisplatin and carboplatin; vinblastine; platinum; etoposide
(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;
vinorelbine; navelbine; novantrone; teniposide; daunomycin;
aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor
RFS 2000; difluoromethylornithine (DMFO); retinoic acid;
capecitabine; and pharmaceutically acceptable salts, acids or
derivatives of any of the above. Also included in this definition
are anti-hormonal agents that act to regulate or inhibit hormone
action on tumors such as anti-estrogens including for example
tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,
4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone,
and toremifene (Fareston); and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
[0183] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-alpha and -beta;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-alpha; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I
and -II; erythropoietin (EPO); osteoinductive factors; interferons
such as interferon-alpha, -beta and -gamma colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis
factor such as TNF-alpha or TNF-beta; and other polypeptide factors
including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0184] The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer
Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382,
615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug
Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this invention include, but are not limited
to, phosphate-containing prodrugs, thiophosphate-containing
prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,
D-amino acid-modified prodrugs, glycosylated prodrugs,
beta-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
[0185] An "angiogenic factor" is a growth factor which stimulates
the development of blood vessels. The preferred angiogenic factor
herein is Vascular Endothelial Growth Factor (VEGF).
[0186] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the polypeptide. The label may be itself be detectable (e.g.,
radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, may catalyze chemical alteration of a substrate
compound or composition which is detectable.
[0187] An "isolated" nucleic acid molecule is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the polypeptide nucleic acid.
An isolated nucleic acid molecule is other than in the form or
setting in which it is found in nature. Isolated nucleic acid
molecules therefore are distinguished from the nucleic acid
molecule as it exists in natural cells. However, an isolated
nucleic acid molecule includes a nucleic acid molecule contained in
cells that ordinarily express the polypeptide where, for example,
the nucleic acid molecule is in a chromosomal location different
from that of natural cells.
[0188] The expression "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0189] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0190] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
II. Modes for Carrying Out the Invention
[0191] A. Multivalent Antibodies
[0192] The invention herein relates to a method for making a
multivalent antibody. Various techniques for generating the
"parent" or "starting" antibody from which the variable domain(s)
of the multivalent antibody may be derived will be described later
in this application.
[0193] The multivalent antibody of particular interest herein is
one which comprises at least three (and preferably four, or more,
e.g. four or five to about eight) antigen binding sites. Generally,
all of the antigen binding sites are "functional" as defined
hereinabove. Preferably, the multivalent antibody does not exist in
nature and is not a native sequence IgM or IgA antibody. The
multivalent antibody herein is preferably not produced in vitro by
chemically cross-linking a pair antibodies (e.g. as in Ghetie et
al. (1997), supra or Wolff et al. (1993), supra). The present
application also provides multivalent antibodies which do not
require introduced cysteine residue(s) in a parent antibody in
order to make the multivalent antibody via disulfide bond(s)
between a pair of Fc regions (e.g. as in Shopes et al. (1992),
supra or Caron et al. (1992), supra).
[0194] In one embodiment, the multivalent antibody comprises a
first polypeptide chain comprising at least two heavy chain (or
light chain) variable domains and a second polypeptide chain
comprising at least two heavy chain (or light chain) variable
domains. Preferably, the first polypeptide chain comprises two
heavy chain variable domains and the second polypeptide chain also
comprises two heavy chain variable domains, which can be combined
with corresponding light chain variable domains (at least two for
each polypeptide chain) to generate four (or more) antigen binding
sites.
[0195] In one preferred embodiment of the invention, the
multivalent antibody comprises a dimerization domain which combines
(1) two (or more) antigen binding sites with (2) one, two (or more)
antigen binding sites. Various dimerization domains are
contemplated herein, but the preferred dimerization domain is an Fc
region or a hinge region. Where the multivalent antibody comprises
an Fc region (e.g. a native sequence or variant Fc region), the Fc
region is preferably "functional" as defined hereinabove and thus
is capable of performing one or more antibody effector functions,
such as ADCC or CDC. Preferably, the multivalent antibody has only
one Fc region or lacks an Fc region.
[0196] Where the multivalent antibody comprises an Fc region,
preferably, the three or more antigen binding sites are provided
amino terminal to the Fc region (rather than at the carboxy
terminus of the Fc region as in Coloma and Morrison, (1997) supra).
This may be achieved by providing a first polypeptide chain
represented by the formula VD1-X1-VD2-X2-Fc, wherein (1) VD1 is a
first heavy or light chain variable domain (preferably a heavy
chain variable domain), (2) VD2 is a second heavy or light chain
variable domain (preferably a heavy chain variable domain), (3) Fc
comprises one chain of an Fc region, and (4) X1 and X2 represent an
optional intervening amino acid or polypeptide. Preferably X1 and
X2 comprise, or consist of, a CH1 domain (where VD1 or VD2 is a
heavy chain variable domain) or a CL domain (where VD1 or VD2 is a
light chain variable domain). Optionally, X1 further comprises a
flexible linker which is generally C-terminal to VD1 (or C-terminal
to CH1 or CL, if present). The flexible linker may comprise a
peptide such as gly-ser, gly-ser-gly-ser (SEQ ID NO:10), ala-ser or
gly-gly-gly-ser (SEQ ID NO:11).
[0197] The multivalent antibody of particular interest herein
comprises three or more (e.g. four or five to about eight) Fab
polypeptides, each capable of binding antigen. The Fab fragments
are preferably provided amino terminal to the Fc region (where the
multivalent antibody has an Fc region). For instance, two or more
Fd fragments may be fused to the amino terminus of one chain of an
Fc region. The polypeptide chain thus engineered may be combined
with (1) another polypeptide chain formed by two or more Fd
fragments fused to the amino terminus of the other chain of the Fc
region, as well as (2) complementary VL domains (e.g. four or more
VL domains which each, optionally, are fused to a CL domain).
Optionally, the antibody comprises a flexible linker between the
two or more Fd fragments. The multivalent antibody may, for
example, comprise a pair of polypeptide chains with the formula (1)
VH-CH1-flexible linker-VH-CH1-Fc chain, or (2) VH-CH1-VH-CH1-Fc
chain (i.e. where there is no flexible linker between the two Fd
fragments).
[0198] The three or more functional antigen binding sites of the
multivalent antibody herein are each preferably formed by a heavy
and light chain variable domain. Thus, where two or more heavy
chain variable domains are fused together (optionally with
intervening amino acid residue(s) as noted above), two or more
complementary light chain variable domain-containing polypeptides
are combined with the heavy chain variable domains (for instance by
co-expressing the fusion protein and the light chain variable
domain polypeptide(s) in the same host cell). Preferably, the
antibody comprises four, or five, or more (e.g. up to about eight)
light chain variable domain polypeptides, which each, optionally,
comprise a CL domain.
[0199] In one embodiment herein, the antibody with three or more
more (e.g. three to about ten, but preferably three or four)
antigen binding sites may comprise a polypeptide chain comprising
three or more (e.g. three to about ten, but preferably three or
four) heavy chain or light chain variable domains, wherein each of
the variable domains is combined with, or associated with, three or
more (e.g. three to about ten, but preferably three or four) light
chain or heavy chain variable domain polypeptides in such a way as
to form the antigen binding sites. Thus, where the polypeptide
chain comprises three or more heavy chain variable domains, it is
combined or associated with three or more corresponding light chain
variable domain polypeptides (e.g. with VL-CL polypeptides).
Alternatively, where the polypeptide chain comprises three or more
light chain variable domains, it is combined or associated with
three or more corresponding heavy chain variable domain
polypeptides (e.g. with VH-CH1 polypeptides). Preferably each of
the three or more antigen binding sites is directed against the
same antigen. Examples of antigens bound by such antibodies include
(1) a receptor in the Tumor Necrosis Factor (TNF) receptor
superfamily (such receptors may be `trimeric receptors`, hence the
antibody need only include only three antigen binding sites as
desired) such as DR4 and DRS; (2) a B cell surface antigen such as
CD20; (3) an ErbB receptor exemplified by the HER2 receptor; or (4)
a cell surface protein expressed by tumor cells. For instance, the
polypeptide chain may comprise three (or four) heavy chain variable
domains which are able to combine with three (or four) light chain
variable domain polypeptides (preferably VL-CL polypeptides) to
generate three (or four) antigen binding sites directed against the
same antigen. Such antibodies are exemplified by those depicted in
FIG. 23D (with three antigen binding sites) and FIG. 23E (with four
antigen binding sites). The multivalent antibody may also comprise
a polypeptide chain comprising the formula: (a) VL-CL-flexible
linker-VL-CL-flexible linker-VL-CL; In this embodiment, the
polypeptide may comprise three to about eight VL-CL polypeptides
joined by flexible linkers. (b) VH-CH.sub.1-flexible
linker-VH-CH1-flexible linker-VH-CH1; In this embodiment, the
polypeptide may comprise three to about eight VH-CH1 polypeptides
joined by flexible linkers. (c) (VL-CL).sub.n, wherein n is three
or more more (e.g. three to about eight, but preferably three or
four); or (d) (VH-CH1).sub.n, wherein n is three or more more (e.g.
three to about eight, but preferably three or four). Preferably,
the polypeptide chain comprises the formula: (a) VH-CH1-flexible
linker-VH-CH1-flexible linker-VH-CH1; (b) VH-CH1-flexible
linker-VH-CH1-flexible linker-VH-CH1-flexible linker-VH-CH1; or (c)
(VH-CH1).sub.n, wherein n is three or four.
[0200] The multivalent antibodies herein have desirable properties
particularly for in vivo therapy and diagnosis. For instance, the
multivalent antibody may be internalized and catabolized by a cell
expressing an antigen, to which the antibody binds, faster than a
bivalent antibody. Thus, the invention provides an immunoconjugate
comprising the multivalent antibody conjugated with a cytotoxic
agent (e.g. one which is active in killing cells once
internalized). Various cytotoxic agents for generating an
immunoconjugate are described herein, but the preferred cytotoxic
agent is a radioactive isotope, a maytansinoid or a
calecheamicin.
[0201] The multivalent antibody, and/or a parent antibody from
which at least one of the multivalent antibody's antigen binding
specificities is derived, may have certain properties. For
instance, the multivalent antibody and/or parent antibody may (1)
be an agonist antibody (e.g. where an antigen bound by the antibody
is a receptor in the TNF receptor family or a B cell surface
antigen); (2) induce apoptosis (for instance, where an antigen
bound by the antibody is an ErbB receptor or a receptor in the TNF
receptor superfamily); (3) bind a cell surface protein (such as a B
cell surface antigen or an ErbB receptor) expressed on tumor cells;
(4) bind a cell surface protein (e.g. Epidermal Growth Factor
Receptor (EGFR), HER2 receptor, ErbB3 receptor, ErbB4 receptor, or
DcR3 receptor) overexpressed by tumor cells; and/or (5) be a growth
inhibitory antibody .
[0202] The multivalent antibody herein may have specificity for
only one antigen, or more than one antigens (e.g. from two to about
three antigens). In one embodiment, the three or more functional
antigen binding sites of the multivalent antibody may all bind the
same antigen (preferably the same epitope on that antigen, in which
case the multivalent antibody would be considered to be
"monospecific"). This application also provides "multispecific"
antibodies. Thus, the three or more functional antigen binding
sites may bind two or more (e.g. from two to about three) different
antigens or epitopes.
[0203] The present application shows that a multivalent antibody
directed against a receptor antigen can be engineered which,
surprisingly, has agonistic and/or apoptosis-inducing capability
which is quantitatively similar to that of the native ligand. By
"quantitatively similar" here is meant that the multivalent
antibody has an activity in an assay which determines agonistic
and/or apoptosis-inducing activity, within about ten fold, and
preferably within about five fold of the agonistic and/or
apoptosis-inducing activity of the native ligand. In this
embodiment, the antibody with agonistic and/or apoptosis-inducing
activity may be one with specificity for a receptor in the TNF
receptor superfamily, e.g. an Apo2L receptor such as DR4, DR5, DcR1
and DcR2 (preferably DR4 or DR5), in which case the activity of the
antibody in an apoptosis assay such as those described in Example 3
below is within about ten fold, e.g. within about five fold, of the
activity of Apo2L in that assay.
[0204] The multivalent antibody herein may, in one embodiment of
the invention, bind a B cell surface antigen. Preferred B cell
surface antigens include CD19, CD20, CD22 and CD40, and most
preferably CD20.
[0205] Various applications for the multivalent antibodies herein
are contemplated and described in more detail below. Where the
multivalent antibody possesses one or more functional Fc regions,
it is anticipated to have the ability to mediate effector functions
(such as ADCC and CDC) and have a longer half-life than multivalent
antibodies lacking an Fc region. Such multivalent antibodies may be
used where killing of cells, such as tumor or cancer cells, is
desired. Other forms of the multivalent antibodies herein which
lack a Fc region may be desirable where a shorter half-life is
desired (e.g. for treating cardiovascular or inflammatory diseases
or disorders, or where the antibody is conjugated with a cytotoxic
agent); where internalization of the antibody is desired (e.g. for
therapy with an immunoconjugate comprising the antibody and a
cytotoxic agent); for improved penetration of a solid tumor; where
expression of the multivalent antibody in a non-mammalian host cell
(e.g. a prokaryotic host cell such as an E. coli host cell) is
desired; for therapy of nononcological diseases or disorders;
and/or to avoid the `first dose affect` observed upon
administration of certain antibodies possessing effector
function(s) to patients. Such forms of the antibody may comprise a
multivalent antibody including a dimerization domain, wherein the
dimerization domain comprises an antibody hinge region fused to a
leucine zipper domain (the leucine zipper domain facilitates
association of the polypeptides which form the dimerization domain,
but may be subsequently proteolytically removed prior to
administration to a patient) (see FIG. 23C); a multivalent antibody
with three antigen binding sites such as that shown in FIG. 23D; or
a multivalent antibody with four antigen binding sites such as that
depicted in FIG. 23E.
