U.S. patent application number 13/835833 was filed with the patent office on 2014-01-09 for binding domain-immunoglobulin fusion proteins.
The applicant listed for this patent is Emergent Product Development Seattle, LLC. Invention is credited to Martha Hayden-Ledbetter, Jeffrey A. LEDBETTER.
Application Number | 20140010809 13/835833 |
Document ID | / |
Family ID | 34658226 |
Filed Date | 2014-01-09 |
United States Patent
Application |
20140010809 |
Kind Code |
A1 |
LEDBETTER; Jeffrey A. ; et
al. |
January 9, 2014 |
BINDING DOMAIN-IMMUNOGLOBULIN FUSION PROTEINS
Abstract
The invention relates to novel binding domain-immunoglobulin
fusion proteins that feature a binding domain for a cognate
structure such as an antigen, a counterreceptor or the like, a
wild-type IgG1, IGA or IgE hinge region polypeptide or a mutant
IgG1 hinge region polypeptide having either zero, one or two
cysteine residues, and immunoglobulin CH2 and CH3 domains, and that
are capable of ADCC and/or CDC while occurring predominantly as
polypeptides that are compromised in their ability to form
disulfide-linked multimers. The fusion proteins can be
recombinantly produced at high express levels. Also provided are
related compositions and methods, including cell surface forms of
the fusion proteins and immunotherapeutic applications of the
fusion proteins and of polynucleotides encoding such fusion
proteins.
Inventors: |
LEDBETTER; Jeffrey A.;
(Shoreline, WA) ; Hayden-Ledbetter; Martha;
(Shoreline, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Emergent Product Development Seattle, LLC |
Seattle |
WA |
US |
|
|
Family ID: |
34658226 |
Appl. No.: |
13/835833 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13396147 |
Feb 14, 2012 |
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13835833 |
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12541062 |
Aug 13, 2009 |
8147835 |
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13396147 |
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10207655 |
Jul 25, 2002 |
7754208 |
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12541062 |
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10053530 |
Jan 17, 2002 |
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10207655 |
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60385691 |
Jun 3, 2002 |
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60367358 |
Jan 17, 2001 |
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Current U.S.
Class: |
424/134.1 ;
435/252.33; 435/254.21; 435/254.23; 435/320.1; 435/328; 530/387.3;
536/23.53 |
Current CPC
Class: |
C07K 2317/622 20130101;
C12N 2510/00 20130101; A61P 29/00 20180101; A61P 35/02 20180101;
A61P 37/00 20180101; A61K 2039/5152 20130101; C12N 5/0636 20130101;
A61P 3/10 20180101; A61P 37/02 20180101; C07K 2317/22 20130101;
C07K 2317/734 20130101; C07K 2319/00 20130101; C07K 2319/30
20130101; C07K 2317/53 20130101; A61K 2039/505 20130101; C07K
16/2878 20130101; A61P 19/02 20180101; A61P 35/00 20180101; C12N
2501/52 20130101; A61K 38/00 20130101; C07K 16/462 20130101; C07K
2317/24 20130101; A61P 17/06 20180101; C07K 16/2818 20130101; C07K
16/3061 20130101; A61K 39/0011 20130101; A61K 2039/5156 20130101;
C12N 2502/11 20130101; C12N 2501/515 20130101; A61P 25/00 20180101;
C07K 16/2809 20130101; A61K 2039/5158 20130101; A61P 1/00 20180101;
A61P 1/04 20180101; C07K 16/2896 20130101; C12N 2501/51 20130101;
C07K 2317/732 20130101; C07K 2317/64 20130101; C12N 2501/23
20130101; A61K 39/001124 20180801 |
Class at
Publication: |
424/134.1 ;
530/387.3; 536/23.53; 435/320.1; 435/328; 435/252.33; 435/254.21;
435/254.23 |
International
Class: |
C07K 16/30 20060101
C07K016/30 |
Claims
1. A recombinant CTLA-4 Ig fusion protein comprising, from
N-terminus to C-terminus, a CTLA-4 domain, an immunoglobulin hinge,
a CH2 immunoglobulin heavy chain constant region, and a CH3
immunoglobulin heavy chain constant region.
2. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
said CTLA-4 domain binds a ligand selected from the group
consisting of CD80 and CD86.
3. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein the
CTLA-4 domain is a CTLA-4 ectodomain polypeptide or extracellular
domain polypeptide.
4. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein the
CTLA-4 domain is an scFv comprising a variable heavy chain and a
variable light chain joined by a linker and the CTLA-4 domain binds
CD80 or CD86.
5. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein the
CTLA-4 domain comprises an amino acid sequence with at least 90%
identity to amino acid sequence of SEQ ID NO:309 or 314.
6. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein the
CTLA-4 domain comprises an amino acid sequence with at least 95%
identity to amino acid sequence of SEQ ID NO:309 or 314.
7. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein the
CTLA-4 domain is encoded by a nucleic acid comprising a nucleic
acid sequence with at least 95% identity to the nucleic acid
sequence of SEQ ID NOS:308 or 313.
8. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein the
recombinant CTLA-4 Ig fusion protein does not contain a CH1
domain.
9. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein the
hinge is an IgG1 hinge.
10. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the IgG1 hinge is a wild-type IgG1 hinge or comprises a mutation at
one, two or three cysteine residues.
11. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the CH2 immunoglobulin heavy chain constant region comprises the
amino acid sequence of SEQ ID NO: 583
12. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the hinge is an IgA hinge.
13. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the IgA hinge is a wild-type IgA hinge or comprises a mutation at
one or two cysteine residues.
14. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the hinge is an IgE hinge.
15. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the CH2 immunoglobulin heavy chain constant region is an IgG1 CH2
immunoglobulin heavy chain constant region and the CH3
immunoglobulin heavy chain constant region is an IgG1 CH3
immunoglobulin heavy chain constant region.
16. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the CH2 immunoglobulin heavy chain constant region is an IgA
immunoglobulin heavy chain constant region and the CH3
immunoglobulin heavy chain constant region is an IgA immunoglobulin
heavy chain constant region.
17. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the CH2 immunoglobulin heavy chain constant region is an IgE
immunoglobulin heavy chain constant region and the CH3
immunoglobulin heavy chain constant region is an IgE immunoglobulin
heavy chain constant region.
18. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the hinge, the CH2 immunoglobulin heavy chain constant region, and
the CH3 immunoglobulin constant region are encoded by a nucleic
acid comprising a nucleic acid sequence with at least 90% identity
to the nucleic acid sequence of SEQ ID NO: 287 or 315.
19. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the immunoglobulin hinge is an IgG1 hinge, the CH2 immunoglobulin
heavy chain constant region is an IgG1 CH2 constant region, and the
CH3 immunoglobulin heavy chain constant region is an IgG1 CH2
constant region.
20. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the immunoglobulin hinge is an IgA hinge, the CH2 immunoglobulin
heavy chain constant region is an IgA CH2 constant region, and the
CH3 immunoglobulin heavy chain constant region is an IgA CH2
constant region.
21. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the CH3 immunoglobulin heavy chain constant region is an IgE CH3
domain with a mutation that prevents association with a J
chain.
22. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the immunoglobulin hinge is an IgE hinge, the CH2 immunoglobulin
heavy chain constant region is an IgE CH2 constant region, and the
CH3 immunoglobulin heavy chain constant region is an IgE CH2
constant region.
23. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the recombinant CTLA-4 Ig fusion protein comprises an amino acid
sequence with at least 95% identity to the amino acid sequence of
SEQ ID NO:307, 316 or 530.
24. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the recombinant CTLA-4 Ig fusion protein mediates antibody
dependent cell-mediated cytoxicity of Reh CD80.10 cells in
vitro.
25. The recombinant CTLA-4 Ig fusion protein of claim 1, wherein
the recombinant CTLA-4 Ig fusion protein is capable of at least one
immunological activity selected from the group consisting of
antibody dependent cell-mediated cytoxicity and complement
fixation.
26. An isolated polynucleotide encoding the recombinant CTLA-4 Ig
fusion protein of claim 1.
27. A recombinant expression construct comprising the
polynucleotide of claim 26.
28. A host cell transformed or transfected with the recombinant
expression construct of claim 27.
29. A CHO or COS cell capable of expressing the recombinant CTLA-4
Ig fusion protein of claim 1.
30. A pharmaceutical composition comprising the recombinant CTLA-4
Ig fusion protein of claim 1 and a physiologically acceptable
carrier.
31. A method of treating a subject having or suspected of having a
malignant condition or a B-cell disorder, comprising administering
to the subject a therapeutically effective amount of a recombinant
CTLA-4 Ig fusion protein of claim 1.
32. The method of claim 31, wherein the malignant condition or
B-cell disorder is selected from the group consisting of rheumatoid
arthritis, myasthenia gravis, Grave's disease, type I diabetes
mellitus, multiple sclerosis and an autoimmune disease.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/396,147, filed Feb. 14, 2012; which is a divisional of U.S.
application Ser. No. 12/541,062, filed Aug. 13, 2009, now U.S. Pat.
No. 8,147,835; which is a continuation of U.S. application Ser. No.
10/207,655, filed Jul. 25, 2002, now U.S. Pat. No. 7,754,208; which
is a Continuation-in-Part of U.S. application Ser. No. 10/053,530,
filed Jan. 17, 2002 (now abandoned); which claims the benefit under
U.S.C. .sctn.119(e) of U.S. Provisional Application No. 60/367,358,
filed Jan. 17, 2001; U.S. application Ser. No. 10/207,655 also
claims the benefit under U.S.C. .sctn.119(e) of U.S. Provisional
Application No. 60/385,691 filed on Jun. 3, 2002. All of the above
applications are incorporated herein by reference in their
entireties.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
EMER.sub.--011.sub.--20US_SubSegList_ST25.txt. The text file is
1,406 KB in size, was created on May 4, 2012, and is being
submitted electronically via EFS-Web.
BACKGROUND
[0003] 1. Technical Field
[0004] The present invention relates generally to immunologically
active, recombinant binding proteins, and in particular, to
molecularly engineered binding domain-immunoglobulin fusion
proteins, including single chain Fv-immunoglobulin fusion proteins.
The present invention also relates to compositions and methods for
treating malignant conditions and B-cell disorders, including
diseases characterized by autoantibody production.
[0005] 2. Description of the Related Art
[0006] An immunoglobulin molecule is a multimeric protein composed
of two identical light chain polypeptides and two identical heavy
chain polypeptides (H2L2) that are joined into a macromolecular
complex by interchain disulfide bonds. Intrachain disulfide bonds
join different areas of the same polypeptide chain, which results
in the formation of loops that, along with adjacent amino acids,
constitute the immunoglobulin domains. At the amino-terminal
portion, each light chain and each heavy chain has a single
variable region that shows considerable variation in amino acid
composition from one antibody to another. The light chain variable
region, V.sub.L, associates with the variable region of a heavy
chain, V.sub.H, to form the antigen binding site of the
immunoglobulin, Fv. Light chains have a single constant region
domain and heavy chains have several constant region domains.
Classes IgG, IgA, and IgD have three constant region domains, which
are designated CH1, CH2, and CH3, and the IgM and IgE classes have
four constant region domains, CH1, CH2, CH3 and CH4. Immunoglobulin
structure and function are reviewed, for example, in Harlow et al.,
Eds., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring
Harbor Laboratory, Cold Spring Harbor (1988).
[0007] The heavy chains of immunoglobulins can be divided into
three functional regions: Fd (fragment comprising VH and CH1),
hinge, and Fc (fragment crystallizable, derived from constant
regions). The Fd region comprises the VH and CH1 domains and in
combination with the light chain forms Fab, the antigen-binding
fragment. The Fc fragment is generally considered responsible for
the effector functions of an immunoglobulin, such as complement
fixation and binding to Fc receptors. The hinge region, found in
IgG, IgA, and IgD classes, acts as a flexible spacer, allowing the
Fab portion to move freely in space. In contrast to the constant
regions, the hinge domains are structurally diverse, varying in
both sequence and length among immunoglobulin classes and
subclasses. For example, three human IgG subclasses, IgG1, IgG2,
and IgG4, have hinge regions of 12-15 amino acids, while
IgG3-derived hinge regions can comprise approximately 62 amino
acids, including around 21 proline residues and around 11 cysteine
residues.
[0008] According to crystallographic studies, the immunoglobulin
hinge region can be further subdivided functionally into three
regions: the upper hinge, the core, and the lower hinge (Shin et
al., Immunological Reviews 130:87 (1992)). The upper hinge includes
amino acids from the carboxyl end of CH1 to the first residue in
the hinge that restricts motion, generally the first cysteine
residue that forms an interchain disulfide bond between the two
heavy chains. The length of the upper hinge region correlates with
the segmental flexibility of the antibody. The core hinge region
contains the inter-heavy chain disulfide bridges, and the lower
hinge region joins the amino terminal end of the CH2 domain and
includes residues in CH2. (Id.) The core hinge region of human IgG1
contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 40) which, when
dimerized by disulfide bond formation, results in a cyclic
octapeptide believed to act as a pivot, thus conferring
flexibility. The hinge region may also contain one or more
glycosylation sites, which include a number of structurally
distinct types of sites for carbohydrate attachment. For example,
IgA1 contains five glycosylation sites within a 17 amino acid
segment of the hinge region, conferring exceptional resistance of
the hinge region polypeptide to intestinal proteases, considered an
advantageous property for a secretory immunoglobulin.
[0009] Conformational changes permitted by the structure and
flexibility of the immunoglobulin hinge region polypeptide sequence
may affect the effector functions of the Fc portion of the
antibody. Three general categories of effector functions associated
with the Fc region include (1) activation of the classical
complement cascade, (2) interaction with effector cells, and (3)
compartmentalization of immunoglobulins. The different human IgG
subclasses vary in the relative efficacies with which they fix
complement, or activate and amplify the steps of the complement
cascade (e.g., Kirschfink, 2001 Immunol. Rev. 180:177; Chakraborti
et al., 2000 Cell Signal 12:607; Kohl et al., 1999 Mol. Immunol.
36:893; Marsh et al., 1999 Curr. Opin. Nephrol. Hypertens. 8:557;
Speth et al., 1999 Wien Klin. Wochenschr. 111:378).
Complement-dependent cytotoxicity (CDC) is believed to be a
significant mechanism for clearance of specific target cells such
as tumor cells. In general, IgG1 and IgG3 most effectively fix
complement, IgG2 is less effective, and IgG4 does not activate
complement. Complement activation is initiated by binding of Clq, a
subunit of the first component Cl in the cascade, to an
antigen-antibody complex. Even though the binding site for Clq is
located in the CH2 domain of the antibody, the hinge region
influences the ability of the antibody to activate the cascade. For
example, recombinant immunoglobulins lacking a hinge region are
unable to activate complement. (Shin et al., 1992) Without the
flexibility conferred by the hinge region, the Fab portion of the
antibody bound to the antigen may not be able to adopt the
conformation required to permit Clq to bind to CH2. (See id.) Hinge
length and segmental flexibility have been correlated with
complement activation; however, the correlation is not absolute.
Human IgG3 molecules with altered hinge regions that are as rigid
as IgG4 can still effectively activate the cascade.
[0010] The absence of a hinge region, or a lack of a functional
hinge region, can also affect the ability of certain human IgG
immunoglobulins to bind Fc receptors on immune effector cells.
Binding of an immunoglobulin to an Fc receptor facilitates
antibody-dependent cell-mediated cytotoxicity (ADCC), which is
presumed to be an important mechanism for the elimination of tumor
cells. The human IgG Fc receptor (FcR) family is divided into three
groups, FcyRI (CD64), which is capable of binding IgG with high
affinity, and Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD 16), both
of which are low affinity receptors. The molecular interaction
between each of the three receptors and an immunoglobulin has not
been defined precisely, but experimental evidence indicates that
residues in the hinge proximal region of the CH2 domain are
important to the specificity of the interaction between the
antibody and the Fe receptor. In addition, IgG1 myeloma proteins
and recombinant IgG3 chimeric antibodies that lack a hinge region
are unable to bind Fc.gamma.RI, likely because accessibility to CH2
is decreased. (Shin et al., Intern. Rev. Immunol. 10:177, 178-79
(1993).)
[0011] Unusual and apparently evolutionarily unrelated exceptions
to the H.sub.2L.sub.2 structure of conventional antibodies occur in
some isotypes of the immunoglobulins found in camelids
(Hamers-Casterman et al., 1993 Nature 363:446; Nguyen et al., 1998
J. Mol. Biol. 275:413) and in nurse sharks (Roux et al., 1998 Proc.
Nat. Acad. Sci. USA 95:11804). These antibodies form their
antigen-binding pocket using the heavy chain variable region alone.
In both species, these variable regions often contain an extended
third complementarity determining region (CDR3) to help compensate
for the lack of a light chain variable region, and there are
frequent disulfide bonds between CDR regions to help stabilize the
binding site [Muyldermans et al., 1994 Prot. Engineer. 7:1129; Roux
et al., 1998]. However, the function of the heavy chain-only
antibodies is unknown, and the evolutionary pressure leading to
their formation has not been identified. Since camelids, including
camels, llamas, and alpacas, also express conventional
H.sub.2L.sub.2 antibodies, the heavy chain-only antibodies do not
appear to be present in these animals simply as an alternative
antibody structure.
[0012] Variable regions (V.sub.HH) of the camelid heavy chain-only
immunoglobulins contain amino acid substitutions at several
positions outside of the CDR regions when compared with
conventional (H.sub.2L.sub.2) heavy chain variable regions. These
amino acid substitutions are encoded in the germ line [Nguyen et
al., 1998 J. Mol. Biol. 275:413] and are located at residues that
normally form the hydrophobic interface between conventional
V.sub.H and V.sub.L domains [Muyldermans et al., 1994 Prot.
Engineer. 7:1129]. Camelid V.sub.HH recombine with IgG2 and IgG3
constant regions that contain hinge, CH2, and CH3 domains but which
lack a CH1 domain [Hamers-Casterman et al., 1993 Nature 363:446].
Interestingly, V.sub.HH are encoded by a chromosomal locus distinct
from the V.sub.H locus [Nguyen, 1998], indicating that camelid B
cells have evolved complex mechanisms of antigen recognition and
differentiation. Thus, for example, llama IgG1 is a conventional
(H.sub.2L.sub.2) antibody isotype in which V.sub.H recombines with
a constant region that contains hinge, CH1, CH2 and CH3 domains,
whereas the llama IgG2 and IgG3 are heavy chain-only isotypes that
lack CH1 domains and that contain no light chains.
[0013] Monoclonal antibody technology and genetic engineering
methods have led to rapid development of immunoglobulin molecules
for diagnosis and treatment of human diseases. Protein engineering
has been applied to improve the affinity of an antibody for its
cognate antigen, to diminish problems related to immunogenicity of
administered recombinant polypeptides, and to alter antibody
effector functions. The domain structure of immunoglobulins is
amenable to recombinant engineering, in that the antigen binding
domains and the domains conferring effector functions may be
exchanged between immunoglobulin classes (e.g., IgG, IgA, IgE) and
subclasses (e.g., IgG1, IgG2, IgG3, etc.).
[0014] In addition, smaller immunoglobulin molecules have been
constructed to overcome problems associated with whole
immunoglobulin therapy. For instance, single chain immunoglobulin
variable region fragment polypeptides (scFv) comprise an
immunoglobulin heavy chain variable domain joined via a short
linker peptide to an immunoglobulin light chain variable domain
(Huston et al. Proc. Natl. Acad. Sci. USA, 85: 5879-83, 1988).
Because of the small size of scFv molecules, they exhibit very
rapid clearance from plasma and tissues and are capable of more
effective penetration into tissues than whole immunoglobulins.
(see, e.g., Jain, 1990 Cancer Res. 50:814s-819s.) An anti-tumor
scFv showed more rapid tumor penetration and more even distribution
through the tumor mass than the corresponding chimeric antibody
(Yokota et al., Cancer Res. 52, 3402-08 (1992)). Fusion of an scFv
to another molecule, such as a toxin, takes advantage of the
specific antigen-binding activity and the small size of an scFv to
deliver the toxin to a target tissue. (Chaudary et al., Nature
339:394 (1989); Batra et al., Mol. Cell. Biol. 11:2200 (1991).)
[0015] Despite the advantages that scFv molecules bring to
serotherapy, several drawbacks to this therapeutic approach exist.
While rapid clearance of scFv may reduce toxic effects in normal
cells, such rapid clearance may prevent delivery of a minimum
effective dose to the target tissue. Manufacturing adequate amounts
of scFv for administration to patients has been challenging due to
difficulties in expression and isolation of scFv that adversely
affect the yield. During expression, scFv molecules lack stability
and often aggregate due to pairing of variable regions from
different molecules. Furthermore, production levels of scFv
molecules in mammalian expression systems are low, limiting the
potential for efficient manufacturing of scFv molecules for therapy
(Davis et al, J. Biol. Chem. 265:10410-18 (1990); Traunecker et
al., EMBO J. 10: 3655-59 (1991)). Strategies for improving
production have been explored, including addition of glycosylation
sites to the variable regions (e.g., U.S. Pat. No. 5,888,773; Jost
et al, J. Biol. Chem. 269: 26267-73 (1994)).
[0016] An additional disadvantage to using scFv for therapy is the
lack of effector function. An scFv that lacks the cytolytic
functions, ADCC and complement dependent-cytotoxicity (CDC), which
are typically associated with immunoglobulin constant regions, may
be ineffective for treating disease. Even though development of
scFv technology began over 12 years ago, currently no scFv products
are approved for therapy. Conjugation or fusion of toxins to scFV
has thus been an alternative strategy to provide a potent,
antigen-specific molecule, but dosing with such conjugates or
chimeras is often limited by excessive and/or non-specific toxicity
having its origin in the toxin moiety of such preparations. Toxic
effects may include supraphysiological elevation of liver enzymes
and vascular leak syndrome, and other undesired effects. In
addition, immunotoxins are themselves highly immunogenic after
being administered to a host, and host antibodies generated against
the immunotoxin limit its potential usefulness in repeated
therapeutic treatments of an individual.
[0017] The benefits of immunoglobulin constant region-associated
effector functions in the treatment of disease has prompted
development of fusion proteins in which immunoglobulin constant
region polypeptide sequences are present and nonimmunoglobulin
sequences are substituted for the antibody variable region. For
example, CD4, the T cell surface protein recognized by HIV, was
recombinantly fused to an immunoglobulin Fc effector domain. (See
Sensel et al., Chem. Immunol. 65:129-158 (1997).) The biological
activity of such a molecule will depend in part on the class or
subclass of the constant region chosen. An IL-2-IgG1 fusion protein
effected complement-mediated lysis of IL-2 receptor-bearing cells.
(See id.) Use of immunoglobulin constant regions to construct these
and other fusion proteins may also confer improved pharmacokinetic
properties.
[0018] Diseases and disorders thought to be amenable to some type
of immunoglobulin therapy include cancer and immune system
disorders. Cancer includes a broad range of diseases, affecting
approximately one in four individuals worldwide. Rapid and
unregulated proliferation of malignant cells is a hallmark of many
types of cancer, including hematological malignancies. Patients
with a hematologic malignant condition have benefited most from
advances in cancer therapy in the past two decades (Multani et al.,
J. Clin. Oncology 16: 3691-3710, 1998). Although remission rates
have increased, most patients still relapse and succumb to their
disease. Barriers to cure with cytotoxic drugs include tumor cell
resistance and the high toxicity of chemotherapy, which prevents
optimal dosing in many patients. New treatments based on targeting
with molecules that specifically bind to a malignant cell,
including monoclonal antibodies (mAbs), can improve effectiveness
without increasing toxicity.
[0019] Since monoclonal antibodies (mAb) were first described in
1975 (Kohler et al., Nature 256:495-97 (1975)), many patients have
been treated with mAbs that specifically bind to tumor antigens, or
antigens expressed on tumor cells. These studies have yielded
important lessons regarding the selection of tumor cell surface
antigens that are tumor antigens suitable for use as immunotherapy
targets. First, it is highly preferable that such a target antigen
is not expressed by normal tissues the preservation of which is
important to host survival. Fortunately, in the case of hematologic
malignancy, malignant cells express many antigens that are not
expressed on the surfaces of stem cells or other essential cells.
Treatment of a hematologic malignant condition using a therapeutic
regimen that depletes both normal and malignant cells of
hematological origin has been acceptable where regeneration of
normal cells from progenitors can occur after therapy has ended.
Second, the target antigen should be expressed on all or virtually
all clonogenic populations of tumor cells, and expression should
persist despite the selective pressure from immunoglobulin therapy.
Thus, a strategy that employs selection of a cell surface idiotype
(e.g., a particular idiotope) as a target for therapy of B cell
malignancy has been limited by the outgrowth of tumor cell variants
with altered surface idiotype expression, even where the antigen
exhibits a high degree of tumor selectivity (Meeker et al., N Engl.
J. Med. 312:1658-65 (1985)). Third, the selected antigen must
traffic properly after an immunoglobulin binds to it. Shedding or
internalization of a cell surface target antigen after an
immunoglobulin binds to the antigen may allow tumor cells to escape
destruction, thus limiting the effectiveness of serotherapy.
Fourth, binding of an immunoglobulin to cell surface target
antigens that transmit or transduce cellular activation signals may
result in improved functional responses to immunotherapy in tumor
cells, and can lead to growth arrest and/or apoptosis. While all of
these properties are important, the triggering of apoptosis after
an immunoglobulin binds to the target antigen may be a critical
factor in achieving successful serotherapy.
[0020] Antigens that have been tested as targets for serotherapy of
B and T cell malignancies include Ig idiotype (Brown et al., Blood
73:651-61 (1989)), CD19 (Hekman et al., Cancer Immunol. Immunother.
32:364-72 (1991); Vlasveld et al., Cancer Immunol. Immunother. 40:
37-47 (1995)), CD20 (Press et al., Blood 69: 584-91 (1987); Maloney
et al., J. Clin. Oncol. 15:3266-74, (1997)) CD21 (Scheinberg et.
al., J. Clin. Oncol. 8:792-803, (1990)), CD5 (Dillman et. al., J.
Biol. Respn. Mod. 5:394-410 (1986)), and CD52 (CAMPATH) (Pawson et
al., J. Clin. Oncol. 15:2667-72, (1997)). Of these, the most
success has been obtained using CD20 as a target for therapy of B
cell lymphomas. Each of the other targets has been limited by the
biological properties of the antigen. For example, surface idiotype
can be altered through somatic mutation, allowing tumor cell
escape. As other examples, CD5, CD21, and CD19 are rapidly
internalized after mAb binding, allowing tumor cells to escape
destruction unless mAbs are conjugated with toxin molecules. CD22
is expressed on only a subset of B cell lymphomas, thereby limiting
its usefulness, while CD52 is expressed on both T cells and B cells
and may therefore generate counterproductive immunosuppression by
effecting selective T cell depletion.
[0021] CD20 fulfills the basic criteria described above for
selection of an appropriate target antigen for therapy of a B cell
malignant condition. Treatment of patients with low grade or
follicular B cell lymphoma using chimeric CD20 mAb induces partial
or complete responses in many patients (McLaughlin et al, Blood
88:90a (abstract, suppl. 1) (1996); Maloney et al, Blood 90:
2188-95 (1997)). However, tumor relapse commonly occurs within six
months to one year. Therefore, further improvements in serotherapy
are needed to induce more durable responses in low grade B cell
lymphoma, and to allow effective treatment of high grade lymphoma
and other B cell diseases.
[0022] One approach to improving CD20 serotherapy has been to
target radioisotopes to B cell lymphomas using mAbs specific for
CD20. While the effectiveness of therapy is increased, associated
toxicity from the long in vivo half-life of the radioactive
antibody increases also, sometimes requiring that the patient
undergo stem cell rescue (Press et al., N. Eng. J. Med. 329:
1219-1224, 1993; Kaminski et al., N. Eng. J. Med. 329:459-65
(1993)). MAbs to CD20 have been cleaved with proteases to yield
F(ab').sub.2 or Fab fragments prior to attachment of the
radioisotope. This improves penetration of the radioisotope
conjugate into the tumor, and shortens the in vivo half-life, thus
reducing the toxicity to normal tissues. However, the advantages of
effector functions, including complement fixation and/or ADCC that
would otherwise be provided by the Fc region of the CD20 mAb, are
lost since the Fab preparations lack immunoglobulin Fc domains.
