U.S. patent application number 14/052942 was filed with the patent office on 2014-03-20 for antibody-drug conjugates.
This patent application is currently assigned to WYETH LLC. The applicant listed for this patent is WYETH LLC. Invention is credited to John Francis DiJoseph, Hans-Peter Gerber, Kiran Manohar Khandke, Kimberly Ann Marquette, Puja Sapra, Lioudmila Gennadievna Tchistiakova.
Application Number | 20140081005 14/052942 |
Document ID | / |
Family ID | 45895455 |
Filed Date | 2014-03-20 |
United States Patent
Application |
20140081005 |
Kind Code |
A1 |
Gerber; Hans-Peter ; et
al. |
March 20, 2014 |
ANTIBODY-DRUG CONJUGATES
Abstract
Disclosed are anti-5T4 antibody drug conjugates and methods for
preparing and using the same.
Inventors: |
Gerber; Hans-Peter;
(Montclair, NJ) ; DiJoseph; John Francis;
(Woodbridge, NJ) ; Khandke; Kiran Manohar;
(Nanuet, NY) ; Marquette; Kimberly Ann;
(Somerville, MA) ; Sapra; Puja; (River Edge,
NJ) ; Tchistiakova; Lioudmila Gennadievna; (Stoneham,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WYETH LLC |
MADISON |
NJ |
US |
|
|
Assignee: |
WYETH LLC
MADISON
NJ
|
Family ID: |
45895455 |
Appl. No.: |
14/052942 |
Filed: |
October 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13616030 |
Sep 14, 2012 |
8586049 |
|
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14052942 |
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Current U.S.
Class: |
530/391.9 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 47/6851 20170801; A61K 2039/505 20130101; A61P 11/00 20180101;
C07K 16/30 20130101; C07K 2317/92 20130101; C07K 2317/732 20130101;
A61K 47/6803 20170801; A61P 15/00 20180101; A61P 35/00 20180101;
A61K 2039/545 20130101 |
Class at
Publication: |
530/391.9 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Claims
1. A process for producing an antibody-drug conjugate of the
formula: Ab-(LU-D)p, wherein: (a) Ab is an anti-5T4 antibody
comprising a VH CDR1 region as shown in SEQ ID NO: 5; a VH CDR2
region as shown in SEQ ID NO: 6; a VH CDR3 region as shown in SEQ
ID NO: 7; a VL CDR1 region as shown in SEQ ID NO: 8; a VL CDR2
region as shown in SEQ ID NO: 9; and, a VL CDR3 region as shown in
SEQ ID NO: 10; (b) LU is a linker unit selected from the group
consisting of maleimidocaproyl and maleimidocaproyl-Val-Cit; (c) D
is a Drug unit selected from the group consisting of MMAE and MMAF;
(d) p is an integer from about 1 to about 8 said process further
comprising: (e) chemically linking LU to D to form LU-D; (f)
conjugating said LU-D to the Ab of (a); and, (g) purifying the
antibody-drug conjugate.
2. The process of claim 1 wherein LU is maleimidocaproyl, D is
MMAF, and p is about 4.
3. The process of claim 1, wherein said anti-5T4 antibody comprises
a VH region of SEQ ID NO: 3 and a VL region of SEQ ID NO: 4.
4. The process of claim 3, wherein said anti-5T4 antibody comprises
a heavy chain having SEQ ID NO: 1 and a light chain having SEQ ID
NO: 2.
Description
[0001] This application claims priority from copending provisional
application No. 61/602,349 filed Feb. 23, 2012, 61/593,549 filed
Feb. 1, 2012 and 61/470,576 filed Apr. 1, 2011 the entire
disclosure of which is hereby incorporated by reference.
FIELD
[0002] The present invention generally relates to anti-5T4
antibody-drug conjugates for the treatment of cancer.
BACKGROUND
[0003] Antibody-drug conjugates (ADCs) combine the binding
specificity of monoclonal antibodies with the potency of
chemotherapeutic agents. The technology associated with the
development of monoclonal antibodies to tumor associated target
molecules, the use of more effective cytotoxic agents, and the
design of chemical linkers to covalently bind these components, has
progressed rapidly in recent years (Ducry L., et al. Bioconjugate
Chemistry, 21:5-13, 2010).
[0004] Promising ADCs such as SGN-75 (US2009/148942) and
trastuzumab-DM1 (US2009/0226465) are currently in clinical trials.
However, as other tumor associated antigens are considered for
targets, numerous challenges remain. Each monoclonal antibody must
be characterized separately, an appropriate linker designed, and a
suitable cytotoxic agent identified that retains its potency upon
delivery to tumor cells. One must consider the antigen density on
the cancer target and whether normal tissues express the target
antigen. Other considerations include whether the entire ADC is
internalized upon binding the target; whether a cytostatic or
cytotoxic drug is preferable when considering possible normal
tissue exposure and/or the type and stage of the cancer being
treated; and, whether the linker connecting the antibody to the
drug payload is a cleavable or a non-cleavable linkage.
Furthermore, the antibody to drug moiety conjugation ratio must be
sufficient without compromising the binding activity of the
antibody and/or the potency of the drug. It is evident that ADCs
are complex biologics and the challenges to develop an effective
ADC remain significant.
[0005] The human 5T4 tumor associated antigen is the target antigen
of the present invention. It has recently been shown that the 5T4
antigen is expressed in high levels on certain highly tumorigenic
cells, also called tumor-initiating cells (WO2010/111659).
Tumor-initiating cells show resistance to standard therapies and
are believed to be responsible for tumor recurrence and metastasis
and therefore present yet another obstacle for ADC development.
[0006] The novel anti-5T4 ADCs of the present invention overcome
the challenges associated with ADC technology and provide highly
specific and potent ADCs that bind to tumor cells expressing the
5T4 antigen and deliver sufficient cytotoxic drug to the cells,
thus providing an innovative and effective treatment for
cancer.
SUMMARY
[0007] In one embodiment, an antibody-drug conjugate of the present
invention has the formula: Ab-(LU-D)p or a pharmaceutically
acceptable salt thereof wherein, Ab is an anti-5T4 antibody or
antigen binding portion thereof, comprising a heavy chain variable
region having a VH CDR1 region as shown in SEQ ID NO: 5, a VH CDR2
region as shown in SEQ ID NO: 6, and a VH CDR3 region as shown in
SEQ ID NO: 7; LU is a linker unit selected from the group
consisting of maleimidocaproyl and maleimidocaproyl-Val-Cit-PABA; p
is an integer from about 1 to about 8; and D is a Drug unit
selected from the group consisting of MMAE, MMAF, and MMAD.
[0008] The present invention further provides anti-5T4
antibody-drug conjugates wherein said anti-5T4 antibody or antigen
binding portion thereof, comprises a heavy chain variable region
having (a) a VH CDR1 region as shown in SEQ ID NO: 5, (b) a VH CDR2
region as shown in SEQ ID NO: 6, and (c) a VH CDR3 region as shown
in SEQ ID NO: 7.
[0009] The present invention further provides an anti-5T4
antibody-drug conjugate wherein said anti-5T4 antibody or antigen
binding portion thereof, comprises a light chain variable region
having (a) a VL CDR1 region as shown in SEQ ID NO: 8, (b) a VL CDR2
region as shown in SEQ ID NO: 9, and (c) a VL CDR3 region as shown
in SEQ ID NO: 10.
[0010] The present invention further provides an anti-5T4
antibody-drug conjugate wherein said anti-5T4 antibody or antigen
binding portion thereof, further comprises a heavy chain variable
region having (a) a VH CDR1 region as shown in SEQ ID NO: 5, (b) a
VH CDR2 region as shown in SEQ ID NO: 6, and (c) a VH CDR3 region
as shown in SEQ ID NO: 7 and a light chain variable region having
(a) a VL CDR1 region as shown in SEQ ID NO: 8, (b) a VL CDR2 region
as shown in SEQ ID NO: 9, and (c) a VL CDR3 region as shown in SEQ
ID NO: 10.
[0011] The present invention further provides an anti-5T4
antibody-drug conjugate wherein said anti-5T4 antibody or antigen
binding portion thereof, comprises the VH region of SEQ ID NO: 3
and the VL region of SEQ ID NO: 4.
[0012] The present invention further provides an anti-5T4
antibody-drug conjugate wherein said anti-5T4 antibody consists of
a heavy chain having SEQ ID NO: 1 and a light chain having SEQ ID
NO: 2.
[0013] The present invention further provides an anti-5T4
antibody-drug conjugate wherein:
(a) said anti-5T4 antibody consists of a heavy chain having SEQ ID
NO:1 and a light chain having SEQ ID NO: 2, (b) said LU is
maleimidocaproyl, (c) said Drug is MMAF, and (d) p is an integer of
about 4.
[0014] The present invention further provides an anti-5T4
antibody-drug conjugate wherein:
(a) said anti-5T4 antibody consists of a heavy chain having SEQ ID
NO:1 and a light chain having SEQ ID NO: 2, (b) said LU is
maleimidocaproyl-Val-Cit-PABA, (c) said Drug is MMAE, and (d) p is
an integer of about 4.
[0015] The present invention further provides an anti-5T4
antibody-drug conjugate wherein:
(a) said anti-5T4 antibody consists of a heavy chain having SEQ ID
NO:1 and a light chain having SEQ ID NO: 2, (b) said LU is
maleimidocaproyl-Val-Cit-PABA, (c) said Drug is MMAD, and (d) p is
an integer from about 1 to about 8.
[0016] The present invention further provides an anti-5T4
antibody-drug conjugate wherein: (a) said anti-5T4 antibody
consists of a heavy chain having SEQ ID NO:15 and a light chain
having SEQ ID NO: 2, (b) said LU is maleimidocaproyl-Val-Cit-PABA,
(c) said Drug is MMAE, and (d) p is an integer of about 1 to about
8.