[0206] B. Antigen Binding Specificity
[0207] The multivalent antibody herein is directed against, or
binds specifically to, one or more target antigen(s). Preferably,
at least one of the antigens bound by the multivalent antibody is a
biologically important polypeptide and administration of the
antibody to a mammal suffering from a disease or disorder can
result in a therapeutic benefit in that mammal. However, antibodies
directed against nonpolypeptide antigens (such as tumor-associated
glycolipid antigens; see U.S. Pat. No. 5,091,178) are also
contemplated.
[0208] Where the antigen is a polypeptide, it may be a
transmembrane molecule (e.g. receptor) or ligand such as a growth
factor. Exemplary antigens include molecules such as renin; a
growth hormone, including human growth hormone and bovine growth
hormone; growth hormone releasing factor; parathyroid hormone;
thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;
insulin A-chain; insulin B-chain; proinsulin; follicle stimulating
hormone; calcitonin; luteinizing hormone; glucagon; clotting
factors such as factor VIIIC, factor IX, tissue factor (TF), and
von Willebrands factor; anti-clotting factors such as Protein C;
atrial natriuretic factor; lung surfactant; a plasminogen
activator, such as urokinase or human urine or tissue-type
plasminogen activator (t-PA); bombesin; thrombin; hemopoietic
growth factor; tumor necrosis factor-alpha and -beta;
enkephalinase; RANTES (regulated on activation normally T-cell
expressed and secreted); human macrophage inflammatory protein
(MIP-1-alpha); a serum albumin such as human serum albumin;
Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;
prorelaxin; mouse gonadotropin-associated peptide; a microbial
protein, such as beta-lactamase; DNase; IgE; a cytotoxic
T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin;
activin; vascular endothelial growth factor (VEGF); receptors for
hormones or growth factors; protein A or D; rheumatoid factors; a
neurotrophic factor such as bone-derived neurotrophic factor
(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6),
or a nerve growth factor such as NGF-.beta.; platelet-derived
growth factor (PDGF); fibroblast growth factor such as aFGF and
bFGF; epidermal growth factor (EGF); transforming growth factor
(TGF) such as TGF-alpha and TGF-beta, including TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, or TGF-.beta.5; insulin-like
growth factor-1 and -II (IGF-1 and IGF-II); des(1-3)-IGF-I (brain
IGF-I), insulin-like growth factor binding proteins; CD proteins
such as CD3, CD4, CD8, CD19, CD20 and CD25 (Tac subunit of the IL-2
receptor); erythropoietin; osteoinductive factors; immunotoxins; a
bone morphogenetic protein (BMP); an interferon such as
interferon-alpha, -beta, and -gamma; colony stimulating factors
(CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g.,
IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface
membrane proteins; decay accelerating factor; viral antigen such
as, for example, a portion of the AIDS envelope; transport
proteins; homing receptors; addressins; regulatory proteins;
integrins such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 or
VCAM; a tumor associated antigen such as HER2, HER3 or HER4
receptor; and fragments of any of the above-listed
polypeptides.
[0209] Preferred molecular targets for antibodies encompassed by
the present invention include leukocyte surface markers or CD
proteins such as CD1a-c, CD2, CD2R, CD3, CD4, CD5, CD6, CD7, CD8,
CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15, CD15s,
CD16, CD16b, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25,
CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36,
CD37, CD38, CD39, CD40, C41, CD42a-d, CD43, CD44, CD44R, CD45,
CD45A, CD45B, CD450, CD46-CD48, CD49a-f, CD50, CD51, CD52,
CD53-CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63, CD64, CDw65,
CD66a-e, CD68-CD74, CDw75, CDw76, CD77, CDw78, CD79a-b, CD80-CD83,
CDw84, CD85-CD89, CDw90, CD91, CDw92, CD93-CD98, CD99, CD99R,
CD100, CDw101, CD102-CD106, CD107a-b, CDw108, CDw109, CD115,
CDw116, CD117, CD119, CD120a-b, CD121a-b, CD122, CDw124,
CD126-CD129, and CD130; members of the ErbB receptor family such as
the EGF receptor, HER2 receptor, HER3 receptor or HER4 receptor;
prostate specific antigen(s); cell adhesion molecules such as
IIb/IIIa, LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM,
.alpha.4/.beta.7 integrin, and .alpha.v/.beta.3 integrin including
either .alpha. or .beta. subunits thereof (e.g. anti-CD11a,
anti-CD18 or anti-CD11b antibodies); growth factors such as VEGF;
tissue factor (TF); alpha interferon (.alpha.-IFN); an interleukin,
such as IL-8; IgE; blood group antigens; flk2/flt3 receptor;
obesity (OB) receptor; c-mpl receptor; CTLA-4; protein C etc.
[0210] Soluble antigens or fragments thereof, optionally conjugated
to other molecules, can be used as immunogens for generating
antibodies. For transmembrane molecules, such as receptors,
fragments of these (e.g. the extracellular domain of a receptor)
can be used as the immunogen. Alternatively, cells expressing the
transmembrane molecule can be used as the immunogen. Such cells can
be derived from a natural source (e.g. cancer cell lines) or may be
cells which have been transformed by recombinant techniques to
express the transmembrane molecule. Other antigens and forms
thereof useful for preparing antibodies will be apparent to those
in the art.
[0211] Preferred target antigens for the multivalent antibodies
herein include (1) ErbB receptors, including EGFR, HER2, HER3 and
HER4; (2) receptors in the TNF receptor superfamily, e.g. Apo2L
receptors, such as DR4, DR5, DcR1 and DcR2; (3) B cell surface
antigens, especially CD19, CD20, CD22 and CD40; (4) antigens
expressed by tumor cells; (5) antigens overexpressed by tumor cells
(e.g. ErbB receptors; DcR3 receptors); (6) receptors activated by
multimeric (e.g. dimeric or trimeric) ligands (e.g. receptors in
the TNF receptor superfamily; VEGF receptors, etc.). In one
embodiment, three or more (e.g. four to about eight) of the antigen
binding sites of the multivalent antibody may all be directed
against the same antigenic determinant or epitope on one of the
above antigens.
[0212] The present application also provides multispecific
antibodies, i.e., antibodies that have binding specificities for at
least two different epitopes or antigenic determinants.
Multispecific antibodies (e.g. bispecific antibodies; BsAbs) have
significant potential in a wide range of clinical applications as
targeting agents for in vitro and in vivo immunodiagnosis and
therapy, and for diagnostic immunoassays.
[0213] Bispecific antibodies have been very useful in probing the
functional properties of cell surface molecules and in defining the
ability of the different Fc receptors to mediate cytotoxicity
(Fanger et al., Crit. Rev. Immunol. 12:101-124 (1992)). Nolan et
al., Biochem. Biophys. Acta. 1040:1-11 (1990) describe other
diagnostic applications for BsAbs. In particular, BsAbs can be
constructed to immobilize enzymes for use in enzyme immunoassays.
To achieve this, one arm of the BsAb can be designed to bind to a
specific epitope on the enzyme so that binding does not cause
enzyme inhibition, the other arm of the BsAb binds to the
immobilizing matrix ensuring a high enzyme density at the desired
site. Examples of such diagnostic BsAbs include the rabbit
anti-IgG/anti-ferritin BsAb described by Hammerling et al., J. Exp.
Med. 128:1461-1473 (1968) which was used to locate surface
antigens. BsAbs having binding specificities for Horse Radish
Peroxidase (HRP) as well as a hormone have also been developed.
Another potential immunochemical application for BsAbs involves
their use in two-site immunoassays. For example, two BsAbs are
produced binding to two separate epitopes on the analyte
protein--one BsAb binds the complex to an insoluble matrix, the
other binds an indicator enzyme (see Nolan et al., supra).
[0214] Multispecific antibodies can also be used for in vitro or in
vivo immunodiagnosis of various diseases such as cancer
(Songsivilai et al., Clin. Exp. Immunol. 79:315 (1990)). To
facilitate this diagnostic use of the BsAb, one arm of the BsAb can
bind a tumor associated antigen and the other arm can bind a
detectable marker such as a chelator which tightly binds a
radionuclide. Using this approach, Le Doussal et al. made a BsAb
useful for radioimmunodetection of colorectal and thryoid
carcinomas which had one arm which bound a carcinoembryonic antigen
(CEA) and another arm which bound diethylenetriaminepentacetic acid
(DPTA). See Le Doussal et al., Int. J. Cancer Suppl. 7:58-62 (1992)
and Le Doussal et al., J. Nucl. Med. 34:1662-1671 (1993). Stickney
et al. similarly describe a strategy for detecting colorectal
cancers expressing CEA using radioimmunodetection. These
investigators describe a BsAb which binds CEA as well as
hydroxyethylthiourea-benzyl-EDTA (EOTUBE). See Stickney et al.,
Cancer Res. 51:6650-6655 (1991).
[0215] Multispecific antibodies can also be used for human therapy
in redirected cytotoxicity by providing one arm which binds a
target (e.g. pathogen or tumor cell) and another arm which binds a
cytotoxic trigger molecule, such as the T-cell receptor or an Fc
gamma receptor. Accordingly, multispecific antibodies can be used
to direct a patient's cellular immune defense mechanisms
specifically to the tumor cell or infectious agent. Using this
strategy, it has been demonstrated that bispecific antibodies which
bind to the Fc gamma RIII (i.e. CD16) can mediate tumor cell
killing by natural killer (NK) cell/large granular lymphocyte (LGL)
cells in vitro and are effective in preventing tumor growth in
vivo. Segal et al., Chem. Immunol. 47:179 (1989) and Segal et al.,
Biologic Therapy of Cancer 2(4) DeVita et al. eds. J. B.
Lippincott, Philadelphia (1992) p. 1. Similarly, a bispecific
antibody having one arm which binds Fc gamma RIII and another which
binds to the HER2 receptor has been developed for therapy of
ovarian and breast tumors that overexpress the HER2 antigen.
(Hseih-Ma et al. Cancer Research 52:6832-6839 (1992) and Weiner et
al. Cancer Research 53:94-100 (1993)). Bispecific antibodies can
also mediate killing by T cells. Normally, the bispecific
antibodies link the CD3 complex on T cells to a tumor-associated
antigen. A fully humanized F(ab').sub.2 BsAb consisting of anti-CD3
linked to anti-p185.sup.HER2 has been used to target T cells to
kill tumor cells overexpressing the HER2 receptor. Shalaby et al.,
J. Exp. Med. 175(1):217 (1992). Bispecific antibodies have been
tested in several early phase clinical trials with encouraging
results. In one trial, 12 patients with lung, ovarian or breast
cancer were treated with infusions of activated T-lymphocytes
targeted with an anti-CD3/anti-tumor (MOC31) bispecific antibody.
deLeij et al. Bispecific Antibodies and Targeted Cellular
Cytotoxicity, Romet-Lemonne, Fanger and Segal Eds., Lienhart (1991)
p. 249. The targeted cells induced considerable local lysis of
tumor cells, a mild inflammatory reaction, but no toxic side
effects or anti-mouse antibody responses. In a very preliminary
trial of an anti-CD3/anti-CD19 bispecific antibody in a patient
with B cell malignancy, significant reduction in peripheral tumor
cell counts was also achieved. Clark et al. Bispecific Antibodies
and Targeted Cellular Cytotoxicity, Romet-Lemonne, Fanger and Segal
Eds., Lienhart (1991) p. 243. See also Kroesen et al., Cancer
Immunol. Immunother. 37:400-407 (1993), Kroesen et al., Br. J.
Cancer 70:652-661 (1994) and Weiner et al., J. Immunol. 152:2385
(1994) concerning therapeutic applications for multispecific
antibodies.
[0216] Multispecific antibodies may also be used as fibrinolytic
agents or vaccine adjuvants. Furthermore, these antibodies may be
used in the treatment of infectious diseases (e.g. for targeting of
effector cells to virally infected cells such as HIV or influenza
virus or protozoa such as Toxoplasma gondii), used to deliver
immunotoxins to tumor cells, or target immune complexes to cell
surface receptors (see Fanger et al., supra).
[0217] Various multispecific antibodies are contemplated herein.
For instance, the multispecific antibody may bind two or more
different epitopes on an antigen of interest. Alternatively, the
multispecfic antibody may have specificity for (1) an antigen
expressed by a target cell (e.g. where the target cell is a tumor
cell) and (2) a triggering molecule on a leukocyte, such as a
T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG
(Fc gamma R), such as Fc gamma RI (CD64), Fc gamma RII (CD32) and
Fc gamma RIII (CD16) so as to focus cellular defense mechanisms to
the antigen-expressing cell. Multispecific antibodies may also be
used to localize cytotoxic agents to cells which express the target
antigen. These antibodies possess an target antigen-binding arm and
an arm which binds the cytotoxic agent (e.g. saporin,
interferon-alpha, vinca alkaloid, ricin A chain, methotrexate or
radioactive isotope hapten).
[0218] WO 96/16673 describes a bispecific anti-HER2/anti-Fc gamma
RIII antibody and U.S. Pat. No. 5,837,234 discloses a bispecific
anti-HER2/anti-Fc gamma RI antibody. A bispecific anti-HER2/Fc
alpha antibody is shown in WO98/02463. U.S. Pat. No. 5,821,337
teaches a bispecific anti-HER2/anti-CD3 antibody.