Therefore, for improved delivery of radioisotopes, a strategy is
needed to make a CD20 mAb derivative that retains Fc-dependent
effector functions but which is smaller in size, thereby increasing
tumor penetration and shortening mAb half-life.
[0023] CD20 was the first human B cell lineage-specific surface
molecule identified by a monoclonal antibody, but the function of
CD20 in B cell biology is still incompletely understood. CD20 is a
non-glycosylated, hydrophobic 35 kDa B cell transmembrane
phosphoprotein that has both amino and carboxy ends situated in the
cytoplasm (Einfeld et al, EMBO J. 7:711-17 (1988)). Natural ligands
for CD20 have not been identified. CD20 is expressed by all normal
mature B cells, but is not expressed by precursor B cells.
[0024] CD20 mAbs deliver signals to normal B cells that affect
viability and growth (Clark et al., Proc. Natl. Acad. Sci. USA
83:4494-98 (1986)), and extensive cross-linking of CD20 can induce
apoptosis in B lymphoma cell lines (Shan et al., Blood 91:1644-52
(1998)). Cross-linking of CD20 on the cell surface increases the
magnitude and enhances the kinetics of signal transduction, for
example, as detected by measuring tyrosine phosphorylation of
cellular substrates (Deans et al., J. Immunol. 146:846-53 (1993)).
Significantly, apoptosis in Ramos B lymphoma cells can also be
induced by FcR cross-linking CD20 mAbs bound to the Ramos cell
surfaces, by the addition of Fc-receptor positive cells (Shan et
al., Blood 91: 1644-52 (1998)). Therefore, in addition to cellular
depletion by complement and ADCC mechanisms, Fc-receptor binding by
CD20 mAbs in vivo can promote apoptosis of malignant B cells by
CD20 cross-linking. This theory is consistent with experiments
showing that effectiveness of CD20 therapy of human lymphoma in a
SCID mouse model was dependent upon Fc-receptor binding by the CD20
mAb (Funakoshi et al., J Immunotherapy 19:93-101 (1996)).
[0025] The CD20 polypeptide contains four transmembrane domains
(Einfeld et al., EMBO J. 7: 711-17, (1988); Stamenkovic et al., J.
Exp. Med. 167:1975-80 (1988); Tedder et. al., J. Immunol.
141:4388-4394 (1988)). The multiple membrane spanning domains
prevent CD20 internalization after antibody binding. This property
of CD20 was recognized as an important feature for effective
therapy of B cell malignancies when a murine CD20 mAb, IFS, was
injected into patients with B cell lymphoma, resulting in
significant depletion of malignant cells and partial clinical
responses (Press et al., Blood 69: 584-91 (1987)).
[0026] Because normal mature B cells also express CD20, normal B
cells are depleted during CD20 antibody therapy (Reff, M. E. et al,
Blood 83: 435-445, 1994). However, after treatment is completed,
normal B cells are regenerated from CD20 negative B cell
precursors; therefore, patients treated with anti-CD20 therapy do
not experience significant immunosuppression. Depletion of normal B
cells may also be beneficial in diseases that involve inappropriate
production of autoantibodies or other diseases where B cells may
play a role. A chimeric mAb specific for CD20, consisting of heavy
and light chain variable regions of mouse origin fused to human
IgG1 heavy chain and human kappa light chain constant regions,
retained binding to CD20 and the ability to mediate ADCC and to fix
complement (Liu et al., J. Immunol. 139:3521-26 (1987); Robinson et
al., U.S. Pat. No. 5,500,362). This work led to development of a
chimeric CD20 mAb, Rituximab.TM., currently approved by the U.S.
Food and Drug Administration for approval for therapy of B cell
lymphomas. While clinical responses are frequently observed after
treatment with Rituximab.TM., patients often relapse after about
6-12 months.
[0027] High doses of Rituximab.TM. are required for intravenous
injection because the molecule is large, approximately 150 kDa, and
diffusion is limited into the lymphoid tissues where many tumor
cells reside. The mechanism of anti-tumor activity of Rituximab.TM.
is thought to be a combination of several activities, including
ADCC, complement fixation, and triggering of signals that promote
apoptosis in malignant B cells. The large size of Rituximab.TM.
prevents optimal diffusion of the molecule into lymphoid tissues
that contain malignant B cells, thereby limiting these anti-tumor
activities. As discussed above, cleavage of CD20 mAbs with
proteases into Fab or F(ab').sub.2 fragments makes them smaller and
allows better penetration into lymphoid tissues, but the effector
functions important for anti-tumor activity are lost. While CD20
mAb fragments may be more effective than intact antibody for
delivery of radioisotopes, it would be desirable to construct a
CD20 mAb derivative that retains the effector functions of the Fc
portion, but that has a smaller molecular size, facilitating better
tumor penetration and resulting in a shorter half-life.
[0028] CD20 is expressed by many malignant cells of B cell origin,
including B cell lymphoma and chronic lymphocytic leukemia (CLL).
CD20 is not expressed by malignancies of pre-B cells, such as acute
lymphoblastic leukemia. CD20 is therefore a good target for therapy
of B cell lymphoma, CLL, and other diseases in which B cells are
involved in the pathogenesis and/or progression of disease. Other B
cell disorders include autoimmune diseases in which autoantibodies
are produced during or after the differentiation of B cells into
plasma cells. Examples of B cell disorders include autoimmune
thyroid disease, including Graves' disease and Hashimoto's
thyroiditis, rheumatoid arthritis, systemic lupus erythematosus
(SLE), Sjogrens syndrome, immune thrombocytopenic purpura (ITP),
multiple sclerosis (MS), myasthenia gravis (MG), psoriasis,
scleroderma, and inflammatory bowel disease, including Crohn's
disease and ulcerative colitis.
[0029] In view of the foregoing, there is clearly a need for
improved compositions and methods to treat malignant conditions in
general, and in particular B cell disorders. As described in
greater detail herein, the compositions and methods of the present
invention overcome the limitations of the prior art by providing a
binding domain-immunoglobulin fusion protein that specifically
binds to an antigen and that is capable of mediating ADCC or
complement fixation. Furthermore, the compositions and methods
offer other related advantages.
BRIEF SUMMARY
[0030] It is an aspect of the present invention to provide a
binding domain-immunoglobulin fusion protein, comprising (a) a
binding domain polypeptide that is fused to an immunoglobulin hinge
region polypeptide, wherein said hinge region polypeptide is
selected from the group consisting of (i) a wild-type human IgG1
immunoglobulin hinge region polypeptide, (ii) a mutated human IgG1
immunoglobulin hinge region polypeptide that is derived from a
wild-type immunoglobulin hinge region polypeptide having three or
more cysteine residues, wherein said mutated human IgG1
immunoglobulin hinge region polypeptide contains two cysteine
residues and wherein a first cysteine of the wild-type hinge region
is not mutated, (iii) a mutated human IgG1 immunoglobulin hinge
region polypeptide that is derived from a wild-type immunoglobulin
hinge region polypeptide having three or more cysteine residues,
wherein said mutated human IgG1 immunoglobulin hinge region
polypeptide contains no more than one cysteine residue, and (iv) a
mutated human IgG1 immunoglobulin hinge region polypeptide that is
derived from a wild-type immunoglobulin hinge region polypeptide
having three or more cysteine residues, wherein said mutated human
IgG1 immunoglobulin hinge region polypeptide contains no cysteine
residues; (b) an immunoglobulin heavy chain CH2 constant region
polypeptide that is fused to the hinge region polypeptide; and (c)
an immunoglobulin heavy chain CH3 constant region polypeptide that
is fused to the CH2 constant region polypeptide, wherein (1) the
binding domain-immunoglobulin fusion protein is capable of at least
one immunological activity selected from the group consisting of
antibody dependent cell-mediated cytotoxicity and complement
fixation, and (2) the binding domain polypeptide is capable of
specifically binding to an antigen.
[0031] In certain embodiments the immunoglobulin hinge region
polypeptide is a mutated hinge region polypeptide and exhibits a
reduced ability to dimerize, relative to a wild-type human
immunoglobulin G hinge region polypeptide. In certain embodiments
the binding domain polypeptide comprises at least one
immunoglobulin variable region polypeptide that is selected an
immunoglobulin light chain variable region polypeptide or an
immunoglobulin heavy chain variable region polypeptide. In certain
further embodiments the binding domain-immunoglobulin fusion
protein comprises an immunoglobulin heavy chain variable region
polypeptide, wherein the heavy chain variable region polypeptide is
a human immunoglobulin heavy chain variable region polypeptide
comprising a mutation at an amino acid at a location corresponding
to amino acid position 11 in the VH domain, or amino acid position
155 in SEQ ID NO: 11, position 158 in SEQ ID NO: 12, position 154
in SEQ ID NO:13, or position 159 in SEQ ID NO 14, or amino acid
position 11 in the VH domain polypeptides listed in SEQ ID NOS:
341, 354, 465, 471, 477, 548. In certain embodiments the
immunoglobulin variable region polypeptide is derived from a human
immunoglobulin, and in certain other embodiments the immunoglobulin
variable region polypeptide comprises a humanized immunoglobulin
polypeptide sequence. In certain embodiments the immunoglobulin
variable region polypeptide is derived from a murine
immunoglobulin.
[0032] According to certain embodiments of the present invention,
the binding domain polypeptide comprises (a) at least one
immunoglobulin light chain variable region polypeptide; (b) at
least one immunoglobulin heavy chain variable region polypeptide;
and (c) at least one linker polypeptide that is fused to the
polypeptide of (a) and to the polypeptide of (b). In certain
further embodiments the immunoglobulin light chain variable region
and heavy chain variable region polypeptides are derived from human
immunoglobulins, and in certain other further embodiments the
linker polypeptide comprises at least one polypeptide having as an
amino acid sequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 39]. In other
embodiments the linker polypeptide comprises at least three repeats
of a polypeptide having as an amino acid sequence
Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 39]. In other embodiments the
linker comprises a glycosylation site, which in certain further
embodiments is an asparagine-linked glycosylation site, an 0-linked
glycosylation site, a C-mannosylation site, a glypiation site or a
phosphoglycation site. In another embodiment at least one of the
immunoglobulin heavy chain CH2 constant region polypeptide and the
immunoglobulin heavy chain CH3 constant region polypeptide is
derived from a human immunoglobulin heavy chain. In another
embodiment the immunoglobulin heavy chain constant region CH2 and
CH3 polypeptides are of an isotype that is human IgG or human IgA.
In certain other embodiments the antigen is CD19, CD20, CD22, CD37,
CD40, L6, CD2, CD28, CD30, CD40, CD50 (ICAM3), CD54 (ICAM1), CD80,
CD86, B7-H1, CD134 (0X40), CD137 (41BB), CD152 (CTLA-4), CD153
(CD30 ligand), CD154 (CD40 ligand), ICOS, CD19, CD3, CD4, CD25,
CD8, CD11b, CD14, CD25, CD56 or CD69. In another embodiment the
binding domain polypeptide comprises a CD154 extracellular domain.
In still another embodiment the binding domain polypeptide
comprises a CD154 extracellular domain and at least one
immunoglobulin variable region polypeptide. In another embodiment
the binding domain polypeptide comprises a CTLA-4 extracellular
domain, and in further embodiments at least one of the
immunoglobulin heavy chain constant region polypeptides selected
from a CH2 constant region polypeptide and a CH3 constant region
polypeptide is a human IgG1 constant region polypeptide. In another
further embodiment at least one of the immunoglobulin heavy chain
constant region polypeptides selected from a CH2 constant region
polypeptide and a CH3 constant region polypeptide is a human IgA
constant region polypeptide.
[0033] Turning to another embodiment, the present invention
provides a binding domain-immunoglobulin fusion protein, comprising
(a) a binding domain polypeptide that is fused to an immunoglobulin
hinge region polypeptide; (b) an immunoglobulin heavy chain CH2
constant region polypeptide that is fused to the hinge region
polypeptide; and (c) an immunoglobulin heavy chain CH3 constant
region polypeptide that is fused to the CH2 constant region
polypeptide, wherein (1) the binding domain polypeptide comprises a
CTLA-4 extracellular domain that is capable of specifically binding
to at least one CTLA-4 ligand selected from the group consisting of
CD80 and CD86, (2) the immunoglobulin hinge region polypeptide
comprises a polypeptide that is selected from the group consisting
of a human IgA hinge region polypeptide and a human IgG1 hinge
region polypeptide, (3) the immunoglobulin heavy chain CH2 constant
region polypeptide comprises a polypeptide that is selected from
the group consisting of a human IgA heavy chain CH2 constant region
polypeptide and a human IgG1 heavy chain CH2 constant region
polypeptide, (4) the immunoglobulin heavy chain CH3 constant region
polypeptide comprises a polypeptide that is selected from the group
consisting of a human IgA heavy chain CH3 constant region
polypeptide and a human IgG1 heavy chain CH3 constant region
polypeptide, and (5) the binding domain-immunoglobulin fusion
protein is capable of at least one immunological activity selected
from the group consisting of antibody dependent cell-mediated
cytotoxicity and complement fixation.
[0034] In another embodiment the present invention provides a
binding domain-immunoglobulin fusion protein, comprising (a) a
binding domain polypeptide that is fused to an immunoglobulin hinge
region polypeptide, wherein said hinge region polypeptide comprises
a human IgE hinge region polypeptide; (b) an immunoglobulin heavy
chain CH2 constant region polypeptide that is fused to the hinge
region polypeptide, wherein said CH2 constant region polypeptide
comprises a human IgE CH2 constant region polypeptide; and (c) an
immunoglobulin heavy chain CH3 constant region polypeptide that is
fused to the CH2 constant region polypeptide, wherein said CH3
constant region polypeptide comprises a human IgE CH3 constant
region polypeptide wherein (1) the binding domain-immunoglobulin
fusion protein is capable of at least one immunological activity
selected from antibody dependent cell-mediated cytotoxicity and
induction of an allergic response mechanism, and (2) the binding
domain polypeptide is capable of specifically binding to an
antigen. In a further embodiment the binding domain-immunoglobulin
fusion protein comprises a human IgE CH4 constant region
polypeptide. In another further embodiment the antigen is a tumor
antigen.
[0035] In certain other embodiments the present invention provides
a binding domain-immunoglobulin fusion protein, comprising (a) a
binding domain polypeptide that is fused to an immunoglobulin hinge
region polypeptide, wherein the binding domain polypeptide is
capable of specifically binding to at least one antigen that is
present on an immune effector cell and wherein the hinge region
polypeptide comprises a polypeptide selected from the group
consisting of a human IgA hinge region polypeptide, a human IgG
hinge region polypeptide, and a human IgE hinge region polypeptide;
(b) an immunoglobulin heavy chain CH2 constant region polypeptide
that is fused to the hinge region polypeptide, wherein said CH2
constant region polypeptide comprises a polypeptide selected from
the group consisting of a human IgA CH2 constant region
polypeptide, a human IgG CH2 constant region polypeptide, and a
human IgE CH2 constant region polypeptide; (c) an immunoglobulin
heavy chain CH3 constant region polypeptide that is fused to the
CH2 constant region polypeptide, wherein said CH3 constant region
polypeptide comprises a polypeptide selected from the group
consisting of a human IgA CH3 constant region polypeptide, a human
IgG CH3 constant region polypeptide, and a human IgE CH3 constant
region polypeptide; and (d) a plasma membrane anchor domain
polypeptide. In a further embodiment the membrane anchor domain
polypeptide comprises a transmembrane domain polypeptide. In
another further embodiment the membrane anchor domain polypeptide
comprises a transmembrane domain polypeptide and a cytoplasmic tail
polypeptide. In a still further embodiment the cytoplasmic tail
polypeptide comprises an apoptosis signaling polypeptide sequence,
which in a still further embodiment is derived from a receptor
death domain polypeptide. In a further embodiment the death domain
polypeptide comprises a polypeptide selected from an ITIM domain,
an ITAM domain, FADD, TRADD, RAIDD, CD95 (FAS/Apo-1), TNFR1 or DR5.
In another embodiment the apoptosis signaling polypeptide sequence
comprises a polypeptide sequence derived from a caspase polypeptide
that is caspase-3 or caspase-8. In another embodiment the plasma
membrane anchor domain polypeptide comprises a
glycosyl-phosphatidylinositol-linkage polypeptide sequence. In
another embodiment the antigen that is present on an immune
effector cell is CD2, CD28, CD30, CD40, LD50 (ICAM3), CD54 (ICAM1),
CD80, CD86, B7-H1, CD134 (OX40), CD137 (41BB), CD152 (CTLA-4),
CD153 (CD30 ligand), CD154 (CD40 ligand), ICOS, CD19, CD20, CD22,
CD37, L6, CD3, CD4, CD25, CD8, CD11b, CD14, CD25, CD56 or CD69. In
another embodiment the human IgG is human IgG1.
[0036] The invention provides, in another embodiment, a binding
domain-immunoglobulin fusion protein, comprising (a) a binding
domain polypeptide that is fused to an immunoglobulin hinge region
polypeptide, wherein the binding domain polypeptide is capable of
specifically binding to at least one antigen that is present on a
cancer cell surface and wherein the hinge region polypeptide
comprises a polypeptide selected from the group consisting of a
human IgA hinge region polypeptide, a human IgG hinge region
polypeptide, and a human IgE hinge region polypeptide; (b) an
immunoglobulin heavy chain CH2 constant region polypeptide that is
fused to the hinge region polypeptide, wherein the CH2 constant
region polypeptide comprises a polypeptide that is a human IgA CH2
constant region polypeptide, a human IgG CH2 constant region
polypeptide, or a human IgE CH2 constant region polypeptide; (c) an
immunoglobulin heavy chain CH3 constant region polypeptide that is
fused to the CH2 constant region polypeptide, wherein the CH3
constant region polypeptide comprises a polypeptide that is a human
IgA CH3 constant region polypeptide, a human IgG CH3 constant
region polypeptide, or a human IgE CH3 constant region polypeptide;
and (d) a plasma membrane anchor domain polypeptide. In a further
embodiment the membrane anchor domain polypeptide comprises a
transmembrane domain polypeptide. In another embodiment the
membrane anchor domain polypeptide comprises a transmembrane domain
polypeptide and a cytoplasmic tail polypeptide. In another
embodiment the membrane anchor domain polypeptide comprises a
glycosyl-phosphatidylinositol-linkage polypeptide sequence. In
another embodiment the human IgG is human IgG1.
[0037] In another embodiment the present invention provides a
binding domain-immunoglobulin fusion protein, comprising (a) a
binding domain polypeptide that is fused to an immunoglobulin hinge
region polypeptide, wherein said hinge region polypeptide comprises
a wild-type human IgA hinge region polypeptide; (b) an
immunoglobulin heavy chain CH2 constant region polypeptide that is
fused to the hinge region polypeptide, wherein said CH2 constant
region polypeptide comprises a human IgA CH2 constant region
polypeptide; and (c) an immunoglobulin heavy chain CH3 constant
region polypeptide that is fused to the CH2 constant region
polypeptide, wherein the CH3 constant region polypeptide comprises
a polypeptide that is (i) a wild-type human IgA CH3 constant region
polypeptide or (ii) a mutated human IgA CH3 constant region
polypeptide that is incapable of associating with a J chain,
wherein (1) the binding domain-immunoglobulin fusion protein is
capable of at least one immunological activity selected from the
group consisting of antibody dependent cell-mediated cytotoxicity
and complement fixation, and (2) the binding domain polypeptide is
capable of specifically binding to an antigen. In certain further
embodiments the mutated human IgA CH3 constant region polypeptide
that is incapable of associating with a J chain is (i) a
polypeptide comprising an amino acid sequence as set forth in SEQ
ID NOS: 296, 511AA or 295 and 510 (DNA) or (ii) a polypeptide
comprising an amino acid sequence as set forth in SEQ ID NO: 303,
520 for amino acid sequence, and (302, 519 for DNA).
[0038] In certain other embodiments the present invention provides
a binding domain-immunoglobulin fusion protein, comprising (a) a
binding domain polypeptide that is fused to an immunoglobulin hinge
region polypeptide; (b) an immunoglobulin heavy chain CH2 constant
region polypeptide that is fused to the hinge region polypeptide,
wherein the CH2 constant region polypeptide comprises a llama CH2
constant region polypeptide that is a llama IgG1 CH2 constant
region polypeptide, a llama IgG2 CH2 constant region polypeptide or
a llama IgG3 CH2 constant region polypeptide; and (c) an
immunoglobulin heavy chain CH3 constant region polypeptide that is
fused to the CH2 constant region polypeptide, wherein said CH3
constant region polypeptide comprises a llama CH3 constant region
polypeptide that is selected from the group consisting of a llama
IgG1 CH3 constant region polypeptide, a llama IgG2 CH3 constant
region polypeptide and a llama IgG3 CH3 constant region polypeptide
wherein (1) the binding domain-immunoglobulin fusion protein is
capable of at least one immunological activity selected from the
group consisting of antibody dependent cell-mediated cytotoxicity
and induction fixation of complement, and (2) the binding domain
polypeptide is capable of specifically binding to an antigen. In a
further embodiment the immunoglobulin hinge region polypeptide, the
llama CH2 constant region polypeptide and the llama CH3 constant
region polypeptide comprise sequences derived from a llama IgG1
polypeptide and the fusion protein does not include a llama IgG1
CH1 domain. In certain embodiments the invention provides any of
the above described binding domain-immunoglobulin fusion proteins
wherein the hinge region polypeptide is mutated to contain a
glycosylation site, which in certain further embodiments is an
asparagine-linked glycosylation site, an O-linked glycosylation
site, a C-mannosylation site, a glypiation site or a
phosphoglycation site. In certain embodiments the invention
provides any of the above described binding domain-immunoglobulin
fusion proteins wherein the binding domain polypeptide comprises
two or more binding domain polypeptide sequences wherein each of
the binding domain polypeptide sequences is capable of specifically
binding to an antigen.
[0039] The present invention also provides, in certain embodiments,
a binding domain-immunoglobulin fusion protein, comprising (a) a
binding domain polypeptide that is fused to an immunoglobulin hinge
region polypeptide, wherein the hinge region polypeptide comprises
an alternative hinge region polypeptide sequence; (b) an
immunoglobulin heavy chain CH2 constant region polypeptide that is
fused to the hinge region polypeptide; and (c) an immunoglobulin
heavy chain CH3 constant region polypeptide that is fused to the
CH2 constant region polypeptide, wherein: (1) the binding
domain-immunoglobulin fusion protein is capable of at least one
immunological activity selected from the group consisting of
antibody dependent cell-mediated cytotoxicity and complement
fixation, and (2) the binding domain polypeptide is capable of
specifically binding to an antigen.
[0040] Turning to another embodiment there is provided a binding
domain-immunoglobulin fusion protein, comprising (a) a binding
domain polypeptide that is fused to an immunoglobulin hinge region
polypeptide, wherein the binding domain polypeptide is capable of
specifically binding to at least one antigen that is present on a
cancer cell surface and wherein the hinge region polypeptide
comprises an alternative hinge region polypeptide sequence; (b) an
immunoglobulin heavy chain CH2 constant region polypeptide that is
fused to the hinge region polypeptide, wherein said CH2 constant
region polypeptide comprises a polypeptide selected from the group
consisting of a human IgA CH2 constant region polypeptide, a human
IgG CH2 constant region polypeptide, and a human IgE CH2 constant
region polypeptide; (c) an immunoglobulin heavy chain CH3 constant
region polypeptide that is fused to the CH2 constant region
polypeptide, wherein the CH3 constant region polypeptide comprises
a polypeptide that is a human IgA CH3 constant region polypeptide,
a human IgG CH3 constant region polypeptide, or a human IgE CH3
constant region polypeptide; and (d) a plasma membrane anchor
domain polypeptide. In certain further embodiments the alternative
hinge region polypeptide sequence comprises a polypeptide sequence
of at least ten continuous amino acids that are present in a
sequence selected from SEQ ID NOS: 215, 216, 217, 218, 223, 224, 6,
15, 16. 35, 36, 37, 41, 207, 208, 223, 275, 276, 277, 296, 300,
350, 390, 391, 392, 396, 397, 398, 488, 582, 584, 586.
[0041] In certain embodiments the present invention provides an
isolated polynucleotide encoding any one of the above described
binding domain-immunoglobulin fusion proteins, and in other
embodiments the invention provides a recombinant expression
construct comprising any such polynucleotide that is operably
linked to a promoter. In other embodiments there is provided a host
cell transformed or transfected with any such recombinant
expression construct. In a related embodiment there is provided a
method of producing a binding domain-immunoglobulin fusion protein,
comprising the steps of (a) culturing a host cell as just described
under conditions that permit expression of the binding
domain-immunoglobulin fusion protein; and (b) isolating the binding
domain-immunoglobulin fusion protein from the host cell culture. In
another embodiment there is provided a pharmaceutical composition
comprising any one of the above described binding
domain-immunoglobulin fusion proteins in combination with a
physiologically acceptable carrier. In another embodiment the
invention provides a pharmaceutical composition comprising an
isolated polynucleotide encoding any one of the above described
binding domain-immunoglobulin fusion proteins, in combination with
a physiologically acceptable carrier. In another embodiment the
invention provides a method of treating a subject having or
suspected of having a malignant condition or a B-cell disorder,
comprising administering to a patient a therapeutically effective
amount of any of the pharmaceutical compositions just described. In
certain further embodiments the malignant condition or B-cell
disorder is a B-cell lymphoma or a disease characterized by
autoantibody production, and in certain other further embodiments
the malignant condition or B-cell disorder is rheumatoid arthritis,
myasthenia gravis, Grave's disease, type I diabetes mellitus,
multiple sclerosis or an autoimmune disease. In certain other
embodiments the malignant condition is melanoma, carcinoma or
sarcoma.
[0042] It is another aspect of the present invention to provide a
binding domain-immunoglobulin fusion protein, comprising (a) a
binding domain polypeptide that is fused to an immunoglobulin hinge
region polypeptide, wherein said hinge region polypeptide is
selected from the group consisting of (i) a mutated hinge region
polypeptide that contains no cysteine residues and that is derived
from a wild-type immunoglobulin hinge region polypeptide having one
or more cysteine residues, (ii) a mutated hinge region polypeptide
that contains one cysteine residue and that is derived from a
wild-type immunoglobulin hinge region polypeptide having two or
more cysteine residues, (iii) a wild-type human IgA hinge region
polypeptide, (iv) a mutated human IgA hinge region polypeptide that
contains no cysteine residues and that is derived from a wild-type
human IgA region polypeptide, and (v) a mutated human IgA hinge
region polypeptide that contains one cysteine residue and that is
derived from a wild-type human IgA region polypeptide; (b) an
immunoglobulin heavy chain CH2 constant region polypeptide that is
fused to the hinge region polypeptide; and (c) an immunoglobulin
heavy chain CH3 constant region polypeptide that is fused to the
CH2 constant region polypeptide, wherein: (1) the binding
domain-immunoglobulin fusion protein is capable of at least one
immunological activity selected from the group consisting of
antibody dependent cell-mediated cytotoxicity and complement
fixation, and (2) the binding domain polypeptide is capable of
specifically binding to an antigen. In one embodiment the
immunoglobulin hinge region polypeptide is a mutated hinge region
polypeptide and exhibits a reduced ability to dimerize, relative to
a wild-type human immunoglobulin G hinge region polypeptide. In
another embodiment the binding domain polypeptide comprises at
least one immunoglobulin variable region polypeptide that is an
immunoglobulin light chain variable region polypeptide or an
immunoglobulin heavy chain variable region polypeptide. In a
further embodiment the immunoglobulin variable region polypeptide
is derived from a human immunoglobulin.
[0043] In another embodiment the binding domain Fv-immunoglobulin
fusion protein binding domain polypeptide comprises (a) at least
one immunoglobulin light chain variable region polypeptide; (b) at
least one immunoglobulin heavy chain variable region polypeptide;
and (c) at least one linker peptide that is fused to the
polypeptide of (a) and to the polypeptide of (b). In a further
embodiment the immunoglobulin light chain variable region and heavy
chain variable region polypeptides are derived from human
immunoglobulins.