[0017] The present invention provides an anti-5T4 antibody-drug
conjugate wherein said antibody recognizes an epitope on human 5T4
antigen wherein said epitope comprises amino acid residues 173-258
and 282-361 of the amino acid sequence of SEQ ID NO: 11.
[0018] The present invention provides a pharmaceutical composition
comprising an antibody-drug conjugate indicated above and a
pharmaceutically acceptable carrier.
[0019] The present invention further provides a method of treating
a 5T4-positive cancer in a patient in need thereof, comprising
administering to said patient an antibody-drug conjugate indicated
above.
[0020] The present invention further provides a method of treating
a 5T4-positive cancer wherein said cancer is selected from the
group consisting of carcinomas of the bladder, breast, cervix,
colon, endometrium, kidney, lung, esophagus, ovary, prostate,
pancreas, liver, skin, stomach, and testes.
[0021] More preferably, the present invention provides a method of
treating a 5T4-positive cancer wherein said cancer is selected from
the group consisting of colorectal, breast, pancreatic, and
non-small cell lung carcinomas.
[0022] The invention further provides an antibody-drug conjugate
indicated above for use in therapy.
[0023] The invention further provides the use of an antibody-drug
conjugate indicated above for the manufacture of a medicament.
[0024] The invention further provides the use indicated above,
wherein said use is for the treatment of a 5T4-positive cancer and
wherein said cancer is selected from the group consisting of
carcinomas of the bladder, breast, cervix, endometrium, kidney,
lung, esophagus, ovary, prostate, pancreas, skin, stomach, and
testes.
[0025] More preferably, the invention further provides the use
indicated above, wherein said use is for the treatment of a
5T4-positive cancer wherein said cancer is selected from the group
consisting of colorectal, breast, pancreatic, and non-small cell
lung carcinomas.
[0026] The invention further provides a nucleic acid that encodes
an anti-5T4 antibody, a vector comprising said nucleic acid, and a
host cell comprising said vector.
[0027] The invention further provides a process for producing an
anti-5T4 antibody comprising cultivating the host cell comprising
the above mentioned vector and recovering the antibody from the
cell culture.
[0028] The invention further provides a process for producing an
anti-5T4 antibody-drug conjugate comprising: (a) taking the
antibody recovered from the cell culture, (b) chemically linking
said antibody via a linker unit selected from the group consisting
of maleimidocaproyl or maleimidocaproyl-Val-Cit to a Drug unit
selected from the group consisting of MMAE, MMAD, or MMAF, and (c)
purifying the antibody-drug conjugate.
DETAILED DESCRIPTION
[0029] The present invention provides anti-5T4 antibody-drug
conjugates for the treatment of cancer. In order that the present
invention is more readily understood, certain terms are first
defined.
[0030] All amino acid abbreviations used in this disclosure are
those accepted by the United States Patent and Trademark Office as
set forth in 37 C.F.R. .sctn.1.822 (B)(I).
[0031] 5T4 refers to the 5T4 oncofetal antigen, a 72 kDa highly
glycosylated transmenbrance glycoprotein comprising a 42 kDa
non-glycosylated core (see U.S. Pat. No. 5,869,053). Human 5T4 is
expressed in numerous cancer types, including carcinomas of the
bladder, breast, cervix, colon, endometrium, kidney, lung,
esophagus, ovary, prostate, pancreas, liver, skin, stomach, and
testes. Highly tumorigenic cells, also called cancer stem cells or
tumor-initiating cells have been shown to have high levels of 5T4
expression (WO2010/111659). Anti-5T4 antibodies of the invention
include antibodies that specifically bind the human 5T4 antigen
(see US 2007/0231333).
[0032] An "antibody" is an immunoglobulin molecule capable of
specific binding to a target, such as a carbohydrate,
polynucleotide, lipid, polypeptide, etc., through at least one
antigen recognition site, located in the variable region of the
immunoglobulin molecule. As used herein, the term "antibody"
encompasses not only intact polyclonal or monoclonal antibodies,
but also any antigen binding fragment (i.e., "antigen-binding
portion") or single chain thereof, fusion proteins comprising an
antibody, and any other modified configuration of the
immunoglobulin molecule that comprises an antigen recognition site
including, for example without limitation, Fab, Fab', F(ab').sub.2,
an Fd fragment consisting of the VH and CH1 domains, an Fv fragment
consisting of the VL and VH domains of a single arm of an antibody,
an isolated complementarity determining region (CDR), scFv, single
domain antibodies (e.g., shark and camelid antibodies), maxibodies,
minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR
and bis-scFv.
[0033] An antibody includes an antibody of any class, such as IgG,
IgA, or IgM (or sub-class thereof), and the antibody need not be of
any particular class. Depending on the antibody amino acid sequence
of the constant region of its heavy chains, immunoglobulins can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG1,
IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant regions
that correspond to the different classes of immunoglobulins are
called alpha, delta, epsilon, gamma, and mu, respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0034] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. As known in
the art, the variable regions of the heavy and light chain each
consist of four framework regions (FRs) connected by three
complementarity determining regions (CDRs) also known as
hypervariable regions, contribute to the formation of the antigen
binding site of antibodies. If variants of a subject variable
region are desired, particularly with substitution in amino acid
residues outside of a CDR region (i.e., in the framework region),
appropriate amino acid substitution, preferably, conservative amino
acid substitution, can be identified by comparing the subject
variable region to the variable regions of other antibodies which
contain CDR1 and CDR2 sequences in the same canonincal class as the
subject variable region (Chothia and Lesk, J Mol Biol 196(4):
901-917, 1987). When choosing FR to flank subject CDRs, e.g., when
humanizing or optimizing an antibody, FRs from antibodies which
contain CDR1 and CDR2 sequences in the same canonical class are
preferred.
[0035] A "CDR" of a variable domain are amino acid residues within
the variable region that are identified in accordance with the
definitions of the Kabat, Chothia, the cumulation of both Kabat and
Chothia, AbM, contact, and/or conformational definitions or any
method of CDR determination well known in the art. Antibody CDRs
may be identified as the hypervariable regions originally defined
by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
NIH, Washington, D.C. The positions of the CDRs may also be
identified as the structural loop structures originally described
by Chothia and others. See, e.g., Chothia et al., 1989, Nature
342:877-883. Other approaches to CDR identification include the
"AbM definition," which is a compromise between Kabat and Chothia
and is derived using Oxford Molecular's AbM antibody modeling
software (now Accelrys.RTM.), or the "contact definition" of CDRs
based on observed antigen contacts, set forth in MacCallum et al.,
1996, J. Mol. Biol., 262:732-745. In another approach, referred to
herein as the "conformational definition" of CDRs, the positions of
the CDRs may be identified as the residues that make enthalpic
contributions to antigen binding. See, e.g., Makabe et al., 2008,
Journal of Biological Chemistry, 283:1156-1166. Still other CDR
boundary definitions may not strictly follow one of the above
approaches, but will nonetheless overlap with at least a portion of
the Kabat CDRs, although they may be shortened or lengthened in
light of prediction or experimental findings that particular
residues or groups of residues or even entire CDRs do not
significantly impact antigen binding. As used herein, a CDR may
refer to CDRs defined by any approach known in the art, including
combinations of approaches. The methods used herein may utilize
CDRs defined according to any of these approaches. For any given
embodiment containing more than one CDR, the CDRs may be defined in
accordance with any of Kabat, Chothia, extended, AbM, contact,
and/or conformational definitions.
[0036] The term "monoclonal antibody" (Mab) refers to an antibody
that is derived from a single copy or done, including e.g., any
eukaryotic, prokaryotic, or phage done, and not the method by which
it is produced. Preferably, a monoclonal antibody of the invention
exists in a homogeneous or substantially homogeneous
population.
[0037] "Humanized" antibody refers to forms of non-human (e.g.
murine) antibodies that are chimeric immunoglobulins,
immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab',
F(ab').sub.2 or other antigen-binding subsequences of antibodies)
that contain minimal sequence derived from non-human
immunoglobulin. Preferably, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, or rabbit having the desired
specificity, affinity, and capacity.
[0038] The term "chimeric antibody" is intended to refer to
antibodies in which the variable region sequences are derived from
one species and the constant region sequences are derived from
another species, such as an antibody in which the variable region
sequences are derived from a mouse antibody and the constant region
sequences are derived from a human antibody.
[0039] Antibodies of the invention can be produced using techniques
well known in the art, e.g., recombinant technologies, phage
display technologies, synthetic technologies or combinations of
such technologies or other technologies readily known in the art
(see, for example, Jayasena, S. D., Olin. Chem., 45: 1628-50 (1999)
and FeHouse, F. A., et al, J. Mol. Biol., 373(4):924-40
(2007)).
[0040] Tables 1 and 2 below depict preferred CDRs for the
antibodies of the present invention.
TABLE-US-00001 TABLE 1 Antibody LCDR1 LCDR2 LCDR3 A1 KASQSVSNDVA
FATNRYT QQDYSSPWT SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 A3
KASQDVDTAVA WASTRLT QQYSSYPYT SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID
NO: 19
TABLE-US-00002 TABLE 2 Anti- body HCDR1 HCDR2 HCDR3 A1 NFGMN
WINTNTGEPRYAEEFKG DWDGAYFFDY SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7
A1- GYTFTNFGMN WINTNTGEPRYAEEFKG DWDGAYFFDY IgG4 SEQ ID NO: 14 SEQ
ID NO: 6 SEQ ID NO: 7 A3 TYAMN RIRSKSNNYATYYADSVKD QWDYDVRAMNY SEQ
ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24
[0041] The present invention includes an antibody or antigen
binding portion thereof, that comprises:
[0042] a) a light chain variable region comprising: [0043] i) a
LCDR1 having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 8 and 17; [0044] ii) a LCDR2 having an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 9 and 18; and [0045] iii) a LCDR3 having an amino acid
sequence selected from the group consisting of SEQ ID NOs: 10 and
19; and
[0046] b) a heavy chain variable region comprising: [0047] i) a
HCDR1 having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 5 and 22; [0048] ii) a HCDR2 having an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 6 and 23; and [0049] iii) a LCDR1 having an amino acid
sequence selected from the group consisting of SEQ ID NOs: 7 and
24.