[0219] C. Preparation of the Parent Antibody
[0220] In order to generate the multivalent antibody, a "parent" or
"starting" antibody with variable domains directed against an
antigen may be prepared using various methodologies for making
antibodies, such as those described hereinbelow. The sequences of
the variable domains of the starting or parent antibody may be used
in the design of the multivalent antibody herein.
[0221] (i) Polyclonal Antibodies
[0222] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups.
[0223] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later the animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Preferably, the animal is boosted
with the conjugate of the same antigen, but conjugated to a
different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
[0224] (ii) Monoclonal Antibodies
[0225] Monoclonal antibodies are 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. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies.
[0226] For example, the monoclonal antibodies may be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).
[0227] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as hereinabove described to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
[0228] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0229] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987)).
[0230] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0231] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al.,
Anal. Biochem., 107:220 (1980).
[0232] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
[0233] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional antibody purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0234] DNA encoding the monoclonal antibodies is 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 murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce antibody protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. Review
articles on recombinant expression in bacteria of DNA encoding the
antibody include Skerra et al., Curr. Opinion in Immunol.,
5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).
Recombinant expression of antibodies is described in more detail
below.
[0235] In a further embodiment, monoclonal antibodies or antibody
fragments can be isolated from antibody phage libraries generated
using the techniques described in McCafferty et al., Nature,
348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and
Marks et al., J. Mol. Biot., 222:581-597 (1991) describe the
isolation of murine and human antibodies, respectively, using phage
libraries. Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et
al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0236] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy chain and light chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA,
81:6851 (1984)), or by covalently joining to the immunoglobulin
coding sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0237] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0238] (iii) Human Antibodies
[0239] Human monoclonal antibodies may be made via an adaptation of
the hybridoma method first described by Kohler and Milstein by
using human B lymphocytes as the fusion partner. Human B
lymphocytes producing an antibody of interest may, for example, be
isolated from a human individual, after obtaining informed consent.
For instance, the individual may be producing antibodies against an
autoantigen as occurs with certain disorders such as systemic lupus
erythematosus (Shoenfeld et al. J. Clin. Invest. 70:205 (1982)),
immune-mediated thrombocytopenic purpura (ITP) (Nugent et al. Blood
70(1):16-22 (1987)), or cancer. Alternatively, or additionally,
lymphocytes may be immunized in vitro. For instance, one may expose
isolated human periperal blood lymphocytes in vitro to a
lysomotrophic agent (e.g. L-leucine-O-methyl ester, L-glutamic acid
dimethly ester or L-leucyl-L-leucine-O-methyl ester) (U.S. Pat. No.
5,567,610, Borrebaeck et al.); and/or T-cell depleted human
peripheral blood lymphocytes may be treated in vitro with adjuvants
such as 8-mercaptoguanosine and cytokines (U.S. Pat. No. 5,229,275,
Goroff et al.).
[0240] The B lymphocytes recovered from the subject or immunized in
vitro, are then generally immortalized in order to generate a human
monoclonal antibody. Techniques for immortalizing the B lymphocyte
include, but are not limited to: (a) fusion of the human B
lymphocyte with human, murine myelomas or mouse-human heteromyeloma
cells; (b) viral transformation (e.g. with an Epstein-Barr virus;
see Nugent et al., supra, for example); (c) fusion with a
lymphoblastoid cell line; or (d) fusion with lymphoma cells.
[0241] Lymphocytes may be fused with myeloma cells using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)). The hybridoma cells thus prepared are
seeded and grown in a suitable culture medium that preferably
contains one or more substances that inhibit the growth or survival
of the unfused, parental myeloma cells. For example, if the
parental myeloma cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine (HAT medium), which substances prevent the growth of
HGPRT-deficient cells. Suitable human myeloma and mouse-human
heteromyeloma cell lines have been described (Kozbor, J. Immunol.,
133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New
York, 1987)). Culture medium in which hybridoma cells are growing
is assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0242] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A chromatography, gel electrophoresis, dialysis,
or affinity chromatography.
[0243] Human antibodies may also be generated using a non-human
host, such as a mouse, which is capable of producing human
antibodies. As noted above, transgenic mice are now available that
are capable, upon immunization, of producing a full repertoire of
human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (J.sub.H) gene
in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene array in such germ-line mutant mice
will result in the production of human antibodies upon antigen
challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.
USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);
Bruggermann et al., Year in Immuno., 7:33 (1993); U.S. Pat. No.
5,591,669; U.S. Pat. No. 5,589,369; and U.S. Pat. No. 5,545,807.
Human antibodies may also be prepared using SCID-hu mice (Duchosal
et al. Nature 355:258-262 (1992)).
[0244] In another embodiment, the human antibody may be selected
from a human antibody phage display library. The preparation of
libraries of antibodies or fragments thereof is well known in the
art and any of the known methods may be used to construct a family
of transformation vectors which may be introduced into host cells.
Libraries of antibody light and heavy chains in phage (Huse et al.,
Science, 246:1275 (1989)) or of fusion proteins in phage or
phagemid can be prepared according to known procedures. See, for
example, Vaughan et al., Nature Biotechnology 14:309-314 (1996);
Barbas et al., Proc. Natl. Acad. Sci., USA, 88:7978-7982 (1991);
Marks et al., J. Mol. Biol., 222:581-597 (1991); Hoogenboom and
Winter, J. Mol. Biol., 227:381-388 (1992); Barbas et al., Proc.
Natl. Acad. Sci., USA, 89:4457-4461 (1992); Griffiths et al., EMBO
Journal, 13:3245-3260 (1994); de Kruif et al., J. Mol. Biol.,
248:97-105 (1995); WO 98/05344; WO 98/15833; WO 97/47314; WO
97/44491; WO 97/35196; WO 95/34648; U.S. Pat. No. 5,712,089; U.S.
Pat. No. 5,702,892; U.S. Pat. No. 5,427,908; U.S. Pat. No.
5,403,484; U.S. Pat. No. 5,432,018; U.S. Pat. No. 5,270,170; WO
92/06176; WO 99/06587; U.S. Pat. No. 5,514,548; WO97/08320; and
U.S. Pat. No. 5,702,892. The antigen of interest is panned against
the phage library using procedures known in the field for selecting
phage-antibodies which bind to the target antigen
[0245] (iv) Humanized Antibodies
[0246] Methods for humanizing non-human antibodies have been
described in the art. Preferably, a humanized antibody has one or
more amino acid residues introduced into it from a source which is
non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting hypervariable region sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some hypervariable region residues and possibly
some FR residues are substituted by residues from analogous sites
in rodent antibodies.
[0247] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework for the humanized
antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et
al., J. Mol. Biol., 196:901 (1987)). Another method uses a
particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285
(1992); Presta et al., J. Immunol., 151:2623 (1993)).
[0248] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, L e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0249] (v) Antibody Fragments
[0250] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
For example, the antibody fragments can be isolated from the
antibody phage libraries discussed above. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner. In other
embodiments, the antibody of choice is a single chain Fv fragment
(scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.
5,587,458. The antibody fragment may also be a "linear antibody",
e.g., as described in U.S. Pat. No. 5,641,870 for example. Such
linear antibody fragments may be monospecific or bispecific.
[0251] (vi) Antibody Variant Sequences
[0252] Amino acid sequence modification(s) of the antibodies
described herein are contemplated. For example, it may be desirable
to improve the binding affinity and/or other biological properties
of the antibody. Amino acid sequence variants of the antibody are
prepared by introducing appropriate nucleotide changes into the
antibody nucleic acid, or by peptide synthesis. Such modifications
include, for example, deletions from, and/or insertions into and/or
substitutions of, residues within the amino acid sequences of the
antibody. Any combination of deletion, insertion, and substitution
is made to arrive at the final construct, provided that the final
construct possesses the desired characteristics. The amino acid
changes also may alter post-translational processes of the
antibody, such as changing the number or position of glycosylation
sites. Such alterations may be made to the parent antibody and/or
multivalent antibody and/or may be introduced in the multivalent
antibody amino acid sequence at the time that sequence is made.
[0253] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
multivalent antibodies are screened for the desired activity.
[0254] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to a cytotoxic
polypeptide. Other insertional variants of the antibody molecule
include the fusion to the N- or C-terminus of the antibody to an
enzyme (e.g. for ADEPT) or a polypeptide which increases the serum
half-life of the antibody.
[0255] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule replaced by a different residue. The sites of
greatest interest for substitutional mutagenesis include the
hypervariable regions, but FR alterations are also contemplated.
Conservative substitutions are shown in Table 1 under the heading
of "preferred substitutions". If such substitutions result in a
change in biological activity, then more substantial changes,
denominated "exemplary substitutions" in Table 1, or as further
described below in reference to amino acid classes, may be
introduced and the products screened.
TABLE-US-00001 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys;
gin; asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn
glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp
Gly (G) ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val;
met; ala; leu phe; norleucine Leu (L) norleucine; ile; val; ile
met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr thr
Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe; leu ala; norleucine
[0256] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
[0257] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0258] (2) neutral hydrophilic: cys, ser, thr;
[0259] (3) acidic: asp, glu;
[0260] (4) basic: asn, gln, his, lys, arg;
[0261] (5) residues that influence chain orientation: gly, pro;
and
[0262] (6) aromatic: trp, tyr, phe.
[0263] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0264] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant crosslinking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability.
[0265] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g. a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g. 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The multivalent antibodies thus generated are displayed
in a monovalent fashion from filamentous phage particles as fusions
to the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g. binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the antibody and antigen. Such
contact residues and neighboring residues are candidates for
substitution according to the techniques elaborated herein.
[0266] Once such variants are generated, the panel of variants is
subjected to screening as described herein and antibodies with
superior properties in one or more relevant assays may be selected
for further development.
[0267] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[0268] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0269] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0270] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
[0271] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g. so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid modifications in
an Fc region of the antibody, thereby generating a variant Fc
region. 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.
[0272] In one embodiment, the variant Fc region may mediate
antibody-dependent cell-mediated cytotoxicity (ADCC) in the
presence of human effector cells more effectively, or bind an Fc
gamma receptor (Fc.gamma.R) with better affinity, than a native
sequence Fc region. Such Fc region variants may comprise an amino
acid modification at any one or more of positions 256, 290, 298,
312, 326, 330, 333, 334, 360, 378 or 430 of the Fc region, wherein
the numbering of the residues in the Fc region is that of the EU
index as in Kabat.
[0273] The Fc region variant with reduced binding to an Fc.gamma.R
may comprise an amino acid modification at any one or more of amino
acid positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270,
272, 278, 289, 292, 293, 294, 295, 296, 298, 301, 303, 322, 324,
327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416,
419, 434, 435, 437, 438 or 439 of the Fc region, wherein the
numbering of the residues in the Fc region is that of the EU index
as in Kabat.
[0274] For example, the Fc region variant may display reduced
binding to an Fc.gamma.RI and comprise an amino acid modification
at any one or more of amino acid positions 238, 265, 269, 270, 327
or 329 of the Fc region, wherein the numbering of the residues in
the Fc region is that of the EU index as in Kabat.
[0275] The Fc region variant may display reduced binding to an
Fc.gamma.RII and comprise an amino acid modification at any one or
more of amino acid positions 238, 265, 269, 270, 292, 294, 295,
298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419,
435, 438 or 439 of the Fc region, wherein the numbering of the
residues in the Fc region is that of the EU index as in Kabat.
[0276] The Fc region variant of interest may display reduced
binding to an Fc.gamma.RIII and comprise an amino acid modification
at one or more of amino acid positions 238, 239, 248, 249, 252,
254, 265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301,
303, 322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 416, 434,
435 or 437 of the Fc region, wherein the numbering of the residues
in the Fc region is that of the EU index as in Kabat.
[0277] In another embodiment, the Fc region variant displays
improved binding to the Fc.gamma.R and comprises an amino acid
modification at any one or more of amino acid positions 255, 256,
258, 267, 268, 272, 276, 280, 283, 285, 286, 290, 298, 301, 305,
307, 309, 312, 315, 320, 322, 326, 330, 331, 333, 334, 337, 340,
360, 378, 398 or 430 of the Fc region, wherein the numbering of the
residues in the Fc region is that of the EU index as in Kabat.
[0278] For example, the Fc region variant may display increased
binding to an Fc.gamma.RIII and, optionally, may further display
decreased binding to an Fc.gamma.RII. An exemplary such variant
comprises amino acid modification(s) at position(s) 298 and/or 333
of the Fc region, wherein the numbering of the residues in the Fc
region is that of the EU index as in Kabat.
[0279] The Fc region variant may display increased binding to an
Fc.gamma.RII and comprise an amino acid modification at any one or
more of amino acid positions 255, 256, 258, 267, 268, 272, 276,
280, 283, 285, 286, 290, 301, 305, 307, 309, 312, 315, 320, 322,
326, 330, 331, 337, 340, 378, 398 or 430 of the Fc region, wherein
the numbering of the residues in the Fc region is that of the EU
index as in Kabat. Such Fc region variants with increased binding
to an Fc.gamma.RII may optionally further display decreased binding
to an Fc.gamma.RIII and may, for example, comprise an amino acid
modification at any one or more of amino acid positions 268, 272,
298, 301, 322 or 340 of the Fc region, wherein the numbering of the
residues in the Fc region is that of the EU index as in Kabat.