[0044] In another embodiment at least one of the immunoglobulin
heavy chain CH2 constant region polypeptide and the immunoglobulin
heavy chain CH3 constant region polypeptide is derived from a human
immunoglobulin heavy chain. In another embodiment the
immunoglobulin heavy chain constant region CH2 and CH3 polypeptides
are of an isotype selected from human IgG and human IgA. In another
embodiment the antigen is selected from the group consisting of
CD19, CD20, CD37, CD40 and L6. In certain further embodiments of
the above described fusion protein, the linker polypeptide
comprises at least one polypeptide having as an amino acid sequence
Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 39], and in certain other
embodiments the linker polypeptide comprises at least three repeats
of a polypeptide having as an amino acid sequence
Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 39]. In certain embodiments the
immunoglobulin hinge region polypeptide comprises a human IgA hinge
region polypeptide. In certain embodiments the binding domain
polypeptide comprises a CD154 extracellular domain. In certain
embodiments the binding domain polypeptide comprises a CD154
extracellular domain and at least one immunoglobulin variable
region polypeptide.
[0045] In other embodiments the invention provides an isolated
polynucleotide encoding any of the above described binding
domain-immunoglobulin fusion proteins, and in related embodiments
the invention provides a recombinant expression construct
comprising such a polynucleotide, and in certain further
embodiments the invention provides a host cell transformed or
transfected with suc a recombinant expression construct. In another
embodiment the invention provides a method of producing a binding
domain-immunoglobulin fusion protein, comprising the steps of (a)
culturing the host cell as just described, under conditions that
permit expression of the binding domain-immunoglobulin fusion
protein; and (b) isolating the binding domain-immunoglobulin fusion
protein from the host cell culture.
[0046] The present invention also provides in certain embodiments a
pharmaceutical composition comprising a binding
domain-immunoglobulin fusion protein as described above, in
combination with a physiologically acceptable carrier. In another
embodiment there is provided a method of treating a subject having
or suspected of having a malignant condition or a B-cell disorder,
comprising administering to a patient a therapeutically effective
amount of an above described binding domain-immunoglobulin fusion
protein. In certain further embodiments the malignant condition or
B-cell disorder is a B-cell lymphoma or a disease characterized by
autoantibody production, and in certain other further embodiments
the malignant condition or B-cell disorder is rheumatoid arthritis,
myasthenia gravis, Grave's disease, type I diabetes mellitus,
multiple sclerosis or an autoimmune disease.
[0047] These and other aspects of the present invention will become
apparent upon reference to the following detailed description and
attached drawings. All references disclosed herein are hereby
incorporated by reference in their entireties as if each was
incorporated individually.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 shows DNA and deduced amino acid sequences (SEQ ID
NO: 2) of 2H7scFv-Ig, a binding domain-immunoglobulin fusion
protein capable of specifically binding CD20. FIG. 1A shows the
first 780 nucleotides and their associated amino acids. FIG. 1B
shows nucleotides 781-1518 and their associated amino acids.
[0049] FIG. 2 shows production levels of 2H7 scFv-Ig by
transfected, stable CHO lines and generation of a standard curve by
binding of purified 2H7 scFv-Ig to CHO cells expressing CD20.
[0050] FIG. 3 shows SDS-PAGE analysis of multiple preparations of
isolated 2H7scFv-Ig protein.
[0051] FIG. 4 shows complement fixation (FIG. 4A) and mediation of
antibody-dependent cellular cytotoxicity (ADCC, FIG. 4B)) by
2H7scFv-Ig.
[0052] FIG. 5 shows the effect of simultaneous ligation of CD20 and
CD40 on growth of normal B cells.
[0053] FIG. 6 shows the effect of simultaneous ligation of CD20 and
CD40 on CD95 expression (FIG. 6A) and induction of apoptosis (FIG.
6B) in a B lymphoblastoid cell line.
[0054] FIG. 7 shows DNA and deduced amino acid sequences of
2H7scFv-CD154 L2 (FIG. 7A-7B, SEQ ID NOS: 21 and 33) and
2H7scFv-CD154 S4 (FIG. 7C-7D, SEQ ID NOS:22 and 34) binding
domain-immunoglobulin fusion proteins capable of specifically
binding CD20 and CD40.
[0055] FIG. 8 shows binding of 2H7scFv-CD 154 binding
domain-immunoglobulin fusion proteins to CD20+ CHO cells by flow
immunocytofluorimetry.
[0056] FIG. 9 shows binding of Annexin V to B cell lines Ramos,
BJAB, and T51 after binding of 2H7scFv-CD154 binding
domain-immunoglobulin fusion protein to cells.
[0057] FIG. 10 shows effects on proliferation of B cell line T51
following binding of 2H7scFv-CD154 binding domain-immunoglobulin
fusion protein.
[0058] FIG. 11 depicts schematic representations of the structures
of 2H7scFv-Ig fusion proteins referred to as CytoxB or CytoxB
derivatives: CytoxB-MHWTG1C (2H7 scFv, mutant hinge, wild-type
human IgG1 Fc domain), CytoxB-MHMG1C (2H7 scFv, mutant hinge,
mutated human IgG1 Fc domain) and CytoxB-IgAHWTHG 1C (2H7 scFv,
human IgA-derived hinge (SEQ ID NO: 41), wild-type human IgG1 Fc
domain). Arrows indicate position numbers of amino acid residues
believed to contribute to FcR binding and ADCC activity (heavy
arrows), and to complement fixation (light arrows). Note absence of
interchain disulfide bonds.
[0059] FIG. 12 shows SDS-PAGE analysis of isolated CytoxB and
2H7scFv-CD154 binding domain-immunoglobulin fusion proteins.
[0060] FIG. 13 shows antibody dependent cell-mediated cytotoxicity
(ADCC) activity of CytoxB derivatives.
[0061] FIG. 14 shows complement dependent cytotoxicity (CDC) of
CytoxB derivatives.
[0062] FIG. 15 shows serum half-life determinations of
CytoxB-MHWTG1C in macaque blood samples.
[0063] FIG. 16 shows effects of CytoxB-MHIWTG1C on levels of
circulating CD40+ B cells in macaque blood samples.
[0064] FIG. 17 shows production levels of HD37 (CD19-specific)
scFv-Ig by transfected mammalian cell lines and generation of a
standard curve by binding of purified HD37 scFv-Ig to cells
expressing CD19.
[0065] FIG. 18 shows production levels of L6 (carcinoma antigen)
scFv-Ig by transfected, stable CHO lines and generation of a
standard curve by binding of purified L6 scFv-Ig to cells
expressing L6 antigen.
[0066] FIG. 19 shows ADCC activity of binding domain-immunoglobulin
fusion proteins 2H7 scFv-Ig, HD37 scFv-Ig and G28-1 (CD37-specific)
scFv-Ig.
[0067] FIG. 20 shows ADCC activity of L6 scFv-Ig fusion
proteins.
[0068] FIG. 21 shows SDS-PAGE analysis of L6 scFv-Ig and 2H7
scFv-Ig fusion proteins.
[0069] FIG. 22 shows SDS-PAGE analysis of G28-1 scFv-Ig and HD37
scFv-Ig fusion proteins.
[0070] FIG. 23 presents a sequence alignment of immunoglobulin
hinge, CH2 and CH3 domains of human IgG1 (SEQ ID NO: 428) with the
hinge, CH2 and CH3 domains of llama IgG1 (SEQ ID NO: 430), IgG2
(SEQ ID NO: 432), and IgG3 (SEQ ID NO: 434).
[0071] FIG. 24 illustrates migration of purified 2H7 scFv llama IgG
fusion proteins in a 10% SDS polyacrylamide gel. Purified fusion
proteins (5.mu.g per sample) were prepared in non-reducing sample
buffer (lanes 2-5) and in reducing sample buffer (lanes 6-9). Lane
1: molecular weight markers (non-reduced); lanes 2 and 6: 2H7
scFv-llama IgG1 (DNA sequence is set forth in SEQ ID NO: 452; amino
acid sequence is set forth in SEQ ID NO: 453); Lanes 3 and 7: 2H7
scFv-llama IgG2 (DNA sequence is set forth in SEQ ID NO: 454; amino
acid sequence is set forth in SEQ ID NO: 455); lanes 4 and 8: 2H7
scFv-llama IgG3 (DNA sequence is set forth in SEQ ID NO: 456; amino
acid sequence is set forth in SEQ ID NO: 457); and Lanes 5 and 9:
Rituximab (chimeric anti-CD20 antibody (human IgG1 constant
region)).
[0072] FIG. 25 shows binding of 2H7 scFv-llama IgG1 (DNA sequence
is set forth in SEQ ID NO: 452; amino acid sequence is set forth in
SEQ ID NO: 453), 2H7 scFv-llama IgG2 (DNA sequence is set forth in
SEQ ID NO: 454; amino acid sequence is set forth in SEQ ID NO:
455), and 2H7 scFv-llama IgG3 (DNA sequence is set forth in SEQ ID
NO: 456; amino acid sequence is set forth in SEQ ID NO: 457) to
CD20+ CHO cells detected by flow immunocytofluorimetry.
[0073] FIG. 26 depicts CDC activity of 2H7 scFv llama IgG fusion
proteins, 2H7 scFv-llama IgG1 (DNA sequence is set forth in SEQ ID
NO: 452; amino acid sequence is set forth in SEQ ID NO: 453), 2H7
scFv-llama IgG2 (DNA sequence is set forth in SEQ ID NO: 454; amino
acid sequence is set forth in SEQ ID NO: 455), and 2H7 scFv-llama
IgG3 (SEQ ID NO: DNA sequence is set forth in SEQ ID NO: 454; amino
acid sequence is set forth in SEQ ID NO: 455), and 2H7 scFv human
IgG1 (2H7 scFv IgG WTH WTCH2CH3) (DNA sequence is set forth in SEQ
ID NO: 456; amino acid sequence is set forth in SEQ ID NO: 457)
against BJAB cells in the presence of rabbit complement. Rituximab
was included as a positive control.
[0074] FIG. 27 shows ADCC activity of 2H7 scFv llama IgG fusion
proteins, 2H7 scFv-llama IgG1 (DNA sequence is set forth in SEQ ID
NO: 452; amino acid sequence is set forth in SEQ ID NO: 453), 2H7
scFv-llama IgG2 (SEQ ID NO: DNA sequence is set forth in SEQ ID NO:
454; amino acid sequence is set forth in SEQ ID NO: 455), and 2H7
scFv-llama IgG3 (DNA sequence is set forth in SEQ ID NO: 456; amino
acid sequence is set forth in SEQ ID NO: 457). Effector cells
(human PBMC) were combined with target cells (BJAB cells) at three
different ratios, 1:25, 1:50, and 1:100. Rituximab was included as
a positive control. Each data point represents three separate
measurements.
[0075] FIG. 28 shows ADCC activity of 2H7 scFv llama IgG fusion
proteins, 2H7 scFv-llama IgG1 (DNA sequence is set forth in SEQ ID
NO: 452; amino acid sequence is set forth in SEQ ID NO: 453), 2H7
scFv-llama IgG2 (DNA sequence is set forth in SEQ ID NO: 454; amino
acid sequence is set forth in SEQ ID NO: 455), and 2H7 scFv-llama
IgG3 (DNA sequence is set forth in SEQ ID NO: 456; amino acid
sequence is set forth in SEQ ID NO: 457). Effector cells (llama
PBMC) were combined with target cells (BJAB cells) at three
different ratios, 1:25, 1:50, and 1:100. Rituximab was included as
a positive control. Each data point represents three separate
measurements.
[0076] FIG. 29 depicts CDC activity of Reh cells (acute lymphocytic
leukemia) expressing scFv-Ig fusion proteins on the cell surface.
Reh cells were transfected with constructs encoding scFv antibodies
specific for human costimulatory molecules, CD152, CD28, CD40, and
CD20, fused to human IgG1 wild-type hinge-CH2-CH3, which was fused
to human CD80 transmembrane and cytoplasmic tail domains. CDC
activity was measured in the presence and absence of rabbit
complement (plus C' and no C', respectively). The data represent
the average of duplicate samples. Reh anti-hCD 152 scFvIg: Reh
cells transfected with polynucleotide 10A8 scFv IgG MTH (SSS) MT
CH2CH3 (SEQ ID NOs: 270 and 485); Reh anti-hCD28scFvIg: 2E12 scFv
IgG MTH (SSS) MT CH2CH3 (SEQ ID NOs: 268 and 483); Reh
anti-hCD40scFvIg: 4.2.220 scFv IgG MTH (SSS) MT CH2CH3 (SEQ ID NOs:
266 and 481); and Reh anti-hCD20scFvIg: 2H7 scFv IgG MTH (SSS) MT
CH2CH3-CD80.
[0077] FIG. 30 presents ADCC activity of Reh cells that were
transfected with constructs encoding scFv antibodies specific for
human costimulatory molecules, CD152, CD28, CD40, and CD20, as
described for FIG. 29, and for murine CD3, fused to human mutant
IgG1 hinge and mutant CH2 and wild type CH3 (Reh anti-mCD3scFv
designating Reh cells transfected with polynucleotide 500A2 scFv
IgG MTH (SSS) MTCH2WTCH3 SEQ ID NO: 272 and 487)), which was fused
to human CD80 transmembrane and cytoplasmic tail domains. The data
represent the average of quadruplicate samples.
[0078] FIG. 31 lists immunoglobulin constant region constructs that
were used in experiments illustrated in subsequent figures.
[0079] FIG. 32 depicts CDC activity of CTLA-4 Ig fusion proteins,
CTLA-4 IgG WTH (CCC) WTCH2CH3 (SEQ ID NO: 307) (2 .mu.g/ml) and
CTLA-4 IgG MTH MTCH2WTCH3 (SEQ ID NO: 316 and 530) (2 .mu.g/ml), in
the presence and absence of rabbit complement (plus C' and no C',
respectively). The target cells were Reh cells and Reh cells
transfected with CD80 (Reh CD80.10).
[0080] FIG. 33 shows ADCC activity of CTLA-4 Ig fusion proteins,
CTLA-4 IgG WTH (CCC) WTCH2CH3 (SEQ ID NO: 307) (2 .mu.g/ml) and
CTLA-4 IgG MTH MTCH2WTCH3 (SEQ ID NO: 316 and 530) (2 .mu.g/ml).
Effector cells, human PBMC, were added to target cells, Reh or Reh
CD80.1, at the ratios indicated. FIG. 33A presents the level of
natural killing in Reh CD80.1 cells in the absence of any Ig fusion
protein. FIG. 33B presents ADCC mediated by CTLA-4 IgG MTH
MTCH2WTCH3, and FIG. 33C presents ADCC mediated by CTLA-4 IgG WTH
(CCC) WTCH2CH3. Each data point represents the average percent
specific killing measured in four sample wells.
[0081] FIG. 34 illustrates binding of 2H7 (anti-CD20) scFv Ig
fusion proteins to (CD20+) CHO cells by flow
immunocytofluorimetry.
[0082] FIG. 35 presents an immunoblot of 2H7 scFv IgG and IgA
fusion proteins. COS cells were transiently transfected with
various 2H7 scFv Ig fusion protein constructs. The expressed
polypeptides were immune precipitated with protein A, separated in
a non-reducing SDS polyacrylamide gel, and then transferred to a
polyvinyl fluoride membrane. Proteins were detected using an
anti-human IgG (Fc specific) horseradish peroxidase conjugate. Lane
1: vector only; lane 2: 2H7 scFv IgG WTH (CCC) WTCH2CH3 (SEQ ID NO:
15 and 240); lane 3: 2H7 scFv IgG MTH (CSS) WTCH2CH3 (SEQ ID NO:
670); lane 4: 2H7 scFv IgG MTH (SCS) WTCH2CH3 (SEQ ID NO: 671);
lane 5: 2H7 scFv IgAH IgG WTCH2CH3 (SEQ ID NO: 18 and 284); and
lane 6: 2H7 scFv IgG MTH (SSS) WTCH2CH3 (SEQ ID NO: 17, 274 and
489).
[0083] FIG. 36 illustrates binding of 2H7 scFv IgAH IgACH2CH3
polypeptide (SEQ ID NO: 286 and 502) and 2H7 scFv IgAH IgAT4 (SEQ
ID NO: 299) to (CD20+) CHO cells by flow immunocytofluorimetry. The
source of the polypeptides was culture supernatants from
transiently transfected COS cells. COS cells transfected with a
plasmid comprising a sequence encoding 2H7 scFv IgAH IgACH2CH3 were
co-transfected with a plasmid containing nucleotide sequence
encoding human J chain.
[0084] FIG. 37 illustrates ADCC activity of anti-CD20 (2H7) scFv Ig
fusion proteins against BJAB target cells using whole blood as the
source of effector cells. Purified 2H7 scFv Ig fusion proteins were
titrated and combined with .sup.51Cr-labeled BJAB cells
(5.times.10.sup.4) and whole blood (1:4 final dilution). Each data
point represents the average percent specific killing measured in
four sample wells.
[0085] FIG. 38 demonstrates ADCC activity of 2H7 scFv Ig fusion
proteins (5 .mu.g/ml) against .sup.51Cr-labeled BJAB cells at 0.25,
0.125, and 0.625 dilutions of whole blood. Each data point
represents the average percent specific killing measured in four
sample wells.
[0086] FIG. 39 shows a comparison of ADCC activity of 2H7 scFv IgG
MTH (SSS) WTCH2CH3 (5 .mu.g/ml) and 2H7 scFv IgAH IgACH2CH3 (5
.mu.g/ml) when human PBMC are the source of effector cells (FIG.
39A) and when human whole blood is the source of effector cells
(FIG. 39B).
[0087] FIG. 40 presents an immunoblot of 2H7 scFv IgG fusion
proteins. COS cells were transiently transfected with various 2H7
scFv Ig fusion protein constructs. Culture supernatants containing
the expressed polypeptides were separated in a non-reducing SDS
polyacrylamide gel, and then were transferred to a polyvinyl
fluoride membrane. Proteins were detected using an anti-human IgG
(Fc specific) horseradish peroxidase conjugate. Lanes 1-5: purified
2H7 scFv IgG MTH (SSS) WTCH2CH3 at 40 ng, 20 ng, 10 ng/5 ng, and
2.5 ng per lane, respectively. Culture supernatants were separated
in lanes 6-9. Lane 6: 2H7 scFv IgG WTH (CCC) WTCH2CH3; lane 7: 2F17
scFv IgG MTH (CSS) WTCH2CH3; lane 8: 2H7 scFv IgG MTH (SCS)
WTCH2CH3; and lane 9: 2H7 scFv VHSER11 IgG MTH (SSS) WTCH2CH3. The
molecular weight (kDal) of marker proteins is indicated on the left
side of the immunoblot.
[0088] FIG. 41A illustrates cell surface expression of 1D8
(anti-murine 4-1BB) scFv IgG WTH WTCH2CH3-CD80 fusion protein on
K1735 melanoma cells by flow immunofluorimetry. The scFv fusion
protein was detected with phycoerythrin-conjugated F(ab').sub.2
goat anti-human IgG. FIG. 41B depicts growth of tumors in naive C3H
mice transplanted by subcutaneous injection with wild type K1735
melanoma cells (K1735-WT) or with K1735 cells transfected with 1D8
scFv IgG WTH WTCH2CH3-CD80 (K1735-1D8). Tumor growth was monitored
by measuring the size of the tumor. FIG. 41C demonstrates the
kinetics of tumor growth in naive C3H mice injected
intraperitoneally with monoclonal antibodies to remove CD8+, CD4+,
or both CD4+ and CD8+ T cells prior to transplantation of the
animals with K1735-1D8 cells.
[0089] FIG. 42 demonstrates therapy of established K1735-WT tumors
using K1735-1D8 as an immunogen. Six days after mice were
transplanted with K1735-WT tumors, one group (five animals) was
injected subcutaneously with K1735-1D8 cells (open circles) or
irradiated K1735-WT cells (solid squares) on the contralateral
side. A control group of mice received PBS (open squares).
Treatments were repeated on the days indicated by the arrows.
[0090] FIG. 43 shows the growth of tumors in animals that were
injected subcutaneously with 2.times.10.sup.6 K1735-WT cells (solid
squares) and the growth of tumors in animals that were injected
subcutaneously with 2.times.10.sup.6 K1735-WT cells plus
2.times.10.sup.5 K1735-1D8 cells (open triangles).
[0091] FIG. 44 presents a flow cytometry analysis of Ag104 murine
sarcoma tumor cells transfected with 1D8 scFv IgG WTH WTCH2CH3-CD80
isolated after repeated rounds of panning against anti-human IgG.
Transfected cells expressing 1D8 scFv IgG WTH WTCH2CH3-CD80 were
detected with fluoroisothiocyanate (FITC)-conjugated goat
anti-human IgG (depicted in black). Untransfected cells are shown
in gray.
[0092] FIG. 45 illustrates migration of various 2H7 scFv Ig fusion
proteins in a 10% SDS-PAGE gel. 2H7 was the anti-CD20 scFv and
[220] was the anti-CD40 scFv. Lane 1: Bio-Rad prestained molecular
weight standards; lane 2: anti-CD20 scFv IgG MTH (SSS) MTCH2WTCH3;
lane 3: anti-CD20 scFv IgG MTH (SSS) WTCH2CH3; lane 4: 2H7 scFv
IgAH IgG WTCH2CH3; lane 5: anti-CD20-anti-CD40 scFv IgG MTH (SSS)
MTCH2WTCH3; lane 6: Rituximab; lane 7: Novex Multimark.RTM.
molecular weight standards.
[0093] FIG. 46 illustrates effector function as measured in an ADCC
assay of 2H7 Ig fusion proteins that contain a mutant CH2 domain or
wild type CH2 domain. The percent specific killing of BJAB target
cells in the presence of human PBMC effector cells by 2H7 scFv IgG
MTH (SSS) MTCH2WTCH3 (diamonds) was compared to 2H7 scFv IgG MTH
(SSS) WTCH2CH3 (squares) and 2H7 scFv IgAH IgG WTCH2CH3 (triangles)
and Rituximab (circles).
[0094] FIG. 47 shows cell surface expression of an anti-human CD3
scFv IgG WTH WTCH2CH3-CD80 (SEQ ID NO: 560) fusion protein on Reh
cells (FIG. 47A) and T51 lymphoblastoid cells (FIG. 47B) by flow
immunocytofluorimetry.
[0095] FIG. 48 presents the percent specific killing of
untransfected Reh and T51 cells and the percent specific killing of
Reh cells (Reh anti-hCD3) (FIG. 48A) and T51 cells (T51 anti-hCD3)
(FIG. 48B) that were transfected with a construct encoding scFv
antibodies specific for human CD3, fused to human IgG1 wild-type
hinge-CH2-CH3, which was fused to human CD80 transmembrane and
cytoplasmic tail domains (anti-human CD3 scFv IgG WTH WTCH2CH3-CD80
(SEQ ID NO: 560). Human PBMC (effector cells) were combined with
BJAB target cells at the ratios indicated.
[0096] FIG. 49 illustrates binding of 5B9, a murine anti-human
CD137 (4-1BB) monoclonal antibody, and a 5B9 scFv IgG fusion
protein (5B9 scFv IgG MTH (SSS) WTCH2CH3 (SEQ ID NO: 363)) to
stimulated human PBMC. Binding of the 5B9 scFv IgG fusion protein
was detected by flow immunocytofluorimetry using FITC conjugated
goat anti-human IgG. Binding of the 5B9 monoclonal antibody was
detected with FITC conjugated goat anti-mouse IgG.
DETAILED DESCRIPTION
[0097] The present invention is directed to binding
domain-immunoglobulin fusion proteins and to related compositions
and methods, which will be useful in immunotherapeutic and
immunodiagnostic applications, and which offer certain advantages
over antigen-specific polypeptides of the prior art. The fusion
proteins of the present invention are preferably single polypeptide
chains that comprise, in pertinent part, the following fused
domains: a binding domain polypeptide, an immunoglobulin hinge
region polypeptide, an immunoglobulin heavy chain CH2 constant
region polypeptide, and an immunoglobulin heavy chain CH3 constant
region polypeptide. According to certain preferred embodiments the
fusion proteins of the present invention further comprise a plasma
membrane anchor domain. According to certain other preferred
embodiments the fusion proteins of the present invention further
comprise an immunoglobulin heavy chain CH4 constant region
polypeptide. In particularly preferred embodiments, the polypeptide
domains of which the binding domain-immunoglobulin fusion protein
is comprised are, or are derived from, polypeptides that are the
products of human gene sequences, but the invention need not be so
limited and may in fact relate to binding domain-immunoglobulin
fusion proteins as provided herein that are derived from any
natural or artificial source, including genetically engineered
and/or mutated polypeptides.
[0098] The present invention relates in part to the surprising
observation that the binding domain-immunoglobulin fusion proteins
described herein are capable of immunological activity. More
specifically, these proteins retain the ability to participate in
well known immunological effector activities including antibody
dependent cell mediated cytotoxicity (ADCC, e.g., subsequent to
antigen binding on a cell surface, engagement and induction of
cytotoxic effector cells bearing appropriate Fc receptors, such as
natural killer (NK) cells bearing FcR .gamma. III, under
appropriate conditions); and/or complement fixation in complement
dependent cytotoxicity (CDC, e.g., subsequent to antigen binding on
a cell surface, recruitment and activation of cytolytic proteins
that are components of the blood complement cascade; for reviews of
ADCC and CDC see, e.g., Carter, 2001 Nat. Rev. Canc. 1:118; Sulica
et al., 2001 Int. Rev. Immunol. 20:371; Maloney et al., 2002 Semin.
Oncol. 29:2; Sondel et al., 2001; Maloney 2001 Anticanc. Drugs 12
Suppl. 2:1-4; IgA activation of complement by the alternative
pathway is described, for example, in Schneiderman et al., 1990 J.
Immunol. 145:233), despite having structures that would not be
expected to be capable of promoting such effector activities. As
described in greater detail below, ADCC and CDC are unexpected
functions for fusion proteins comprising immunoglobulin heavy chain
regions and having the structures described herein, and in
particular for immunoglobulin fusion proteins comprising
immunoglobulin hinge region polypeptides that are compromised in
their ability to form interchain, homodimeric disulfide bonds.
[0099] Another advantage afforded by the present invention is a
binding domain-immunoglobulin fusion polypeptide that can be
produced in substantial quantities that are typically greater than
those routinely attained with single-chain antibody constructs of
the prior art. In preferred embodiments, the binding
domain-immunoglobulin fusion polypeptides of the present invention
are recombinantly expressed in mammalian expression systems, which
offer the advantage of providing polypeptides that are stable in
vivo (e.g., under physiological conditions). According to
non-limiting theory, such stability may derive in part from
posttranslational modifications, and specifically glycosylation, of
the fusion proteins. Production of the present binding
domain-immunoglobulin fusion proteins via recombinant mammalian
expression has been attained in static cell cultures at a level of
greater than 50 mg protein per liter culture supernatant and has
been routinely observed in such cultures at 10-50 mg/l, such that
preferably at least 10-50 mg/l may be produced under static culture
conditions; also contemplated are enhanced production of the fusion
proteins using art-accepted scale-up methodologies such as "fed
batch" (i.e., non-static) production, where yields of at least
5-500 mg/l, and in some instances at least 0.5-1 gm/l, depending on
the particular protein product, are obtained.
[0100] A binding domain polypeptide according to the present
invention may be any polypeptide that possesses the ability to
specifically recognize and bind to a cognate biological molecule or
complex of more than one molecule or assembly or aggregate, whether
stable or transient, of such a molecule, which includes a protein,
polypeptide, peptide, amino acid, or derivative thereof; a lipid,
fatty acid or the like, or derivative thereof; a carbohydrate,
saccharide or the like or derivative thereof, a nucleic acid,
nucleotide, nucleoside, purine, pyrimidine or related molecule, or
derivative thereof, or the like; or any combination thereof such
as, for example, a glycoprotein, a glycopeptide, a glycolipid, a
lipoprotein, a proteolipid; or any other biological molecule that
may be present in a biological sample. Biological samples may be
provided by obtaining a blood sample, biopsy specimen, tissue
explant, organ culture, biological fluid or any other tissue or
cell preparation from a subject or a biological source. The subject
or biological source may be a human or non-human animal, a primary
cell culture or culture adapted cell line including but not limited
to genetically engineered cell lines that may contain chromosomally
integrated or episomal recombinant nucleic acid sequences,
immortalized or immortalizable cell lines, somatic cell hybrid cell
lines, differentiated or differentiatable cell lines, transformed
cell lines and the like. In certain preferred embodiments of the
invention, the subject or biological source may be suspected of
having or being at risk for having a malignant condition or a
B-cell disorder as provided herein, which in certain further
preferred embodiments may be an autoimmune disease, and in certain
other preferred embodiments of the invention the subject or
biological source may be known to be free of a risk or presence of
such disease.