[0050] A preferred antibody or antigen binding portion thereof, of
the invention comprises:
[0051] a) a LCVR comprising: a LCDR1 of SEQ ID NO: 8, a LCDR2 of
SEQ ID NO: 9, and a LCDR3 of SEQ ID NO: 10; and
[0052] b) a HCVR comprising: a HCDR1 of SEQ ID NO: 5, a HCDR2 of
SEQ ID NO: 6, and a HCDR3 of SEQ ID NO: 7.
[0053] Preferred monoclonal antibodies of the invention are
referred to herein as A1 (a humanized anti-5T4 IgG1 antibody);
A1-IgG4 (a humanized anti-5T4 IgG4 antibody); A3 (a mouse/human
chimeric antibody); and A3hu (a humanized anti-5T4 IgG1 antibody).
The SEQ ID NOs of the amino acid sequences encoding Mabs A1,A1-IgG4
and A3 are provided in Table 3 below:
TABLE-US-00003 TABLE 3 Mab LC HC LCVR LCDR1 LCDR2 LCDR3 HCVR HCDR1
HCDR2 HCDR3 A1 2 1 4 8 9 10 3 5 6 7 A1-IgG4 2 12 4 8 9 10 13 5 6 7
A3 2 15 21 22 23 24 16 17 18 19 A3hu 30 25 31 32 33 34 26 27 28
29
[0054] The phrases "an antibody recognizing an antigen" and "an
antibody specific for an antigen" are used interchangeably herein
with the term "an antibody which binds specifically to an
antigen."
[0055] Anti-5T4 Antibody-Drug conjugate refers to an anti-5T4
antibody or antigen binding portion thereof, as described herein
linked to a cytotoxic drug moiety (D) via a linker unit molecule
(LU).
[0056] Linker Unit (LU): LU describes the direct or indirect
linkage of the antibody to the drug. Attachment of a linker to a
mAb can be accomplished in a variety of ways, such as through
surface lysines, reductive-coupling to oxidized carbohydrates, and
through cysteine residues liberated by reducing interchain
disulfide linkages. A variety of ADC linkage systems are known in
the art, including hydrazone-, disulfide- and peptide-based
linkages.
[0057] Drug (D): A drug is any substance having biological or
detectable activity, for example, therapeutic agents, detectable
labels, binding agents, etc., and prodrugs, which are metabolized
to an active agent in vivo. The terms drug and payload are used
interchangeably. In some embodiments, the Drug is an auristatin,
such as auristatin E (also known in the art as a derivative of
dolastatin-10) or a derivative thereof. The auristatin can be, for
example, an ester formed between auristatin E and a keto acid. For
example, auristatin E can be reacted with paraacetyl benzoic acid
or benzoylvaleric acid to produce AEB and AEVB, respectively. Other
typical auristatins include AFP, MMAF, and MMAE. The synthesis and
structure of exemplary auristatins are described in U.S. Pat. Nos.
6,884,869, 7,098,308, 7,256,257, 7,423,116, 7,498,298 and
7,745,394, each of which is incorporated by reference herein in its
entirety and for all purposes.
[0058] Auristatins have been shown to interfere with microtubule
dynamics and nuclear and cellular division and have anticancer
activity. Auristatins of the present invention bind tubulin and can
exert a cytotoxic or cytostatic effect on a 5T4 expressing cell or
cell line. There are a number of different assays, known in the
art, that can be used for determining whether an auristatin or
resultant antibody-drug conjugate exerts a cytostatic or cytotoxic
effect on a desired cell or cell line. Methods for determining
whether a compound binds tubulin are known in the art. See, for
example, Muller et al., Anal. Chem. 2006, 78, 4390-4397; Hamel et
al., Molecular Pharmacology, 1995 47: 965-976; and Hamel et al.,
The Journal of Biological Chemistry, 1990 265:28, 17141-17149.
[0059] Examples of drugs or payloads are selected from the group
consisting of DM1 (maytansine,
N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- or
N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD
(6-maleimidocaproyl-monomethylauristatin-D or
N-methyl-L-valyl-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-meth-
yl-3-oxo-3-[[(1S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidin-
yl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valinamide),
mc-MMAF (maleimidocaproyl-monomethylauristatin F or
N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valyl--
L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl-(.alpha.R,.-
beta.R,2S)-.beta.-methoxy-.alpha.-methyl-2-pyrrolidinepropanoyl-L-phenylal-
anine) and mc-Val-Cit-PABA-MMAE
(6-maleimidocaproyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethylauristat-
in E or
N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-
-valyl-N-5-(aminocarbonyl)-L-ornithyl]amino]phenyl]methoxy]carbonyl]-N-met-
hyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-
-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-meth-
oxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide). DM1
is a derivative of the tubulin inhibitor maytansine while MMAD,
MMAE, and MMAF are auristatin derivatives. The preferred payloads
of the present invention are selected from the group consisting of
mc-MMAF and mc-Val-Cit-PABA-MMAE.
[0060] The term "epitope" refers to that portion of a molecule
capable of being recognized by and bound by an antibody at one or
more of the antibody's antigen-binding regions. Epitopes often
consist of a chemically active surface grouping of molecules such
as amino acids or sugar side chains and have specific
three-dimensional structural characteristics as well as specific
charge characteristics. The term "antigenic epitope" as used
herein, is defined as a portion of a polypeptide to which an
antibody can specifically bind as determined by any method well
known in the art, for example, by conventional immunoassays. A
"nonlinear epitope" or "conformational epitope" comprises
noncontiguous polypeptides (or amino acids) within the antigenic
protein to which an antibody specific to the epitope binds.
[0061] The term "binding affinity (K.sub.D)" as used herein, is
intended to refer to the dissociation rate of a particular
antigen-antibody interaction. The K.sub.D is the ratio of the rate
of dissociation, also called the "off-rate (k.sub.off)", to the
association rate, or "on-rate (k.sub.on)", Thus, K.sub.D equals
k.sub.off/k.sub.on and is expressed as a molar concentration (M).
It follows that the smaller the K.sub.D, the stronger the affinity
of binding. Therefore, a K.sub.D of 1 .mu.M indicates weak binding
affinity compared to a K.sub.D of 1 nM. K.sub.D values for
antibodies can be determined using methods well established in the
art. One method for determining the K.sub.D of an antibody is by
using surface plasmon resonance (SPR), typically using a biosensor
system such as a Biacore.RTM. system.
[0062] The term "specifically binds" as used herein in reference to
the binding between an antibody and a 5T4 antigen and the antibody
binds the 5T4 antigen with a K.sub.D less than about 30 nM as
determined by SPR at 25.degree. C.
[0063] Pharmaceutically acceptable salt as used herein refers to
pharmaceutically acceptable organic or inorganic salts of a
molecule or macromolecule.
[0064] The term "potency" is a measurement of biological activity
and may be designated as IC.sub.50, or effective concentration of
antibody needed to inhibit 50% of growth of a 5T4 positive cell
line as described in Example 3. Alternatively, potency may refer to
anti-tumor activity as determined in an in vivo tumor xenograph
model as shown in Example 4.
[0065] The terms "polynucleotide" or "nucleic acid molecule", as
used herein, are intended to include DNA molecules and RNA
molecules. A nucleic acid molecule may be single-stranded or
double-stranded, but preferably is double-stranded DNA.
[0066] The polynucleotides that encode the antibodies of the
present invention may include the following: only the coding
sequence for the variant, the coding sequence for the variant and
additional coding sequences such as a functional polypeptide, or a
signal or secretory sequence or a pro-protein sequence; the coding
sequence for the antibody and non-coding sequence, such as introns
or non-coding sequence 5' and/or 3' of the coding sequence for the
antibody. The term `polynucleotide encoding an antibody"
encompasses a polynucleotide which includes additional coding
sequence for the variant but also a polynucleotide which includes
additional coding and/or non-coding sequence. It is known in the
art that a polynucleotide sequence that is optimized for a specific
host cell/expression system can readily be obtained from the amino
acid sequence of the desired protein (see GENEART.RTM. AG,
Regensburg, Germany).
[0067] The polynucleotides encoding the antibodies of the present
invention will typically include an expression control
polynucleotide sequence operably linked to the antibody coding
sequences, including naturally-associated or heterologous promoter
regions known in the art. Preferably, the expression control
sequences will be eukaryotic promoter systems in vectors capable of
transforming or transfecting eukaryotic host cells, but control
sequences for prokaryotic hosts may also be used. Once the vector
has been incorporated into the appropriate host cell line, the host
cell is propagated under conditions suitable for expressing the
nucleotide sequences, and, as desired, for the collection and
purification of the antibodies. Preferred eukaryotic cell lines
include the CHO cell lines, various COS cell lines, HeLa cells,
myeloma cell lines, transformed B-cells, or human embryonic kidney
cell lines. The most preferred host cell is a CHO cell line.
[0068] The present invention encompasses antibodies or
antigen-binding portions thereof that bind to a specific epitope on
the 5T4 antigen. The epitope identified is a nonlinear or
conformational epitope comprising a first contact with the human
5T4 antigen (SEQ ID NO: 11) between amino acid residues 173 and 252
and comprising a second contact between amino acid residues 276 and
355 (see Example 7). Thus, the CDRs and heavy and light chain
variable regions described herein are used to make full-length
antibodies as well as functional fragments and analogs that
maintain the binding affinity of the protein employing the CDRs
specific for the above mentioned epitope of the 5T4 antigen.