[0280] The variant Fc region may alternatively or additionally have
altered neonatal Fc receptor (FcRn) binding affinity. Such variant
Fc regions may comprise an amino acid modification at any one or
more of amino acid positions 238, 252, 253, 254, 255, 256, 265,
272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434,
435, 436, 439 or 447 of the Fc region, wherein the numbering of the
residues in the Fc region is that of the EU index as in Kabat. Fc
region variants with reduced binding to an FcRn may comprise an
amino acid modification at any one or more of amino acid positions
252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435, 436,
439 or 447 of the Fc region, wherein the numbering of the residues
in the Fc region is that of the EU index as in Kabat. The
above-mentioned Fc region variants may, alternatively, display
increased binding to FcRn and comprise an amino acid modification
at any one or more of amino acid positions 238, 256, 265, 272, 286,
303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380,
382, 413, 424 or 434 of the Fc region, wherein the numbering of the
residues in the Fc region is that of the EU index as in Kabat.
[0281] Fc region variants with altered (i.e. improved or
diminished) C1q binding and/or Complement Dependent Cytotoxicity
(CDC) are described in WO99/51642. Such variants may comprise an
amino acid substitution at one or more of amino acid positions 270,
322, 326, 327, 329, 331, 333 or 334 of the Fc region. See, also,
Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No.
5,648,260; U.S. Pat. No. 5,624,821; and WO94/29351 concerning Fc
region variants.
[0282] To increase the serum half life of the antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of an IgG
molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that
is responsible for increasing the in vivo serum half-life of the
IgG molecule.
[0283] (vii) Immunoconjugates
[0284] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g. a small molecule toxin or an
enzymatically active toxin of bacterial, fungal, plant or animal
origin, including fragments and/or variants thereof), or a
radioactive isotope (i.e., a radioconjugate).
[0285] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above.
[0286] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, a maytansine (U.S. Pat. No.
5,208,020), a trichothene, and CC1065 are also contemplated
herein.
[0287] In one preferred embodiment of the invention, the antibody
is conjugated to one or more maytansine molecules (e.g. about 1 to
about 10 maytansine molecules per antibody molecule). Maytansine
may, for example, be converted to May-SS-Me which may be reduced to
May-SH3 and reacted with modified antibody (Chari et al. Cancer
Research 52: 127-131 (1992)) to generate a maytansinoid-antibody
immunoconjugate.
[0288] Another immunoconjugate of interest comprises an antibody
conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations.
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma..sub.1.sup.I, .alpha..sub.2.sup.I,
.alpha..sub.3.sup.I, N-acetyl-.gamma..sub.1.sup.I, PSAG and
.theta..sup.I.sub.1 (Hinman et al. Cancer Research 53: 3336-3342
(1993) and Lode et al. Cancer Research 58: 2925-2928 (1998)). See,
also, U.S. Pat. Nos. 5,714,586; 5,712,374; 5,264,586; and 5,773,001
expressly incorporated herein by reference.
[0289] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0290] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0291] A variety of radioactive isotopes are available for the
production of radioconjugated antibodies. Examples include
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32 and radioactive isotopes of
Lu.
[0292] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoylyethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, dimethyl linker or disulfide-containing linker (Chari et
al. Cancer Research 52: 127-131 (1992)) may be used.
[0293] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g. by recombinant techniques or
peptide synthesis.
[0294] In yet another embodiment, the antibody may be conjugated to
a "receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g. avidin) which is conjugated to a
cytotoxic agent (e.g. a radionucleotide).
[0295] (viii) Antibody Dependent Enzyme Mediated Prodrug Therapy
(ADEPT)
[0296] The antibodies of the present invention may also be used in
ADEPT by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug (e.g. a peptidyl chemotherapeutic agent,
see WO81/01145) to an active anti-cancer drug. See, for example, WO
88/07378 and U.S. Pat. No. 4,975,278.
[0297] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to covert it into its more active, cytotoxic form.
[0298] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and cathepsins (such as cathepsins B and L), that
are useful for converting peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as beta-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; beta-lactamase
useful for converting drugs derivatized with beta-lactams into free
drugs; and penicillin amidases, such as penicillin V amidase or
penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to a tumor cell population.
[0299] The enzymes of this invention can be covalently bound to the
antibodies by techniques well known in the art such as the use of
the heterobifunctional crosslinking reagents discussed above.
Alternatively, fusion proteins comprising at least the antigen
binding region of an antibody of the invention linked to at least a
functionally active portion of an enzyme of the invention can be
constructed using recombinant DNA techniques well known in the art
(see, e.g., Neuberger et al., Nature, 312: 604-608 (1984).
[0300] (ix) Other Antibody Modifications
[0301] Other modifications of the antibody are contemplated herein.
For example, the antibody may be linked to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol. The antibody also 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, Oslo, A., Ed., (1980).
[0302] The antibodies disclosed herein may also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and
4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0303] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al. J.
Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al. J. National Cancer Inst.
81(19)1484 (1989).
[0304] D. Vectors, Host Cells and Recombinant Methods
[0305] The invention also provides isolated nucleic acid encoding
an antibody as disclosed herein, vectors and host cells comprising
the nucleic acid, and recombinant techniques for the production of
the antibody.
[0306] For recombinant production of the antibody, the nucleic acid
encoding it is isolated and inserted into a replicable vector for
further cloning (amplification of the DNA) or for expression. DNA
encoding the antibody is readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the
antibody). Many vectors are available. The vector components
generally include, but are not limited to, one or more of the
following: a signal sequence, an origin of replication, one or more
marker genes, an enhancer element, a promoter, and a transcription
termination sequence.
[0307] (i) Signal Sequence Component
[0308] The multivalent antibody of this invention may be produced
recombinantly not only directly, but also as a fusion polypeptide
with a heterologous polypeptide, which is preferably a signal
sequence or other polypeptide having a specific cleavage site at
the N-terminus of the mature protein or polypeptide. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. For prokaryotic host cells that do not recognize and
process the native multivalent antibody signal sequence, the signal
sequence is substituted by a prokaryotic signal sequence selected,
for example, from the group of the alkaline phosphatase,
penicillinase, Ipp, or heat-stable enterotoxin II leaders. For
yeast secretion the native signal sequence may be substituted by,
e.g., the yeast invertase leader, .alpha. factor leader (including
Saccharomyces and Kluyveromyces .alpha.-factor leaders), or acid
phosphatase leader, the C. albicans glucoamylase leader, or the
signal described in WO 90/13646. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available.
[0309] The DNA for such precursor region is ligated in reading
frame to DNA encoding the multivalent antibody.
[0310] (ii) Origin of Replication Component
[0311] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2.mu. plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells. Generally,
the origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only
because it contains the early promoter).
[0312] (iii) Selection Gene Component
[0313] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli.
[0314] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0315] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the multivalent antibody nucleic acid, such as DHFR,
thymidine kinase, metallothionein-I and -II, preferably primate
metallothionein genes, adenosine deaminase, ornithine
decarboxylase, etc.
[0316] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell when wild-type DHFR is
employed is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity.
[0317] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding multivalent antibody, wild-type DHFR protein,
and another selectable marker such as aminoglycoside
3'-phosphotransferase (APH) can be selected by cell growth in
medium containing a selection agent for the selectable marker such
as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or
G418. See U.S. Pat. No. 4,965,199.
[0318] A suitable selection gene for use in yeast is the trp1 gene
present in the yeast plasmid YRp7 (Stinchcomb et al., Nature,
282:39 (1979)). The trp1 gene provides a selection marker for a
mutant strain of yeast lacking the ability to grow in tryptophan,
for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12
(1977). The presence of the trp1 lesion in the yeast host cell
genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly,
Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are
complemented by known plasmids bearing the Leu2 gene.
[0319] In addition, vectors derived from the 1.6 .mu.m circular
plasmid pKD1 can be used for transformation of Kluyveromyces
yeasts. Alternatively, an expression system for large-scale
production of recombinant calf chymosin was reported for K. lactis.
Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy
expression vectors for secretion of mature recombinant human serum
albumin by industrial strains of Kluyveromyces have also been
disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).
[0320] (iv) Promoter Component
[0321] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the multivalent antibody nucleic acid. Promoters suitable for use
with prokaryotic hosts include the phoA promoter, .beta.-lactamase
and lactose promoter systems, alkaline phosphatase, a tryptophan
(trp) promoter system, and hybrid promoters such as the tac
promoter. However, other known bacterial promoters are suitable.
Promoters for use in bacterial systems also will contain a
Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding
the multivalent antibody.
[0322] Promoter sequences are known for eukaryotes. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CNCAAT region where N may be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors.
[0323] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase or other
glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0324] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657. Yeast enhancers also are advantageously used with yeast
promoters.
[0325] Multivalent antibody transcription from vectors in mammalian
host cells is controlled, for example, by promoters obtained from
the genomes of viruses such as polyoma virus, fowlpox virus,
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
most preferably Simian Virus 40 (SV40), from heterologous mammalian
promoters, e.g., the actin promoter or an immunoglobulin promoter,
from heat-shock promoters, provided such promoters are compatible
with the host cell systems.
[0326] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the rous sarcoma
virus long terminal repeat can be used as the promoter.
[0327] (v) Enhancer Element Component
[0328] Transcription of a DNA encoding the multivalent antibody of
this invention by higher eukaryotes is often increased by inserting
an enhancer sequence into the vector. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the
multivalent antibody-encoding sequence, but is preferably located
at a site 5' from the promoter.
[0329] (vi) Transcription Termination Component
[0330] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding the
multivalent antibody. One useful transcription termination
component is the bovine growth hormone polyadenylation region. See
WO94/11026 and the expression vector disclosed therein.
[0331] (vii) Selection and Transformation of Host Cells
[0332] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0333] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for multivalent antibody-encoding vectors. Saccharomyces
cerevisiae, or common baker's yeast, is the most commonly used
among lower eukaryotic host microorganisms. However, a number of
other genera, species, and strains are commonly available and
useful herein, such as Schizosaccharomyces pombe; Kluyveromyces
hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K.
bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii
(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,
and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP
183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora
crassa; Schwanniomyces such as Schwanniomyces occidentalis; and
filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium, and Aspergillus hosts such as A. nidulans and A.
niger.
[0334] Suitable host cells for the expression of glycosylated
multivalent antibody are derived from multicellular organisms.
Examples of invertebrate cells include plant and insect cells.
Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera
frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster (fruitfly), and
Bombyx mori have been identified. A variety of viral strains for
transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells.
[0335] Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, and tobacco can also be utilized as hosts.
[0336] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells
(Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);
TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
MRC 5 cells; FS4 cells; a human hepatoma line (Hep G2); and myeloma
or lymphoma cells (e.g. Y0, J558L, P3 and NS0 cells) (see U.S. Pat.
No. 5,807,715).
[0337] Host cells are transformed with the above-described
expression or cloning vectors for multivalent antibody production
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0338] (viii) Culturing the Host Cells
[0339] The host cells used to produce the multivalent antibody of
this invention may be cultured in a variety of media. Commercially
available media such as Ham's F10 (Sigma), Minimal Essential Medium
((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium ((DMEM), Sigma) are suitable for culturing the host cells.
In addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. No. 30,985 may be used
as culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0340] (ix) Purification
[0341] When using recombinant techniques, the multivalent antibody
can be produced intracellularly, in the periplasmic space, or
directly secreted into the medium. If the multivalent antibody is
produced intracellularly, as a first step, the particulate debris,
either host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10:163-167 (1992) describe a procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the multivalent
antibody is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0342] The multivalent antibody composition prepared from the cells
can be purified using, for example, hydroxylapatite chromatography,
gel electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc region that is present
in the multivalent antibody. Protein A can be used to purify
antibodies that are based on human .gamma.1, .gamma.2, or .gamma.4
heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).
Protein G is recommended for all mouse isotypes and for human
.gamma.3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to
which the affinity ligand is attached is most often agarose, but
other matrices are available. Mechanically stable matrices such as
controlled pore glass or poly(styrenedivinyl)benzene allow for
faster flow rates and shorter processing times than can be achieved
with agarose. Where the multivalent antibody comprises a C.sub.H3
domain, the Bakerbond ABX.TM. resin (J. T. Baker, Phillipsburg,
N.J.) is useful for purification. Other techniques for protein
purification such as fractionation on an ion-exchange column,
ethanol precipitation, Reverse Phase HPLC, chromatography on
silica, chromatography on heparin SEPHAROSE.TM. chromatography on
an anion or cation exchange resin (such as a polyaspartic acid
column), chromatofocusing, SDS-PAGE, and ammonium sulfate
precipitation are also available depending on the multivalent
antibody to be recovered.
[0343] E. Pharmaceutical Formulations
[0344] Therapeutic formulations of the multivalent antibody are
prepared for storage by mixing the multivalent antibody having the
desired degree of purity with optional physiologically acceptable
carriers, excipients or stabilizers (Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980)), in the form of aqueous
solutions, lyophilized or other dried formulations. Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at
the dosages and concentrations employed, and include 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
TWEENT.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0345] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Examples of combinations of active
compounds are provided in Section G below entitled "In Vivo Uses
for the Multivalent Antibody". Such molecules are suitably present
in combination in amounts that are effective for the purpose
intended.
[0346] The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
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).
[0347] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0348] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the multivalent
antibody, which matrices are in the form of shaped articles, e.g.,
films, or microcapsule. Examples of sustained-release matrices
include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0349] F. Non-Therapeutic Uses for the Multivalent Antibody
[0350] The multivalent antibody of the invention may be used as an
affinity purification agent. In this process, the multivalent
antibody is immobilized on a solid phase such a Sephadex resin or
filter paper, using methods well known in the art. The immobilized
multivalent antibody is contacted with a sample containing the
antigen to be purified, and thereafter the support is washed with a
suitable solvent that will remove substantially all the material in
the sample except the antigen to be purified, which is bound to the
immobilized multivalent antibody. Finally, the support is washed
with another suitable solvent, such as glycine buffer, pH 5.0, that
will release the antigen from the multivalent antibody.