[0101] A binding domain polypeptide may therefore be any naturally
occurring or recombinantly produced binding partner for a cognate
biological molecule as provided herein that is a target structure
of interest, herein referred to as an "antigen" but intended
according to the present disclosure to encompass any target
biological molecule to which it is desirable to have the subject
invention fusion protein specifically bind. Binding
domain-immunoglobulin fusion proteins are defined to be
"immunospecific" or capable of specifically binding if they bind a
desired target molecule such as an antigen as provided herein, with
a K.sub.a of greater than or equal to about 10.sup.4 M-.sup.1,
preferably of greater than or equal to about 10.sup.5 M-.sup.1,
more preferably of greater than or equal to about 10.sup.6 M-.sup.1
and still more preferably of greater than or equal to about
10.sup.7 M-.sup.1. Affinities of binding domain-immunoglobulin
fusion proteins according to the present invention can be readily
determined using conventional techniques, for example those
described by Scatchard et al., Ann. N.Y. Acad. Sci. 51:660 (1949).
Such determination of fusion protein binding to target antigens of
interest can also be performed using any of a number of known
methods for identifying and obtaining proteins that specifically
interact with other proteins or polypeptides, for example, a yeast
two-hybrid screening system such as that described in U.S. Pat. No.
5,283,173 and U.S. Pat. No. 5,468,614, or the equivalent.
[0102] Preferred embodiments of the subject invention binding
domain-immunoglobulin fusion protein comprise binding domains that
include at least one immunoglobulin variable region polypeptide,
such as all or a portion or fragment of a heavy chain or a light
chain V-region, provided it is capable of specifically binding an
antigen or other desired target structure of interest as described
herein. In other preferred embodiments the binding domain comprises
a single chain immunoglobulin-derived Fv product, which may include
all or a portion of at least one immunoglobulin light chain
V-region and all or a portion of at least one immunoglobulin heavy
chain V-region, and which further comprises a linker fused to the
V-regions; preparation and testing such constructs are described in
greater detail herein and are well known in the art. As described
herein and as also known in the art, immunoglobulins comprise
products of a gene family the members of which exhibit a high
degree of sequence conservation, such that amino acid sequences of
two or more immunoglobulins or immunoglobulin domains or regions or
portions thereof (e.g., VH domains, VL domains, hinge regions, CH2
constant regions, CH3 constant regions) can be aligned and analyzed
to identify portions of such sequences that correspond to one
another, for instance, by exhibiting pronounced sequence homology.
Determination of sequence homology may be readily determined with
any of a number of sequence alignment and analysis tools, including
computer algorithms well known to those of ordinary skill in the
art, such as Align or the BLAST algorithm (Altschul, J. Mol. Biol.
219:555-565, 1991; Henikoff and Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915-10919, 1992), which is available at the NCBI website
(http://www/ncbi.nlm.nih.gov/cgi-bin/BLAST). Default parameters may
be used.
[0103] Portions of a particular immunoglobulin reference sequence
and of any one or more additional immunoglobulin sequences of
interest that may be compared to the reference sequence are
regarded as "corresponding" sequences, regions, fragments or the
like, based on the convention for numbering immunoglobulin amino
acid positions according to Kabat, Sequences of Proteins of
Immunological Interest, (5.sup.th ed. Bethesda, Md.: Public Health
Service, National Institutes of Health (1991)). For example,
according to this convention, the immunoglobulin family to which an
immunoglobulin sequence of interest belongs is determined based on
conservation of variable region polypeptide sequence invariant
amino acid residues, to identify a particular numbering system for
the immunoglobulin family, and the sequence(s) of interest can then
be aligned to assign sequence position numbers to the individual
amino acids which comprise such sequence(s). Preferably at least
70%, more preferably at least 80%-85% or 86%-89%, and still more
preferably at least 90%, 92%, 94%, 96%, 98% or 99% of the amino
acids in a given amino acid sequence of at least 1000, more
preferably 700-950, more preferably 350-700, still more preferably
100-350, still more preferably 80-100, 70-80, 60-70, 50-60, 40-50
or 30-40 consecutive amino acids of a sequence, are identical to
the amino acids located at corresponding positions in a reference
sequence such as those disclosed by Kabat (1991) or in a similar
compendium of related immunoglobulin sequences, such as may be
generated from public databases (e.g., Genbank, SwissProt, etc.)
using sequence alignment tools as described above. In certain
preferred embodiments, an immunoglobulin sequence of interest or a
region, portion, derivative or fragment thereof is greater than 95%
identical to a corresponding reference sequence, and in certain
preferred embodiments such a sequence of interest may differ from a
corresponding reference at no more than 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10 amino acid positions.
[0104] For example, in certain embodiments the present invention is
directed to a binding domain-immunoglobulin fusion protein
comprising in pertinent part a human immunoglobulin heavy chain
variable region polypeptide comprising a mutation at an amino acid
at a location corresponding to amino acid position 11 in the VH
domain, or amino acid position 155 in SEQ ID NO: 11, position 158
in SEQ ID NO: 12, position 154 in SEQ ID NO:13, or position 159 in
SEQ ID NO 14, or amino acid position 11 in the VH domain
polypeptides listed in SEQ ID NOS: 341, 354, 465, 471, 477, 548
which comprises a murine VH-derived sequence, and regarding which
it is noteworthy that at a relatively limited number of
immunoglobulin VH sequence positions, including position 11, amino
acid conservation is observed in the overwhelming majority of VH
sequences analyzed across mammalian species lines (e.g., Leul 1,
Va137, Gly44, Leu45, Trp47; Nguyen et al., 1998 J. Mol. Biol.
275:413). These amino acid side chains are located at the surface
of the variable domain (VH), where they may contact residues of the
CH1 (Leull) and the VL domains (Va137, Gly44, Leu45, and Trp47) and
may, in the absence of light chains, contribute to stability and
solubility of the protein (see, e.g., Chothia et al., 1985 J. Mol.
Biol. 186:651; Muyldermans et al., 1994 Prot. Engineer. 7:1129;
Desmyter et al., 1996 Nat. Struct. Biol. 3:803; Davies et al., 1994
FEES Lett. 339:285). As another example, by reference to
immunoglobulin sequence compendia and databases such as those cited
above, the relatedness of two or more immunoglobulin sequences to
each other can readily and without undue experimentation be
established in a manner that permits identification of the animal
species of origin, the class and subclass (e.g., isotype) of a
particular immunoglobulin or immunoglobulin region polypeptide
sequence. Any immunoglobulin variable region polypeptide sequence,
including VH and/or VL and/or single-chain variable region (sFv)
sequences or other V region-derived sequences or the like, may be
used as a binding domain. Preferred embodiments include
immunoglobulin V region polypeptide sequences derived from
monoclonal antibodies such as murine or other rodent antibodies, or
monoclonal antibodies derived from other sources such as goat,
rabbit, equine, bovine, camelid or other species, including
transgenic animals, and also including human or humanized
monoclonal antibodies. Non-limiting examples include variable
region polypeptide sequences derived from mAb such as those
described in greater detail in the Examples below, for instance,
CD20-specific murine monoclonal antibodies (e.g., 2H7), mAb L6
(specific for a carbohydrate-defined epitope and available from
American Type Culture Collection, Manassas, Va., as hybridoma
HB8677), and mAb specific for CD28 (e.g., mAb 2E12), CD40, CD80,
CD137 (e.g., mAb 5B9 or mAb 1D8 which recognizes the human
homologue of CD137, 41BB) and CD152 (CTLA-4).
[0105] Other binding domain polypeptides may comprise any protein
or portion thereof that retains the ability to specifically bind an
antigen as provided herein, including non-immunoglobulins.
Accordingly the invention contemplates fusion proteins comprising
binding domain polypeptides that are derived from polypeptide
ligands such as hormones, cytokines, chemokines, and the like; cell
surface or soluble receptors for such polypeptide ligands; lectins;
intercellular adhesion receptors such as specific leukocyte
integrins, selectins, immunoglobulin gene superfamily members,
intercellular adhesion molecules (ICAM-1, -2, -3) and the like;
histocompatibility antigens; etc.
[0106] Examples of cell surface receptors that may provide a
binding domain polypeptide, and that may also be selected as the
target molecule or antigen to which a binding domain-Ig fusion
protein of the present invention desirably binds, include the
following, or the like: HER1 (e.g., GenBank Accession Nos. U48722,
SEG HEGFREXS, K03193), HER2 (Yoshino et al., 1994 J. Immunol.
152:2393; Disis et al., 1994 Canc. Res. 54:16; see also, e.g.,
GenBank Acc. Nos. X03363, M17730, SEG HUMHER20), HER3 (e.g.,
GenBank Acc. Nos. U29339, M34309), HER4 (Plowman et al., 1993
Nature 366:473; see also e.g., GenBank Acc. Nos. L07868, T64105),
epidermal growth factor receptor (EGFR) (e.g., GenBank Acc. Nos.
U48722, SEG HEGFREXS, K03193), vascular endothelial cell growth
factor (e.g., GenBank No. M32977), vascular endothelial cell growth
factor receptor (e.g., GenBank Acc. Nos. AF022375, 1680143, U48801,
X62568), insulin-like growth factor-I (e.g., GenBank Acc. Nos.
X00173, X56774, X56773, X06043, see also European Patent No. GB
2241703), insulin-like growth factor-II (e.g., GenBank Acc. Nos.
X03562, X00910, SEG HUMGFIA, SEG HUMGFI2, M17863, M17862),
transferrin receptor (Trowbridge and Omary, 1981 Proc. Nat. Acad.
USA 78:3039; see also e.g., GenBank Acc. Nos. X01060, Ml 1507),
estrogen receptor (e.g., GenBank Acc. Nos. M38651, X03635, X99101,
U47678, M12674), progesterone receptor (e.g., GenBank Acc. Nos.
X51730, X69068, M15716), follicle stimulating hormone receptor
(FSH-R) (e.g., GenBank Acc. Nos. 234260, M65085), retinoic acid
receptor (e.g., GenBank Ace. Nos. L12060, M60909, X77664, X57280,
X07282, X06538), MUC-1 (Barnes et al., 1989 Proc. Nat. Acad. Sci.
USA 86:7159; see also e.g., GenBank Acc. Nos. SEG MUSMUCIO, M65132,
M64928) NY-ESO-1 (e.g., GenBank Acc. Nos. AJ003149, U87459), NA
17-A (e.g., European Patent No. WO 96/40039), Melan-A/MART-1
(Kawakami et al., 1994 Proc. Nat. Acad. Sci. USA 91:3515; see also
e.g., GenBank Acc. Nos. U06654, U06452), tyrosinase (Topalian et
al., 1994 Proc. Nat. Acad. Sci. USA 91:9461; see also e.g., GenBank
Acc. Nos. M26729, SEG HUMTYRO, see also Weber et al., J. Clin.
Invest (1998) 102:1258), Gp-100 (Kawakami et al., 1994 Proc. Nat.
Acad. Sci. USA 91:3515; see also e.g., GenBank Acc. No. 573003, see
also European Patent No. EP 668350; Adema et al., 1994 J. Biol.
Chem. 269:20126), MAGE (van den Bruggen et al., 1991 Science
254:1643; see also, e.g., GenBank Acc. Nos. U93163, AF064589,
U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690,
U10689, U10688, U10687, U10686, U10685, L18877, U10340, U10339,
L18920, UO3735, M77481), BAGE (e.g., GenBank Acc. No. U19180, see
also U.S. Pat. Nos. 5,683,886 and 5,571,711), GAGE (e.g., GenBank
Acc. Nos. AF055475, AF055474, AF055473, U19147, U19146, U19145,
U19144, U19143, U19142), any of the CTA class of receptors
including in particular HOM-MEL-40 antigen encoded by the SSX2 gene
(e.g., GenBank Acc. Nos. X86175, U90842, U90841, X86174),
carcinoembyonic antigen (CEA, Gold and Freedman, 1985 J. Exp. Med.
121:439; see also e.g., GenBank Acc. Nos. SEG HUMCEA, M59710,
M59255, M29540), and PyLT (e.g., GenBank Acc. Nos. J02289,
J02038).
[0107] Additional cell surface receptors that may be sources of
binding domain polypeptides or that may be cognate antigens include
the following, or the like: CD2 (e.g., GenBank Acc. Nos. Y00023,
SEG_HUMCD2, M16336, M16445, SEG_MUSCD2, M14362), 4-1BB (CDw137,
Kwon et al., 1989 Proc. Nat. Acad. Sci. USA 86:1963, 4-1BB ligand
(Goodwin et al., 1993 Eur. J. Immunol. 23:2361; Melero et al., 1998
Eur. J. Immunol. 3:116), CD5 (e.g., GenBank Acc. Nos. X78985,
X89405), CD10 (e.g., GenBank Acc. Nos. M81591, X76732) CD27 (e.g.,
GenBank Acc. Nos. M63928, L24495, L08096), CD28 (June et al., 1990
Immunol. Today 11:211; see also, e.g., GenBank Acc. Nos. J02988,
SEG HUMCD28, M34563), CD152/CTLA-4 (e.g., GenBank Acc. Nos. L15006,
X05719, SEG HUMIGCTL), CD40 (e.g., GenBank Acc. Nos. M83312, SEG
MUSC040A0, Y10507, X67878, X96710, U15637, L07414),
interferon-.gamma. (IFN-.gamma.; see, e.g., Farrar et al. 1993 Ann.
Rev. Immunol. 11:571 and references cited therein, Gray et al. 1982
Nature 295:503, Rinderknecht et al. 1984 J. Biol. Chem. 259:6790,
DeGrado et al. 1982 Nature 300:379), interleukin-4 (IL-4; see,
e.g., 53.sup.rd Forum in Immunology, 1993 Research in Immunol.
144:553-643; Banchereau et al., 1994 in The Cytokine Handbook,
2.sup.nd ed., A. Thomson, ed., Academic Press, NY, p. 99; Keegan et
al., 1994 J Leukocyt. Biol. 55:272, and references cited therein),
interleukin-17 (IL-17) (e.g., GenBank Acc. Nos. U32659, U43088) and
interleukin-17 receptor (IL-17R) (e.g., GenBank Acc. Nos. U31993,
U58917). Notwithstanding the foregoing, the present invention
expressly does not encompass any immunoglobulin fusion protein that
is disclosed in U.S. Pat. No. 5,807,734, U.S. Pat. No. 5,795,572 or
U.S. Pat. No. 5,807,734.
[0108] Additional cell surface receptors that may be sources of
binding domain polypeptides or that may be cognate antigens include
the following, or the like: CD59 (e.g., GenBank Acc. Nos. SEG
HUMCD590, M95708, M34671), CD48 (e.g., GenBank Acc. Nos. M59904),
CD58/LFA-3 (e.g., GenBank Acc. No. A25933, Y00636, E12817; see also
JP 1997075090-A), CD72 (e.g., GenBank Acc. Nos. AA311036, 540777,
L35772), CD70 (e.g., GenBank Acc. Nos. Y13636, S69339), CD80/B7.1
(Freeman et al., 1989 J. Immunol. 43:2714; Freeman et al., 1991 J.
Exp. Med. 174:625; see also e.g., GenBank Acc. Nos. U33208,
1683379), CD86/B7.2 (Freeman et al., 1993 J. Exp. Med. 178:2185,
Boriello et al., 1995 J. Immunol. 155:5490; see also, e.g., GenBank
Acc. Nos. AF099105, SEG_MMB72G, U39466, U04343, SEG_HSB725, L25606,
L25259), B7-H1/B7-DC (e.g., Genbank Acc. Nos. NM.sub.--014143,
AF177937, AF317088; Dong et al., 2002 Nat. Med. June 24 [epub ahead
of print], PMID 12091876; Tseng et al., 2001 J. Exp. Med. 193:839;
Tamura et al., 2001 Blood 97:1809; Dong et al., 1999 Nat. Med.
5:1365), CD40 ligand (e.g., GenBank Acc. Nos. SEG HUMCD40L, X67878,
X65453, L07414), IL-17 (e.g., GenBank Acc. Nos. U32659, U43088),
CD43 (e.g., GenBank Acc. Nos. X52075, J04536), ICOS (e.g., Genbank
Acc. No. AH011568), CD3 (e.g., Genbank Acc. Nos. NM 000073 (gamma
subunit), NM 000733 (epsilon subunit), X73617 (delta subunit)), CD4
(e.g., Genbank Acc. No. NM 000616), CD25 (e.g., Genbank Acc. No. NM
000417), CD8 (e.g., Genbank Acc. No. M12828), CD11b (e.g., Genbank
Acc. No. J03925), CD14 (e.g., Genbank Acc. No. XM 039364), CD56
(e.g., Genbank Acc. No. U63041), CD69 (e.g., Genbank Acc. No. NM
001781) and VLA-4 (a-4(37) (e.g., GenBank Acc. Nos. L12002, X16983,
L20788, U97031, L24913, M68892, M95632). The following cell surface
receptors are typically associated with B cells: CD19 (e.g.,
GenBank Acc. Nos. SEG_HUMCD19WO, M84371, SEG MUSCD19W, M62542),
CD20 (e.g., GenBank Acc. Nos. SEG_HUMCD20, M62541), CD22 (e.g.,
GenBank Acc. Nos. 1680629, Y10210, X59350, U62631, X52782, L16928),
CD30 (e.g., Genbank Acc. Nos. M83554, D86042), CD153 (CD30 ligand,
e.g., GenBank Acc. Nos. L09753, M83554), CD37 (e.g., GenBank Acc.
Nos. SEG MMCD37X, X14046, X53517), CD50 (ICAM-3, e.g., GenBank Acc.
No. NM 002162), CD106 (VCAM-1) (e.g., GenBank Acc. Nos. X53051,
X67783, SEG_MMVCAM1C, see also U.S. Pat. No. 5,596,090), CD54
(ICAM-1) (e.g., GenBank Acc. Nos. X84737, 582847, X06990, J03132,
SEG MUSICAMO), interleukin-12 (see, e.g., Reiter et al, 1993 Crit.
Rev. Immunol. 13:1, and references cited therein), CD134 (OX40,
e.g., GenBank Acc. No. AJ277151), CD137 (41BB, e.g., GenBank Acc.
No. L12964, NM.sub.--001561), CD83 (e.g., GenBank Acc. Nos.
AF001036, AL021918), DEC-205 (e.g., GenBank Acc. Nos. AF011333,
U19271).
[0109] Binding domain-immunoglobulin fusion proteins of the present
invention comprise a binding domain polypeptide that, according to
certain particularly preferred embodiments, is capable of
specifically binding at least one antigen that is present on an
immune effector cell. According to non-limiting theory, such
binding domain-Ig fusion proteins may advantageously recruit
desired immune effector cell function(s) in a therapeutic context,
where it is well known that immune effector cells having different
specialized immune functions can be distinguished from one another
on the basis of their differential expression of a wide variety of
cell surface antigens, such as many of the antigens described
herein to which binding domain polypeptides can specifically bind.
Immune effector cells include any cell that is capable of directly
mediating an activity which is a component of immune system
function, including cells having such capability naturally or as a
result of genetic engineering.
[0110] In certain embodiments an immune effector cell comprises a
cell surface receptor for an immunoglobulin, such as a receptor for
an immunoglobulin constant region and including the class of
receptors commonly referred to as "Fc receptors" (FcR). A number of
FcR have been structurally and/or functionally characterized and
are well known in the art, including FcR having specific abilities
to interact with a restricted subset of immunoglobulin heavy chain
isotypes, or that interact with Fc domains with varying affinities,
and/or which may be expressed on restricted subsets of immune
effector cells under certain conditions (e.g., Kijimoto-Ochichai et
al., 2002 Cell Mol. Life. Sci. 59:648; Davis et al., 2002 Curr.
Top. Microbiol. Immunol. 266:85; Pawankar, 2001 Curr. Opin. Allerg.
Clin. Immunol. 1:3; Radaev et al., 2002 Mol. Immunol. 38:1073;
Wurzburg et al., 2002 Mol. Immunol. 38:1063; Sulica et al., 2001
Int. Rev. Immunol. 20:371; Underhill et al., 2002 Ann. Rev.
Immunol. 20:825; Coggeshall, 2002 Curr. Dir. Autoimm. 5:1; Mimura
et al., 2001 Adv. Exp. Med. Biol. 495:49; Baumann et al., 2001 Adv.
Exp. Med. Biol. 495:219; Santoso et al., 2001 Ital. Heart J. 2:811;
Novak et al., 2001 Curr. Opin. Immunol. 13:721; Fossati et al.,
2001 Eur. J. Clin. Invest. 31:821).
[0111] Cells that are capable of mediating ADCC are preferred
examples of immune effector cells according to the present
invention. Other preferred examples include natural killer (NK)
cells, tumor-infiltrating T lymphocytes (TIL), cytotoxic T
lymphocytes (CTL), and granulocytic cells such as cells that
comprise allergic response mechanisms. Immune effector cells thus
include, but are not limited to, cells of hematopoietic origins
including cells at various stages of differentiation within myeloid
and lymphoid lineages and which may (but need not) express one or
more types of functional cell surface FcR, such as T lymphocytes, B
lymphocytes, NK cells, monocytes, macrophages, dendritic cells,
neutrophils, basophils, eosinophils, mast cells, platelets,
erythrocytes, and precursors, progenitors (e.g., hematopoietic stem
cells), quiescent, activated and mature forms of such cells. Other
immune effector cells may include cells of non-hematopoietic origin
that are capable of mediating immune functions, for example,
endothelial cells, keratinocytes, fibroblasts, osteoclasts,
epithelial cells and other cells. Immune effector cells may also
include cells that mediate cytotoxic or cytostatic events, or
endocytic, phagocytic, or pinocytotic events, or that effect
induction of apoptosis, or that effect microbial immunity or
neutralization of microbial infection, or cells that mediate
allergic, inflammatory, hypersensitivity and/or autoimmune
reactions.
[0112] Allergic response mechanisms are well known in the art and
include an antigen (e.g., allergen)-specific component such as an
immunoglobulin (e.g., IgE), as well as the cells and mediators
which comprise sequelae to allergen-immunoglobulin (e.g., IgE)
encounters (e.g., Ott et al., 2000 J. Allerg. Clin. Immunol.
106:429; Barnes, 2000 J. Allerg. Clin. Immunol. 106:5; Togias, 2000
J. Allerg. Clin. Immunol. 105:S599; Akdis et al., 2000 Int. Arch.
Allerg. Immunol. 121:261; Beach, 2000 Occup. Med. 15:455).
Particularly with regard to binding domain-immunoglobulin fusion
proteins of the present invention that interact with FcR, certain
embodiments of the present invention contemplate fusion proteins
that comprise one or more IgE-derived domains and that are capable
of inducing an allergic response mechanism that comprises
IgE-specific FcR, as also noted above and as described in the cited
references. Without wishing to be bound by theory, and as disclosed
herein, fusion proteins of the present invention may comprise
portions of IgE heavy chain Fc domain polypeptides, whether
expressed as cell surface proteins (e.g., with a plasma membrane
anchor domain) or as soluble proteins (e.g., without a plasma
membrane anchor domain). Further according to non-limiting theory,
recruitment and induction of an allergic response mechanism (e.g.,
an FcR-epsilon expressing immune effector cell) may proceed as the
result of either or both of the presence of an IgE Fc domain (e.g.,
that is capable of triggering an allergic mechanism by FcR
crosslinking) and the presence of the cognate antigen to which the
binding domain specifically binds. The present invention therefore
exploits induction of allergic response mechanisms in heretofore
unappreciated contexts, such as treatment of a malignant condition
or a B-cell disorder as described herein.
[0113] An immunoglobulin hinge region polypeptide, as discussed
above, includes any hinge peptide or polypeptide that occurs
naturally, as an artificial peptide or as the result of genetic
engineering and that is situated in an immunoglobulin heavy chain
polypeptide between the amino acid residues responsible for forming
intrachain immunoglobulin-domain disulfide bonds in CH1 and CH2
regions; hinge region polypeptides for use in the present invention
may also include a mutated hinge region polypeptide. Accordingly,
an immunoglobulin hinge region polypeptide may be derived from, or
may be a portion or fragment of (i.e., one or more amino acids in
peptide linkage, typically 15-115 amino acids, preferably 95-110,
80-94, 60-80, or 5-65 amino acids, preferably 10-50, more
preferably 15-35, still more preferably 18-32, still more
preferably 20-30, still more preferably 21, 22, 23, 24, 25, 26, 27,
28 or 29 amino acids) an immunoglobulin polypeptide chain region
classically regarded as having hinge function, as described above,
but a hinge region polypeptide for use in the instant invention
need not be so restricted and may include amino acids situated
(according to structural criteria for assigning a particular
residue to a particular domain that may vary, as known in the art)
in an adjoining immunoglobulin domain such as a CH1 domain or a CH2
domain, or in the case of certain artificially engineered
immunoglobulin constructs, an immunoglobulin variable region
domain.
[0114] Wild-type immunoglobulin hinge region polypeptides include
any naturally occurring hinge region that is located between the
constant region domains, CH1 and CH2, of an immunoglobulin. The
wild-type immunoglobulin hinge region polypeptide is preferably a
human immunoglobulin hinge region polypeptide, preferably
comprising a hinge region from a human IgG, IgA or IgE
immunoglobulin, and more preferably, a hinge region polypeptide
from a wild-type or mutated human IgG1 isotype as described herein.
As is known to the art, despite the tremendous overall diversity in
immunoglobulin amino acid sequences, immunoglobulin primary
structure exhibits a high degree of sequence conservation in
particular portions of immunoglobulin polypeptide chains, notably
with regard to the occurrence of cysteine residues which, by virtue
of their sulfhydryl groups, offer the potential for disulfide bond
formation with other available sulfhydryl groups. Accordingly, in
the context of the present invention wild-type immunoglobulin hinge
region polypeptides may be regarded as those that feature one or
more highly conserved (e.g., prevalent in a population in a
statistically significant manner) cysteine residues, and in certain
preferred embodiments a mutated hinge region polypeptide may be
selected that contains zero or one cysteine residue and that is
derived from such a wild-type hinge region.
[0115] In certain preferred embodiments wherein the hinge region
polypeptide is a mutated human IgG1 immunoglobulin hinge region
polypeptide that is derived from a wild-type hinge region, it is
noted that the wild-type human IgG1 hinge region polypeptide
sequence comprises three non-adjacent cysteine residues, referred
to as a first cysteine of the wild-type hinge region, a second
cysteine of the wild-type hinge region and a third cysteine of the
wild-type hinge region, respectively, proceeding along the hinge
region sequence from the polypeptide N-terminus toward the
C-terminus. Accordingly, in certain such embodiments of the present
invention, the mutated human IgG1 immunoglobulin hinge region
polypeptide contains two cysteine residues and the first cysteine
of the wild-type hinge region is not mutated. In certain other
embodiments of the present invention the mutated human IgG1
immunoglobulin hinge region polypeptide contains no more than one
cysteine residue, and in certain other embodiments the mutated
human IgG1 immunoglobulin hinge region polypeptide contains no
cysteine residues.
[0116] The binding domain-immunoglobulin fusion proteins of the
present invention expressly do not contemplate any fusion protein
that is disclosed in U.S. Pat. No. 5,892,019. For example, and as
disclosed in U.S. Pat. No. 5,892,019, a mutated human IgG1 hinge
region described therein has a substitution or deletion of the
first IgG1 hinge region cysteine residue, but retains both of the
second and third IgG1 hinge region cysteine residues that
correspond to the second and third cysteines of the wild-type IgG1
hinge region sequence. This reference discloses that the first
cysteine residue of the wild-type IgG1 hinge region is replaced to
prevent interference, by the first cysteine residue, with proper
assembly of the single chain immunoglobulin-like polypeptide
described therein into an immunoglobulin-like dimer. As also
disclosed in this reference, the second and third cysteines of the
IgG1 hinge region are retained to provide interchain disulfide
linkage between two heavy chain constant regions to promote dimer
formation, which further according to U.S. Pat. No. 5,892,019
results in an immunoglobulin-like dimer having effector function
such as ADCC capability.