[0069] The binding affinity of antibodies of the present invention
is determined using SPR (Example 6). In these experiments the 5T4
antigens are immobilized at low densities onto a BIAcore.RTM. chip
and antibodies are flowed past. Build up of mass at the surface of
the chip is measured. This analytical method allows the
determination in real time of both on and off rates to obtain
affinity (K.sub.D) for binding. The humanized antibodies of the
present invention have a K.sub.D of between about 0.30 and about 30
nM; about 0.30 and about 20 nM; about 0.30 and about 10 nM; about
0.5 and about 7 nM; about 1.0 and about 5 nM; and about 1.0 and
about 3 nM.
Conjugation of Drugs to an Antibody
[0070] The drug has, or is modified to include, a group reactive
with a conjugation point on the antibody. For example, a drug can
be attached by alkylation (e.g., at the epsilon-amino group lysines
or the N-terminus of antibodies), reductive amination of oxidized
carbohydrate, transesterification between hydroxyl and carboxyl
groups, amidation at amino groups or carboxyl groups, and
conjugation to thiols. In some embodiments, the number of drug
moieties, p, conjugated per antibody molecule ranges from an
average of 1 to 8; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to
2. In some embodiments, p ranges from an average of 2 to 8, 2 to 7,
2 to 6, 2 to 5, 2 to 4 or 2 to 3. In other embodiments, p is an
average of 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, p ranges
from an average of about 1 to about 8; about 1 to about 7, about 1
to about 6, about 1 to about 5, about 1 to about 4, about 1 to
about 3, or about 1 to about 2. In some embodiments, p ranges from
about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2
to about 5, about 2 to about 4 or about 2 to about 3. For examples
of chemistries that can be used for conjugation, see, e.g., Current
Protocols in Protein Science (John Wiley & Sons, Inc.), Chapter
15 (Chemical Modifications of Proteins) (the disclosure of which is
incorporated by reference herein in its entirety.)
[0071] For example, when chemical activation of the protein results
in formation of free thiol groups, the protein may be conjugated
with a sulfhydryl reactive agent. In one aspect, the agent is one
which is substantially specific for free thiol groups. Such agents
include, for example, malemide, haloacetamides (e.g., iodo, bromo
or chloro), haloesters (e.g., iodo, bromo or chloro), halomethyl
ketones (e.g., iodo, bromo or chloro), benzylic halides (e.g.,
iodide, bromide or chloride), vinyl sulfone and pyridylthio.
[0072] Linkers
[0073] The drug can be linked to an antibody by a linker. Suitable
linkers include, for example, cleavable and non-cleavable linkers.
A cleavable linker is typically susceptible to cleavage under
intracellular conditions. Suitable cleavable linkers include, for
example, a peptide linker cleavable by an intracellular protease,
such as lysosomal protease or an endosomal protease. In exemplary
embodiments, the linker can be a dipeptide linker, such as a
valine-citrulline (val-cit), a phenylalanine-lysine (phe-lys)
linker, or
maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl
(mc-Val-Cit-PABA) linker. Another linker is
Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate
(smcc). Sulfo-smcc conjugation occurs via a maleimide group which
reacts with sulfhydryls (thiols, --SH), while its Sulfo-NHS ester
is reactive toward primary amines (as found in Lysine and the
protein or peptide N-terminus). Yet another linker is
maleimidocaproyl (mc). Other suitable linkers include linkers
hydrolyzable at a specific pH or a pH range, such as a hydrazone
linker. Additional suitable cleavable linkers include disulfide
linkers. The linker may be covalently bound to the antibody to such
an extent that the antibody must be degraded intracellularly in
order for the drug to be released e.g. the mc linker and the
like.
[0074] A linker can include a group for linkage to the antibody.
For example, linker can include an amino, hydroxyl, carboxyl or
sulfhydryl reactive groups (e.g., malemide, haloacetamides (e.g.,
iodo, bromo or chloro), haloesters (e.g., iodo, bromo or chloro),
halomethyl ketones (e.g., iodo, bromo or chloro), benzylic halides
(e.g., iodide, bromide or chloride), vinyl sulfone and
pyridylthio). See generally Wong, Chemistry of Protein Conjugation
and Cross-linking; CRC Press, Inc., Boca Raton, 1991.
[0075] Immunotherapy
[0076] For immunotherapy, an antibody can be conjugated to a
suitable drug, such as a cytotoxic or cytostatic agent, an
immunosuppressive agent, a radioisotope, a toxin, or the like. The
conjugate can be used for inhibiting the multiplication of a tumor
cell or cancer cell, causing apoptosis in a tumor or cancer cell,
or for treating cancer in a patient. The conjugate can be used
accordingly in a variety of settings for the treatment of animal
cancers. The conjugate can be used to deliver a drug to a tumor
cell or cancer cell. Without being bound by theory, in some
embodiments, the conjugate binds to or associates with a
cancer-cell or a tumor-associated antigen, and the conjugate and/or
drug can be taken up inside a tumor cell or cancer cell through
receptor-mediated endocytosis. The antigen can be attached to a
tumor cell or cancer cell or can be an extracellular matrix protein
associated with the tumor cell or cancer cell. Once inside the
cell, one or more specific peptide sequences within the conjugate
(e.g., in a linker) are hydrolytically cleaved by one or more
tumor-cell or cancer-cell-associated proteases, resulting in
release of the drug. The released drug is then free to migrate
within the cell and induce cytotoxic or cytostatic or other
activities. In some embodiments, the drug is cleaved from the
antibody outside the tumor cell or cancer cell, and the drug
subsequently penetrates the cell, or acts at the cell surface.
[0077] Therapy for Cancer
[0078] As discussed above, cancers, including, but not limited to,
a tumor, metastasis, or other disease or disorder characterized by
uncontrolled cell growth, can be treated or prevented by
administration of a protein-drug conjugate.
[0079] In other embodiments, methods for treating or preventing
cancer are provided, including administering to a patient in need
thereof an effective amount of a conjugate and a chemotherapeutic
agent. In some embodiments, the chemotherapeutic agent is that with
which treatment of the cancer has not been found to be refractory.
In some embodiments, the chemotherapeutic agent is that with which
the treatment of cancer has been found to be refractory. The
conjugate can be administered to a patient that has also undergone
a treatment, such as surgery for treatment for the cancer. In
another embodiment, the additional method of treatment is radiation
therapy.
[0080] Multi-Drug Therapy for Cancer
[0081] Methods for treating cancer include administering to a
patient in need thereof an effective amount of an antibody-drug
conjugate and another therapeutic agent that is an anti-cancer
agent. Suitable anticancer agents include, but are not limited to,
methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine,
hydroxyurea, cytarabine, cyclophosphamide, ifosfamide,
nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine,
procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide,
5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin,
doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin,
mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine,
paclitaxel, calicheamicin, and docetaxel.
[0082] The ADCs of the present invention can be in the form of a
pharmaceutical composition for administration that are formulated
to be appropriate for the selected mode of administration, and
pharmaceutically acceptable diluent or excipients, such as buffers,
surfactants, preservatives, solubilizing agents, isotonicity
agents, stabilizing agents, carriers, and the like. Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 18.sup.th
ed., 1995, incorporated herein by reference, provides a compendium
of formulation techniques as are generally known to
practitioners.
[0083] These pharmaceutical compositions may be administered by any
means known in the art that achieve the generally intended purpose
to treat cancer. The preferred route of administration is
parenteral, defined herein as referring to modes of administration
that include but not limited to intravenous, intramuscular,
intraperitoneal, subcutaneous, and intraarticular injection and
infusion. The dosage administered will be dependent upon the age,
health, and weight of the recipient, kind of concurrent treatment,
if any, frequency of treatment, and the nature of the effect
desired.
[0084] Compositions within the scope of the invention include all
compositions wherein an ADC is present in an amount that is
effective to achieve the desired medical effect for treating
cancer. While individual needs may vary from one patient to
another, the determination of the optimal ranges of effective
amounts of all of the components is within the ability of the
clinician of ordinary skill.
Example 1
Preparation of an anti-5T4 ADC
[0085] 5T4-A1 antibody drug conjugate (ADC) is prepared via partial
reduction of the mAb with tris(2-carboxyethyl)phosphine (TCEP)
followed by reaction of reduced Cys residues with the desired
maleimide terminated linker-payload. In particular, 5T4-A1 mAb is
partially reduced via addition of 2.8 molar excess of
tris(2-carboxyethyl)phosphine (TCEP) in 100 mM HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer), pH 7.0
and 1 mM diethylenetriaminepentaacetic acid (DTPA) for 2 h at
37.degree. C. The desired linker-payload is then added to the
reaction mixture at a linker-payload/mAb-thiol molar ratio of 5.5
(maleimidocapronic-monomethylauristatin F [mc-MMAF]) or 8
(maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl-monomethylau-
ristatin E [mc-Val-Cit-PABA-MMAE]) and reacted for an additional 1
h at 25.degree. C. in the presence of 15% v/v of dimethylacetamide
(DMA). After the 1 h incubation period, N-ethylmaleimide (4.5 fold
excess for mc-MMAF and 2 fold excess for mc-Val-Cit-PABA-MMAE) is
added to cap the unreacted thiols and is allowed to react for 15
minutes followed by addition of 6 fold excess L-Cys to quench any
unreacted linker-payload. The reaction mixture is dialyzed
overnight at 4.degree. C. in phosphate buffered saline (PBS), pH
7.4, and purified via SEC (AKTA explorer, Superdex 200 10/30 GL
column). The ADC is further characterized via size exclusion
chromatography (SEC) for purity, hydrophobic interaction
chromatography (HIC), and liquid chromatography electrospray
ionisation tandem mass spectrometry (LC-ESI MS) to calculate
loading, and the concentration is determined via UV
spectrophotometer.
Example 2
Binding Studies
[0086] Cells expressing the 5T4 antigen, and the negative control
Raji cells, are plated at a density of 500,000 cells/well on
non-tissue culture treated 96 well plates and kept on ice.