[0351] The multivalent antibody may also be useful in diagnostic
assays, e.g., for detecting expression of an antigen of interest in
specific cells, tissues, or serum.
[0352] For diagnostic applications, the multivalent antibody
typically will be labeled with a detectable moiety. Numerous labels
are available which can be generally grouped into the following
categories:
[0353] (a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I. The multivalent antibody can be labeled
with the radioisotope using the techniques described in Current
Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed.
Wiley-Interscience, New York, N.Y., Pubs. (1991) for example and
radioactivity can be measured using scintillation counting.
[0354] (b) Fluorescent labels such as rare earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. The fluorescent labels can be conjugated to the
multivalent antibody using the techniques disclosed in Current
Protocols in Immunology, supra, for example. Fluorescence can be
quantified using a fluorimeter.
[0355] (c) Various enzyme-substrate labels are available and U.S.
Pat. No. 4,275,149 provides a review of some of these. The enzyme
generally catalyzes a chemical alteration of the chromogenic
substrate that can be measured using various techniques. For
example, the enzyme may catalyze a color change in a substrate,
which can be measured spectrophotometrically. Alternatively, the
enzyme may alter the fluorescence or chemiluminescence of the
substrate. Techniques for quantifying a change in fluorescence are
described above. The chemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit
light which can be measured (using a chemiluminometer, for example)
or donates energy to a fluorescent acceptor. Examples of enzymatic
labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase,
and the like. Techniques for conjugating enzymes to antibodies are
described in O'Sullivan et al., Methods for the Preparation of
Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in
Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic
press, New York, 73:147-166 (1981).
[0356] Examples of enzyme-substrate combinations include, for
example
[0357] (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase
as a substrate, wherein the hydrogen peroxidase oxidizes a dye
precursor (e.g., orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));
[0358] (ii) alkaline phosphatase (AP) with para-Nitrophenyl
phosphate as chromogenic substrate; and
[0359] (iii) .beta.-D-galactosidase (.beta.-D-Gal) with a
chromogenic substrate (e.g., p-nitrophenyl-.beta.-D-galactosidase)
or fluorogenic substrate
4-methylumbelliferyl-.beta.-D-galactosidase.
[0360] Numerous other enzyme-substrate combinations are available
to those skilled in the art. For a general review of these, see
U.S. Pat. Nos. 4,275,149 and 4,318,980.
[0361] Sometimes, the label is indirectly conjugated with the
multivalent antibody. The skilled artisan will be aware of various
techniques for achieving this. For example, the multivalent
antibody can be conjugated with biotin and any of the three broad
categories of labels mentioned above can be conjugated with avidin,
or vice versa. Biotin binds selectively to avidin and thus, the
label can be conjugated with the multivalent antibody in this
indirect manner. Alternatively, to achieve indirect conjugation of
the label with the multivalent antibody, the multivalent antibody
is conjugated with a small hapten (e.g., digoxin) and one of the
different types of labels mentioned above is conjugated with an
anti-hapten multivalent antibody (e.g., anti-digoxin antibody).
Thus, indirect conjugation of the label with the multivalent
antibody can be achieved.
[0362] In another embodiment of the invention, the multivalent
antibody need not be labeled, and the presence thereof can be
detected using a labeled antibody which binds to the multivalent
antibody.
[0363] The multivalent antibody of the present invention may be
employed in any known assay method, such as competitive binding
assays, direct and indirect sandwich assays, and
immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual
of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
[0364] The multivalent antibody may also be used for in vivo
diagnostic assays. Generally, the multivalent antibody is labeled
with a radionuclide (such as .sup.111In, .sup.99Tc, .sup.14C,
.sup.131I, .sup.125I, .sup.3H, .sup.32P or .sup.35S) so that the
antigen or cells expressing it can be localized using
immunoscintiography.
[0365] G. In Vivo Uses for the Multivalent Antibody
[0366] It is contemplated that the multivalent antibody of the
present invention may be used to treat a mammal e.g. a patient
suffering from, or predisposed to, a disease or disorder who could
benefit from administration of the multivalent antibody.
[0367] Where the antibody binds an ErbB receptor, such as HER2,
conditions to be treated therewith include benign or malignant
tumors; leukemias and lymphoid malignancies; other disorders such
as neuronal, glial, astrocytal, hypothalamic, glandular,
macrophagal, epithelial, stromal, blastocoelic, inflammatory,
angiogenic and immunologic disorders. Generally, the disease or
disorder to be treated with the antibody that binds an ErbB
receptor is cancer.
[0368] Examples of cancer to be treated herein 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, lung cancer including small-cell lung
cancer, non-small cell lung cancer, adenocarcinoma of the lung and
squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer including
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,
breast cancer, colon cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney or renal
cancer, liver cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma as well as head and neck cancer.
[0369] The cancer will generally comprise cells that express an
antigen bound by the antibody, such that the antibody is able to
bind to the cancer. In one embodiment, the cancer may be
characterized by overexpression of the antigen (e.g. overexpression
of an ErbB receptor). To determine expression of the antigen by the
cancer, various diagnostic/prognostic assays are available. In one
embodiment, antigen overexpression may be analyzed by INC, e.g.
using the HERCEPTEST.RTM. (Dako) where the antigen is HER2. In the
HER2 IHC test, parrafin embedded tissue sections from a tumor
biopsy may be subjected to the IHC assay and accorded a HER2
protein staining intensity criteria as follows: [0370] Score 0 no
staining is observed or membrane staining is observed in less than
10% of tumor cells. [0371] Score 1+ a faint/barely perceptible
membrane staining is detected in more than 10% of the tumor cells.
The cells are only stained in part of their membrane. [0372] Score
2+ a weak to moderate complete membrane staining is observed in
more than 10% of the tumor cells. [0373] Score 3+ a moderate to
strong complete membrane staining is observed in more than 10% of
the tumor cells.
[0374] Those tumors with 0 or 1+ scores for HER2 overexpression
assessment may be characterized as not overexpressing HER2, whereas
those tumors with 2+ or 3+ scores may be characterized as
overexpressing HER2.
[0375] Alternatively, or additionally, FISH assays such as the
INFORM.TM. (sold by Ventana, Ariz.) or PATHVISION.TM. (Vysis, Ill.)
may be carried out on formalin-fixed, paraffin-embedded tumor
tissue to determine the extent (if any) of antigen overexpression
by the tumor.
[0376] In one embodiment, the cancer will be one which expresses
(and may overexpress) an ErbB receptor selected from the group
consisting of EGFR, ErbB3 and ErbB4. Examples of cancers which may
express/overexpress EGFR, ErbB3 or ErbB4 include squamous cell
cancer, lung cancer including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung and squamous carcinoma
of the lung, cancer of the peritoneum, hepatocellular cancer,
gastric or stomach cancer including gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney or renal cancer, liver cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma
as well as head and neck cancer as well as glioblastomas.
[0377] The cancer to be treated herein may be one characterized by
excessive activation of an ErbB receptor, e.g. EGFR. Such excessive
activation may be attributable to overexpression or increased
production of the ErbB receptor or an ErbB ligand. In one
embodiment of the invention, a diagnostic or prognostic assay will
be performed to determine whether the patient's cancer is
characterized by excessive activation of an ErbB receptor. For
example, ErbB gene amplification and/or overexpression of an ErbB
receptor in the cancer may be determined. Various assays for
determining such amplification/overexpression are available in the
art and include the IHC, FISH and shed antigen assays described
above. Alternatively, or additionally, levels of an ErbB ligand,
such as TGF-alpha, in or associated with the tumor may be
determined according to known procedures. Such assays may detect
protein and/or nucleic acid encoding it in the sample to be tested.
In one embodiment, ErbB ligand levels in the tumor may be
determined using immunohistochemistry (1HC); see, for example,
Scher et al. Clin. Cancer Research 1:545-550 (1995). Alternatively,
or additionally, one may evaluate levels of ErbB ligand-encoding
nucleic acid in the sample to be tested; e.g. via fluorescent in
situ hybridization or FISH, southern blotting, or polymerase chain
reaction (PCR) techniques.
[0378] Moreover, ErbB receptor or ErbB ligand overexpression or
amplification may be evaluated using an in vivo diagnostic assay,
e.g. by administering a molecule (such as an antibody) which binds
the molecule to be detected and is tagged with a detectable label
(e.g. a radioactive isotope) and externally scanning the patient
for localization of the label.
[0379] Where the antibody binds a B cell surface antigen, the
antibody may be used to treat a B cell lymphoma (including low
grade/follicular non-Hodkin's lymphoma (NHL); small lymphocytic
(SL) NHL; intermediate grade/follicular NHL; intermediate grade
diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic
NHL; high grade small non-cleaved cell NHL; bulky disease NHL;
mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; and chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD).
[0380] The antibody, e.g. the anti-B cell surface antigen antibody,
may also be used to treat an autoimmune disease. Examples of
autoimmune diseases or disorders include, but are not limited to,
inflammatory responses such as inflammatory skin diseases including
psoriasis and dermatitis (e.g. atopic dermatitis); systemic
scleroderma and sclerosis; responses associated with inflammatory
bowel disease (such as Crohn's disease and ulcerative colitis);
respiratory distress syndrome (including adult respiratory distress
syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis;
colitis; glomerulonephritis; allergic conditions such as eczema and
asthma and other conditions involving infiltration of T cells and
chronic inflammatory responses; atherosclerosis; leukocyte adhesion
deficiency; rheumatoid arthritis; systemic lupus erythematosus
(SLE); diabetes mellitus (e.g. Type I diabetes mellitus or insulin
dependent diabetes mellitis); multiple sclerosis; Reynaud's
syndrome; autoimmune thyroiditis; allergic encephalomyelitis;
Sjorgen's syndrome; juvenile onset diabetes; and immune responses
associated with acute and delayed hypersensitivity mediated by
cytokines and T-lymphocytes typically found in tuberculosis,
sarcoidosis, polymyositis, granulomatosis and vasculitis;
pernicious anemia (Addison's disease); diseases involving leukocyte
diapedesis; central nervous system (CNS) inflammatory disorder;
multiple organ injury syndrome; hemolytic anemia (including, but
not limited to cryoglobinemia or Coombs positive anemia);
myasthenia gravis; antigen-antibody complex mediated diseases;
anti-glomerular basement membrane disease; antiphospholipid
syndrome; allergic neuritis; Graves' disease; Lambert-Eaton
myasthenic syndrome; pemphigoid bullous; pemphigus; autoimmune
polyendocrinopathies; Reiter's disease; stiff-man syndrome; Behcet
disease; giant cell arteritis; immune complex nephritis; IgA
nephropathy; IgM polyneuropathies; immune thrombocytopenic purpura
(ITP) or autoimmune thrombocytopenia etc.
[0381] Antibodies directed against B cell surface antigens may also
be used to block an immune response to a foreign antigen. By
"foreign antigen" here is meant a molecule or molecules which
is/are not endogenous or native to a mammal which is exposed to it.
The foreign antigen may elicit an immune response, e.g. a humoral
and/or T cell mediated response in the mammal. Generally, the
foreign antigen will provoke the production of antibodies
thereagainst. Examples of foreign antigens contemplated herein
include immunogenic therapeutic agents, e.g. proteins such as
antibodies, particularly antibodies comprising non-human amino acid
residues (e.g. rodent, chimeric/humanized, and primatized
antibodies); toxins (optionally conjugated to a targeting molecule
such as an antibody, wherein the targeting molecule may also be
immunogenic); gene therapy viral vectors, such as retroviruses and
adenoviruses; grafts; infectious agents (e.g. bacteria and virus);
alloantigens (i.e. an antigen that occurs in some, but not in other
members of the same species) such as differences in blood types,
human lymphocyte antigens (HLA), platelet antigens, antigens
expressed on transplanted organs, blood components, pregnancy (Rh),
and hemophilic factors (e.g. Factor VIII and Factor IX).
[0382] The anti-B cell surface antigen antibody may also be used to
desenzitize a mammal awaiting transplantation.
[0383] Antibodies directed against a receptor in the TNF receptor
superfamily may be employed to activate or stimulate apoptosis in
cancer cells.
[0384] In certain embodiments, an immunoconjugate comprising the
antibody conjugated with a cytotoxic agent is administered to the
patient. Preferably, the immunoconjugate and/or antigen to which it
is bound is/are internalized by the cell, resulting in increased
therapeutic efficacy of the immunoconjugate in killing the cancer
cell to which it binds. In a preferred embodiment, the cytotoxic
agent targets or interferes with nucleic acid in the cancer cell.
Examples of such cytotoxic agents include any of the
chemotherapeutic agents noted herein (such as a maytansinoid or a
calicheamicin), a radioactive isotope, or a ribonuclease or a DNA
endonuclease. As noted above, the multivalent antibody may also be
used for ADEPT.
[0385] The present application contemplates combining the
multivalent antibody (or immunoconjugate thereof) with one or more
other therapeutic agent(s), especially for treating cancer. For
instance, the multivalent antibody may be co-administered with
another multivalent antibody (or multivalent antibodies), a
monovalent or bivalent antibody (or antibodies), chemotherapeutic
agent(s) (including cocktails of chemotherapeutic agents), other
cytotoxic agent(s), anti-angiogenic agent(s), cytokines, and/or
growth inhibitory agent(s). Where the multivalent antibody induces
apoptosis, it may be particularly desirable to combine the
multivalent antibody with one or more other therapeutic agent(s)
which also induce apoptosis. For instance, pro-apoptotic antibodies
(e.g. bivalent or multivalent antibodies) directed against B cell
surface antigens (e.g. RITUXAN.RTM., ZEVALIN.RTM. or BEXXAR.RTM.
anti-CD20 antibodies) may be combined with (1) pro-apoptotic
antibodies (e.g. bivalent or multivalent antibodies directed
against a receptor in the TNF receptor superfamily, such as
anti-DR4 or anti-DR5 antibodies) or (2) with cytokines in the TNF
family of cytokines (e.g. Apo2L). Likewise, anti-ErbB antibodies
(e.g. HERCEPTIN.RTM. anti-HER2 antibody) may be combined with (1)
and/or (2). Alternatively, or additionally, the patient may receive
combined radiation therapy (e.g. external beam irradiation or
therapy with a radioactive labelled agent, such as an antibody).