[0117] By contrast and as described herein, the binding
domain-immunogloblin fusion proteins of the present invention,
which are capable of ADCC, are not so limited and may comprise, in
pertinent part, (i) a wild-type human IgG1 immunoglobulin hinge
region polypeptide, (ii) a mutated human IgG1 immunoglobulin hinge
region polypeptide that is derived from a wild-type immunoglobulin
hinge region polypeptide having three or more cysteine residues,
wherein the mutated human IgG1 immunoglobulin hinge region
polypeptide contains two cysteine residues and wherein a first
cysteine of the wild-type hinge region is not mutated, (iii) a
mutated human IgG1 immunoglobulin hinge region polypeptide that is
derived from a wild-type immunoglobulin hinge region polypeptide
having three or more cysteine residues, wherein the mutated human
IgG1 immunoglobulin hinge region polypeptide contains no more than
one cysteine residue, or (iv) a mutated human IgG1 immunoglobulin
hinge region polypeptide that is derived from a wild-type
immunoglobulin hinge region polypeptide having three or more
cysteine residues, wherein the mutated human IgG1 immunoglobulin
hinge region polypeptide contains no cysteine residues. In
particular, the present invention thus offers unexpected advantages
associated with retention by the fusion proteins described herein
of the ability to mediate ADCC even where the ability to dimerize
via IgG1 hinge region interchain disulfide bonds is ablated or
compromised by the removal or replacement of one, two or three
hinge region cysteine residues, and even where the first cysteine
of the IgG1 hinge region is not mutated.
[0118] A mutated immunoglobulin hinge region polypeptide may
comprise a hinge region that has its origin in an immunoglobulin of
a species, of an immunoglobulin isotype or class, or of an
immunoglobulin subclass that is different from that of the CH2 and
CH3 domains. For instance, in certain embodiments of the invention,
the binding domain-immunoglobulin fusion protein may comprise a
binding domain polypeptide that is fused to an immunoglobulin hinge
region polypeptide comprising a wild-type human IgA hinge region
polypeptide, or a mutated human IgA hinge region polypeptide that
contains zero or only one cysteine residues, as described herein,
or a wild-type human IgG1 hinge region polypeptide or a wild-type
human IgE hinge region polypeptide or a mutated human IgG1 hinge
region polypeptide that is mutated to contain zero, one or two
cysteine residues wherein the first cysteine of the wild-type hinge
region is not mutated, as also described herein. Such a hinge
region polypeptide may be fused to an immunoglobulin heavy chain
CH2 region polypeptide from a different Ig isotype or class, for
example an IgA or an IgE or an IgG subclass, which in certain
preferred embodiments will be the IgG1 subclass and in certain
other preferred embodiments may be any one of the IgG2, IgG3 or
IgG4 subclasses.
[0119] For example, and as described in greater detail below, in
certain embodiments of the present invention an immunoglobulin
hinge region polypeptide is selected which is derived from a
wild-type human IgA hinge region that naturally comprises three
cysteines, where the selected hinge region polypeptide is truncated
relative to the complete hinge region such that only one of the
cysteine residues remains (e.g., SEQ ID NOS:35-36). Similarly, in
certain other embodiments of the invention, the binding
domain-immunoglobulin fusion protein comprises a binding domain
polypeptide that is fused to an immunoglobulin hinge region
polypeptide comprising a mutated hinge region polypeptide in which
the number of cysteine residues is reduced by amino acid
substitution or deletion, for example a mutated IgG1 hinge region
containing zero, one or two cysteine residues as described herein.
A mutated hinge region polypeptide may thus be derived from a
wild-type immunoglobulin hinge region that contains one or more
cysteine residues. In certain embodiments, a mutated hinge region
polypeptide may contain zero or only one cysteine residue, wherein
the mutated hinge region polypeptide is derived from a wild type
immunoglobulin hinge region that contains, respectively, one or
more or two or more cysteine residues. In the mutated hinge region
polypeptide, the cysteine residues of the wild-type immunoglobulin
hinge region are preferably substituted with amino acids that are
incapable of forming a disulfide bond. In one embodiment of the
invention, the mutated hinge region polypeptide is derived from a
human IgG wild-type hinge region polypeptide, which may include any
of the four human IgG isotype subclasses, IgG1, IgG2, IgG3 or IgG4.
In certain preferred embodiments, the mutated hinge region
polypeptide is derived from a human IgG1 wild-type hinge region
polypeptide. By way of example, a mutated hinge region polypeptide
derived from a human IgG1 wild-type hinge region polypeptide may
comprise mutations at two of the three cysteine residues in the
wild-type immunoglobulin hinge region, or mutations at all three
cysteine residues.
[0120] The cysteine residues that are present in a wild-type
immunoglobulin hinge region and that are removed by mutagenesis
according to particularly preferred embodiments of the present
invention include cysteine residues that form, or that are capable
of forming, interchain disulfide bonds. Without wishing to be bound
by theory, the present invention contemplates that mutation of such
hinge region cysteine residues, which are believed to be involved
in formation of interchain disulfide bridges, reduces the ability
of the subject invention binding domain-immunoglobulin fusion
protein to dimerize (or form higher oligomers) via interchain
disulfide bond formation, while surprisingly not ablating the
ability of the fusion protein to promote antibody dependent
cell-mediated cytotoxicity (ADCC) or to fix complement. In
particular, the Fc receptors (FcR) which mediate ADCC (e.g.,
FcRIII, CD16) exhibit low affinity for immunoglobulin Fc domains,
suggesting that functional binding of Fc to FcR requires avidity
stabilization of the Fc-FcR complex by virtue of the dimeric
structure of heavy chains in a conventional antibody, and/or FcR
aggregation and cross-linking by a conventional Ab Fc structure.
(Sonderman et al., 2000 Nature 406:267; Radaev et al., 2001 J.
Biol. Chem. 276:16469; Radaev et al., 2001 J. Biol. Chem.
276:16478; Koolwijk et al., 1989 J. Immunol. 143:1656; Kato et al.,
2000 Immunol. Today 21:310.) Hence, the binding
domain-immunoglobulin fusion proteins of the present invention
provide the advantages associated with single-chain immunoglobulin
fusion proteins while also unexpectedly retaining immunological
activity. Similarly, the ability to fix complement is typically
associated with immunoglobulins that are dimeric with respect to
heavy chain constant regions such as those that comprise Fc, while
the binding domain-immunoglobulin fusion proteins of the present
invention, which may, due to the replacement or deletion of hinge
region cysteine residues or due to other structural modifications
as described herein, have compromised or ablated abilities to form
interchain disulfide bonds, exhibit the unexpected ability to fix
complement. Additionally, according to certain embodiments of the
present invention wherein a binding domain-immunoglobulin fusion
protein may comprise one or more of a human IgE hinge region
polypeptide, a human IgE CH2 constant region polypeptide, a human
IgE CH3 constant region polypeptide, and a human IgE CH4 constant
region polypeptide, the invention fusion proteins unexpectedly
retain the immunological activity of mediating ADCC and/or of
inducing an allergic response mechanism.
[0121] Selection of an immunoglobulin hinge region polypeptide
according to certain embodiments of the subject invention binding
domain-immunoglobulin fusion proteins may relate to the use of an
"alternative hinge region" polypeptide sequence, which includes a
polypeptide sequence that is not necessarily derived from any
immunoglobulin hinge region sequence per se. Instead, an
alternative hinge region refers to a hinge region polypeptide that
comprises an amino acid sequence of at least ten consecutive amino
acids, and in certain embodiments at least 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21-25, 26-30, 31-50, 51-60, 71-80, 81-90, or 91-110
amino acids that is present in a sequence selected from any one of
SEQ ID NOS: 215, 216, 217, 218, 223, 224, 6, 15, 16. 35, 36, 37,
41, 207, 208, 223, 275, 276, 277, 296, 300, 350, 390, 391, 392,
396, 397, 398, 488, 582, 584, 586, for example a polypeptide
sequence derived from a region located between intrachain
disulfide-generated immunoglobulin-like loop domains of
immunoglobulin gene superfamily members such as CD2 (e.g., Genbank
Acc. No. NM.sub.--001767), CD4 (e.g., Genbank Acc. No.
NM.sub.--000616), CD5 (e.g., Genbank Acc. No. BC027901), CD6 (e.g.,
Genbank Acc. No. NM.sub.--006725), CD7 (e.g., Genbank Acc. Nos.
XM.sub.--046782, BC009293, NM.sub.--006137) or CD8 (e.g., Genbank
Acc. No. M12828), or other Ig superfamily members. By way of
non-limiting example, an alternative hinge region may provide a
glycosylation site as provided herein, or may provide a human
gene-derived polypeptide sequence for purposes of enhancing the
degree of "humanization" of a fusion protein, or may comprise an
amino acid sequence that reduces the ability of a fusion protein to
form multimers or oligomers or aggregates or the like. Certain
alternative hinge region polypeptide sequences as described herein
may be derived from the polypeptide sequences of immunoglobulin
gene superfamily members that are not actual immunoglobulins per
se. For instance and according to non-limiting theory, certain
polypeptide sequences that are situated between intrachain
disulfide-generated immunoglobulin loop domain of immunoglobulin
gene super-family member proteins may be used as alternative hinge
region polypeptides as provided herein, or may be further modified
for such use.
[0122] As noted above, binding domain-immunoglobulin fusion
proteins are believed, according to non-limiting theory, to be
compromised in their ability to dimerize via interchain disulfide
bond formation, and further according to theory, this property is a
consequence of a reduction in the number of cysteine residues that
are present in the immunoglobulin hinge region polypeptide selected
for inclusion in the construction of the fusion protein.
Determination of the relative ability of a polypeptide to dimerize
is well within the knowledge of the relevant art, where any of a
number of established methodologies may be applied to detect
protein dimerization (see, e.g., Scopes, Protein Purification:
Principles and Practice, 1987 Springer-Verlag, New York). For
example, biochemical separation techniques for resolving proteins
on the basis of molecular size (e.g., gel electrophoresis, gel
filtration chromatography, analytical ultracentrifugation, etc.),
and/or comparison of protein physicochemical properties before and
after introduction of sulfhydryl-active (e.g., iodoacetamide,
N-ethylmaleimide) or disulfide-reducing (e.g., 2-mercaptoethanol,
dithiothreitol) agents, or other equivalent methodologies, may all
be employed for determining a degree of polypeptide dimerization or
oligomerization, and for determining possible contribution of
disulfide bonds to such potential quarternary structure. In certain
embodiments, the invention relates to a binding
domain-immunoglobulin fusion protein that exhibits a reduced (i.e.,
in a statistically significant manner relative to an appropriate
IgG-derived control) ability to dimerize, relative to a wild-type
human immunoglobulin G hinge region polypeptide as provided herein.
Accordingly, those familiar with the art will be able readily to
determine whether a particular fusion protein displays such reduced
ability to dimerize.
[0123] Compositions and methods for preparation of immunoglobulin
fusion proteins are well known in the art, as described for
example, in U.S. Pat. No. 5,892,019, which discloses recombinant
antibodies that are the products of a single encoding
polynucleotide but which are not binding domain-immunoglobulin
fusion proteins according to the present invention.
[0124] For an immunoglobulin fusion protein of the invention which
is intended for use in humans, the constant regions will typically
be of human sequence origin, to minimize a potential anti-human
immune response and to provide appropriate effector functions.
Manipulation of sequences encoding antibody constant regions is
described in the PCT publication of Morrison and Oi, WO 89/07142.
In particularly preferred embodiments, the CH1 domain is deleted
and the carboxyl end of the binding domain, or where the binding
domain comprises two immunoglobulin variable region polypeptides,
the second (i.e., more proximal to the C-terminus) variable region
is joined to the amino terminus of CH2 through the hinge region. A
schematic diagram depicting the structures of two exemplary binding
domain-immunoglobulin fusion proteins is shown in FIG. 11, where it
should be noted that in particularly preferred embodiments no
interchain disulfide bonds are present, and in other embodiments a
restricted number of interchain disulfide bonds may be present
relative to the number of such bonds that would be present if
wild-type hinge region polypeptides were instead present, and that
in other embodiments the fusion protein comprises a mutated hinge
region polypeptide that exhibits a reduced ability to dimerize,
relative to a wild-type human IgG hinge region polypeptide. Thus,
the isolated polynucleotide molecule codes for a single chain
immunoglobulin fusion protein having a binding domain that provides
specific binding affinity for a selected antigen.
[0125] The invention also contemplates in certain embodiments
binding domain-immunoglobulin fusion proteins as provided herein
that comprise fused polypeptide sequences or portions thereof
derived from a plurality of genetic sources, for example, according
to molecular "domain swapping" paradigms. Those having familiarity
with the art will readily appreciate that selection of such
polypeptide sequences for assembly into a binding
domain-immunoglobulin fusion protein may involve determination of
what are appropriate portions of each component polypeptide
sequence, based on structural and/or functional properties of each
such sequence (see, e.g., Carayannopoulos et al., 1996 J. Exp. Med.
183:1579; Marlow et al., Eds., Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor (1988)). The
component polypeptide sequences of which the fusion protein is
comprised may therefore comprise intact or full-length binding
domain, immunoglobulin, linker and/or plasma membrane anchor domain
polypeptide sequences, or truncated versions or variants thereof as
provided herein. According to these and related embodiments of the
invention, any two or more of the candidate component polypeptides
of which the subject invention fusion protein may be comprised will
be derived from independent sources, such as from immunoglobulin
sequences of differing allotype, isotype, subclass, class, or
species of origin (e.g., xenotype). Thus, as a non-limiting
example, a binding domain polypeptide (or its constituent
polypeptides such as one or more variable region polypeptides
and/or a linker polypeptide), a hinge region polypeptide,
immunoglobulin heavy chain CH2 and CH3 constant region polypeptides
and optionally an immunoglobulin heavy chain CH4 constant region
polypeptide as may be obtained from an IgM or IgE heavy chain, and
a plasma membrane anchor domain polypeptide may all be separately
obtained from distinct genetic sources and engineered into a
chimeric or fusion protein using well known techniques and
according to methodologies described herein.
[0126] Accordingly, a binding domain-immunoglobulin fusion protein
according to certain embodiments of the present invention may also
therefore comprise in pertinent part an immunoglobulin heavy chain
CH3 constant region polypeptide that is a wild-type IgA CH3
constant region polypeptide, or alternatively, that is a mutated
IgA CH3 constant region polypeptide that is incapable of
associating with a J chain; preferably the IgA CH3 constant region
polypeptides are of human origin. By way of brief background, IgA
molecules are known to be released into secretory fluids by a
mechanism that involves association of IgA into disulfide-linked
polymers (e.g., dimers) via a J chain polypeptide (e.g., Genbank
Acc. Nos. XM 059628, M12378, M12759; Johansen et al., 1999 Eur. J.
Immunol. 29:1701) and interaction of the complex so formed with
another protein that acts as a receptor for polymeric
immunoglobulin, and which is known as transmembrane secretory
component (S C; Johansen et al., 2000 Sc. J. Immunol. 52:240; see
also, e.g., Sorensen et al., 2000 Int. Immunol. 12:19; Yoo et al.,
1999 J. Biol. Chem. 274:33771; Yoo et al., 2002 J. Immunol. Meth.
261:1; Corthesy, 2002 Trends Biotechnol. 20:65; Symersky et al.,
2000 Mol. Immunol. 37:133; Crottet et al., 1999 Biochem. J.
341:299). Interchain disulfide bond formation between IgA Fc
domains and J chain is mediated through a penultimate cysteine
residue in an 18-amino acid C-terminal extension that forms part of
the IgA heavy chain constant region CH3 domain polypeptide (Yoo et
al., 1999; Sorensen et al., 2000). Certain embodiments of the
subject invention fusion proteins therefore contemplate inclusion
of the wild-type IgA heavy chain constant region polypeptide
sequence, which is capable of associating with J chain. Certain
other embodiments of the invention, however, contemplate fusion
proteins that comprise a mutated IgA CH3 constant region
polypeptide that is incapable of associating with a J chain.
According to such embodiments, two or more residues from the
C-terminus of an IgA CH3 constant region polypeptide such as a
human IgA CH3 constant region polypeptide may be deleted to yield a
truncated CH3 constant region polypeptide as provided herein. In
preferred embodiments and as described in greater detail below, a
mutated human IgA CH3 constant region polypeptide that is incapable
of associating with a J chain comprises such a C-terminal deletion
of either four or 18 amino acids. However, the invention need not
be so limited, such that the mutated IgA CH3 constant region
polypeptide may comprise a deletion of 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-25, 26-30 or more amino
acids, so long as the fusion protein is capable of specifically
binding an antigen and of at least one immunological activity as
provided herein. Alternatively, the invention also contemplates
fusion proteins that comprise a mutated IgA CH3 constant region
polypeptide that is incapable of associating with a J chain by
virtue of replacement of the penultimate cysteine, or by chemical
modification of that amino acid residue, in a manner that prevents
interchain disulfide bond formation. Methods for determining
whether a fusion protein can associate with a J chain will be known
to those having familiarity with the art and are described herein,
including in the cited references.
[0127] As also described herein and according to procedures known
in the art, the fusion protein may further be tested routinely for
immunological activity, for instance, in ADCC or CDC assays. As an
illustrative example, a fusion protein according to such an
embodiment may comprise a binding domain polypeptide derived from a
human heavy chain variable region polypeptide sequence, a human
IgA-derived immunoglobulin hinge region polypeptide sequence, a
human IgG1 immunoglobulin heavy chain CH2 constant region
polypeptide sequence, a human IgG2 immunoglobulin heavy chain CH3
constant region polypeptide sequence, and optionally a human IgE
immunoglobulin heavy chain CH4 constant region polypeptide sequence
and/or a human TNF-.alpha. receptor type 1 (TNFR1) plasma membrane
anchor domain polypeptide sequence that comprises a cytoplasmic
tail polypeptide which is capable of apoptotic signaling. The
invention therefore contemplates these and other embodiments
according to the present invention in which two or more polypeptide
sequences that are present in a fusion protein have independent
genetic origins.
[0128] As noted above, in certain embodiments the binding
protein-immunoglobulin fusion protein comprises at least one
immunoglobulin variable region polypeptide, which may be a light
chain or a heavy chain variable region polypeptide, and in certain
embodiments the fusion protein comprises at least one such light
chain V-region and one such heavy chain V-region and at least one
linker peptide that is fused to each of the V-regions. Construction
of such binding domains, for example single chain Fv domains, is
well known in the art and is described in greater detail in the
Examples below, and has been described, for example, in U.S. Pat.
No. 5,892,019 and references cited therein; selection and assembly
of single-chain variable regions and of linker polypeptides that
may be fused to each of a heavy chain-derived and a light
chain-derived V region (e.g., to generate a binding domain that
comprises a single-chain Fv polypeptide) is also known to the art
and described herein and, for example, in U.S. Pat. No. 5,869,620,
U.S. Pat. No. 4,704,692 and U.S. Pat. No. 4,946,778. In certain
embodiments all or a portion of an immunoglobulin sequence that is
derived from a non-human source may be "humanized" according to
recognized procedures for generating humanized antibodies, i.e.,
immunoglobulin sequences into which human Ig sequences are
introduced to reduce the degree to which a human immune system
would perceive such proteins as foreign (see, e.g., U.S. Pat. Nos.
5,693,762; 5,585,089; 4,816,567; 5,225,539; 5,530,101; and
references cited therein).
[0129] Binding domain-immunoglobulin fusion proteins as described
herein may, according to certain embodiments, desirably comprise
sites for glycosylation, e.g., covalent attachment of carbohydrate
moieties such as monosaccharides or oligosaccharides. Incorporation
of amino acid sequences that provide substrates for polypeptide
glycosylation is within the scope of the relevant art, including,
for example, the use of genetic engineering or protein engineering
methodologies to obtain a polypeptide sequence containing the
classic Asn-X-Ser/Thr site for N-(asparagine)-linked glycosylation,
or a sequence containing Ser or Thr residues that are suitable
substrates for 0-linked glycosylation, or sequences amenable to
C-mannosylation, glypiation/glycosylphosphatidylinositol
modification, or phosphoglycation, all of which can be identified
according to art-established criteria (e.g., Spiro, 2002 Glybiology
12:43R). Without wishing to be bound by theory, glycosylated fusion
proteins having particular amino acid sequences may beneficially
possess attributes associated with one or more of improved
solubility, enhanced stability in solution, enhanced physiological
stability, improved bioavailability including in vivo
biodistribution, and superior resistance to proteases, all in a
statistically significant manner, relative to fusion proteins
having the same or highly similar amino acid sequences but lacking
glycosyl moieties. In certain preferred embodiments the subject
invention fusion protein comprises a glycosylation site that is
present in a linker as provided herein, and in certain other
preferred embodiments the subject invention fusion protein
comprises a glycosylation site that is present in a hinge region
polypeptide sequence as provided herein.
[0130] In certain preferred embodiments of the present invention,
the binding domain-immunoglobulin fusion protein is a protein or
glycoprotein that is capable of being expressed by a host cell such
that it localizes to the cell surface. Binding
domain-immunoglobulin fusion proteins that localize to the cell
surface may do so by virtue of having naturally present or
artificially introduced structural features that direct the fusion
protein to the cell surface (e.g., Nelson et al. 2001 Trends Cell
Biol. 11:483; Ammon et al., 2002 Arch. Physiol. Biochem. 110:137;
Kasai et al., 2001 J. Cell Sci. 114:3115; Watson et al., 2001 Am.
J. Physiol. Cell Physiol. 281:C215; Chatteijee et al., 2001 Biol.
Chem. 275:24013) including by way of illustration and not
limitation, secretory signal sequences, leader sequences, plasma
membrane anchor domain polypeptides such as hydrophobic
transmembrane domains (e.g., Heuck et al., 2002 Cell Biochem.
Biophys. 36:89; Sadlish et al., 2002 Biochem J. 364:777; Phoenix et
al., 2002 Mol. Membra. Biol. 19:1; Minke et al., 2002 Physiol. Rev.
82:429) or glycosylphosphatidylinositol attachment sites
("glypiation" sites, e.g., Chatteijee et al., 2001 Cell Mol. Life.
Sci. 58:1969; Hooper, 2001 Proteomics 1:748; Spiro, 2002 Glycobiol.
12:43R), cell surface receptor binding domains, extracellular
matrix binding domains, or any other structural feature that causes
the fusion protein to localize to the cell surface. Particularly
preferred are fusion proteins that comprise a plasma membrane
anchor domain which includes a transmembrane polypeptide domain,
typically comprising a membrane spanning domain which includes a
hydrophobic region capable of energetically favorable interaction
with the phospholipid fatty acyl tails that form the interior of
the plasma membrane bilayer. Such features are well known to those
of ordinary skill in the art, who will further be familiar with
methods for introducing nucleic acid sequences encoding these
features into the subject expression constructs by genetic
engineering, and with routine testing of such constructs to verify
cell surface localization of the product.
[0131] According to certain further embodiments, a plasma membrane
anchor domain polypeptide comprises such a transmembrane domain
polypeptide and also comprises a cytoplasmic tail polypeptide,
which refers to a region or portion of the polypeptide sequence
that contacts the cytoplasmic face of the plasma membrane and/or is
in contact with the cytosol or other cytoplasmic components. A
large number of cytoplasmic tail polypeptides are known that
comprise the intracellular portions of plasma membrane
transmembrane proteins, and discrete functions have been identified
for many such polypeptides, including biological signal
transduction (e.g., activation or inhibition of protein kinases,
protein phosphatases, G-proteins, cyclic nucleotides and other
second messengers, ion channels, secretory pathways), biologically
active mediator release, stable or dynamic association with one or
more cytoskeletal components, cellular differentiation, cellular
activation, mitogenesis, cytostasis, apoptosis and the like (e.g.,
Maher et al., 2002 Immunol. Cell Biol. 80:131; El Far et al., 2002
Biochem J. 365:329; Teng et al., 2002 Genome Biol. 2REVIEWS:3012;
Simons et al., 2001 Cell Signal 13:855; Furie et al., 2001 Thromb.
Haemost. 86:214; Gaffen, 2001 Cytokine 14:63; Dittel, 2000 Arch.
Immunol. Ther. Exp. (Warsz.) 48:381; Parnes et al., 2000 Immunol.
Rev. 176:75; Moretta et al., 2000 Semin. Immunol. 12:129; Ben
Ze'ev, 1999 Ann. N.Y. Acad. Sci. 886:37; Marsters et al., Recent
Prog. Horm. Res. 54:225).
[0132] In the context of methods of using binding
domain-immunoglobulin fusion proteins for the treatment of a
malignant condition or a B-cell disorder as provided herein, the
present invention contemplates certain embodiments wherein a
binding domain-immunoglobulin fusion protein that comprises a
plasma membrane anchor domain polypeptide is expressed at a cell
surface and further comprises a cytoplasmic tail polypeptide which
comprises an apoptosis signaling polypeptide sequence. A number of
apoptosis signaling polypeptide sequences are known to the art, as
reviewed, for example, in When Cells Die: A Comprehensive
Evaluation of Apoptosis and Programmed Cell Death (R. A. Lockshin
et al., Eds., 1998 John Wiley & Sons, New York; see also, e.g.,
Green et al., 1998 Science 281:1309 and references cited therein;
Ferreira et al., 2002 Clin. Canc. Res. 8:2024; Gurumurthy et al.,
2001 Cancer Metastas. Rev. 20:225; Kanduc et al., 2002 Int. J.
Oncol. 21:165). Typically an apoptosis signaling polypeptide
sequence comprises all or a portion of, or is derived from, a
receptor death domain polypeptide, for instance, FADD (e.g.,
Genbank Ace. Nos. U24231, U43184, AF009616, AF009617,
NM.sub.--012115), TRADD (e.g., Genbank Ace. No. NM.sub.--003789),
RAIDD (e.g., Genbank Ace. No. U87229), CD95 (FAS/Apo-1; e.g.,
Genbank Ace. Nos. X89101, NM.sub.--003824, AF344850, AF344856),
TNF-a-receptor-1 (TNFR1, e.g., Genbank Ace. Nos. 563368, AF040257),
DR5 (e.g., Genbank Ace. No. AF020501, AF016268, AF012535), an ITIM
domain (e.g., Genbank Ace. Nos. AF081675, BC015731, NM 006840,
NM.sub.--006844, NM.sub.--006847, XM.sub.--017977; see, e.g.,
Billadeau et al., 2002 J. Clin. Invest. 109:161), an ITAM domain
(e.g., Genbank Ace. Nos. NM 005843, NM 003473, BCO30586; see, e.g.,
Billadeau et al., 2002), or other apoptosis-associated receptor
death domain polypeptides known to the art, for example, TNFR2
(e.g., Genbank Ace. No. L49431, L49432), caspase/procaspase-3
(e.g., Genbank Ace. No. XM.sub.--54686), caspase/procaspase-8
(e.g., AF380342, NM.sub.--004208, NM.sub.--001228, NM.sub.--033355,
NM.sub.--033356, NM.sub.--033357, NM.sub.--033358),
caspase/procaspase-2 (e.g., Genbank Ace. No. AF314174, AF314175),
etc.
[0133] Cells in a biological sample that are suspected of
undergoing apoptosis may be examined for morphological,
permeability or other changes that are indicative of an apoptotic
state. For example by way of illustration and not limitation,
apoptosis in many cell types may cause altered morphological
appearance such as plasma membrane blebbing, cell shape change,
loss of substrate adhesion properties or other morphological
changes that can be readily detected by a person having ordinary
skill in the art, for example by using light microscopy. As another
example, cells undergoing apoptosis may exhibit fragmentation and
disintegration of chromosomes, which may be apparent by microscopy
and/or through the use of DNA-specific or chromatin-specific dyes
that are known in the art, including fluorescent dyes. Such cells
may also exhibit altered plasma membrane permeability properties as
may be readily detected through the use of vital dyes (e.g.,
propidium iodide, trypan blue) or by the detection of lactate
dehydrogenase leakage into the extracellular milieu. These and
other means for detecting apoptotic cells by morphologic criteria,
altered plasma membrane permeability and related changes will be
apparent to those familiar with the art.
[0134] In another embodiment of the invention wherein a binding
domain-immunoglobulin fusion protein that is expressed at a cell
surface comprises a plasma membrane anchor domain having a
transmembrane domain and a cytoplasmic tail that comprises an
apoptosis signaling polypeptide, cells in a biological sample may
be assayed for translocation of cell membrane phosphatidylserine
(PS) from the inner to the outer leaflet of the plasma membrane,
which may be detected, for example, by measuring outer leaflet
binding by the PS-specific protein annexin. (Martin et al., J. Exp.