Dilutions of the A1 and A1-IgG4 antibodies or A1-mcMMAF ADC are
made in 3% bovine serum albumin BSA in Dulbecco's phosphate
buffered saline (DPBS) and added to the plate at a final
concentration of 10 .mu.g/mL. The plates are then incubated on ice
for 1 hour followed by 2 washes. The secondary antibody, PE
(phycoerythrin) conjugated Goat Anti-Human IgG Fc is added to the
wells. After 30 minutes of incubation at 4.degree. C., the mean
fluorescence intensity is then measured using a flow cytometer.
[0087] The data in Table 4 indicates that the A1 antibody binds a
diverse panel of 5T4 positive cell lines. The data in Table 5
indicates that similar binding on several different cell lines is
observed with the A1 and A1-IgG4 antibodies as well as the
A1-mcMMAF ADC.
TABLE-US-00004 TABLE 4 A1 antibody Human Cell Lines Mean
Fluorescent Intensity MDAMB435/5T4 (melanoma) 15000 MDAMB468
(breast) 3000 MDAMD361-DYT2 (breast) 4500 NCI-H157 (lung) 4100 A431
(epithelial) 2000 Caki (kidney) 2500 PC3mm2 (prostate) 4500 PC14PE6
(lung) 3200 Panc1 (pancreatic) 3500 BxPC3 (pancreatic 1000 Su8686
(pancreatic) 3700 H1975 1600 37622A-(Primary Lung cancer cells)
10600 Raji (negative control) <100
TABLE-US-00005 TABLE 5 Mean Fluorescent Intensity A1- IgG Cell
Lines A1 mcMMAF A1-IGG4 A1-IGG4-CM control MDAMD361- 7000 6900 5500
4800 <100 DYT2 A431 3900 3400 2400 2000 <100 MDAMB468 3500
2800 2500 1800 <100 PC3mm2 3400 2700 2100 1500 <100 Raji
<200 <200 <200 <100 <100
Example 3
Cytotoxicity Assay
[0088] Cell lines expressing 5T4, and the negative control Raji
cell line, are cultured with increasing concentrations of ADC.
After four days, viability of each culture is assessed. IC.sub.50
values are calculated by logistic non-linear regression and are
presented as ng Ab/mL. A1-mcMMAF, A1-vcMMAE, A3-mcMMAF and
A3-mcMMAE are shown to inhibit the growth of 5T4 expressing cell
lines (MDAMB435/5T4, MDAMB468, and MDAMB361DYT2), while being
inactive on 5T4 negative cells (Raji), Table 6.
TABLE-US-00006 TABLE 6 IC.sub.50 (ng Ab/ml) ADC MDAMB435/5T4
MDAMB361DYT2 MDAMB468 Raji (5T4-) A1-mcMMAF 1.3 104.2 534.2
>45,000 A1-vcMMAE 6.8 157.7 7667 >45,000 A3-mcMMAF 0.3 31.0
27.7 >45,000 A3-vcMMAE 3.5 86.8 160.6 >45,000 Nonbinding
21258 ~50,000 73059 >45,000 Ab-mcMMAF Nonbinding 7979 27650
23819 >45,000 Ab-vcMMAE
[0089] Additionally, 5T4+ primary lung tumor 37622a cells are
isolated and grown in culture. Cells are cultured with increasing
concentrations of ADC. Ten days later, viability of each culture is
assessed using the MTS method. IC.sub.50 values were calculated by
logistic non-linear regression and are presented as ng Ab/ml.
A1-mcMMAF, A1-vcMMAE, A3-mcMMAF, and A3-vcMMAE inhibit the growth
of the primary lung tumor cells, Table 7.
TABLE-US-00007 TABLE 7 37622a primary lung ADC IC.sub.50 (ng Ab/ml)
A1-mcMMAF 504.1 A1-vcMMAE 443.1 A3-mcMMAF 77.2 A3-vcMMAE 78.1
Nonbinding Ab- >45,000 mcMMAF
Example 4
Subcutaneous Xenograft Model
[0090] Female, athymic (nude) mice (or another strain of
immunosupressed mice) are injected s.c. with MDAMB435/5T4, MDAMB361
DYT2, or H1975 tumor cells. Mice with staged tumors, approximately
0.1 to 0.3 g (n=6 to 10 mice/treatment group) are administered
intravenously Q4Dx4 with normal saline (vehicle), A1-mcMMAF,
A1-vcMMAE, A1-mcMMAD, A1-smccDM1, A3-mcMMAF, A3-vcMMAE, or a
nonbinding control antibody conjugated to either mcMMAF or vcMMAE,
at the dose of 3 mg Ab/kg. All ADCs are dosed based on Ab content.
Tumors are measured at least once a week and their size
(mm.sup.2.+-.SEM) is calculated as mm.sup.2=0.5.times.(tumor
width.sup.2).times.(tumor length).
[0091] The data in Table 8 indicates that A1-mcMMAF, A1-vcMMAE,
A1-vcMMAD, A3-mcMMAF, and A3-vcMMAE inhibit the growth of
MDAMB435/5T4 xenografts while A1-mcMMAD and A1-smccDM1 were not
active in this model.
[0092] The data in Table 9 indicates that A1-mcMMAF, A1-vcMMAE,
A1-vcMMAD, A1-smccDM1, A3-mcMMAF, and A3-vcMMAE inhibit the growth
of MDAMB361 DYT2 xenografts while A1-mcMMAD was not active in this
model.
[0093] The data in Table 10 indicates that A1-mcMMAF, A1-vcMMAE,
A1-vcMMAD, A3-mcMMAF, and A3-vcMMAE inhibit the growth of H1975
xenografts while A1-mcMMAD and A1-smccDM1 were not active in this
model.
TABLE-US-00008 TABLE 8 MDAMB435/5T4 xenografts Compound Tumor
volume (mm.sup.3, x .+-. sem) (3 mg/kg Q4dx4) Day 0 Day 17 Day 42
Day 65 Day 85 Vehicle 169 .+-. 8 531 .+-. 73 1255 .+-. 190 GT GT A1
168 v 15 53 .+-. 12 67 .+-. 56 174 .+-. 119 364 .+-. 278 mcMMAF A1
168 .+-. 8 4 .+-. 4 10 .+-. 10 91 .+-. 91 200 .+-. 200 vcMMAE A1
168 .+-. 12 390 .+-. 112 GT GT GT mcMMAD A1 174 .+-. 10 429 .+-. 62
1255 .+-. 227 1781 .+-. 388 GT smccDM1 A1 169 .+-. 12 17 .+-. 7 0
.+-. 0 0 .+-. 0 0 .+-. 0 vcMMAD A3 174 .+-. 12 105 .+-. 27 216 .+-.
143 448 .+-. 220 GT mcMMAF A3 172 .+-. 13 0 .+-. 0 0 .+-. 0 0 .+-.
0 0 .+-. 0 vcMMAE Nonbinding Ab 170 .+-. 11 100 .+-. 15 314 .+-.
121 838 .+-. 381 GT mcMMAF Nonbinding Ab 172 .+-. 11 168 .+-. 53
461 .+-. 178 GT GT vcMMAE GT = group terminated due to large tumor
size
TABLE-US-00009 TABLE 9 MDAMB361DYT2 xenografts Compound Tumor
volume (mm.sup.3, x .+-. sem) (3 mg/kg Q4dx4) Day 0 Day 19 Day 47
Day 90 Day 131 Vehicle 353 .+-. 10 363 .+-. 58 558 .+-. 149 1117
.+-. 348 GT A1 348 .+-. 14 76 .+-. 32 0 .+-. 0 7 .+-. 7 11 .+-. 11
mcMMAF A1 356 .+-. 11 86 .+-. 8 0 .+-. 0 9 .+-. 9 34 .+-. 27 vcMMAE
A1 352 .+-. 26 130 .+-. 15 0 .+-. 0 0 .+-. 0 0 .+-. 0 vcMMAD A3 342
.+-. 23 128 .+-. 10 79 .+-. 30 105 .+-. 49 353 .+-. 234 mcMMAF A3
354 .+-. 21 111 .+-. 20 21 .+-. 21 72 .+-. 72 155 .+-. 155 vcMMAE
A1 347 .+-. 15 380 .+-. 66 775 .+-. 199 GT GT mcMMAD A1 352 .+-. 26
130 .+-. 15 0 .+-. 0 0 .+-. 0 0 .+-. 0 vcMMAD A1 353 .+-. 25 123
.+-. 9 51 .+-. 25 98 .+-. 41 330 .+-. 146 smccDM1 Nonbinding Ab 342
.+-. 38 407 .+-. 93 869 .+-. 198 GT GT mcMMAF Nonbinding Ab 344
.+-. 20 303 .+-. 78 346 .+-. 185 595 .+-. 362 GT vcMMAE GT = group
terminated due to large tumor size
TABLE-US-00010 TABLE 10 Dose H1975 Xenografts (mg/kg) Tumor volume
(mm.sup.3, x .+-. sem) Compound Q4dx4 Day 0 Day 8 Day 15 Day 22 Day
40 Vehicle 423 .+-. 14 1154 .+-. 136 2229 .+-. 240 GT GT A1 3 425
.+-. 14 619 .+-. 46 519 .+-. 45 581 .+-. 79 2840 .+-. 207 mcMMAF A1
3 425 .+-. 12 702 .+-. 45 929 .+-. 90 926 .+-. 116 GT vcMMAE A1 3
427 .+-. 18 739 .+-. 59 467 .+-. 19 240 .+-. 14 625 .+-. 317 vcMMAD
A3 3 426 .+-. 10 980 .+-. 79 1343 .+-. 140 1261 .+-. 203 GT mcMMAF
A3 3 431 .+-. 14 944 .+-. 52 993 .+-. 71 GT GT vcMMAE A1 3 427 .+-.
16 837 .+-. 69 1468 .+-. 139 GT GT mcMMAD A1 3 423 .+-. 18 901 .+-.
83 1852 .+-. 167 GT GT smccDMI Nonbinding 3 423 .+-. 16 1026 .+-.