Such combined therapies noted above include combined administration
(where the two or more agents are included in the same or separate
formulations), and separate administration, in which case,
administration of the multivalent antibody can occur prior to,
and/or following, administration of the adjunct therapy or
therapies.
[0386] The multivalent antibody (and adjunct therapeutic agent)
is/are administered by any suitable means, including parenteral,
subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and,
if desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration. In
addition, the multivalent antibody is suitably administered by
pulse infusion, particularly with declining doses of the
multivalent antibody. Preferably the dosing is given by injections,
most preferably intravenous or subcutaneous injections, depending
in part on whether the administration is brief or chronic.
[0387] Aside from administration of the antibody protein to the
patient, the present application contemplates administration of the
antibody by gene therapy. Such administration of nucleic acid
encoding the antibody is encompassed by the expression
"administering a therapeutically effective amount of an antibody".
See, for example, WO96/07321 published Mar. 14, 1996 concerning the
use of gene therapy to generate intracellular antibodies.
[0388] There are two major approaches to getting the nucleic acid
(optionally contained in a vector) into the patient's cells; in
vivo and ex vivo. For in vivo delivery the nucleic acid is injected
directly into the patient, usually at the site where the antibody
is required. For ex vivo treatment, the patient's cells are
removed, the nucleic acid is introduced into these isolated cells
and the modified cells are administered to the patient either
directly or, for example, encapsulated within porous membranes
which are implanted into the patient (see, e.g. U.S. Pat. Nos.
4,892,538 and 5,283,187). There are a variety of techniques
available for introducing nucleic acids into viable cells. The
techniques vary depending upon whether the nucleic acid is
transferred into cultured cells in vitro, or in vivo in the cells
of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro include the use of
liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate precipitation method, etc. A
commonly used vector for ex vivo delivery of the gene is a
retrovirus. The currently preferred in vivo nucleic acid transfer
techniques include transfection with viral vectors (such as
adenovirus, Herpes simplex I virus, or adeno-associated virus) and
lipid-based systems (useful lipids for lipid-mediated transfer of
the gene are DOTMA, DOPE and DC-Chol, for example). In some
situations it is desirable to provide the nucleic acid source with
an agent that targets the target cells, such as an antibody
specific for a cell surface membrane protein or the target cell, a
ligand for a receptor on the target cell, etc. Where liposomes are
employed, proteins which bind to a cell surface membrane protein
associated with endocytosis may be used for targeting and/or to
facilitate uptake, e.g. capsid proteins or fragments thereof tropic
for a particular cell type, antibodies for proteins which undergo
internalization in cycling, and proteins that target intracellular
localization and enhance intracellular half-life. The technique of
receptor-mediated endocytosis is described, for example, by Wu et
al., J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc.
Natl. Acad. Sci. USA 87:3410-3414 (1990). For review of the
currently known gene marking and gene therapy protocols see
Anderson et al., Science 256:808-813 (1992). See also WO 93/25673
and the references cited therein.
[0389] For the prevention or treatment of disease, the appropriate
dosage of multivalent antibody will depend on the type of disease
to be treated, the severity and course of the disease, whether the
multivalent antibody is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the multivalent antibody, and the discretion of the
attending physician. The multivalent antibody is suitably
administered to the patient at one time or over a series of
treatments.
[0390] Depending on the type and severity of the disease, about 1
.mu.g/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of multivalent antibody
is an initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. A typical daily dosage might range from
about 1 .mu.g/kg to 100 mg/kg or more, depending on the factors
mentioned above. For repeated administrations over several days or
longer, depending on the condition, the treatment is sustained
until a desired suppression of disease symptoms occurs. However,
other dosage regimens may be useful. The progress of this therapy
is easily monitored by conventional techniques and assays.
[0391] The multivalent antibody composition will 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 "therapeutically
effective amount" of the multivalent antibody to be administered
will be governed by such considerations, and is the minimum amount
necessary to prevent, ameliorate, or treat a disease or disorder.
The multivalent antibody 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 multivalent antibody present in the
formulation, the type of disorder or treatment, and other factors
discussed above. These are generally used in the same dosages and
with administration routes as used hereinbefore or about from 1 to
99% of the heretofore employed dosages.
[0392] H. Articles of Manufacture
[0393] In another embodiment of the invention, an article of
manufacture containing materials useful for the treatment of the
disorders described above 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, etc. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition which is effective for treating the
condition 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 a multivalent antibody. The
label or package insert indicates that the composition is used for
treating the condition of choice, such as cancer. Moreover, the
article of manufacture may comprise (a) a first container with a
composition contained therein, wherein the composition comprises a
multivalent antibody; and (b) a second container with a composition
contained therein, wherein the composition comprises a further
cytotoxic agent. The article of manufacture in this embodiment of
the invention may further comprises a package insert indicating
that the first and second antibody compositions can be used to
treat cancer. 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 and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
and syringes.
[0394] I. Deposit of Materials
[0395] The following hybridoma cell lines have been deposited with
the American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. 20110-2209, USA (ATCC):
TABLE-US-00002 Antibody Designation ATCC No. Deposit Date 7C2
(anti-HER2) ATCC HB-12215 Oct. 17, 1996 7F3 (anti-HER2) ATCC
HB-12216 Oct. 17, 1996 4D5 (anti-HER2) ATCC CRL 10463 May 24, 1990
2C4 (anti-HER2) ATCC HB-12697 Apr. 8, 1999 3F11.39.7 (anti-DR5)
HB-12456 Jan. 13, 1998 3H3.14.5 (anti-DR5) HB-12534 Jun. 2, 1998
3D5.1.10 (anti-DR5) HB-12536 Jun. 2, 1998 3H1.18.10 (anti-DR5)
HB-12535 Jun. 2, 1998 4E7.24.3 (anti-DR4) HB-12454 Jan. 13, 1998
4H6.17.8 (anti-DR4) HB-12455 Jan. 13, 1998
[0396] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of this invention. All literature and patent
citations mentioned herein are expressly incorporated by
reference.
Example 1
Construction of Multivalent Antibodies
[0397] The construct used to generate a tetravalent anti-HER2
antibody, called an "Octopus antibody" (OctHER2), is illustrated in
FIG. 5 herein. The backbone of this Octopus antibody is the
recombinant, humanized monoclonal antibody 4D5 variant 8 (rhuMAb
4D5-8) (U.S. Pat. No. 5,821,337, Carter et al., expressly
incorporated herein by reference). The heavy chain of rhuMAb 4D5-8
was subcloned into the pRK5 vector (EP 307,247, published Mar. 15,
1989). The VH-CH1 region of the heavy chain was removed by
mutagenesis, and three unique restriction sites (BamHI; NheI;
BspEI) were inserted. These sites were incorporated into PCR
primers designed to amplify the VH-CH1 region from different
antibodies. The resulting fragments were subcloned into the vector
to create the Octopus heavy chain. Co-expression of the Octopus
heavy chain with the appropriate light chain in a pRK5 vector in
mammalian cell transfections results in the completed Octopus
antibody (FIG. 4).
[0398] Octopus constructs containing flexible linkers inserted
between the tandem Fd regions were are also engineered. Through
mutagenesis, DNA encoding either "gly-ser" (flex 1 linker) or
"gly-ser-gly-ser" (SEQ ID NO:10) (flex 2 linker) was inserted
between the DNA encoding the VH-CH1 regions of the heavy chain.
Example 2
Evaluation of Anti-HER2 Octopus Antibodies
[0399] OctHER2 was expressed in transiently transfected 293 cells
(Graham et al. J. Gen. Virol. 36:59-72 (1977)) and purified over a
Protein A sepharose column. The complete antibody is approximately
245 kDa, as compared to the 150 kDa molecular weight of the parent
antibody. The Octopus heavy chain is 75 kDa (without carbohydrate),
and the light chain is 30 kDa.
Antigen binding
[0400] Binding of OctHER2 to antigen, HER2 extracellular domain
(HER2ECD), was analyzed using a HER2ELISA assay (Sias et al. J.
Immunol. Methods 132:73-80 (1990)). Ninety-six well plates were
coated with the HER2 extracellular domain (ECD) (WO90/14357), and
incubated with different dilutions of anti-HER2 antibodies. After
washing to remove unbound antibody, a secondary
peroxidase-conjugated antibody was then added to detect the
anti-HER2 antibody bound to the ECD. The appropriate substrate was
then added, and the wells were visualized and then quantitated on a
plate reader at 562 nm.
[0401] The ELISA results for OctHER2, bivalent human IgG1 anti-HER2
antibody rhuMAb 4D5-8 expressed by 293 cells, or bivalent anti-HER2
antibody HERCEPTIN.RTM. (commercially available from Genentech,
Inc., South San Francisco, USA), are shown in FIGS. 6A-C. OctHER2
binds the HER2ECD similar to HERCEPTIN.RTM. when analyzed in an
ELISA assay. The rhuMAb 4D5-8 expressed by 293 cells binds
identically to the vialed HERCEPTIN.RTM. (produced by Chinese
Hamster Ovary (CHO) cells), indicating that 293 cells do not
substantially alter the antigen binding capability of the
antibodies.
[0402] Ultracentrifugation analysis was used to determine whether
OctHER2 was capable of binding target with all four antigen binding
sites. Different amounts of the HER2 extracellular domain (ECD)
(WO90/14357) were titrated in with the Octopus antibody, and based
upon these ratios, the average molecular weight of the complexes
was calculated assuming that the Octopus antibody either had four
fully functional binding sites, or three functional binding sites.
These theoretical values (circles, assuming OctHER2 has four
functional binding sites; and squares, assuming OctHER2 has three
functional binding sites) were compared to the actual experimental
values obtained (triangles). The experimental values depicted in
FIG. 7 more closely follow the curve representing four binding
sites, however the drift observed is an indicator that all four
sites probably do not bind with the same affinity.
Biological Function
[0403] Antiproliferation Assays: OctHER2 was compared to
HERCEPTIN.RTM. in functional assays measuring growth inhibition of
HER2 overexpressing tumor cell lines. The growth inhibition assay
described in Lewis et al. Cancer Immuno. Immunother. 37:255-263
(1993) was used. Briefly, serial dilutions of OctHER2 and
HERCEPTIN.RTM. were added to the media of plated cells which were
then allowed to continue growing for five days. After this time,
the media was removed and the cells were stained with crystal
violet and quantitated by spectrophotometry. Crystal violet is a
colorimetric dye that stains cells, thus allowing measurement of
cell growth after treatment.
[0404] In 3+ HER2 overexpressing cells (on which HERCEPTIN.RTM. is
very effective), OctHER2 was similar to slightly better at
inhibiting growth of SKBR3 cells (FIG. 8A), however was not as
effective on BT474 cells (FIG. 8B). Interestingly, OctHER2
inhibited more effectively than HERCEPTIN.RTM. a 2+ overexpressing
cell line, MDA 361 (FIG. 8B).
[0405] As shown in FIG. 9, the flexible linker Octopus constructs
(OctHER2flex1, OctHER2flex2) inhibited cell growth more effectively
than HERCEPTIN.RTM..
[0406] Internalization Assays: In order to assess the application
of the Octopus antibody for immunotoxin therapy, its
internalization capabilities were evaluated. For antibody arming or
immunotoxin therapy, a cytotoxic agent is conjugated with or fused
to the antibody and the immunotoxin thus produced binds
specifically to its cellular target; the thus-bound cell
internalizes the antibody, and catabolizes or degrades the antibody
releasing the toxin which kills the cell.
[0407] In the internalization assays performed herein, the antibody
was radioiodinated, and incubated for varying times with the cells.
This was followed by measurements of the amount of intact, unbound
antibody in the supernatant, the amount bound to the cell surface,
the amount internalized, and finally, the amount catabolized and
degraded.
[0408] The results of internalization assays performed with respect
to a 3+ overexpressing cell line (SKBR3) and a 2+ overexpressing
cell line (MDA453) (the solid lines represent 2+ HER2
overexpressors, and the dashed lines, 3+overexpressors) are
depicted in FIGS. 10A-B. These results indicate that OctHER2,
surprisingly, internalized and catabolized twice as fast as
HERCEPTIN.RTM. in both cell lines. The rapid internalization and
catabolism displayed by the Octopus antibody is ideal for an armed
antibody. In comparison to unbound HERCEPTIN.RTM., there is very
little free Octopus antibody in a 2+overexpressing cell. Once
again, these results suggest that the Octopus antibody would be an
excellent candidate for conjugating cytotoxic agents for tumor
delivery.
[0409] Electron Microscopy Autoradiography: To confirm that the
Octopus antibody was being internalized and degraded in the
appropriate vesicles, and not just nonspecifically, Electron
Microscopy (EM) autoradiography was used. The Octopus antibody was
iodinated and incubated with the cells in the same fashion as in
the internalization assays. The results depicted in FIGS. 11A-C
confirm that the Octopus antibody was being internalized into the
correct vesicles (early endosome, FIG. 11B; and lysosome, FIG.