Med. 182:1545, 1995; Fadok et al., J. Immunol. 148:2207, 1992.) In
still other related embodiments of the invention, including
embodiments wherein the binding domain-immunoglobulin fusion
protein is expressed at the cell surface and comprises a plasma
membrane anchor domain having an apoptosis signaling polypeptide
and also including embodiments wherein the binding
domain-immunoglobulin fusion protein is a soluble protein that
lacks a membrane anchor domain and that is capable of inducing
apoptosis, a cellular response to an apoptogen is determined by an
assay for induction of specific protease activity in any member of
a family of apoptosis-activated proteases known as the caspases
(see, e.g., Green et al., 1998 Science 281:1309). Those having
ordinary skill in the art will be readily familiar with methods for
determining caspase activity, for example by determination of
caspase-mediated cleavage of specifically recognized protein
substrates. These substrates may include, for example,
poly-(ADP-ribose) polymerase (PARP) or other naturally occurring or
synthetic peptides and proteins cleaved by caspases that are known
in the art (see, e.g., Ellerby et al., 1997 J. Neurosci. 17:6165).
The synthetic peptide Z-Tyr-Val-Ala-Asp-AFC (SEQ ID NO: ______),
wherein "Z" indicates a benzoyl carbonyl moiety and AFC indicates
7-amino-4-trifluoromethylcoumarin (Kluck et al., 1997 Science
275:1132; Nicholson et al., 1995 Nature 376:37), is one such
substrate. Other non-limiting examples of substrates include
nuclear proteins such as U1-70 kDa and DNA-PKcs (Rosen and
Casciola-Rosen, 1997 J. Cell. Biochem. 64:50; Cohen, 1997 Biochem.
J. 326:1). Cellular apoptosis may also be detected by determination
of cytochrome c that has escaped from mitochondria in apoptotic
cells (e.g., Liu et al., Cell 86:147, 1996). Such detection of
cytochrome c may be performed spectrophotometrically,
immunochemically or by other well established methods for
determining the presence of a specific protein. Persons having
ordinary skill in the art will readily appreciate that there may be
other suitable techniques for quantifying apoptosis.
[0135] Once a binding domain-immunoglobulin fusion protein as
provided herein has been designed, DNAs encoding the polypeptide
may be synthesized via oligonucleotide synthesis as described, for
example, in Sinha et al., Nucleic Acids Res., 12, 4539-4557 (1984);
assembled via PCR as described, for example in Innis, Ed., PCR
Protocols, Academic Press (1990) and also in Better et al. J. Biol.
Chem. 267, 16712-16118 (1992); cloned and expressed via standard
procedures as described, for example, in Ausubel et al., Eds.,
Current Protocols in Molecular Biology, John Wiley & Sons, New
York (1989) and also in Robinson et al., Hum. Antibod Hybridomas,
2, 84-93 (1991); and tested for specific antigen binding activity,
as described, for example, in Harlow et al., Eds., Antibodies: A
Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold
Spring Harbor (1988) and Munson et al., Anal. Biochem., 107,
220-239 (1980).
[0136] The preparation of single polypeptide chain binding
molecules of the Fv region, single-chain Fv molecules, is described
in U.S. Pat. No. 4,946,778, which is incorporated herein by
reference. In the present invention, single-chain Fv-like molecules
are synthesized by encoding a first variable region of the heavy or
light chain, followed by one or more linkers to the variable region
of the corresponding light or heavy chain, respectively. The
selection of appropriate linker(s) between the two variable regions
is described in U.S. Pat. No. 4,946,778 (see also, e.g., Huston et
al., 1993 Int. Rev. Immunol. 10:195). An exemplary linker described
herein is (Gly-Gly-Gly-Gly-Ser)3. The linker is used to convert the
naturally aggregated but chemically separate heavy and light chains
into the amino terminal antigen binding portion of a single
polypeptide chain, wherein this antigen binding portion will fold
into a structure similar to the original structure made of two
polypeptide chains and thus retain the ability to bind to the
antigen of interest. The nucleotide sequences encoding the variable
regions of the heavy and light chains, joined by a sequence
encoding a linker, are joined to a nucleotide sequence encoding
antibody constant regions. The constant regions are those which
permit the resulting polypeptide to form interchain disulfide bonds
to form a dimer, and which contain desired effector functions, such
as the ability to mediate antibody-dependent cellular cytotoxicity
(ADCC). For an immunoglobulin-like molecule of the invention which
is intended for use in humans, the constant regions will typically
be substantially human to minimize a potential anti-human immune
response and to provide appropriate effector functions.
Manipulation of sequences encoding antibody constant regions is
described in the PCT publication of Morrison and Oi, WO 89/07142,
which is incorporated herein by reference. In preferred
embodiments, the CH1 domain is deleted and the carboxyl end of the
binding domain polypeptide (e.g., an immunoglobulin variable region
polypeptide) is joined to the amino terminus of CH2 via a hinge
region polypeptide as provided herein.
[0137] As described above, the present invention provides
recombinant expression constructs capable of directing the
expression of binding domain-immunoglobulin fusion proteins as
provided herein. The amino acids, which occur in the various amino
acid sequences referred to herein, are identified according to
their well known three-letter or single-letter abbreviations. The
nucleotides, which occur in the various DNA sequences or fragments
thereof referred herein, are designated with the standard single
letter designations used routinely in the art. A given amino acid
sequence may also encompass similar amino acid sequences having
only minor changes, for example by way of illustration and not
limitation, covalent chemical modifications, insertions, deletions
and substitutions, which may further include conservative
substitutions. Amino acid sequences that are similar to one another
may share substantial regions of sequence homology. In like
fashion, nucleotide sequences may encompass substantially similar
nucleotide sequences having only minor changes, for example by way
of illustration and not limitation, covalent chemical
modifications, insertions, deletions and substitutions, which may
further include silent mutations owing to degeneracy of the genetic
code. Nucleotide sequences that are similar to one another may
share substantial regions of sequence homology.
[0138] The presence of a malignant condition in a subject refers to
the presence of dysplastic, cancerous and/or transformed cells in
the subject, including, for example neoplastic, tumor, non-contact
inhibited or oncogenically transformed cells, or the like (e.g.,
melanoma, carcinomas such as adenocarcinoma, squamous cell
carcinoma, small cell carcinoma, oat cell carcinoma, etc., sarcomas
such as chondrosarcoma, osteosarcoma, etc.) which are known to the
art and for which criteria for diagnosis and classification are
established. In preferred embodiments contemplated by the present
invention, for example, such cancer cells are malignant
hematopoietic cells, such as transformed cells of lymphoid lineage
and in particular, B-cell lymphomas and the like; cancer cells may
in certain preferred embodiments also be epithelial cells such as
carcinoma cells. The invention also contemplates B-cell disorders,
which may include certain malignant conditions that affect B-cells
(e.g., B-cell lymphoma) but which is not intended to be so limited,
and which is also intended to encompass autoimmune diseases and in
particular, diseases, disorders and conditions that are
characterized by autoantibody production.
[0139] Autoantibodies are antibodies that react with self antigens.
Autoantibodies are detected in several autoimmune diseases (i.e., a
disease, disorder or condition wherein a host immune system
generates an inappropriate anti-"self" immune reaction) where they
are involved in disease activity. The current treatments for these
autoimmune diseases are immunosuppressive drugs that require
continuing administration, lack specificity, and cause significant
side effects. New approaches that can eliminate autoantibody
production with minimal toxicity will address an unmet medical need
for a spectrum of diseases that affect many people. The subject
invention binding domain-immunoglobulin fusion protein is designed
for improved penetration into lymphoid tissues. Depletion of B
lymphocytes interrupts the autoantibody production cycle, and
allows the immune system to reset as new B lymphocytes are produced
from precursors in the bone marrow.
[0140] A number of diseases have been identified for which
beneficial effects are believed, according to non-limiting theory,
to result from B cell depletion therapy; a brief description of
several exemplars of these diseases follows.
[0141] Autoimmune thyroid disease includes Graves' disease and
Hashimoto's thyroiditis. In the United States alone, there are
about 20 million people who have some form of autoimmune thyroid
disease. Autoimmune thyroid disease results from the production of
autoantibodies that either stimulate the thyroid to cause
hyperthyroidism (Graves' disease) or destroy the thyroid to cause
hypothyroidism (Hashimoto's thyroiditis). Stimulation of the
thyroid is caused by autoantibodies that bind and activate the
thyroid stimulating hormone (TSH) receptor. Destruction of the
thyroid is caused by autoantibodies that react with other thyroid
antigens.
[0142] Current therapy for Graves' disease includes surgery,
radioactive iodine, or antithyroid drug therapy. Radioactive iodine
is widely used, since antithyroid medications have significant side
effects and disease recurrence is high. Surgery is reserved for
patients with large goiters or where there is a need for very rapid
normalization of thyroid function. There are no therapies that
target the production of autoantibodies responsible for stimulating
the TSH receptor. Current therapy for Hashimoto's thyroiditis is
levothyroxine sodium, and therapy is usually lifelong because of
the low likelihood of remission. Suppressive therapy has been shown
to shrink goiters in Hashimoto's thyroiditis, but no therapies that
reduce autoantibody production to target the disease mechanism are
known.
[0143] Rheumatoid arthritis (RA) is a chronic disease characterized
by inflammation of the joints, leading to swelling, pain, and loss
of function. RA effects an estimated 2.5 million people in the
United States. RA is caused by a combination of events including an
initial infection or injury, an abnormal immune response, and
genetic factors. While autoreactive T cells and B cells are present
in RA, the detection of high levels of antibodies that collect in
the joints, called rheumatoid factor, is used in the diagnosis of
RA. Current therapy for RA includes many medications for managing
pain and slowing the progression of the disease. No therapy has
been found that can cure the disease. Medications include
nonsteroidal antiinflammatory drugs (NSAIDS), and disease modifying
antirheumatic drugs (DMARDS). NSAIDS are effective in benign
disease, but fail to prevent the progression to joint destruction
and debility in severe RA. Both NSAIDS and DMARDS are associated
with significant side effects. Only one new DMARD, Leflunomide, has
been approved in over 10 years. Leflunomide blocks production of
autoantibodies, reduces inflammation, and slows progression of RA.
However, this drug also causes severe side effects including
nausea, diarrhea, hair loss, rash, and liver injury.
[0144] Systemic Lupus Erythematosus (SLE) is an autoimmune disease
caused by recurrent injuries to blood vessels in multiple organs,
including the kidney, skin, and joints. SLE effects over 500,000
people in the United States. In patients with SLE, a faulty
interaction between T cells and B cells results in the production
of autoantibodies that attack the cell nucleus. These include
anti-double stranded DNA and anti-Sm antibodies. Autoantibodies
that bind phospholipids are also found in about half of SLE
patients, and are responsible for blood vessel damage and low blood
counts. Immune complexes accumulate the kidneys, blood vessels, and
joints of SLE patients, where they cause inflammation and tissue
damage. No treatment for SLE has been found to cure the disease.
NSAIDS and DMARDS are used for therapy depending upon the severity
of the disease.
[0145] Plasmapheresis with plasma exchange to remove autoantibodies
can cause temporary improvement in SLE patients. There is general
agreement that autoantibodies are responsible for SLE, so new
therapies that deplete the B cell lineage, allowing the immune
system to reset as new B cells are generated from precursors, offer
hope for long lasting benefit in SLE patients.
[0146] Sjogren's syndrome is an autoimmune disease characterized by
destruction of the body's moisture producing glands. Sjogren's
syndrome is one of the most prevalent autoimmune disorders,
striking up to 4 million people in the United States. About half of
people with Sjogren's also have a connective tissue disease, such
as rheumatoid arthritis, while the other half have primary
Sjogren's with no other concurrent autoimmune disease.
Autoantibodies, including anti-nuclear antibodies, rheumatoid
factor, anti-fodrin, and anti-muscarinic receptor are often present
in patients with Sjogren's syndrome. Conventional therapy includes
corticosteroids.
[0147] Immune Thrombocytopenic purpura (ITP) is caused by
autoantibodies that bind to blood platelets and cause their
destruction. Some cases of ITP are caused by drugs, and others are
associated with infection, pregnancy, or autoimmune disease such as
SLE. About half of all cases are classified as "idiopathic",
meaning the cause is unknown. The treatment of ITP is determined by
the severity of the symptoms. In some cases, no therapy is needed.
In most cases, immunosuppressive drugs are used, including
corticosteroids or intravenous infusions of immune globulin to
deplete T cells. Another treatment that usually results in an
increased number of platelets is removal of the spleen, the organ
that destroys antibody-coated platelets. More potent
immunosuppressive drugs, including cyclosporine, cyclophosphamide,
or azathioprine, are used for patients with severe cases. Removal
of autoantibodies by passage of patients' plasma over a Protein A
column is used as a second line treatment in patients with severe
disease.
[0148] Multiple Sclerosis (MS) is an autoimmune disease
characterized by inflammation of the central nervous system and
destruction of myelin, which insulates nerve cell fibers in the
brain, spinal cord, and body. Although the cause of MS is unknown,
it is widely believed that autoimmune T cells are primary
contributors to the pathogenesis of the disease. However, high
levels of antibodies are present in the cerebral spinal fluid of
patients with MS, and some theories predict that the B cell
response leading to antibody production is important for mediating
the disease. No B cell depletion therapies have been studies in
patients with MS. There is no cure for MS. Current therapy is
corticosteroids, which can reduce the duration and severity of
attacks, but do not affect the course of MS over time. New
biotechnology interferon (IFN) therapies for MS have recently been
approved.
[0149] Myasthenia Gravis (MG) is a chronic autoimmune neuromuscular
disorder that is characterized by weakness of the voluntary muscle
groups. MG effects about 40,000 people in the United States. MG is
caused by autoantibodies that bind to acetylcholine receptors
expressed at neuromuscular junctions. The autoantibodies reduce or
block acetylcholine receptors, preventing the transmission of
signals from nerves to muscles. There is no known cure for MG.
Common treatments include immunosuppression with corticosteroids,
cyclosporine, cyclophosphamide, or azathioprine. Surgical removal
of the thymus is often used to blunt the autoimmune response.
Plasmapheresis, used to reduce autoantibody levels in the blood, is
effective in MG, but is short-lived because the production of
autoantibodies continues. Plasmapheresis is usually reserved for
severe muscle weakness prior to surgery.
[0150] Psoriasis affects approximately five million people. It is
an autoimmune inflammation of the skin and joints. Psoriasis is
associated with arthritis (psoriatic arthritis) in up to 30% of
patients with psoriasis. Many treatments are used, including
steroids, UV light retenoids, vitamin D derivatives, cyclosporine,
and methotrexate.
[0151] Scleroderma is a chronic autoimmune disease of the
connective tissue that is also known as systemic sclerosis.
Scleroderma is characterized by an overproduction of collagen,
resulting in a thickening of the skin. Approximately 300,000 people
in the United States have scleroderma.
[0152] Inflammatory Bowel Disease, including Crohn's disease and
ulcerative colitis, is an autoimmune disease of the digestive
system.
[0153] The present invention further relates to constructs encoding
binding domain-immunoglobulin fusion proteins, and in particular to
methods for administering recombinant constructs encoding such
proteins that may be expressed, for example, as fragments, analogs
and derivatives of such polypeptides. The terms "fragment,"
"derivative" and "analog" when referring to binding
domain-immunoglobulin fusion polypeptides or fusion proteins,
refers to any binding domain-immunoglobulin fusion polypeptide or
fusion protein that retains essentially the same biological
function or activity as such polypeptide. Thus, an analog includes
a proprotein which can be activated by cleavage of the proprotein
portion to produce an active binding domain-immunoglobulin fusion
polypeptide.
[0154] A fragment, derivative or analog of an binding
domain-immunoglobulin fusion polypeptide or fusion protein,
including binding domain-immunoglobulin fusion polypeptides or
fusion proteins encoded by the cDNAs referred to herein, may be (i)
one in which one or more of the amino acid residues are substituted
with a conserved or non-conserved amino acid residue (preferably a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which additional amino acids are
fused to the binding domain-immunoglobulin fusion polypeptide,
including amino acids that are employed for detection or specific
functional alteration of the binding domain-immunoglobulin fusion
polypeptide or a proprotein sequence. Such fragments, derivatives
and analogs are deemed to be within the scope of those skilled in
the art from the teachings herein.
[0155] The polypeptides of the present invention include binding
domain-immunoglobulin fusion polypeptides and fusion proteins
having binding domain polypeptide amino acid sequences that are
identical or similar to sequences known in the art, or fragments or
portions thereof. For example by way of illustration and not
limitation, a human CD154 molecule extracellular domain [SEQ ID
NO:______] is contemplated for use according to the instant
invention, as are polypeptides having at least 70% similarity
(preferably a 70% identity) and more preferably 90% similarity
(more preferably a 90% identity) to the reported polypeptide and
still more preferably a 95% similarity (still more preferably a 95%
identity) to the reported polypeptides and to portions of such
polypeptides, wherein such portions of a binding
domain-immunoglobulin fusion polypeptide generally contain at least
30 amino acids and more preferably at least 50 amino acids.
Extracellular domains include portions of a cell surface molecule,
and in particularly preferred embodiments cell surface molecules
that are integral membrane proteins or that comprise a plasma
membrane spanning transmembrane domain, that extend beyond the
outer leaflet of the plasma membrane phospholipid bilayer when the
molecule is expressed at a cell surface, preferably in a manner
that exposes the extracellular domain portion of such a molecule to
the external environment of the cell, also known as the
extracellular milieu. Methods for determining whether a portion of
a cell surface molecule comprises an extracellular domain are well
known to the art and include experimental determination (e.g.,
direct or indirect labeling of the molecule, evaluation of whether
the molecule can be structurally altered by agents to which the
plasma membrane is not permeable such as proteolytic or lipolytic
enzymes) or topological prediction based on the structure of the
molecule (e.g., analysis of the amino acid sequence of a
polypeptide) or other methodologies.
[0156] As known in the art, "similarity" between two polypeptides
is determined by comparing the amino acid sequence and conserved
amino acid substitutes thereto of the polypeptide to the sequence
of a second polypeptide. Fragments or portions of the nucleic acids
encoding polypeptides of the present invention may be used to
synthesize full-length nucleic acids of the present invention. As
used herein, "% identity" refers to the percentage of identical
amino acids situated at corresponding amino acid residue positions
when two or more polypeptide are aligned and their sequences
analyzed using a gapped BLAST algorithm (e.g., Altschul et al.,
1997 Nucl. Ac. Res. 25:3389) which weights sequence gaps and
sequence mismatches according to the default weightings provided by
the National Institutes of Health/NCBI database (Bethesda, Md.; see
wvvw.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast).
[0157] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally occurring nucleic
acid or polypeptide present in a living animal is not isolated, but
the same nucleic acid or polypeptide, separated from some or all of
the co-existing materials in the natural system, is isolated. Such
nucleic acids could be part of a vector and/or such nucleic acids
or polypeptides could be part of a composition, and still be
isolated in that such vector or composition is not part of its
natural environment.
[0158] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region "leader and trailer" as well as
intervening sequences (introns) between individual coding segments
(exons).
[0159] As described herein, the invention provides binding
domain-immunoglobulin fusion proteins encoded by nucleic acids that
have the binding domain coding sequence fused in frame to an
additional immunoglobulin domain encoding sequence to provide for
expression of a binding domain polypeptide sequence fused to an
additional functional polypeptide sequence that permits, for
example by way of illustration and not limitation, detection,
functional alteration, isolation and/or purification of the fusion
protein. Such fusion proteins may permit functional alteration of a
binding domain by containing additional immunoglobulin-derived
polypeptide sequences that influence behavior of the fusion
product, for example (and as described above) by reducing the
availability of sulfhydryl groups for participation in disulfide
bond formation, and by conferring the ability to potentiate ADCC
and/or CDC.
[0160] Modification of the polypeptide may be effected by any means
known to those of skill in this art. The preferred methods herein
rely on modification of DNA encoding the fusion protein and
expression of the modified DNA. DNA encoding one of the binding
domain-immunoglobulin fusions discussed above may be mutagenized
using standard methodologies, including those described below. For
example, cysteine residues that may otherwise facilitate multimer
formation or promote particular molecular conformations can be
deleted from a polypeptide or replaced, e.g., cysteine residues
that are responsible for aggregate formation. If necessary, the
identity of cysteine residues that contribute to aggregate
formation may be determined empirically, by deleting and/or
replacing a cysteine residue and ascertaining whether the resulting
protein aggregates in solutions containing physiologically
acceptable buffers and salts. In addition, fragments of binding
domain-immunoglobulin fusions may be constructed and used. As noted
above, the counterreceptor/ligand binding domains for many
candidate binding domain-immunoglobulin fusion proteins have been
delineated, such that one having ordinary skill in the art may
readily select appropriate polypeptide domains for inclusion in the
encoded products of the instant expression constructs.
[0161] Conservative substitutions of amino acids are well-known and
may be made generally without altering the biological activity of
the resulting binding domain-immunoglobulin fusion protein
molecule. For example, such substitutions are generally made by
interchanging within the groups of polar residues, charged
residues, hydrophobic residues, small residues, and the like. If
necessary, such substitutions may be determined empirically merely
by testing the resulting modified protein for the ability to bind
to the appropriate cell surface receptors in in vitro biological
assays, or to bind to appropriate antigens or desired target
molecules.
[0162] The present invention further relates to nucleic acids which
hybridize to binding domain-immunoglobulin fusion protein encoding
polynucleotide sequences as provided herein, or their complements,
as will be readily apparent to those familiar with the art, if
there is at least 70%, preferably 80-85%, more preferably at least
90%, and still more preferably at least 95%, 96%, 97%, 98% or 99%
identity between the sequences. The present invention particularly
relates to nucleic acids which hybridize under stringent conditions
to the binding domain-immunoglobulin fusion encoding nucleic acids
referred to herein. As used herein, the term "stringent conditions"
means hybridization will occur only if there is at least 90-95% and
preferably at least 97% identity between the sequences. The nucleic
acids which hybridize to binding domain-immunoglobulin fusion
encoding nucleic acids referred to herein, in preferred
embodiments, encode polypeptides which retain substantially the
same biological function or activity as the binding
domain-immunoglobulin fusion polypeptides encoded by the cDNAs of
the references cited herein.
[0163] As used herein, to "hybridize" under conditions of a
specified stringency is used to describe the stability of hybrids
formed between two single-stranded nucleic acid molecules.
Stringency of hybridization is typically expressed in conditions of
ionic strength and temperature at which such hybrids are annealed
and washed. Typically "high", "medium" and "low" stringency
encompass the following conditions or equivalent conditions
thereto: high stringency: 0.1.times.SSPE or SSC, 0.1% SDS,
65.degree. C.; medium stringency: 0.2.times.SSPE or SSC, 0.1% SDS,
50.degree. C.; and low stringency: 1.0.times.SSPE or SSC, 0.1% SDS,
50.degree. C. As known to those having ordinary skill in the art,
variations in stringency of hybridization conditions may be
achieved by altering the time, temperature and/or concentration of
the solutions used for prehybridization, hybridization and wash
steps, and suitable conditions may also depend in part on the
particular nucleotide sequences of the probe used, and of the
blotted, proband nucleic acid sample. Accordingly, it will be
appreciated that suitably stringent conditions can be readily
selected without undue experimentation where a desired selectivity
of the probe is identified, based on its ability to hybridize to
one or more certain proband sequences while not hybridizing to
certain other proband sequences.
[0164] The nucleic acids of the present invention, also referred to
herein as polynucleotides, may be in the form of RNA or in the form
of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
The DNA may be double-stranded or single-stranded, and if single
stranded may be the coding strand or non-coding (anti-sense)
strand. A coding sequence which encodes an binding
domain-immunoglobulin fusion polypeptide for use according to the
invention may be identical to the coding sequence known in the art
for any given binding domain-immunoglobulin fusion, or may be a
different coding sequence, which, as a result of the redundancy or
degeneracy of the genetic code, encodes the same binding
domain-immunoglobulin fusion polypeptide.
[0165] The nucleic acids which encode binding domain-immunoglobulin
fusion polypeptides for use according to the invention may include,
but are not limited to: only the coding sequence for the binding
domain-immunoglobulin fusion polypeptide; the coding sequence for
the binding domain-immunoglobulin fusion polypeptide and additional
coding sequence; the coding sequence for the binding
domain-immunoglobulin fusion polypeptide (and optionally additional
coding sequence) and non-coding sequence, such as introns or
non-coding sequences 5' and/or 3' of the coding sequence for the
binding domain-immunoglobulin fusion polypeptide, which for example
may further include but need not be limited to one or more
regulatory nucleic acid sequences that may be a regulated or
regulatable promoter, enhancer, other transcription regulatory
sequence, repressor binding sequence, translation regulatory
sequence or any other regulatory nucleic acid sequence. Thus, the
term "nucleic acid encoding" or "polynucleotide encoding" a binding
domain-immunoglobulin fusion protein encompasses a nucleic acid
which includes only coding sequence for a binding
domain-immunoglobulin fusion polypeptide as well as a nucleic acid
which includes additional coding and/or non-coding sequence(s).
[0166] Nucleic acids and oligonucleotides for use as described
herein can be synthesized by any method known to those of skill in
this art (see, e.g., WO 93/01286, U.S. application Ser. No.
07/723,454; U.S. Pat. No. 5,218,088; U.S. Pat. No. 5,175,269; U.S.
Pat. No. 5,109,124). Identification of oligonucleotides and nucleic
acid sequences for use in the present invention involves methods
well known in the art. For example, the desirable properties,
lengths and other characteristics of useful oligonucleotides are
well known. In certain embodiments, synthetic oligonucleotides and
nucleic acid sequences may be designed that resist degradation by
endogenous host cell nucleolytic enzymes by containing such
linkages as: phosphorothioate, methylphosphonate, sulfone, sulfate,
ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and
other such linkages that have proven useful in antisense
applications (see, e.g., Agrwal et al., Tetrahedron Lett.
28:3539-3542 (1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665
(1971); Stec et al., Tetrahedron Lett. 26:2191-2194 (1985); Moody
et al., Nucl. Acids Res. 12:4769-4782 (1989); Uznanski et al.,
Nucl. Acids Res. (1989); Letsinger et al., Tetrahedron 40:137-143
(1984); Eckstein, Annu. Rev. Biochem. 54:367-402 (1985); Eckstein,
Trends Biol. Sci. 14:97-100 (1989); Stein In:
Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression,
Cohen, Ed., Macmillan Press, London, pp. 97-117 (1989); Jager et
al., Biochemistry 27:7237-7246 (1988)).
[0167] In one embodiment, the present invention provides truncated
components (e.g., binding domain polypeptide, hinge region
polypeptide, linker, etc.) for use in a binding
domain-immunoglobulin fusion protein, and in another embodiment the
invention provides nucleic acids encoding a binding
domain-immunoglobulin fusion protein having such truncated
components. A truncated molecule may be any molecule that comprises
less than a full length version of the molecule. Truncated
molecules provided by the present invention may include truncated
biological polymers, and in preferred embodiments of the invention
such truncated molecules may be truncated nucleic acid molecules or
truncated polypeptides. Truncated nucleic acid molecules have less
than the full length nucleotide sequence of a known or described
nucleic acid molecule, where such a known or described nucleic acid
molecule may be a naturally occurring, a synthetic or a recombinant
nucleic acid molecule, so long as one skilled in the art would
regard it as a full length molecule. Thus, for example, truncated
nucleic acid molecules that correspond to a gene sequence contain
less than the full length gene where the gene comprises coding and
non-coding sequences, promoters, enhancers and other regulatory
sequences, flanking sequences and the like, and other functional
and non-functional sequences that are recognized as part of the
gene. In another example, truncated nucleic acid molecules that
correspond to a mRNA sequence contain less than the full length
mRNA transcript, which may include various translated and
non-translated regions as well as other functional and
non-functional sequences.