68 1861 .+-. 224 GT GT Ab- mcMMAF Nonbinding 3 427 .+-. 13 1213
.+-. 67 1959 .+-. 139 GT GT Ab-vcMMAE GT = group terminated due to
large tumor size
[0094] Alternatively, nude mice with 37622a primary tumor cell
xenografts established subcutaneously are treated iv Q4Dx4 with
A1-mcMMAF, A1-mcMMAD, A1-vcMMAD, or A3-mcMMAF at the dose of 3 mg
Ab/kg and the tumor growth is monitored over the period of 96 days.
Table 11 demonstrates that A1-mcMMAF, A1-vcMMAD and A3-mcMMAF
inhibit the growth of 37622a primary tumor xenografts compared to
vehicle control treated animals while A1-mcMMAD was not active in
this model.
TABLE-US-00011 TABLE 11 37622a Primary Tumor Xenografts Tumor
volume (mm.sup.3, x .+-. sem) Dose (mg/kg) Compound Q4dx4 Day 1 Day
22 Day 46 Day 68 Day 96 Vehicle 111 .+-. 18 503 .+-. 155 1174 .+-.
247 GT GT A1- 3 111 .+-. 18 67 .+-. 11 124 .+-. 47 233 .+-. 105 357
.+-. 150 mcMMAF A1- 3 127 .+-. 28 376 .+-. 119 862 .+-. 377 GT GT
mcMMAD A1- 3 108 .+-. 14 52 .+-. 14 13 .+-. 5 50 .+-. 37 160 .+-.
121 vcMMAD A3- 3 131 .+-. 28 99 .+-. 26 211 .+-. 128 463 .+-. 210
GT mcMMAF GT = group terminated due to large tumor size
[0095] Unexpectedly, the data in Tables 8-11 show that ADCs with
the same antibody and drug payload but with different linkers had a
dissimilar efficacy profile i.e. A1-mcMMAD vs A1-vcMMAD in all four
xenograft models. In addition, the data show that ADCs with the
same antibody and linker but with different drug payloads also had
a different efficacy profile i.e. A1-mcMMAF vs A1-mcMMAD, in all
four xenograft models. Thus, the drug MMAD is effective in all four
xenograft models when linked to the A1 antibody by the vc linker
but has no activity in any of the xenograft models tested when
linked by the mc linker. In contrast, the drug MMAF is highly
effective in all 4 xenograft models when linked to the A1 antibody
with the mc linker while the chemically related drug MMAD has no
activity in all 4 xenograft models when linked to the same antibody
by the same linker.
[0096] Yet another unexpected observation is seen with the ADC
A1-smccDM1 (Tables 8-10). This ADC was very effective against the
MDAMB361 DYT2 xenograft but had essentially no effect against the
MDAMB435/5T4 and the H1975 xenografts even though all the
xenografts have a high expression of the 5T4 target antigen. This
data illustrates that the effectiveness of the linker-payload could
not be predicted even when the same high affinity antibody is
utilized or even when the same ADC is used.
Example 5
Antibody Dependent Cell-Mediated Cytotoxicity (ADCC)
ADCC Assay:
[0097] Blood from a healthy volunteer is collected into a BD
Vacutainer CPT cell preparation tube with sodium heparin. Human
peripheral blood mononucleocytes (PBMC) are harvested and
resuspended in assay buffer (RPMI 1640 supplemented with 10 mM
HEPES) at 2.5.times.10.sup.7 cells/ml. Target cells (MDAMB435/5T4
or MDAMB435/neo) are seeded at a density of 1.times.10.sup.4
cells/well in a 96 well assay plate. A1 antibody or A1-mcMMAF are
added, then human PBMC effector cells (5.times.10.sup.5) are
dispensed into the wells for an effector:target cell ratio (E:T) of
50:1. The assay plate is incubated at 37.degree. C. for 4 hours for
ADCC activity. The plate is harvested by adding equal volume of
CytoTox-One reagent (Promega). Stop solution (Promega; 50 ul) is
added to each well and lactate dehydrogenase release was quantified
by measuring fluorescence intensity. As a positive control, 2 .mu.l
of lysis buffer per well is added to generate a maximum LDH release
(100% cytotoxicity) in control wells. Percent cytotoxicity is
calculated using the following equation:
% Specific Cytotoxicity = experimental - effector spontaneous -
target spontaneous target maximum - target spontaneous .times. 100
##EQU00001##
[0098] Where "experimental" corresponds to the signal measured in
one of the experimental conditions, "effector spontaneous"
corresponds to the signal measured in the presence of PBMC alone,
"target spontaneous" corresponds to the signal measured in the
presence of target cells alone, and "target maximum" corresponds to
the signal measured in the presence of detergent-lysed target cells
alone.
[0099] The ADCC activity of A1-IgG1 Ab and A1-mcMMAF compared to
A1-IgG4 Ab is shown in Table 12. Both the A1 antibody and A1-mcMMAF
demonstate comparable ADCC activity indicating that the ADCC
activity of A1-mcMMAF may contribute to its anti-tumor
activity.
TABLE-US-00012 TABLE 12 Compound % Cytotoxicity A1-IgG1 37 .+-. 8
A1-mcMMAF 34 .+-. 1 A1-IgG4 9 .+-. 5
Example 6
Binding Affinity
[0100] Surface plasmon resonance (SPR) analysis is performed
utilizing the BIAcore.RTM. to determine the affinity constants for
A1-IgG1 and A1-IgG4 binding to either human or cynomolgus 5T4 at pH
6.0 and pH 7.4. BIAcore.RTM. technology utilizes changes in the
refractive index at the surface layer upon binding of the huA1
antibody variants to the human 5T4 protein immobilized on the
surface layer. Binding is detected by SPR of laser light refracting
from the surface. Analysis of the signal kinetics on-rate and
off-rate allows the discrimination between non-specific and
specific interactions. The 5T4 proteins used for this analysis
consisted of the human or cynomolgus 5T4 ectodomain fused to the
human IgG1-Fc domain and low densities (45.1 and 45.4 RU for human
and cynomolgus respectively) are immobilized onto a CM5 chip to
accurately measure affinity constants.
[0101] The measurement of specific binding to the 5T4 ectodomain is
attained by subtracting binding to a reference surface that had
only human IgG1-Fc protein immobilized onto the CM5 chip at the
same density to that on the 5T4-Fc surfaces. Next, various
concentrations of A1, A1-IgG4, or A3 antibodies in either HBS-EP pH
7.4 or MES-EP pH 6.0 buffer are injected over the surface. The
surface is regenerated two times with Glycine pH 1.7+0.05%
Surfactant P20 (GE Healthcare, BR-1000-54) between injection
cycles.
[0102] Results show that the A1 has a slightly higher affinity for
human 5T4 using the low-density 5T4 surface at both pH 6.0 and pH
7.4 relative to A1-IgG4 (1.5-fold and 1.2-fold respectively, Table
13). Additionally, A1 exhibited slightly better binding to
cynomolgus 5T4 at both pH 6.0 and pH 7.4 compared to A1-IgG4
(1.7-fold and 1.2-fold respectively) and both A1 and A1-IgG4 bound
human 5T4, 3-4 fold better than cynomolgus 5T4 (Table 12).
TABLE-US-00013 TABLE 13 Antibody Antigen pH ka (1/Ms) kd (1/s) KD
(nM) A1-IgG1 hu5T4 6.0 4.31E+05 4.59E-04 1.06 A1-IgG4 hu5T4 6.0
6.26E+05 8.93E-04 1.43 A1-IgG1 cyno5T4 6.0 2.33E+05 6.41E-04 2.76
A1-IgG4 cyno5T4 6.0 2.02E+05 9.50E-04 4.70 A1-IgG1 hu5T4 7.4
2.75E+05 1.32E-04 0.48 A1-IgG4 hu5T4 7.4 3.28E+05 1.72E-04 0.52
A1-IgG1 cyno5T4 7.4 1.51E+05 2.73E-04 1.80 A1-IgG4 cyno5T4 7.4
1.81E+05 3.82E-04 2.11
[0103] Comparing the A1 and A3 antibodies, it is apparent that the
A1 antibody binds human and cynomolgus 5T4 better at pH 7.4
relative to pH 6.0 while the A3 antibody exhibits enhanced binding
at pH 6.0 compared to pH 7.4, Table 14.
TABLE-US-00014 TABLE 14 Antibody Antigen pH ka (1/Ms)on kd (1/s)off
KD (nM) A1 hu5T4 6.0 4.31E+05 4.59E-04 1.06 A3 hu5T4 6.0 3.51E+05
4.17E-05 0.12 A1 cyno5T4 6.0 2.33E+05 6.41E-04 2.76 A3 cyno5T4 6.0
4.58E+05 1.87E-04 0.41 A1 hu5T4 7.4 2.75E+05 1.32E-04 0.48 A3 hu5T4
7.4 1.79E+05 3.06E-05 0.17 A1 cyno5T4 7.4 1.51E+05 2.73E-04 1.80 A3
cyno5T4 1.98E+05 1.62E-04 0.82 1.98E+05
Example 7
Epitope Mapping Using 5T4 Chimeras
[0104] To identify the epitopes to which each of the A1 and A3
antibodies bind, an enzyme linked immunosorbent assay (ELISA) is
performed using (1) 5T4 ectodomain Fc construct and (2) human/mouse
5T4 chimera constructs transiently expressed in COS-1 cells. The
ectodomain includes the amino-terminal region, two leucine-rich
repeats, and the intervening hydrophilic region. Mouse and rat 5T4
ectodomains contain a 6 amino acid direct repeat within their
hydrophilic region.
[0105] Fusion proteins containing a 5T4 ectodomain and a Fc
constant region from human IgG1 are prepared using human 5T4 (amino
acids 1-355), mouse 5T4 (amino acids 1-361), rat 5T4 (amino acids
1-361), cynomologus monkey 5T4 (amino acids 1-355), chimpanzee 5T4
(amino acids 1-355), and black-tailed marmoset (amino acids 1-355).