11C). Additionally, the percentage of internalization observed with
OctHER2 and HERCEPTIN.RTM. in these assays matched with the
measurements in the internalization assays.
Example 3
Evaluation of Anti-DR5 Octopus Antibodies
[0410] DR5 a member of the TNF receptor superfamily that binds the
trimeric Apo2L/TRAIL (Apo2L). After Apo2L receptors bind Apo2L and
are clustered, death domains in the cytosolic region of the
receptors induce caspases to trigger cellular apoptosis. Two
versions of anti-DR5 Octopus constructs were made: one from 16E2,
an anti-DR5 cloned from a single-chain human Fv phage library (see
WO98/51793, expressly incorporated herein by reference); the second
anti-DR5 Octopus antibody was made from Mab 3H3.14.5 (the "3H3"
antibody; ATCC HB-12534, WO99/64461), a murine anti-DR5MAb that
induces apoptosis when it is crosslinked. Since anti-Death receptor
monoclonal antibodies may require crosslinking to trigger
apoptosis, they are candidates for the Octopus antibody construct.
The anti-DR5 Octopus antibodies were prepared by replacing the
variable domains of the OctHER2 construct described above with the
VL and VH domains from 16E2 or 3H3.
[0411] The anti-DR5 Octopus antibodies were analyzed in apoptosis
assays using either crystal violet or alamarBlue staining. Briefly,
serial dilutions of the Octopus antibody or Apo2L were added to the
media of plated cells which were then allowed to continue growing
for 24 hours. After this time, the media was either removed and the
cells were stained with crystal violet, or alamarBlue was added to
the media and incubated briefly with the cells. Crystal violet
stains the cells, whereas alamarBlue detects metabolic activity in
the culture media, thus these dyes allow for measurement of cells
that survive treatment. Staining by both colorimetric dyes, crystal
violet and alamarBlue, was quantitated by spectrophotometry.
[0412] As shown in FIGS. 12A-E, the 16E2 Octopus, surprisingly,
induces apoptosis with comparable potency to Apo2L in lung
(SK-MES-1; HOP 92) and colon (HCT116; COLO 205) tumor cell lines,
however does not cause apoptosis on normal control cell line
(HUMEC). The apoptosis induced by the 16E2 Octopus is
caspase-dependent.
[0413] The anti-DR516E2 Octopus was also effective in vivo in
inducing apoptosis and shrinking a colon tumor, human COLO205, in
athymic nude mice. As shown in FIG. 13A-D, histology slides of
tumor tissues stained with hematoxylin and eosin from mice treated
with the 16E2 Octopus or Apo2L induced similar levels of apoptotic
cells.
[0414] The 16E2 Octopus-treated mice also demonstrated significant
decrease in tumor volume, similar to that measured for the Apo2L
and two bivalent anti-DR5 mAbs, 16E2 and 3H3, as shown in FIG. 14.
Mice that did not receive any anti-DR5 antibodies or Apo2L
(Vehicle) showed dramatic increase in their tumor volume due to
uncontrolled growth.
[0415] The apoptotic activity of the material used in the mouse
studies was confirmed in an in vitro apoptotic assay in FIG. 15.
The anti-DR516E2 Octopus and the Apo2L used in the study were
compared to an Apo2L standard positive control and an anti-IgE MAb
(E25) negative control in an alamarBlue apoptosis assay.
[0416] FIG. 16 demonstrates that another anti-DR5 Octopus, 3H3
Octopus, is capable of inducing apoptosis similar to the 16E2
Octopus. Additionally, FIG. 16 shows that the apoptotic activity of
the Octopus antibody is not lot dependent, as several 16E2 Octopus
antibodies prepared on different dates retain similar function.
[0417] In FIGS. 17A and B, the apoptotic activity of both the 16E2
and 3H3 Octopus antibodies is better than Apo2L on a lung tumor
cell line, SK-MES-1 (FIG. 17A), and a T cell tumor line, Jurkat
(FIG. 17B). The anti-DR5 Octopus antibodies may be more effective
at clustering DR5 on the tumor cell surface than Apo2L.
[0418] The 16E2 Octopus was analyzed in a 2-day and 6-day screen
against the National Cancer Institute (NCI) panel of human tumor
cell lines in comparison with the Apo2L. FIGS. 18A-C depict the
2-day dose response curves showing the effects of the 16E2 Octopus
and Apo2L on the growth of several human leukemia, non-small cell
lung cancer, colon cancer, central nervous system (CNS) cancer,
melanoma, ovarian cancer, renal cancer, prostate cancer and breast
cancer tumor cell lines, while FIGS. 19A-C show dose response
curves from the 6 day screens. Comparable results were observed for
16E2 Octopus and Apo2L against most of the tumor cell lines, again
indicating that the anti-DR5 Octopus functions similar to Apo2L.
Similar inhibition of the lung and colon cancer cell lines
confirmed the previous in vitro and in vivo results from apoptosis
assays comparing 16E2 Octopus and Apo2L on cell lines of these
cancers. The ability of 16E2 Octopus to kill certain tumor cell
lines was unexpected; for example, a CNS cancer cell line, SF-295
(FIG. 19B), as well as two renal cancer cell lines, ACHN and RXF393
(FIG. 19C).
[0419] The results of the NCI tumor panel screens are depicted
quantitatively in FIGS. 20 A and B (2-day results) and FIGS. 21A
and B (6-day results) which summarize the effect of 16E2 Octopus
compared to Apo2L on growth inhibition (GI50), stasis (TGI), and
toxicity (LC50) of the treated tumor cell lines. Again, these
results suggest that 16E2 Octopus may be effective against more
types of cancer than previously observed.
Example 4
Evaluation of Anti-CD20 Octopus Antibody
[0420] In an effort to improve the potency of the chimeric
anti-CD20 antibody C2B8 (RITUXAN.RTM.; U.S. Pat. No. 5,736,137,
expressly incorporated herein by reference), one approach being
investigated is the ability of the antibody to trigger apoptosis of
tumor cells. The apoptosis assay in Koopman et al. Blood
84:1415-1420 (1994) was performed. An Octopus anti-CD20 antibody
(OctCD20) was prepared by using the C2B8 VL and VH domains in the
preparation of an anti-CD20 Octopus antibody. The OctCD20 antibody
was expressed in 293 cells and purified via Protein A sepharose
chromatograpy as described for the previous examples.
[0421] As shown in FIG. 22, RITUXAN.RTM. alone does not trigger
much apoptosis of a non-Hodgkins lymphoma B cell line, Wil-2,
unless it is crosslinked with anti-human IgG (RITUXAN.RTM.-IgG).
OctCD20, however, is capable of inducing apoptosis in Wil-2 cells
independent of crosslinking. The level of apoptosis observed with
OctCD20 is lower than that of crosslinked RITUXAN.RTM., however,
suggesting that the apoptotic activity of OctCD20 could be
improved, perhaps through the use of the flexible linkers.
Example 5
Construction of Further Multivalent Antibodies
[0422] Versions of the Octopus antibodies of Example 2 (anti-HER2),
Example 3 (anti-DR5) and Example 4 (anti-CD20) with an antibody
hing region dimerization domain (designated "Octopus F(ab').sub.2"
herein) were engineered. The anti-HER2 Octopus F(ab').sub.2
construct was engineered by replacing the Fc region of the heavy
chain cDNA with sequence encoding a leucine-zipper motif which,
when expressed as protein, dimerizes to effectively join the
Octopus Fab arms (FIG. 23C). The octopus F(ab').sub.2 can maintain
the leucine zipper motif, or that motif can, e.g., be
proteolytically removed as desired. As depicted in FIG. 24, PCR was
used to amplify the duplicate VH/CH1 domains and to insert a
restriction site onto the end of the Octopus heavy chain cDNA
(NotI) to permit in-frame subcloning into a vector (VG15)
containing a leucine-zipper motif. PCR was again utilized to add
another restriction site downstream of the heavy chain termination
codon (XhoI) to allow subcloning into the pRK vector for expression
in mammalian cells. The VH/CH1 domains of anti-DR5Mab16E2 and
anti-CD20 Mab C2B8 were substituted into the Oct F(ab)'.sub.2 heavy
chain backbone using the unique restriction sites BamHI, NheI, and
BspEI.
[0423] "POPoctopus" antibodies were created by linking together Fab
domains in tandem repeats to form linear Fab multimers. "POPoct-3"
contains three linked Fab domains (FIG. 23D), while "POPoct-4" has
four Fab repeats (FIG. 23E). Anti-HER2 (rhuMab 4D5), anti-DR5
(16E2), and anti-CD20 (C2B8) POPoct-3 constructs were generated, as
were anti-HER2 (rhuMab 4D5) and anti-DR5 (16E2) POPoct-4
constructs. POPoct-3 antibodies were engineered both with and
without flex 1 linkers.
[0424] FIG. 25 depicts the construction of the POPoct-3 heavy chain
cDNA. PCR was used to amplify the VH/CH1 domain adding a 5'-BspEI
site and a 3'-NotI site. This sequence was digested and along with
BamHI/BspEI digested Octopus heavy chain, ligated into a pRK vector
to yield an Octopus heavy chain containing sequence for three
VH/CH1 domains. The BspEI site encodes for a serine and a glycine
residue.
[0425] To engineer the POPoct-4 antibody (FIG. 26), site-directed
oligomutagenesis was used to introduce a silent mutation, resulting
in the elimination of the NheI restriction site in-between the
duplicate VH/CH1 domains on the Octopus heavy chain cDNA.
Oligomutagenesis was again employed to add a NheI restriction site
immediately downstream of the second VH/CH.sub.1 sequence. This
cDNA along with the POPoct-3 construct were digested with
BamHI/NheI restriction endonucleases, and ligated together with the
pRK vector to produce a heavy chain cDNA containing sequence for
four VH/CH1 domains.
[0426] The different Octopus heavy chains were transiently
cotransfected with the appropriate light chain cDNAs into 293
mammalian cells to express antibodies containing either three Fab
domains (POPoct-3 Fab) or four Fab domains (full-length Octopus;
Octopus F(ab)'.sub.2; POPoct-4 Fab). While native IgG Mabs and
full-length Octopus antibodies were purified over Protein A
sepharose, Octopus F(ab)'.sub.2 and POPoct-3 and -4 were purified
over Protein G sepharose columns.
[0427] The Octopus F(ab)'.sub.2 is approximately 200 kDa (FIG. 23F,
lane 4), smaller than the 240 kDa of the full-length Octopus
antibody (FIG. 23F, lane 3), but larger than the 150 kDa native IgG
Mab (FIG. 23F, lanes 1 and 2). At approximately 140 kDa (FIG. 23F,
lane 5), POPoct-3 is slightly smaller than native IgG Mab, while
POPoct-4 is slightly larger at 190 kDa. The heavy chain of the
Octopus F(ab)'.sub.2 (FIG. 23G, lane 4) is approximately the same
size as the native IgG Mab heavy chain (FIG. 23G, lanes 1 and 2) at
55 kDa. The POPoct-3 heavy chain (FIG. 23G, lane 5) is similar in
size to the full-length Octopus heavy chain (FIG. 23G, lane 3),
while at approximately 97 kDa the POPoct-4 has the largest heavy
chain
Example 6
Evaluation of Anti-HER2Multivalent Antibodies
[0428] Antiproliferation Assays OctHER F(ab)'.sub.2, POPoct-3 HER2,
OctHER2, OctHER2 flex 1, and rhuMAb 4D5 (HERCEPTIN.RTM.) were added
to the 3+HER2 over-expressing tumor cell line, BT474, at equimolar
concentrations and evaluated for their ability to inhibit cell
growth as measured by crystal violet staining. The results of these
assays are shown in FIG. 27. Although all of the antibodies induced
some cytostasis of the BT474 cells, POPoct-3HER2 and rhuMAb 4D5
showed the most efficacy and inhibited growth equivalently, while
OctHER2F(ab)'.sub.2 lost potency rapidly as its concentration
decreased. OctHER2 flex1 demonstrated a slight but consistent
improvement over OctHER2 (n=6), suggesting that improved
flexibility may result in better access of the Fab to the HER2
target.
[0429] OctHER2, OctHER2 flex-1, POPoct-3HER2, POPoct-3HER2 flex-1
and rhuMAb 4D5 (HERCEPTIN.RTM.) were also evaluated at equimolar
concentrations on another 3+HER2 over-expressing cell line, SKBR3,
in crystal violet cytostasis assays. The results of this assay are
depicted in FIG. 28. On this cell line, all Octopus constructs
tested inhibited cell growth equivalently, and better than rhuMab
4D5 (n=4). Any improvement in efficacy due to the flexible-linker
in between the Fab arms of OctHER2 or POPoct-3 was less evident on
this cell line.
[0430] Internalization Assays: POPoct-3HER2 was compared to OctHER
and HERCEPTIN.RTM. in internalization assays on two 3+HER2
over-expressing tumor cell lines, SKBR3 and BT474, to assess its
candidacy for applications in immunotoxin therapies. Although
structurally different than the full-length OctHER2 antibody,
POPoct-3HER2 was internalized and catabolized identically to
OctHER2 by both cell lines (FIGS. 29A and B) and at twice the rate
of HERCEPTIN.RTM..
Example 7
Evaluation of Anti-DR5Multivalent Antibodies
[0431] Apoptosis assays: Multivalent versions of the anti-DR516E2
MAb were evaluated in this example. Oct1DR5, OctDR5flex-1,
OctDR5F(ab)'.sub.2, POPoct-3DR5, POPoct-3DR5flex-1 and POPoct-4 DR5
were added at equimolar concentrations to the colon tumor cell line
COLO205 and analyzed in crystal violet apoptosis assays in
comparison to the 16E2 MAb (n=4). The results are shown in FIGS.