[0168] In other preferred embodiments, truncated molecules are
polypeptides that comprise less than the full length amino acid
sequence of a particular protein or polypeptide component. As used
herein "deletion" has its common meaning as understood by those
familiar with the art, and may refer to molecules that lack one or
more of a portion of a sequence from either terminus or from a
non-terminal region, relative to a corresponding full length
molecule, for example, as in the case of truncated molecules
provided herein. Truncated molecules that are linear biological
polymers such as nucleic acid molecules or polypeptides may have
one or more of a deletion from either terminus of the molecule or a
deletion from a non-terminal region of the molecule, where such
deletions may be deletions of 1-1500 contiguous nucleotide or amino
acid residues, preferably 1-500 contiguous nucleotide or amino acid
residues and more preferably 1-300 contiguous nucleotide or amino
acid residues, including deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31-40, 41-50, 51-74, 75-100, 101-150, 151-200,
201-250 or 251-299 contiguous nucleotide or amino acid residues. In
certain particularly preferred embodiments truncated nucleic acid
molecules may have a deletion of 270-330 contiguous nucleotides. In
certain other particularly preferred embodiments truncated
polypeptide molecules may have a deletion of 80-140 contiguous
amino acids.
[0169] The present invention further relates to variants of the
herein referenced nucleic acids which encode fragments, analogs
and/or derivatives of a binding domain-immunoglobulin fusion
polypeptide. The variants of the nucleic acids encoding binding
domain-immunoglobulin fusion may be naturally occurring allelic
variants of the nucleic acids or non-naturally occurring variants.
As is known in the art, an allelic variant is an alternate form of
a nucleic acid sequence which may have at least one of a
substitution, a deletion or an addition of one or more nucleotides,
any of which does not substantially alter the function of the
encoded binding domain-immunoglobulin fusion polypeptide.
[0170] Variants and derivatives of binding domain-immunoglobulin
fusion may be obtained by mutations of nucleotide sequences
encoding binding domain-immunoglobulin fusion polypeptides or any
portion thereof. Alterations of the native amino acid sequence may
be accomplished by any of a number of conventional methods.
Mutations can be introduced at particular loci by synthesizing
oligonucleotides containing a mutant sequence, flanked by
restriction sites enabling ligation to fragments of the native
sequence. Following ligation, the resulting reconstructed sequence
encodes an analog having the desired amino acid insertion,
substitution, or deletion.
[0171] Alternatively, oligonucleotide-directed site-specific
mutagenesis procedures can be employed to provide an altered gene
wherein predetermined codons can be altered by substitution,
deletion or insertion. Exemplary methods of making such alterations
are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al.
(Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19);
Smith et al. (Genetic Engineering: Principles and Methods
BioTechniques, January 1985, 12-19); Smith et al. (Genetic
Engineering: Principles and Methods, Plenum Press, 1981); Kunkel
(Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methods
in Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and
4,737,462.
[0172] As an example, modification of DNA may be performed by
site-directed mutagenesis of DNA encoding the protein combined with
the use of DNA amplification methods using primers to introduce and
amplify alterations in the DNA template, such as PCR splicing by
overlap extension (SOE). Site-directed mutagenesis is typically
effected using a phage vector that has single- and double-stranded
forms, such as M13 phage vectors, which are well-known and
commercially available. Other suitable vectors that contain a
single-stranded phage origin of replication may be used (see, e.g.,
Veira et al., Meth. Enzymol. 15:3, 1987). In general, site-directed
mutagenesis is performed by preparing a single-stranded vector that
encodes the protein of interest (e.g., all or a component portion
of a given binding domain-immunoglobulin fusion protein). An
oligonucleotide primer that contains the desired mutation within a
region of homology to the DNA in the single-stranded vector is
annealed to the vector followed by addition of a DNA polymerase,
such as E. coli DNA polymerase I (Klenow fragment), which uses the
double stranded region as a primer to produce a heteroduplex in
which one strand encodes the altered sequence and the other the
original sequence. The heteroduplex is introduced into appropriate
bacterial cells and clones that include the desired mutation are
selected. The resulting altered DNA molecules may be expressed
recombinantly in appropriate host cells to produce the modified
protein.
[0173] Equivalent DNA constructs that encode various additions or
substitutions of amino acid residues or sequences, or deletions of
terminal or internal residues or sequences not needed for
biological activity are also encompassed by the invention. For
example, and as discussed above, sequences encoding Cys residues
that are not desirable or essential for biological activity can be
altered to cause the Cys residues to be deleted or replaced with
other amino acids, preventing formation of incorrect intramolecular
disulfide bridges upon renaturation.
[0174] Host organisms include those organisms in which recombinant
production of binding domain-immunoglobulin fusion products encoded
by the recombinant constructs of the present invention may occur,
such as bacteria (for example, E. coli), yeast (for example,
Saccharomyces cerevisiae and Pichia pastoris), insect cells and
mammals, including in vitro and in vivo expression. Host organisms
thus may include organisms for the construction, propagation,
expression or other steps in the production of the compositions
provided herein; hosts also include subjects in which immune
responses take place, as described above. Presently preferred host
organisms are E. coli bacterial strains, inbred murine strains and
murine cell lines, and human cells, subjects and cell lines.
[0175] The DNA construct encoding the desired binding
domain-immunoglobulin fusion is introduced into a plasmid for
expression in an appropriate host. In preferred embodiments, the
host is a bacterial host. The sequence encoding the ligand or
nucleic acid binding domain is preferably codon-optimized for
expression in the particular host. Thus, for example, if a human
binding domain-immunoglobulin fusion is expressed in bacteria, the
codons would be optimized for bacterial usage. For small coding
regions, the gene can be synthesized as a single oligonucleotide.
For larger proteins, splicing of multiple oligonucleotides,
mutagenesis, or other techniques known to those in the art may be
used. The sequences of nucleotides in the plasmids that are
regulatory regions, such as promoters and operators, are
operationally associated with one another for transcription. The
sequence of nucleotides encoding a binding domain-immunoglobulin
fusion protein may also include DNA encoding a secretion signal,
whereby the resulting peptide is a precursor protein. The resulting
processed protein may be recovered from the periplasmic space or
the fermentation medium.
[0176] In preferred embodiments, the DNA plasmids also include a
transcription terminator sequence. As used herein, a "transcription
terminator region" is a sequence that signals transcription
termination. The entire transcription terminator may be obtained
from a protein-encoding gene, which may be the same or different
from the inserted binding domain-immunoglobulin fusion encoding
gene or the source of the promoter. Transcription terminators are
optional components of the expression systems herein, but are
employed in preferred embodiments.
[0177] The plasmids used herein include a promoter in operative
association with the DNA encoding the protein or polypeptide of
interest and are designed for expression of proteins in a suitable
host as described above (e.g., bacterial, murine or human)
depending upon the desired use of the plasmid (e.g., administration
of a vaccine containing binding domain-immunoglobulin fusion
encoding sequences). Suitable promoters for expression of proteins
and polypeptides herein are widely available and are well known in
the art. Inducible promoters or constitutive promoters that are
linked to regulatory regions are preferred. Such promoters include,
but are not limited to, the T7 phage promoter and other T7-like
phage promoters, such as the T3, T5 and SP6 promoters, the trp,
1pp, and lac promoters, such as the lacUV5, from E. coli; the P10
or polyhedrin gene promoter of baculovirus/insect cell expression
systems (see, e.g., U.S. Pat. Nos. 5,243,041, 5,242,687, 5,266,317,
4,745,051, and 5,169,784) and inducible promoters from other
eukaryotic expression systems. For expression of the proteins such
promoters are inserted in a plasmid in operative linkage with a
control region such as the lac operon.
[0178] Preferred promoter regions are those that are inducible and
functional in E. coli. Examples of suitable inducible promoters and
promoter regions include, but are not limited to: the E. coli lac
operator responsive to isopropyl 13-D-thiogalactopyranoside (IPTG;
see Nakamura et al., Cell 18:1109-1117, 1979); the metallothionein
promoter metal-regulatory-elements responsive to heavy-metal (e.g.,
zinc) induction (see, e.g., U.S. Pat. No. 4,870,009 to Evans et
al.); the phage T7lac promoter responsive to IPTG (see, e.g., U.S.
Pat. No. 4,952,496; and Studier et al., Meth. Enzymol. 185:60-89,
1990) and the TAC promoter.
[0179] The plasmids may optionally include a selectable marker gene
or genes that are functional in the host. A selectable marker gene
includes any gene that confers a phenotype on bacteria that allows
transformed bacterial cells to be identified and selectively grown
from among a vast majority of untransformed cells. Suitable
selectable marker genes for bacterial hosts, for example, include
the ampicillin resistance gene (Amp.sup.r), tetracycline resistance
gene (Tc.sup.r) and the kanamycin resistance gene (Kan.sup.r). The
kanamycin resistance gene is presently preferred.
[0180] The plasmids may also include DNA encoding a signal for
secretion of the operably linked protein. Secretion signals
suitable for use are widely available and are well known in the
art. Prokaryotic and eukaryotic secretion signals functional in E.
coli may be employed. The presently preferred secretion signals
include, but are not limited to, those encoded by the following E.
coli genes: ompA, ompT, ompF, ompC, beta-lactamase, and alkaline
phosphatase, and the like (von Heijne, J. Mol. Biol. 184:99-105,
1985). In addition, the bacterial pelB gene secretion signal (Lei
et al., J. Bacteriol. 169:4379, 1987), the phoA secretion signal,
and the cek2 functional in insect cell may be employed. The most
preferred secretion signal is the E. coli ompA secretion signal.
Other prokaryotic and eukaryotic secretion signals known to those
of skill in the art may also be employed (see, e.g., von Heijne, J.
Mol. Biol. 184:99-105, 1985). Using the methods described herein,
one of skill in the art can substitute secretion signals that are
functional in either yeast, insect or mammalian cells to secrete
proteins from those cells.
[0181] Preferred plasmids for transformation of E. coli cells
include the pET expression vectors (e.g., pET-11a, pET-12a-c,
pET-15b; see U.S. Pat. No. 4,952,496; available from Novagen,
Madison, Wis.). Other preferred plasmids include the pKK plasmids,
particularly pKK 223-3, which contains the tac promoter (Brosius et
al., Proc. Natl. Acad. Sci. 81:6929, 1984; Ausubel et al., Current
Protocols in Molecular Biology; U.S. Pat. Nos. 5,122,463,
5,173,403, 5,187,153, 5,204,254, 5,212,058, 5,212,286, 5,215,907,
5,220,013, 5,223,483, and 5,229,279). Plasmid pKK has been modified
by replacement of the ampicillin resistance gene with a kanamycin
resistance gene. (Available from Pharmacia; obtained from pUC4K,
see, e.g., Vieira et al. (Gene 19:259-268, 1982; and U.S. Pat. No.
4,719,179.) Baculovirus vectors, such as pBlueBac (also called
pJVETL and derivatives thereof), particularly pBlueBac III (see,
e.g., U.S. Pat. Nos. 5,278,050, 5,244,805, 5,243,041, 5,242,687,
5,266,317, 4,745,051, and 5,169,784; available from Invitrogen, San
Diego) may also be used for expression of the polypeptides in
insect cells. Other plasmids include the pIN-IllompA plasmids (see
U.S. Pat. No. 4,575,013; see also Duffaud et al., Meth. Enz.
153:492-507, 1987), such as pIN-IIIompA2.
[0182] Preferably, the DNA molecule is replicated in bacterial
cells, preferably in E. coli. The preferred DNA molecule also
includes a bacterial origin of replication, to ensure the
maintenance of the DNA molecule from generation to generation of
the bacteria. In this way, large quantities of the DNA molecule can
be produced by replication in bacteria. Preferred bacterial origins
of replication include, but are not limited to, the fl-ori and col
El origins of replication. Preferred hosts contain chromosomal
copies of DNA encoding T7 RNA polymerase operably linked to an
inducible promoter, such as the lacUV promoter (see U.S. Pat. No.
4,952,496). Such hosts include, but are not limited to, lysogens E.
coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS, HMS174(DE3) and
BL21(DE3). Strain BL21(DE3) is preferred. The pLys strains provide
low levels of T7 lysozyme, a natural inhibitor of T7 RNA
polymerase.
[0183] The DNA molecules provided may also contain a gene coding
for a repressor protein. The repressor protein is capable of
repressing the transcription of a promoter that contains sequences
of nucleotides to which the repressor protein binds. The promoter
can be derepressed by altering the physiological conditions of the
cell. For example, the alteration can be accomplished by adding to
the growth medium a molecule that inhibits the ability to interact
with the operator or with regulatory proteins or other regions of
the DNA or by altering the temperature of the growth media.
Preferred repressor proteins include, but are not limited to the E.
coli lad repressor responsive to IPTG induction, the temperature
sensitive X c1857 repressor, and the like. The E. coli lad
repressor is preferred.
[0184] In general, recombinant constructs of the subject invention
will also contain elements necessary for transcription and
translation. In particular, such elements are preferred where the
recombinant expression construct containing nucleic acid sequences
encoding binding domain-immunoglobulin fusion proteins is intended
for expression in a host cell or organism. In certain embodiments
of the present invention, cell type preferred or cell type specific
expression of a cell binding domain-immunoglobulin fusion encoding
gene may be achieved by placing the gene under regulation of a
promoter. The choice of the promoter will depend upon the cell type
to be transformed and the degree or type of control desired.
Promoters can be constitutive or active and may further be cell
type specific, tissue specific, individual cell specific, event
specific, temporally specific or inducible. Cell-type specific
promoters and event type specific promoters are preferred. Examples
of constitutive or nonspecific promoters include the SV40 early
promoter (U.S. Pat. No. 5,118,627), the SV40 late promoter (U.S.
Pat. No. 5,118,627), CMV early gene promoter (U.S. Pat. No.
5,168,062), and adenovirus promoter. In addition to viral
promoters, cellular promoters are also amenable within the context
of this invention. In particular, cellular promoters for the
so-called housekeeping genes are useful. Viral promoters are
preferred, because generally they are stronger promoters than
cellular promoters. Promoter regions have been identified in the
genes of many eukaryotes including higher eukaryotes, such that
suitable promoters for use in a particular host can be readily
selected by those skilled in the art.
[0185] Inducible promoters may also be used. These promoters
include MMTV LTR (PCT WO 91/13160), inducible by dexamethasone;
metallothionein promoter, inducible by heavy metals; and promoters
with cAMP response elements, inducible by cAMP. By using an
inducible promoter, the nucleic acid sequence encoding a binding
domain-immunoglobulin fusion protein may be delivered to a cell by
the subject invention expression construct and will remain
quiescent until the addition of the inducer. This allows further
control on the timing of production of the gene product.
[0186] Event-type specific promoters are active or up-regulated
only upon the occurrence of an event, such as tumorigenicity or
viral infection. The HIV LTR is a well known example of an
event-specific promoter. The promoter is inactive unless the tat
gene product is present, which occurs upon viral infection. Some
event-type promoters are also tissue-specific.
[0187] Additionally, promoters that are coordinately regulated with
a particular cellular gene may be used. For example, promoters of
genes that are coordinately expressed may be used when expression
of a particular binding domain-immunoglobulin fusion
protein-encoding gene is desired in concert with expression of one
or more additional endogenous or exogenously introduced genes. This
type of promoter is especially useful when one knows the pattern of
gene expression relevant to induction of an immune response in a
particular tissue of the immune system, so that specific
immunocompetent cells within that tissue may be activated or
otherwise recruited to participate in the immune response.
[0188] In addition to the promoter, repressor sequences, negative
regulators, or tissue-specific silencers may be inserted to reduce
non-specific expression of binding domain-immunoglobulin fusion
protein encoding genes in certain situations, such as, for example,
a host that is transiently immunocompromised as part of a
therapeutic strategy. Multiple repressor elements may be inserted
in the promoter region. Repression of transcription is independent
on the orientation of repressor elements or distance from the
promoter. One type of repressor sequence is an insulator sequence.
Such sequences inhibit transcription (Dunaway et al., Mol Cell Biol
17: 182-9, 1997; Gdula et al., Proc Natl Acad Sci USA 93:9378-83,
1996, Chan et al., J Virol 70: 5312-28, 1996; Scott and Geyer, EMBO
J. 14:6258-67, 1995; Kalos and Fournier, Mol Cell Riot 15:198-207,
1995; Chung et al., Cell 74: 505-14, 1993) and will silence
background transcription.
[0189] Repressor elements have also been identified in the promoter
regions of the genes for type II (cartilage) collagen, choline
acetyltransferase, albumin (Hu et al., J. Cell Growth Differ.
3(9):577-588, 1992), phosphoglycerate kinase (PGK-2) (Misuno et
al., Gene 119(2):293-297, 1992), and in the
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.
(Lemaigre et al., Mol. Cell. Biol. 11(2):1099-1106.) Furthermore,
the negative regulatory element Tse-1 has been identified in a
number of liver specific genes, and has been shown to block cAMP
response element--(CRE) mediated induction of gene activation in
hepatocytes. (B o s hart et al., Cell 61(5):905-916, 1990.)
[0190] In preferred embodiments, elements that increase the
expression of the desired product are incorporated into the
construct. Such elements include internal ribosome binding sites
(IRES; Wang and Siddiqui, Curr. Top. Microbiol. Immunol 203:99,
1995; Ehrenfeld and Semler, Curr. Top. Microbiol. Immunol. 203:65,
1995; Rees et al., Biotechniques 20:102, 1996; Sugimoto et al.,
Biotechnology 12:694, 1994). IRES increase translation efficiency.
As well, other sequences may enhance expression. For some genes,
sequences especially at the 5' end inhibit transcription and/or
translation. These sequences are usually palindromes that can form
hairpin structures. Any such sequences in the nucleic acid to be
delivered are generally deleted. Expression levels of the
transcript or translated product are assayed to confirm or
ascertain which sequences affect expression. Transcript levels may
be assayed by any known method, including Northern blot
hybridization, RNase probe protection and the like. Protein levels
may be assayed by any known method, including ELISA, western blot,
immunocytochemistry or other well known techniques.
[0191] Other elements may be incorporated into the binding
domain-immunoglobulin fusion protein encoding constructs of the
present invention. In preferred embodiments, the construct includes
a transcription terminator sequence, including a polyadenylation
sequence, splice donor and acceptor sites, and an enhancer. Other
elements useful for expression and maintenance of the construct in
mammalian cells or other eukaryotic cells may also be incorporated
(e.g., origin of replication). Because the constructs are
conveniently produced in bacterial cells, elements that are
necessary for, or that enhance, propagation in bacteria are
incorporated. Such elements include an origin of replication, a
selectable marker and the like.
[0192] As provided herein, an additional level of controlling the
expression of nucleic acids encoding binding domain-immunoglobulin
fusion proteins delivered to cells using the constructs of the
invention may be provided by simultaneously delivering two or more
differentially regulated nucleic acid constructs. The use of such a
multiple nucleic acid construct approach may permit coordinated
regulation of an immune response such as, for example,
spatiotemporal coordination that depends on the cell type and/or
presence of another expressed encoded component. Those familiar
with the art will appreciate that multiple levels of regulated gene
expression may be achieved in a similar manner by selection of
suitable regulatory sequences, including but not limited to
promoters, enhancers and other well known gene regulatory
elements.
[0193] The present invention also relates to vectors, and to
constructs prepared from known vectors that include nucleic acids
of the present invention, and in particular to "recombinant
expression constructs" that include any nucleic acids encoding
binding domain-immunoglobulin fusion proteins and polypeptides
according to the invention as provided above; to host cells which
are genetically engineered with vectors and/or constructs of the
invention and to methods of administering expression constructs
comprising nucleic acid sequences encoding such binding
domain-immunoglobulin fusion polypeptides and fusion proteins of
the invention, or fragments or variants thereof, by recombinant
techniques. Binding domain-immunoglobulin fusion proteins can be
expressed in virtually any host cell under the control of
appropriate promoters, depending on the nature of the construct
(e.g., type of promoter, as described above), and on the nature of
the desired host cell (e.g., whether postmitotic terminally
differentiated or actively dividing; e.g., whether the expression
construct occurs in host cell as an episome or is integrated into
host cell genome). Appropriate cloning and expression vectors for
use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989); as noted above, in
particularly preferred embodiments of the invention, recombinant
expression is conducted in mammalian cells that have been
transfected or transformed with the subject invention recombinant
expression construct.
[0194] Typically, the constructs are derived from plasmid vectors.
A preferred construct is a modified pNASS vector (Clontech, Palo
Alto, Calif.), which has nucleic acid sequences encoding an
ampicillin resistance gene, a polyadenylation signal and a T7
promoter site. Other suitable mammalian expression vectors are well
known (see, e.g., Ausubel et al., 1995; Sambrook et al., supra; see
also, e.g., catalogues from Invitrogen, San Diego, Calif.; Novagen,
Madison, Wis.; Pharmacia, Piscataway, N.J.; and others). Presently
preferred constructs may be prepared that include a dihydrofolate
reductase (DHFR) encoding sequence under suitable regulatory
control, for promoting enhanced production levels of the binding
domain-immunoglobulin fusion protei, which levels result from gene
amplification following application of an appropriate selection
agent (e.g., methetrexate).
[0195] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, and a promoter derived from a
highly-expressed gene to direct transcription of a downstream
structural sequence, as described above. The heterologous
structural sequence is assembled in appropriate phase with
translation initiation and termination sequences. Thus, for
example, the binding domain-immunoglobulin fusion protein encoding
nucleic acids as provided herein may be included in any one of a
variety of expression vector constructs as a recombinant expression
construct for expressing a binding domain-immunoglobulin fusion
polypeptide in a host cell. In certain preferred embodiments the
constructs are included in formulations that are administered in
vivo. Such vectors and constructs include chromosomal,
nonchromosomal and synthetic DNA sequences, e.g., derivatives of
SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors
derived from combinations of plasmids and phage DNA, viral DNA,
such as vaccinia, adenovirus, fowl pox virus, and pseudorabies, or
replication deficient retroviruses as described below. However, any
other vector may be used for preparation of a recombinant
expression construct, and in preferred embodiments such a vector
will be replicable and viable in the host.
[0196] The appropriate DNA sequence(s) may be inserted into the
vector by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Standard techniques for cloning, DNA
isolation, amplification and purification, for enzymatic reactions
involving DNA ligase, DNA polymerase, restriction endonucleases and
the like, and various separation techniques are those known and
commonly employed by those skilled in the art. A number of standard
techniques are described, for example, in Ausubel et al. (1993
Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc.
& John Wiley & Sons, Inc., Boston, Mass.); Sambrook et al.
(1989 Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory,
Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning, Cold
Spring Harbor Laboratory, Plainview, N.Y.); Glover (Ed.) (1985 DNA
Cloning Vol. I and II, IRL Press, Oxford, UK); Hames and Higgins
(Eds.), (1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK);
and elsewhere.
[0197] The DNA sequence in the expression vector is operatively
linked to at least one appropriate expression control sequences
(e.g., a constitutive promoter or a regulated promoter) to direct
mRNA synthesis. Representative examples of such expression control
sequences include promoters of eukaryotic cells or their viruses,
as described above. Promoter regions can be selected from any
desired gene using CAT (chloramphenicol transferase) vectors or
other vectors with selectable markers. Eukaryotic promoters include
CMV immediate early, HSV thymidine kinase, early and late SV40,
LTRs from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art, and preparation of certain particularly
preferred recombinant expression constructs comprising at least one
promoter or regulated promoter operably linked to a nucleic acid
encoding an binding domain-immunoglobulin fusion polypeptide is
described herein.
[0198] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes may be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 by that
act on a promoter to increase its transcription. Examples include
the SV40 enhancer on the late side of the replication origin by 100
to 270, a cytomegalovirus early promoter enhancer, the polyoma
enhancer on the late side of the replication origin, and adenovirus
enhancers.
[0199] As provided herein, in certain embodiments the vector may be
a viral vector such as a retroviral vector. (Miller et al., 1989
BioTechniques 7:980; Coffin and Varmus, 1996 Retroviruses, Cold
Spring Harbor Laboratory Press, NY.) For example, retroviruses from
which the retroviral plasmid vectors may be derived include, but
are not limited to, Moloney Murine Leukemia Virus, spleen necrosis
virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma
virus, avian leukosis virus, gibbon ape leukemia virus, human
immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma
Virus, and mammary tumor virus.
[0200] Retroviruses are RNA viruses which can replicate and
integrate into the genome of a host cell via a DNA intermediate.
This DNA intermediate, or provirus, may be stably integrated into
the host cell DNA. According to certain embodiments of the present
invention, an expression construct may comprise a retrovirus into
which a foreign gene that encodes a foreign protein is incorporated
in place of normal retroviral RNA. When retroviral RNA enters a
host cell coincident with infection, the foreign gene is also
introduced into the cell, and may then be integrated into host cell
DNA as if it were part of the retroviral genome. Expression of this
foreign gene within the host results in expression of the foreign
protein.
[0201] Most retroviral vector systems which have been developed for
gene therapy are based on murine retroviruses. Such retroviruses
exist in two forms, as free viral particles referred to as virions,
or as proviruses integrated into host cell DNA. The virion form of
the virus contains the structural and enzymatic proteins of the
retrovirus (including the enzyme reverse transcriptase), two RNA
copies of the viral genome, and portions of the source cell plasma
membrane containing viral envelope glycoprotein. The retroviral
genome is organized into four main regions: the Long Terminal
Repeat (LTR), which contains cis-acting elements necessary for the
initiation and termination of transcription and is situated both 5'
and 3' of the coding genes, and the three coding genes gag, pol,
and env. These three genes gag, pol, and env encode, respectively,
internal viral structures, enzymatic proteins (such as integrase),
and the envelope glycoprotein (designated gp70 and pl5e) which
confers infectivity and host range specificity of the virus, as
well as the "R" peptide of undetermined function.
[0202] Separate packaging cell lines and vector producing cell
lines have been developed because of safety concerns regarding the
uses of retroviruses, including their use in expression constructs
as provided by the present invention. Briefly, this methodology
employs the use of two components, a retroviral vector and a
packaging cell line (PCL). The retroviral vector contains long
terminal repeats (LTRs), the foreign DNA to be transferred and a
packaging sequence (y). This retroviral vector will not reproduce
by itself because the genes which encode structural and envelope
proteins are not included within the vector genome. The PCL
contains genes encoding the gag, poi, and env proteins, but does
not contain the packaging signal "y". Thus, a PCL can only form
empty virion particles by itself. Within this general method, the
retroviral vector is introduced into the PCL, thereby creating a
vector-producing cell line (VCL). This VCL manufactures virion
particles containing only the retroviral vector's (foreign) genome,
and therefore has previously been considered to be a safe
retrovirus vector for therapeutic use.
[0203] "Retroviral vector construct" refers to an assembly which
is, within preferred embodiments of the invention, capable of
directing the expression of a sequence(s) or gene(s) of interest,
such as binding domain-immunoglobulin fusion encoding nucleic acid
sequences. Briefly, the retroviral vector construct must include a
5' LTR, a tRNA binding site, a packaging signal, an origin of
second strand DNA synthesis and a 3' LTR. A wide variety of
heterologous sequences may be included within the vector construct,
including for example, sequences which encode a protein (e.g.,
cytotoxic protein, disease-associated antigen, immune accessory
molecule, or replacement gene), or which are useful as a molecule
itself (e.g., as a ribozyme or antisense sequence).
[0204] Retroviral vector constructs of the present invention may be
readily constructed from a wide variety of retroviruses, including
for example, B, C, and D type retroviruses as well as spumaviruses
and lentiviruses (see, e.g., RNA Tumor Viruses, Second Edition,
Cold Spring Harbor Laboratory, 1985). Such retroviruses may be
readily obtained from depositories or collections such as the
American Type Culture Collection ("ATCC"; Rockville, Md.), or
isolated from known sources using commonly available techniques.
Any of the above retroviruses may be readily utilized in order to
assemble or construct retroviral vector constructs, packaging
cells, or producer cells of the present invention given the
disclosure provided herein, and standard recombinant techniques
(e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d
ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS
82:488, 1985).
[0205] Suitable promoters for use in viral vectors generally may
include, but are not limited to, the retroviral LTR; the SV40
promoter; and the human cytomegalovirus (CMV) promoter described in
Miller, et al., Biotechniques 7:980-990 (1989), or any other
promoter (e.g., cellular promoters such as eukaryotic cellular
promoters including, but not limited to, the histone, pol III, and
.beta.-actin promoters). Other viral promoters which may be
employed include, but are not limited to, adenovirus promoters,
thymidine kinase (TK) promoters, and B 19 parvovirus promoters. The
selection of a suitable promoter will be apparent to those skilled
in the art from the teachings contained herein, and may be from
among either regulated promoters or promoters as described
above.