The binding results with human/mouse 5T4 chimera constructs are
summarized in Table 14, which indicates specific binding, partial
binding, or lack of binding, by the A1 and A3 antibodies.
[0106] Table 15 refers to binding ability of the antibodies to the
various human/mouse chimeras and the nomenclature is designated by
mouse 5T4 content. When no binding is observed, this indicates
where the antibody binds human 5T4 since these antibodies do not
bind mouse 5T4. For example, the A3 antibody has the most
N-terminal binding epitope (between 83-163) and this is shown by
lack of binding to the 5T4 chimera that has residues 83-163
replaced by mouse 5T4, hence A3 can no longer bind. Based upon
these results, it is determined that humanized A1 antibody has a
first contact with human 5T4 between amino acid residues 173 and
252 and a second contact with human 5T4 between amino acid residues
276 and 355. The A3 antibody binds the first leucine-rich repeat
region of human 5T4 between amino acid residues 83 through 163. The
number of amino acid residues corresponds to the human 5T4 antigen
amino acid sequence of SEQ ID NO: 11.
TABLE-US-00015 TABLE 15 Antibody 5T4 Antigen Construct A1 A3
Human/mouse 83-163 + - Human/mouse 173-361 - + Human/mouse 173-258
+/- + Human/mouse 282-361 +/- +
Example 8
Comparison of A1-mcMMAF ADC with A1-IgG4-CM ADC A1-mcMMAF is
compared to A1-IgG4-AcBut calicheamicin (A1-IGG4-CM) for both
safety and efficacy. A1-4-CM is comprised of the A1-IgG4 antibody
conjugated with the linker, AcBut [-(4' acetylphenoxy)butanoic
acid], to a calicheamicin payload. The calicheamicins are potent
antitumor agents of a class of enediyne antibiotics derived from
the bacterium Micromonospora echinospora.
[0107] The cell binding activity of A1 Ab, A1-IgG4 Ab, A1-mcMMAF
ADC and A1-IgG4-CM ADC are compared using several 5T4 positive cell
lines (see Example 2, Table 5). The data indicates that similar
binding is observed with the A1 and A1-IgG4 antibodies as well as
the A1-mcMMAF ADC, all of which have a higher mean fluorescent
intensity than A1-IgG4-CM for all the 5T4 positive cell lines
tested.
[0108] A1-mcMMAF and A1-IgG4-CM are tested side-by-side in the
MDAMB435/5T4 subcutaneous xenograft model. Both ADCs are given iv
(Q4dx2) when the tumors reach approximately 200 mm.sup.2 in size.
The anti-tumor activity of A1-IgG4-CM at a dose of 3 mg/kg is
similar to the anti-tumor activity of A1-mcMMAF administered at
dose of 10 mg/kg (Table 16). Based upon these results, the
anti-tumor activity of A1-IgG4-CM is approximately 3.3 fold more
potent than A1-mcMMAF.
TABLE-US-00016 TABLE 16 Tumor volume (mm.sup.3, x .+-. sem) Dose
(mg/kg) Compound Q4dx4 Day 0 Day 7 Day 21 Day 31 Day 45 Vehicle 0
123 .+-. 8 195 .+-. 36 402 .+-. 56 635 .+-. 111 1309 .+-. 332 A1- 3
124 .+-. 11 121 .+-. 8 166 .+-. 29 227 .+-. 42 361 .+-. 89 mcMMAF
A1- 10 123 .+-. 14 76 .+-. 11 0 .+-. 0 3 .+-. 3 2 .+-. 1 mcMMAF
A1-IGG4- 3 121 .+-. 12 140 .+-. 15 32 .+-. 10 24 .+-. 10 26 .+-. 15
CM
[0109] It could be expected that the 3.3 fold enhanced potency of
A1-IgG4-CM over that of A1-mcMMAF would translate into a 3.3 fold
enhanced safety margin of A1-mcMMAF over that of A1-IgG4-CM in an
animal toxicity study. However, when the safety profile of
A1-IgG4-CM in cynomolgus macques is reviewed, it is determined that
A1-IgG4-CM is at least 100 fold more toxic than A1-mcMMAF in the
cynomolgus macque. When A1-IgG4-CM is administered at 0.032, 0.095
and 0.32 mg Ab/kg/cycle (2, 6, 20 .mu.g calicheamicin/kg/cycle) to
male (n=3) and female (n=3) cynomolgus macques, toxicity is
observed at each dose level. After 2 cycles (2 doses), 4 out of 6
animals in the 0.095 treatment group are either euthanized or found
dead. On the other hand, no deaths are observed at dosages up to 10
mg/kg with A1-mcMMAF (247 .mu.g mcMMAF/kg/cycle), after 2 cycles (2
doses), over the same 4 week time period. In summary, the 10 mg/kg
dosage group of A1-mcMMAF is safe while the 0.096 mg/kg dosage
group of A1-IgG4-CM is deemed toxic when both are administered
twice to cynomolgus macques in a 4 week observation period.
[0110] Unexpectedly, these results demonstrate a 105 fold
(10/0.095=105) safety margin of A1-mcMMAF over that of A1-IgG4-CM,
rather than the expected 3.3 fold safety margin based on the
relative anti-tumor potency of each ADC. This data reveals the
unpredictable nature of antibody-drug conjugates that utilize
antibodies to the same antigen target but are conjugated to a
different drug payload.
Example 9
A1-mcMMAF Mouse PK/PD Modeling and Clinical Dose Predictions
[0111] PK/PD modeling has been used to quantify the tumor response
of A1-mcMMAF in mouse xenograft studies, in order to determine
efficacious concentration across cell lines. The transit
compartment tumor kill PK/PD model used was previously described by
Simeoni et al. (Simeoni et al, Cancer Res, 64:1094, (2004). The
model has been modified to account for linear, exponential and
logistic growth of tumor, and saturative killing by the drug. PK/PD
model parameters include:
[0112] k.sub.g ex exponential growth
[0113] k.sub.g logistic growth
[0114] w.sub.0 initial tumor volume
[0115] tau transduction rate
[0116] k.sub.max maximum kill rate
[0117] kC.sub.50 concentration at half max kill rate
[0118] The PK/PD modeling results are used to calculate the Tumor
Static Concentration (TSC, Equation 1). This is the drug
concentration where tumor growth is equal to tumor death rates and
tumor volume remains unchanged. TSC can be defined as the minimal
concentration required for efficacy. TSC is used to give guidance
on clinical dose selection, with concentrations of >TSC required
for efficacy in the clinic.
[0119] For A1-mcMMAF, mouse PK was determined in a separate study
(3 mg/kg IV, female athymic nu/nu mice). Mouse xenograft studies
were completed using 3 different 5T4 cell lines with A1-mcMMAF
administered at dose levels between 1 and 30 mg/kg every 4 days:
cell line MDAMB435/5T4 (dosed at 1, 3, 10, and 30 mg/kg), cell line
H1975 (dosed at 1, 3, and 10 mg/kg) and cell line 37622A (dosed at
1 and 10 mg/kg). PK/PD modeling was performed as described and TSCs
are reported in Table 17.
[0120] Mouse PK/PD parameters for each xenograft cell line were
combined with predicted human PK of A1-mcMMAF to simulate doses
required for efficacy in the clinic. Using this methodology,
A1-mcMMAF has a predicted minimally efficacious clinical dose of
about 0.22 to about 2.3 mg/kg Q3 weeks [every three weeks] (Table
17).
[0121] In an embodiment of the present invention, dose ranges can
be in the range from about 0.18 mg/kg to about 2.7 mg/kg, from
about 0.22 mg/kg to about 2.6 mg/kg, from about 0.27 mg/kg to about
2.5 mg/kg, from about 0.32 mg/kg to about 2.3 mg/kg, from about
0.37 mg/kg to about 2.15 mg/kg, from about 0.42 mg/kg to about 2.10
mg/kg, from about 0.47 mg/kg to about 2.05 mg/kg, from about 0.52
mg/kg to about 2.00 mg/kg, from about 0.57 mg/kg to about 1.95
mg/kg, from about 0.62 mg/kg to about 1.90 mg/kg, from about 0.67
mg/kg to about 1.85 mg/kg, from about 0.72 mg/kg to about 1.80
mg/kg, from about 0.82 mg/kg to about 1.70 mg/kg, from about 0.92
mg/kg to about 1.60 mg/kg, from about 1.02 mg/kg to about 1.50
mg/kg, from about 1.12 mg/kg to about 1.40 mg/kg, or from about
1.20 mg/kg to about 1.30 mg/kg, with dosing at Q3 weeks.
Preferably, dose ranges can be in the range from about 0.22 mg/kg
to about 2.3 mg/kg.