30A and B. All Octopus antibodies induced more apoptosis than the
16E2 MAb, with the order of efficacy from most potent to least:
OctDR5flex-1>OctDR5=POPoct-4 DR5=POPoct-3flex-1 DR5=POPoct-3DR5
>OctDR5F(ab)'.sub.2>16E2 MAb. OctDR5flex-1 showed increased
potency compared to OctDR5, especially at lower concentrations
(FIG. 30A), indicating that flexibility between the Fab arms
improves efficacy. POPoct-3flex-1DR5 induced equivalent levels of
apoptosis as OctHER (FIG. 30A) and showed similar efficacy to
POPoct16-3 and POPoct16-4 (FIG. 30B).
[0432] Cell signaling: Apo2L binds to the death receptors and
triggers cellular apoptosis through the caspase signaling pathway.
As shown in FIGS. 31A and B, the anti-DR5 Octopus antibodies were
shown to induce apoptosis through the same signaling pathway as
Apo2L. Oct16E2 triggered similar levels of apoptosis as APO2L on
the lung tumor cell line SK-MES-1 (FIG. 31A, dashed lines), but
after the addition of ZVAD, an inhibitor of caspase 3 and 9,
cellular apoptosis triggered by both Apo2L and Oct16E2 was
inhibited (FIG. 31B solid lines). Further evidence that the
anti-DR5 Octopus antibodies signaled through the same pathway as
Apo2L was obtained by DISC (Death Induced Signaling Complex)
analyses (FIG. 31B). BJAB cells, a B-cell lymphoma line that
expresses DR5, was incubated at two different concentrations of two
anti-DR5 Octopus antibodies, Oct16E2 and Oct3H3, for varying times.
Purification of the antibody-DR5 complexes was followed Western
blot analysis to identify the signaling molecules that copurified
with the complexes. As with Apo2L, the signaling molecules caspase
8 and FADD associated with DR5 after the receptor was bound by both
Oct16E2 and Oct3H3 (FIG. 31B).
Example 8
Evaluation of Anti-CD20 Octopus Antibody
[0433] Apoptosis assays: As shown in FIG. 22, RITUXAN.RTM. did not
efficiently trigger apoptosis in vitro on the B-cell lymphoma cell
line WIL-2 unless first crosslinked by anti-IgG antibody. OctCD20
was capable of inducing apoptosis of WIL-2 cells independent of
crosslinking, at levels higher than RITUXAN.RTM. alone, yet
slightly lower than anti-IgG-crosslinked RITUXAN.RTM.. When
crosslinked with anti-IgG antibody, OctCD20 induced more apoptosis
of the WIL-2 cells than crosslinked RITUXAN.RTM. (FIG. 32). Since
one potential explanation for the efficacy of RITUXAN.RTM. in vivo
is that the antibody is being crosslinked by either complement or
Fc.gamma.R bearing cells, this observation suggests that OctCD20
will be even more efficacious in vivo.
[0434] OctCD20 F(ab)'.sub.2, POPoct-3CD20 and POPoct-3CD20flex-1
were tested at various concentrations in apoptosis assays with
WIL-2 cells, and the optimal doses are shown in the maximum
response curves in FIG. 33. The Octopus antibodies were compared to
the anti-CD20 antibody 1F5 (Clark et al. supra), which functions
similar to RITUXAN.RTM. in that it does not induce apoptosis unless
crosslinked with anti-IgG antibody. Both Octopus antibodies tested
induced either similar (OctCD20 F(ab)'.sub.2) or higher
(POPoct-3CD20, POPoct-3CD20flex-1) levels of apoptosis than
crosslinked IF5 anti-CD20. Additionally, the Octopus antibodies
were efficacious at considerably lower concentrations than the
crosslinked anti-CD20.
[0435] When crosslinked anti-CD20 antibodies are added to the B
cell lymphoma line WIL-2S, a homotypic adhesion of the cells is
observed. This cell clumping is one indication that the cells have
been activated through CD20. The Octopus anti-CD20 antibodies
induce this same homotypic adhesion phenomenon independent of
crosslinker, and as shown in FIG. 34 with POPoct-3CD20, at much
lower concentrations than crosslinked IF5 anti-CD20.
[0436] Apoptosis induction by the various anti-CD20 antibodies was
further assessed using blood from a patient with chronic
lymphocytic leukemia (CLL). PBL's were separated out using dextran
sedimentation, washed and plated in serum-free lymphocyte medium
treated overnight with no sample, 1F5 (20 .mu.g/ml),
1F5+cross-linking mouse anti-IgG (100 .mu.g/ml), OctCD20
F(ab').sub.2 at approx 0.5 or 1.0 .mu.g/mland POPoct-3 CD20 at 0.5
.mu.g/ml.
[0437] An apoptosis assay was performed using annexin and PI
staining. The percentage of apoptotic cells were:
TABLE-US-00003 Untreated 38.5% 1F5 37.1% 1F5 X-linked with anti-IgG
25.1% POPoct-3 CD20 (0.5 .mu.g) 50.2% OctCD20 F(ab').sub.2 (0.5
.mu.g) 37.7% OctCD20 F(ab').sub.2 (1.0 .mu.g) 48.6%
[0438] The data indicate that multivalent anti-CD20 antibodies
(especially POPoct-3 CD20) enhance apoptosis in a dose-dependent
manner.
[0439] Internalization Assays: OctCD20 was also evaluated as a
candidate for immunotoxin therapy in internalization assays on
three B-cell lymphoma lines, DB, WIL-2, and Ramos, and compared to
RITUXAN.RTM.. As shown in FIG. 35, twice as much OctCD20 was
internalized by the cells as compared to RITUXAN.RTM., which was
not internalized by the cells at appreciable levels. The higher
avidity that would be expected for the multivalent antibodies due
to the increased number of binding sites is evident in the fact
that more OctCD20 remains bound to the cell surface of the cells
over time as compared to RITUXAN.RTM..
Sequence CWU 1
1
111218PRTHomo sapiens 1Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro1 5 10 15Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val 20 25 30Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys 35 40 45Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr 50 55 60Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser65 70 75Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr 80 85 90Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys 95 100 105Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr 110 115 120Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser 125 130 135Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val140 145 150Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr 155 160 165Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys 170 175 180Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 185 190 195Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 200 205 210Ser Leu Ser
Leu Ser Pro Gly Lys2152218PRTHomo sapiens 2Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro1 5 10 15Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys 35 40 45Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 50 55 60Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser65 70 75Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr 80 85 90Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys 95 100 105Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 110 115 120Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 125 130 135Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val140 145 150Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 155 160 165Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 170 175
180Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 185
190 195Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
200 205 210Ser Leu Ser Leu Ser Pro Gly Lys2153217PRTHomo sapiens
3Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro1 5 10
15Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 20 25
30Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe 35 40
45Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 50 55
60Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val65 70
75Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 80 85
90Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr 95
100 105Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
110 115 120Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu 125 130 135Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu140 145 150Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro 155 160 165Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu 170 175 180Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys 185 190 195Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser 200 205 210Leu Ser Leu Ser Pro Gly
Lys2154218PRTHomo sapiens 4Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro1 5 10 15Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 20 25 30Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Gln 35 40 45Phe Lys Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 50 55 60Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Phe Arg Val Val Ser65 70 75Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr 80 85 90Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 95 100 105Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr 110 115 120Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 125 130 135Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val140 145 150Glu Trp Glu Ser Ser Gly
Gln Pro Glu Asn Asn Tyr Asn Thr Thr 155 160 165Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 170 175 180Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser 185 190 195Cys Ser Val
Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys 200 205 210Ser Leu
Ser Leu Ser Pro Gly Lys2155218PRTHomo sapiens 5Pro Ala Pro Glu Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro1 5 10 15Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu Val Gln 35 40 45Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr 50 55 60Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser65 70 75Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 80 85 90Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 95 100 105Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 110 115 120Thr Leu Pro
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 125 130 135Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val140 145 150Glu
Trp Glx Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 155 160
165Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 170
175 180Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
185 190 195Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys 200 205 210Ser Leu Ser Leu Ser Leu Gly Lys2156215PRTMus
musculus 6Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys
Pro1 5 10 15Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys
Val 20 25 30Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser
Trp 35 40 45Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro
Arg 50 55 60Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu
Pro65 70 75Ile Met His Gln Asp Cys Leu Asn Gly Lys Glu Phe Lys Cys
Arg 80 85 90Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile
Ser 95 100 105Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr
Ile Pro 110 115 120Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser
Leu Thr Cys 125 130 135Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr
Val Glu Trp Gln140 145 150Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys
Asn Thr Gln Pro Ile 155 160 165Met Asp Thr Asp Gly Ser Tyr Phe Val
Tyr Ser Lys Leu Asn Val 170 175 180Gln Lys Ser Asn Trp Glu Ala Gly
Asn Thr Phe Thr Cys Ser Val 185 190 195Leu His Glu Gly Leu His Asn
His His Thr Glu Lys Ser Leu Ser 200 205 210His Ser Pro Gly
Lys2157218PRTMus musculus 7Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser
Val Phe Ile Phe Pro1 5 10 15Pro Lys Ile Lys Asp Val Leu Met Ile Ser
Leu Ser Pro Ile Val 20 25 30Thr Cys Val Val Val Asp Val Ser Glu Asp
Asp Pro Asp Val Gln 35 40 45Ile Ser Trp Phe Val Asn Asn Val Glu Val
His Thr Ala Gln Thr 50 55 60Gln Thr His Arg Glu Asp Tyr Asn Ser Thr
Leu Arg Val Val Ser65 70 75Ala Leu Pro Ile Gln His Gln Asp Trp Met
Ser Gly Lys Glu Phe 80 85 90Lys Cys Lys Val Asn Asn Lys Asp Leu Pro
Ala Pro Ile Glu Arg 95 100 105Thr Ile Ser Lys Pro Lys Gly Ser Val
Arg Ala Pro Gln Val Tyr 110 115 120Val Leu Pro Pro Pro Glu Glu Glu
Met Thr Lys Lys Gln Val Thr 125 130 135Leu Thr Cys Met Val Thr Asp
Phe Met Pro Glu Asp Ile Tyr Val140 145 150Glu Trp Thr Asn Asn Gly
Lys Thr Glu Leu Asn Tyr Lys Asn Thr 155 160 165Glu Pro Val Leu Asp
Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys 170 175 180Leu Arg Val Glu
Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser 185 190 195Cys Ser Val
Val His Glu Gly Leu His Asn His His Thr Thr Lys 200 205 210Ser Phe
Ser Arg Thr Pro Gly Lys2158218PRTMus musculus 8Pro Ala Pro Asn Leu
Glu Gly Gly Pro Ser Val Phe Ile Phe Pro1 5 10 15Pro Asn Ile Lys Asp
Val Leu Met Ile Ser Leu Thr Pro Lys Val 20 25 30Thr Cys Val Val Val
Asp Val Ser Glu Asp Asp Pro Asp Val Gln 35 40 45Ile Ser Trp Phe Val
Asn Asn Val Glu Val His Thr Ala Gln Thr 50 55 60Gln Thr His Arg Glu
Asp Tyr Asn Ser Thr Ile Arg Val Val Ser65 70 75His Leu Pro Ile Gln
His Gln Asp Trp Met Ser Gly Lys Glu Phe 80 85 90Lys Cys Lys Val Asn
Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg 95 100 105Thr Ile Ser Lys
Pro Lys Gly Leu Val Arg Ala Pro Gln Val Tyr 110 115 120Thr Leu Pro
Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser 125 130 135Leu Thr
Cys Leu Val Val Gly Phe Asn Pro Gly Asp Ile Ser Val140 145 150Glu
Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr 155 160
165Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys 170
175 180Leu Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser
185 190 195Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys
Lys 200 205 210Thr Ile Ser Arg Ser Pro Gly Lys2159218PRTMus
musculus 9Pro Pro Gly Asn Ile Leu Gly Gly Pro Ser Val Phe Ile Phe
Pro1 5 10 15Pro Lys Pro Lys Asp Ala Leu Met Ile Ser Leu Thr Pro Lys
Val 20 25 30Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val
His 35 40 45Val Ser Trp Phe Val Asp Asn Lys Glu Val His Thr Ala Trp
Thr 50 55 60Gln Pro Arg Glu Ala Gln Tyr Asn Ser Thr Phe Arg Val Val
Ser65 70 75Ala Leu Pro Ile Gln His Gln Asp Trp Met Arg Gly Lys Glu
Phe 80 85 90Lys Cys Lys Val Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu
Arg 95 100 105Thr Ile Ser Lys Pro Lys Gly Arg Ala Gln Thr Pro Gln
Val Tyr 110 115 120Thr Ile Pro Pro Pro Arg Glu Gln Met Ser Lys Lys
Lys Val Ser 125 130 135Leu Thr Cys Leu Val Thr Asn Phe Phe Ser Glu
Ala Ile Ser Val140 145 150Glu Trp Glu Arg Asn Gly Glu Leu Glu Gln
Asp Tyr Lys Asn Thr 155 160 165Pro Pro Ile Leu Asp Ser Asp Gly Thr
Tyr Phe Leu Tyr Ser Lys 170 175 180Leu Thr Val Asp Thr Asp Ser Trp
Leu Gln Gly Glu Ile Phe Thr 185 190 195Cys Ser Val Val His Glu Ala
Leu His Asn His His Thr Gln Lys 200 205 210Asn Leu Ser Arg Ser Pro
Gly Lys215104PRTArtificial SequenceSequence is synthesized. 10Gly
Ser Gly Ser1114PRTArtificial SequenceSequence is synthesized. 11Gly
Gly Gly Ser1
* * * * *