Equation 1 if k gEx k g w 0 .ltoreq. 1 , T S C = k gEx k C 50 k max
- k gEx 1.1 if k gEx k g w 0 > 1 , T S C = k g k C 50 w 0 k max
- k g 1.2 ##EQU00002##
TABLE-US-00017 TABLE 17 TSC Predicted Stasis Dose [80% confidence]
[80% confidence] Cell Line (ug/ml) (mg/kg Q3 weeks) MDAMB435/5T4
1.1 [0.9, 1.4] 0.22 [0.18, 0.28] 37622A 5.1 [2.1, 9.9] 1.1 [0.6,
2.0] H1975 11.6 [9.6, 14.1] 2.3 [2.0, 2.7]
Sequence CWU 1
1
341449PRTArtificial Sequence"Synthetic Sequence Humanized A1 human
IgG1 heavy chain 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Tyr Thr Phe Thr Asn Phe 20 25 30 Gly Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Asn Thr Asn
Thr Gly Glu Pro Arg Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Trp Asp Gly Ala Tyr Phe Phe Asp Tyr Trp Gly Gln Gly 100
105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225
230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys
2214PRTArtificial SequenceSynthetic Sequence Humanized A1 human
Kappa light chain 2Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Ala Thr Asn Arg
Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Trp 85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100
105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn
Arg Gly Glu Cys 210 3119PRTArtificial SequenceSynthetic Sequence
A1-VH 3Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe
Thr Asn Phe 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Asn Thr Asn Thr Gly Glu
Pro Arg Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp
Trp Asp Gly Ala Tyr Phe Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr
Leu Val Thr Val Ser Ser 115 4107PRTArtificial SequenceSynthetic
Sequence A1-VL 4Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Ala Thr Asn Arg Tyr
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Trp 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 55PRTArtificial
SequenceSynthetic Sequence A1-HC CDR1 5Asn Phe Gly Met Asn 1 5
617PRTArtificial SequenceSynthetic Sequence A1-HC CDR2 6Trp Ile Asn
Thr Asn Thr Gly Glu Pro Arg Tyr Ala Glu Glu Phe Lys 1 5 10 15 Gly
710PRTArtificial SequenceSynthetic Sequence A1-HC CDR3 7Asp Trp Asp
Gly Ala Tyr Phe Phe Asp Tyr 1 5 10 811PRTArtificial
SequenceSynthetic Sequence A1-LC-CDR1 8Lys Ala Ser Gln Ser Val Ser
Asn Asp Val Ala 1 5 10 97PRTArtificial SequenceSynthetic Sequence
A1-LC-CDR2 9Phe Ala Thr Asn Arg Tyr Thr 1 5 109PRTArtificial
SequenceSynthetic Sequence A1-LC-CDR3 10Gln Gln Asp Tyr Ser Ser Pro
Trp Thr 1 5 11420PRTArtificial SequenceSynthetic Sequence Human 5T4
antigen 11Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly
Arg Leu 1 5 10 15 Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp
Val Ser Ser Ser 20 25 30 Ser Pro Thr Ser Ser Ala Ser Ser Phe Ser
Ser Ser Ala Pro Phe Leu 35 40 45 Ala Ser Ala Val Ser Ala Gln Pro
Pro Leu Pro Asp Gln Cys Pro Ala 50 55 60 Leu Cys Glu Cys Ser Glu
Ala Ala Arg Thr Val Lys Cys Val Asn Arg 65 70 75 80 Asn Leu Thr Glu
Val Pro Thr Asp Leu Pro Ala Tyr Val Arg Asn Leu 85 90 95 Phe Leu
Thr Gly Asn Gln Leu Ala Val Leu Pro Ala Gly Ala Phe Ala 100 105 110
Arg Arg Pro Pro Leu Ala Glu Leu Ala Ala Leu Asn Leu Ser Gly Ser 115
120 125 Arg Leu Asp Glu Val Arg Ala Gly Ala Phe Glu His Leu Pro Ser
Leu 130 135 140 Arg Gln Leu Asp Leu Ser His Asn Pro Leu Ala Asp Leu
Ser Pro Phe 145 150 155 160 Ala Phe Ser Gly Ser Asn Ala Ser Val Ser
Ala Pro Ser Pro Leu Val 165 170 175 Glu Leu Ile Leu Asn His Ile Val
Pro Pro Glu Asp Glu Arg Gln Asn 180 185 190 Arg Ser Phe Glu Gly Met
Val Val Ala Ala Leu Leu Ala Gly Arg Ala 195 200 205 Leu Gln Gly Leu
Arg Arg Leu Glu Leu Ala Ser Asn His Phe Leu Tyr 210 215 220 Leu Pro
Arg Asp Val Leu Ala Gln Leu Pro Ser Leu Arg His Leu Asp 225 230 235
240 Leu Ser Asn Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn
245 250 255 Leu Thr His Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu
Lys Val 260 265 270 Leu His Asn Gly Thr Leu Ala Glu Leu Gln Gly Leu
Pro His Ile Arg 275 280 285 Val Phe Leu Asp Asn Asn Pro Trp Val Cys
Asp Cys His Met Ala Asp 290 295 300 Met Val Thr Trp Leu Lys Glu Thr
Glu Val Val Gln Gly Lys Asp Arg 305 310 315 320 Leu Thr Cys Ala Tyr
Pro Glu Lys Met Arg Asn Arg Val Leu Leu Glu 325 330 335 Leu Asn Ser
Ala Asp Leu Asp Cys Asp Pro Ile Leu Pro Pro Ser Leu 340 345 350 Gln
Thr Ser Tyr Val Phe Leu Gly Ile Val Leu Ala Leu Ile Gly Ala 355 360
365 Ile Phe Leu Leu Val Leu Tyr Leu Asn Arg Lys Gly Ile Lys Lys Trp
370 375 380 Met His Asn Ile Arg Asp Ala Cys Arg Asp His Met Glu Gly
Tyr His 385 390 395 400 Tyr Arg Tyr Glu Ile Asn Ala Asp Pro Arg Leu
Thr Asn Leu Ser Ser 405 410 415 Asn Ser Asp Val 420
12446PRTArtificial SequenceSynthetic Sequence Humanized A1 human
IgG4m heavy chain 12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Tyr Thr Phe Thr Asn Phe 20 25 30 Gly Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Asn Thr Asn
Thr Gly Glu Pro Arg Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Trp Asp Gly Ala Tyr Phe Phe Asp Tyr Trp Gly Gln Gly 100
105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly
Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225
230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345
350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 13119PRTArtificial
SequenceSynthetic Sequence human IgG4m VH (A1-IGG4-VH) 13Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Phe 20
25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Trp Ile Asn Thr Asn Thr Gly Glu Pro Arg Tyr Ala
Glu Glu Phe 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Trp Asp Gly Ala
Tyr Phe Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val
Ser Ser 115 145PRTArtificial SequenceSynthetic Sequence
A1-IgG4-VH-CDR1 14Asn Phe Gly Met Asn 1 5 15452PRTArtificial
SequenceSynthetic Sequence Chimeric A3 heavy chain (muA3-huIgG1)
15Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1
5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr
Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala
Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser
Arg Asp Asp Ser Gln Ser Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn
Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg Gln
Trp Asp Tyr Asp Val Arg Ala Met Asn Tyr Trp 100 105 110 Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His
Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265
270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390
395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450
16122PRTArtificial SequenceSynthetic Sequence Chimeric A3 VH 16Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10
15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr
Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg
Asp Asp Ser Gln Ser Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu
Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg Gln Trp
Asp Tyr Asp Val Arg Ala Met Asn Tyr Trp 100 105 110 Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 175PRTArtificial SequenceSynthetic
Sequence Chimeric A3 VH-CDR1 17Thr Tyr Ala Met Asn 1 5
1819PRTArtificial SequenceSynthetic Sequence Chimeric A3 VH-CDR2
18Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1
5 10 15 Val Lys Asp 1911PRTArtificial SequenceSynthetic Sequence
Chimeric A3 VH-CDR3 19Gln Trp Asp Tyr Asp Val Arg Ala Met Asn Tyr 1
5 10 20214PRTArtificial SequenceSynthetic Sequence Chimeric A3
light chain (muA3-huKappa) 20Asp Ile Val Met Thr Gln Ser His Ile
Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys
Lys Ala Ser Gln Asp Val Asp Thr Ala 20 25 30 Val Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala
Ser Thr Arg Leu Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70
75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro
Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr
Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195
200 205 Phe Asn Arg Gly Glu Cys 210 21107PRTArtificial
SequenceSynthetic Sequence Chimeric A3 VL 21Asp Ile Val Met Thr Gln
Ser His Ile Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser
Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Thr Ala 20 25 30 Val Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45
Tyr Trp Ala Ser Thr Arg Leu Thr Gly Val Pro Asp Arg Phe Thr Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln
Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser
Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 2211PRTArtificial SequenceSynthetic Sequence Chimeric A3
VL-CDR1 22Lys Ala Ser Gln Asp Val Asp Thr Ala Val Ala 1 5 10
237PRTArtificial SequenceSynthetic Sequence Chimeric A3 VL-CDR2
23Trp Ala Ser Thr Arg Leu Thr 1 5 249PRTArtificial
SequenceSynthetic Sequence Chimeric A3 VL-CDR3 24Gln Gln Tyr Ser
Ser Tyr Pro Tyr Thr 1 5 25452PRTArtificial SequenceSynthetic
Sequence Humanized A3 human IgG1 heavy chain 25Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys
Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg Gln Trp Asp Tyr Asp Val
Arg Ala Met Asn Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170
175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295
300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420
425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu 435 440 445 Ser Pro Gly Lys 450 26122PRTArtificial
SequenceSynthetic Sequence Humanized A3 VH 26Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys
Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg Gln Trp Asp Tyr Asp Val
Arg Ala Met Asn Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 275PRTArtificial SequenceSynthetic Sequence
Humanized A3 VH-CDR1 27Thr Tyr Ala Met Asn 1 5 2819PRTArtificial
SequenceSynthetic Sequence Humanized A3 VH-CDR2 28Arg Ile Arg Ser
Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys
Asp 2911PRTArtificial SequenceSynthetic Sequence Humanized A3
VH-CDR3 29Gln Trp Asp Tyr Asp Val Arg Ala Met Asn Tyr 1 5 10
30214PRTArtificial SequenceSynthetic Sequence Humanized A3 human
Kappa light chain 30Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Asp Val Asp Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg
Leu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100
105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn
Arg Gly Glu Cys 210 31107PRTArtificial SequenceSynthetic Sequence
Humanized A3 VL 31Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Asp Val Asp Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg
Leu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
3211PRTArtificial SequenceSynthetic Sequence Humanized A3 VL-CDR1
32Lys Ala Ser Gln Asp Val Asp Thr Ala Val Ala 1 5 10
337PRTArtificial SequenceSynthetic Sequence Humanized A3 VL-CDR2
33Trp Ala Ser Thr Arg Leu Thr 1 5 349PRTArtificial
SequenceSynthetic Sequence Humanized A3 VL-CDR3 34Gln Gln Tyr Ser
Ser Tyr Pro Tyr Thr 1 5
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