[0206] As described above, the retroviral plasmid vector is
employed to transduce packaging cell lines to form producer cell
lines. Examples of packaging cells which may be transfected
include, but are not limited to, the PE501, PA317, .psi.-2,
.psi.-AM, PAl2, T19-14X, VT-19-17-H2, CRE, .psi.CRIP, GP+E-86,
GP+envAml2, and DAN cell lines as described in Miller, Human Gene
Therapy, 1:5-14 (1990). The vector may transduce the packaging
cells through any means known in the art. Such means include, but
are not limited to, electroporation, the use of liposomes, and
CaPO.sub.4 precipitation. In one alternative, the retroviral
plasmid vector may be encapsulated into a liposome, or coupled to a
lipid, and then administered to a host.
[0207] The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequence(s)
encoding the binding domain-immunoglobulin fusion polypeptides or
fusion proteins. Such retroviral vector particles then may be
employed, to transduce eukaryotic cells, either in vitro or in
vivo. The transduced eukaryotic cells will express the nucleic acid
sequence(s) encoding the binding domain-immunoglobulin fusion
polypeptide or fusion protein. Eukaryotic cells which may be
transduced include, but are not limited to, embryonic stem cells,
as well as hematopoietic stem cells, hepatocytes, fibroblasts,
circulating peripheral blood mononuclear and polymorphonuclear
cells including myelomonocytic cells, lymphocytes, myoblasts,
tissue macrophages, dendritic cells, Kupffer cells, lymphoid and
reticuloendothelia cells of the lymph nodes and spleen,
keratinocytes, endothelial cells, and bronchial epithelial
cells.
[0208] As another example of an embodiment of the invention in
which a viral vector is used to prepare the recombinant binding
domain-immunoglobulin fusion expression construct, in one preferred
embodiment, host cells transduced by a recombinant viral construct
directing the expression of binding domain-immunoglobulin fusion
polypeptides or fusion proteins may produce viral particles
containing expressed binding domain-immunoglobulin fusion
polypeptides or fusion proteins that are derived from portions of a
host cell membrane incorporated by the viral particles during viral
budding.
[0209] In another aspect, the present invention relates to host
cells containing the above described recombinant binding
domain-immunoglobulin fusion expression constructs. Host cells are
genetically engineered (transduced, transformed or transfected)
with the vectors and/or expression constructs of this invention
which may be, for example, a cloning vector, a shuttle vector or an
expression construct. The vector or construct may be, for example,
in the form of a plasmid, a viral particle, a phage, etc. The
engineered host cells can be cultured in conventional nutrient
media modified as appropriate for activating promoters, selecting
transformants or amplifying particular genes such as genes encoding
binding domain-immunoglobulin fusion polypeptides or binding
domain-immunoglobulin fusion proteins. The culture conditions for
particular host cells selected for expression, such as temperature,
pH and the like, will be readily apparent to the ordinarily skilled
artisan.
[0210] The host cell can be a higher eukaryotic cell, such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell,
or the host cell can be a prokaryotic cell, such as a bacterial
cell. Representative examples of appropriate host cells according
to the present invention include, but need not be limited to,
bacterial cells, such as E. coli, Streptomyces, Salmonella
tvphimurium; fungal cells, such as yeast; insect cells, such as
Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS or
293 cells; adenoviruses; plant cells, or any suitable cell already
adapted to in vitro propagation or so established de novo. The
selection of an appropriate host is deemed to be within the scope
of those skilled in the art from the teachings herein.
[0211] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences, for example as described herein regarding
the preparation of binding domain-immunoglobulin fusion expression
constructs. DNA sequences derived from the SV40 splice, and
polyadenylation sites may be used to provide the required
nontranscribed genetic elements. Introduction of the construct into
the host cell can be effected by a variety of methods with which
those skilled in the art will be familiar, including but not
limited to, for example, calcium phosphate transfection,
DEAE-Dextran mediated transfection, or electroporation (Davis et
al., 1986 Basic Methods in Molecular Biology).
[0212] The present invention binding domain-immunoglobulin fusion
proteins, or compositions comprising one or more polynucleotides
encoding same as described herein, (for example, to be administered
under conditions and for a time sufficient to permit expression of
a binding domain-immunoglobulin fusion protein in a host cell in
vivo or in vitro), may be formulated into pharmaceutical
compositions for administration according to well known
methodologies. Pharmaceutical compositions generally comprise one
or more recombinant expression constructs, and/or expression
products of such constructs, in combination with a pharmaceutically
acceptable carrier, excipient or diluent. Such carriers will be
nontoxic to recipients at the dosages and concentrations employed.
For nucleic acid-based formulations, or for formulations comprising
expression products of the subject invention recombinant
constructs, about 0.01 .mu.g/kg to about 100 mg/kg body weight will
be administered, typically by the intradermal, subcutaneous,
intramuscular or intravenous route, or by other routes. A preferred
dosage is about 1 .mu.g/kg to about 1 mg/kg, with about 5 .mu.g/kg
to about 200 .mu.g/kg particularly preferred. It will be evident to
those skilled in the art that the number and frequency of
administration will be dependent upon the response of the host.
"Pharmaceutically acceptable carriers" for therapeutic use are well
known in the pharmaceutical art, and are described, for example, in
Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R.
Gennaro edit. 1985). For example, sterile saline and
phosphate-buffered saline at physiological pH may be used.
Preservatives, stabilizers, dyes and even flavoring agents may be
provided in the pharmaceutical composition. For example, sodium
benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be
added as preservatives. Id. at 1449. In addition, antioxidants and
suspending agents may be used. Id.
[0213] "Pharmaceutically acceptable salt" refers to salts of the
compounds of the present invention derived from the combination of
such compounds and an organic or inorganic acid (acid addition
salts) or an organic or inorganic base (base addition salts). The
compounds of the present invention may be used in either the free
base or salt forms, with both forms being considered as being
within the scope of the present invention.
[0214] The pharmaceutical compositions that contain one or more
binding domain-immunoglobulin fusion protein encoding constructs
(or their expressed products) may be in any form which allows for
the composition to be administered to a patient. For example, the
composition may be in the form of a solid, liquid or gas (aerosol).
Typical routes of administration include, without limitation, oral,
topical, parenteral (e.g., sublingually or buccally), sublingual,
rectal, vaginal, and intranasal. The term parenteral as used herein
includes subcutaneous injections, intravenous, intramuscular,
intrasternal, intracavernous, intrathecal, intrameatal,
intraurethral injection or infusion techniques. The pharmaceutical
composition is formulated so as to allow the active ingredients
contained therein to be bioavailable upon administration of the
composition to a patient. Compositions that will be administered to
a patient take the form of one or more dosage units, where for
example, a tablet may be a single dosage unit, and a container of
one or more compounds of the invention in aerosol form may hold a
plurality of dosage units.
[0215] For oral administration, an excipient and/or binder may be
present. Examples are sucrose, kaolin, glycerin, starch dextrins,
sodium alginate, carboxymethylcellulose and ethyl cellulose.
Coloring and/or flavoring agents may be present. A coating shell
may be employed.
[0216] The composition may be in the form of a liquid, e.g., an
elixir, syrup, solution, emulsion or suspension. The liquid may be
for oral administration or for delivery by injection, as two
examples. When intended for oral administration, preferred
compositions contain, in addition to one or more binding
domain-immunoglobulin fusion construct or expressed product, one or
more of a sweetening agent, preservatives, dye/colorant and flavor
enhancer. In a composition intended to be administered by
injection, one or more of a surfactant, preservative, wetting
agent, dispersing agent, suspending agent, buffer, stabilizer and
isotonic agent may be included.
[0217] A liquid pharmaceutical composition as used herein, whether
in the form of a solution, suspension or other like form, may
include one or more of the following adjuvants: sterile diluents
such as water for injection, saline solution, preferably
physiological saline, Ringer's solution, isotonic sodium chloride,
fixed oils such as synthetic mono or digylcerides which may serve
as the solvent or suspending medium, polyethylene glycols,
glycerin, propylene glycol or other solvents; antibacterial agents
such as benzyl alcohol or methyl paraben; antioxidants such as
ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The parenteral preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic. Physiological saline is a preferred
adjuvant. An injectable pharmaceutical composition is preferably
sterile.
[0218] It may also be desirable to include other components in the
preparation, such as delivery vehicles including but not limited to
aluminum salts, water-in-oil emulsions, biodegradable oil vehicles,
oil-in-water emulsions, biodegradable microcapsules, and liposomes.
Examples of immunostimulatory substances (adjuvants) for use in
such vehicles include N-acetylmuramyl-L-alanine-D-isoglutamine
(MDP), lipopoly-saccharides (LPS), glucan, IL-12, GM-CSF, gamma
interferon and IL-15.
[0219] While any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this invention, the type of carrier will vary depending on the mode
of administration and whether a sustained release is desired. For
parenteral administration, such as subcutaneous injection, the
carrier preferably comprises water, saline, alcohol, a fat, a wax
or a buffer. For oral administration, any of the above carriers or
a solid carrier, such as mannitol, lactose, starch, magnesium
stearate, sodium saccharine, talcum, cellulose, glucose, sucrose,
and magnesium carbonate, may be employed. Biodegradable
microspheres (e.g., polylactic galactide) may also be employed as
carriers for the pharmaceutical compositions of this invention.
Suitable biodegradable microspheres are disclosed, for example, in
U.S. Pat. Nos. 4,897,268 and 5,075,109. In this regard, it is
preferable that the microsphere be larger than approximately 25
microns.
[0220] Pharmaceutical compositions may also contain diluents such
as buffers, antioxidants such as ascorbic acid, low molecular
weight (less than about 10 residues) polypeptides, proteins, amino
acids, carbohydrates including glucose, sucrose or dextrins,
chelating agents such as EDTA, glutathione and other stabilizers
and excipients. Neutral buffered saline or saline mixed with
nonspecific serum albumin are exemplary appropriate diluents.
Preferably, product is formulated as a lyophilizate using
appropriate excipient solutions (e.g., sucrose) as diluents.
[0221] As described above, the subject invention includes
compositions capable of delivering nucleic acid molecules encoding
binding domain-immunoglobulin fusion proteins. Such compositions
include recombinant viral vectors (e.g., retroviruses (see WO
90/07936, WO 91/02805, WO 93/25234, WO 93/25698, and WO 94/03622),
adenovirus (see Berkner, Biotechniques 6:616-627, 1988; Li et al.,
Hum. Gene Ther. 4:403-409, 1993; Vincent et al., Nat. Genet.
5:130-134, 1993; and Kolls et al., Proc. Natl. Acad. Sci. USA
91:215-219, 1994), pox virus (see U.S. Pat. No. 4,769,330; U.S.
Pat. No. 5,017,487; and WO 89/01973)), recombinant expression
construct nucleic acid molecules complexed to a polycationic
molecule (see WO 93/03709), and nucleic acids associated with
liposomes (see Wang et al., Proc. Natl. Acad. Sci. USA 84:7851,
1987). In certain embodiments, the DNA may be linked to killed or
inactivated adenovirus (see Curiel et al., Hum. Gene Ther.
3:147-154, 1992; Cotton et al., Proc. Natl. Acad. Sci. USA 89:6094,
1992). Other suitable compositions include DNA-ligand (see Wu et
al., J. Biol. Chem. 264:16985-16987, 1989) and lipid-DNA
combinations (see Feigner et al., Proc. Natl. Acad. Sci. USA
84:7413-7417, 1989).
[0222] In addition to direct in vivo procedures, ex vivo procedures
may be used in which cells are removed from a host, modified, and
placed into the same or another host animal. It will be evident
that one can utilize any of the compositions noted above for
introduction of binding domain-immunoglobulin fusion proteins or of
binding domain-immunoglobulin fusion protein encoding nucleic acid
molecules into tissue cells in an ex vivo context. Protocols for
viral, physical and chemical methods of uptake are well known in
the art.
[0223] Accordingly, the present invention is useful for treating a
patient having a B-cell disorder or a malignant condition, or for
treating a cell culture derived from such a patient. As used
herein, the term "patient" refers to any warm-blooded animal,
preferably a human. A patient may be afflicted with cancer or a
malignant condition, such as B-cell lymphoma, or may be normal
(i.e., free of detectable disease and infection). A "cell culture"
includes any preparation amenable to ex vivo treatment, for example
a preparation containing immunocompetent cells or isolated cells of
the immune system (including, but not limited to, T cells,
macrophages, monocytes, B cells and dendritic cells). Such cells
may be isolated by any of a variety of techniques well known to
those of ordinary skill in the art (e.g., Ficoll-hypaque density
centrifugation). The cells may (but need not) have been isolated
from a patient afflicted with a B-cell disorder or a malignant
condition, and may be reintroduced into a patient after
treatment.
[0224] A liquid composition intended for either parenteral or oral
administration should contain an amount of binding
domain-immunoglobulin fusion protein encoding construct or
expressed product such that a suitable dosage will be obtained.
Typically, this amount is at least 0.01 wt % of a binding
domain-immunoglobulin fusion construct or expressed product in the
composition. When intended for oral administration, this amount may
be varied to be between 0.1 and about 70% of the weight of the
composition. Preferred oral compositions contain between about 4%
and about 50% of binding domain-immunoglobulin fusion construct or
expressed product(s). Preferred compositions and preparations are
prepared so that a parenteral dosage unit contains between 0.01 to
1% by weight of active compound.
[0225] The pharmaceutical composition may be intended for topical
administration, in which case the carrier may suitably comprise a
solution, emulsion, ointment or gel base. The base, for example,
may comprise one or more of the following: petrolatum, lanolin,
polyethylene glycols, beeswax, mineral oil, diluents such as water
and alcohol, and emulsifiers and stabilizers. Thickening agents may
be present in a pharmaceutical composition for topical
administration. If intended for transdermal administration, the
composition may include a transdermal patch or iontophoresis
device. Topical formulations may contain a concentration of the
binding domain-immunoglobulin fusion construct or expressed product
of from about 0.1 to about 10% w/v (weight per unit volume).
[0226] The composition may be intended for rectal administration,
in the form, e.g., of a suppository which will melt in the rectum
and release the drug. The composition for rectal administration may
contain an oleaginous base as a suitable nonirritating excipient.
Such bases include, without limitation, lanolin, cocoa butter and
polyethylene glycol.
[0227] In the methods of the invention, the binding
domain-immunoglobulin fusion encoding constructs or expressed
product(s) may be administered through use of insert(s), bead(s),
timed-release formulation(s), patch(es) or fast-release
formulation(s).
[0228] The following Examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Cloning of the 2H7 Variable Regions and Construction and Sequencing
of 2H7ScFV-Ig
[0229] This Example illustrates the cloning of cDNA molecules that
encode the heavy chain and light chain variable regions of the
monoclonal antibody 2H7. This Example also demonstrates the
construction, sequencing, and expression of 2H7scFv-Ig.
[0230] Hybridoma cells expressing 2H7 monoclonal antibody that
specifically bound to CD20 were provided by Ed Clark at the
University of Washington, Seattle, Wash. Prior to harvesting,
hybridoma cells were kept in log phase growth for several days in
RPMI 1640 media Invitrogen/Life Technologies, Gaithersburg, Md.)
supplemented with glutamine, pyruvate, DMEM non-essential amino
acids, and penicillin-streptomycin. Cells were pelleted by
centrifugation from the culture medium, and 2.times.10.sup.7 cells
were used to prepare RNA. RNA was isolated from the 2H7-producing
hybridoma cells using the Pharmingen (San Diego, Calif.) total RNA
isolation kit (Catalog #45520K) according to the manufacturer's
instructions accompanying the kit. One microgram (1 ug) of total
RNA was used as template to prepare cDNA by reverse transcription.
The RNA and 300 ng random primers were combined and denatured at
72.degree. C. for 10 minutes prior to addition of enzyme.
Superscript II reverse transcriptase (Life Technologies) was added
to the RNA plus primer mixture in a total volume of 25 .mu.l in the
presence of 5.times. second strand buffer and 0.1 M DTT provided
with the enzyme. The reverse transcription reaction was allowed to
proceed at 42.degree. C. for one hour.
[0231] The 2H7 cDNA generated in the randomly primed reverse
transcriptase reaction and V region specific primers were used to
amplify by PCR the variable regions for the light and heavy chain
of the 2H7 antibody. The V region specific primers were designed
using the published sequence (Genbank accession numbers M17954 for
V.sub.L and M17953 for V.sub.H) as a guide. The two variable chains
were designed with compatible end sequences so that an scFv could
be assembled by ligation of the two V regions after amplification
and restriction enzyme digestion.
[0232] A (gly.sub.4ser).sub.3 peptide linker to be inserted between
the two V regions was incorporated by adding the extra nucleotides
to the antisense primer for the V.sub.L of 2H7. A Sac I restriction
site was also introduced at the junction between the two V regions.
The sense primer used to amplify the 2H7 V.sub.L, that included a
HindIll restriction site and the light chain leader peptide was
5'-gtc aag ctt gcc gcc atg gat ttt caa gtg cag att ttt cag c-3'
(SEQ ID NO: 23). The antisense primer was 5'-gtc gtc gag ctc cca
cct cct cca gat cca cca ccg ccc gag cca ccg cca cct ttc agc tcc agc
ttg gtc cc-3' (SEQ ID NO: 24). The reading frame of the V region is
indicated as a bold, underlined codon. The Hind III and Sac I sites
are indicated by underlined italicized sequences.
[0233] The V.sub.H domain was amplified without a leader peptide,
but included a 5' Sac I restriction site for fusion to the V.sub.L
and a Bell restriction site at the 3' end for fusion to various
tails, including the human IgG1 Fc domain and the truncated forms
of CD40 ligand, CD154. The sense primer was 5'-gct get gag ctc tca
ggc tta tct aca gca agt ctg g-3' (SEQ ID NO: 25). The Sac I site is
indicated in italicized and underlined font, and the reading frame
of the codon for the first amino acid of the V.sub.H domain is
indicated in bold, underlined type. The antisense primer was 5'-gtt
gtc tga tca gag acg gtg acc gtg gtc cc-3'(SEQ ID NO: 26). The Bell
site is indicated in italicized, underlined type, and the last
serine of the VH domain sequence is indicated in bold, underlined
type.
[0234] The scFv-Ig was assembled by inserting the 2H7 scFv
HindIII-Bc1I fragment into pUC19 containing the human IgG1 hinge,
CH2, and CH3 regions, which was digested with restriction enzymes,
HindIll and BclI. After ligation, the ligation products were
transformed into DH5.alpha. bacteria. Positive clones were screened
for the properly inserted fragments using the Sac I site at the
VL-VH junction of 2H7 as a diagnostic site. The 2H7scFv-Ig cDNA was
subjected to cycle sequencing on a PE 9700 thermocycler using a
25-cycle program by denaturing at 96.degree. C. for 10 seconds,
annealing at 50.degree. C. for 30 seconds, and extending at
72.degree. C. for 4 minutes. The sequencing primers were pUC
forward and reverse primers and an internal primer that annealed to
the CH2 domain human in the IgG constant region portion. Sequencing
reactions were performed using the Big Dye Terminator Ready
Sequencing Mix (PE-Applied Biosystems, Foster City, Calif.)
according to the manufacturer's instructions. Samples were
subsequently purified using Centrisep columns (Catalog #CS-901,
Princeton Separations, Adelphia, N.J.), the eluates dried in a
Savant vacuum dryer, denatured in Template Suppression Reagent
(PE-ABI), and analyzed on an ABI 310 Genetic Analyzer (PE-Applied
Biosystems). The sequence was edited, translated, and analyzed
using Vector Nti version 6.0 (Informax, North Bethesda, Md.). FIG.
1 shows the cDNA and predicted amino acid sequence of the
2H7scFv-Ig construct.
Example 2
Expression of 2H7 scFv-Ig in Stable CHO Cell Lines
[0235] This Example illustrates expression of 2H7scFv-Ig in a
eukaryotic cell line and characterization of the expressed
2H7scFv-Ig by SDS-PAGE and by functional assays, including ADCC and
complement fixation.
[0236] The 2H7scFv-Ig HindIII-XbaI (.about.1.6 kb) fragment with
correct sequence was inserted into the mammalian expression vector
pDl8, and DNA from positive clones was amplified using QIAGEN
plasmid preparation kits (QIAGEN, Valencia, Calif.). The
recombinant plasmid DNA (100 ng) was then linearized in a
nonessential region by digestion with AscI, purified by phenol
extraction, and resuspended in tissue culture media, Excell 302
(Catalog #14312-79P, JRH Biosciences, Lenexa, Kans.). Cells for
transfection, CHO DG44 cells, were kept in logarithmic growth, and
10.sup.7 cells harvested for each transfection reaction. Linearized
DNA was added to the CHO cells in a total volume of 0.8 ml for
electroporation.
[0237] Stable production of the 2H7 scFv-Ig fusion protein (SEQ. ID
NO:10) was achieved by electroporation of a selectable, amplifiable
plasmid, pDl8, containing the 2H7 scFv-Ig cDNA under the control of
the CMV promoter, into Chinese Hamster Ovary (CHO) cells (all cell
lines from American Type Culture Collection, Manassas, Va., unless
otherwise noted). The 2H7 expression cassette was subcloned
downstream of the CMV promoter into the vector multiple cloning
site as a .about.1.6 kb HindIII-XbaI fragment. The pD 18 vector is
a modified version of pcDNA3 encoding the DHFR selectable marker
with an attenuated promoter to increase selection pressure for the
plasmid. Plasmid DNA was prepared using Qiagen maxiprep kits, and
purified plasmid was linearized at a unique AscI site prior to
phenol extraction and ethanol precipitation. Salmon sperm DNA
(Sigma Aldrich, St. Louis, Mo.) was added as carrier DNA, and 100
.mu.g each of plasmid and carrier DNA was used to transfect 107 CHO
DG44 cells by electroporation. Cells were grown to logarithmic
phase in Excell 302 media (JRH Biosciences) containing glutamine (4
mM), pyruvate, recombinant insulin, penicillin-streptomycin, and
2.times.DMEM nonessential amino acids (all from Life Technologies,
Gaithersburg, Md.), hereafter referred to as "Excell 302 complete"
media. Media for untransfected cells also contained HT (diluted
from a 100.times. solution of hypoxanthine and thymidine)
(Invitrogen/Life Technologies). Media for transfections under
selection contained varying levels of methotrexate (Sigma-Aldrich)
as selective agent, ranging from 50 nM to 5 .mu.M. Electroporations
were performed at 275 volts, 950 .mu.f. Transfected cells were
allowed to recover overnight in non-selective media prior to
selective plating in 96 well flat bottom plates (Costar) at varying
serial dilutions ranging from 125 cells/well to 2000 cells/well.
Culture media for cell cloning was Excell 302 complete, containing
100 nM methotrexate. Once clonal outgrowth was sufficient, serial
dilutions of culture supernatants from master wells were screened
for binding to CD20-CHO transfected cells. The clones with the
highest production of the fusion protein were expanded into T25 and
then T75 flasks to provide adequate numbers of cells for freezing
and for scaling up production of the 2H7scFvIg. Production levels
were further increased in cultures from three clones by progressive
amplification in methotrexate containing culture media. At each
successive passage of cells, the Excell 302 complete media
contained an increased concentration of methotrexate, such that
only the cells that amplified the DHFR plasmid could survive.
[0238] Supernatants were collected from CHO cells expressing the
2H7scFv-Ig, filtered through 0.2 .mu.m PES express filters
(Nalgene, Rochester, N.Y.) and were passed over a Protein A-agarose
(IPA 300 crosslinked agarose) column (Repligen, Needham, Mass.).
The column was washed with PBS, and then bound protein was eluted
using 0.1 M citrate buffer, pH 3.0. Fractions were collected and
eluted protein was neutralized using 1M Tris, pH 8.0, prior to
dialysis overnight in PBS. Concentration of the purified 2H7scFv-Ig
(SEQ ID NOs: 1 and 11) was determined by absorption at 280 nm. An
extinction coefficient of 1.77 was determined using the protein
analysis tools in the Vector Nti Version 6.0 Software package
(Informax, North Bethesda, Md.). This program uses the amino acid
composition data to calculate extinction coefficients.
[0239] Production levels of 2H7scFv-Ig by transfected, stable CHO
cells were analyzed by flow cytometry. Purified 2H7scFv-Ig to CHO
cells was allowed to bind to CHO cells that expressed CD20 (CD20
CHO) and analyzed by flow cytometry using a fluorescein-conjugated
anti-human IgG second step reagent (Catalog Numbers H10101 and
H10501, CalTag, Burlingame, Calif.). FIG. 2 (top) shows a standard
curve generated by titration of 2H7scFv-Ig binding to CD20 CHO. At
each concentration of 2H7scFv-Ig, the mean brightness of the
fluorescein signal in linear units is shown. Supernatants collected
from T flasks containing stable CHO cell clones expressing
2H7scFv-Ig were then allowed to bind to CD20 CHO and the binding
was analyzed by flow cytometry. The fluorescein signal generated by
2H7scFv-Ig contained in the supernatants was measured and the
2H7scFv-Ig concentration in the supernatants was calculated from
the standard curve (FIG. 2, bottom).
[0240] Purified 2H7scFv-Ig (SEQ ID NOs: 1 and 11) was analyzed by
electrophoresis on SDS-Polyacrylamide gels. Samples of 2H7scFv-Ig,
purified by independent Protein A Agarose column runs, were boiled
in SDS sample buffer without reduction of disulfide bonds and
applied to SDS 10% Tris-BIS gels (Catalog #NP0301, Novex, Carlsbad,
Calif.). Twenty micrograms of each purified batch was loaded on the
gels.
[0241] The proteins were visualized after electrophoresis by
Coomassie Blue staining (Pierce Gel Code Blue Stain Reagent,
Catalog #24590, Pierce, Rockford, Ill.), and destaining in
distilled water. Molecular weight markers were included on the same
gel (Kaleidoscope Prestained Standards, Catalog #161-0324, Bio-Rad,
Hercules, Calif.). The results are presented in FIG. 3. The numbers
above the lanes designate independent purification batches. The
molecular weights in kilodaltons of the size markers are indicated
on the left side of the figure. Further experiments with
alternative sample preparation conditions indicated that reduction
of disulfide bonds by boiling the protein in SDS sample buffer
containing DTT or 2-mercaptoethanol caused the 2H7scFv-Ig to
aggregate.
[0242] Any number of other immunological parameters may be
monitored using routine assays that are well known in the art.
These may include, for example, antibody dependent cell-mediated
cytotoxicity (ADCC) assays, secondary in vitro antibody responses,
flow immunocytofluorimetric analysis of various peripheral blood or
lymphoid mononuclear cell subpopulations using well established
marker antigen systems, immunohistochemistry or other relevant
assays. These and other assays may be found, for example, in Rose
et al. (Eds.), Manual of Clinical Laboratory Immunology, 5.sup.th
Ed., 1997 American Society of Microbiology, Washington, D.C.
[0243] The ability of 2H7scFv-Ig to kill CD20 positive cells in the
presence of complement was tested using B cell lines Ramos and
Bjab. Rabbit complement (Pel-Freez, Rogers, Ak.) was used in the
assay at a final dilution of 1/10. Purified 2H7scFv-Ig was
incubated with B cells and complement for 45 minutes at 37.degree.
C., followed by counting of live and dead cells by trypan blue
exclusion. The results in FIG. 4A show that in the presence of
rabbit complement, 2H7scFv-Ig lysed B cells expressing CD20.
[0244] The ability of 2H7scFv-Ig to kill CD20 positive cells in the
presence of peripheral blood mononuclear cells (PBMC) was tested by
measuring the release of .sup.51 Cr from labeled Bjab cells in a
4-hour assay using a 100:1 ratio of PBMC to Bjab cells. The results
shown in FIG. 4B indicated that 2H7scFv-Ig can mediate antibody
dependent cellular cytotoxicity (ADCC) because the release of
.sup.51Cr was higher in the presence of both PBMC and 2H7scFv-Ig
than in the presence of either PBMC or 2H7scFv-Ig alone.
Example 3
Effect of Simultaneous Ligation of CD20 and CD400N Growth of Normal
B Cells, and on CD95 Expression, and Induction of Apoptosis
[0245] This example illustrates the effect of cross-linking of CD20
and CD40 expressed on the cell surface on cell proliferation.
[0246] Dense resting B cells were isolated from human tonsil by a
Percoll step gradient and T cells were removed by E-rosetting.
Proliferation of resting, dense tonsillar B cells was measured by
uptake of .sup.3-[H]-thymidine during the last 12 hours of a 4-day
experiment. Proliferation was measured in quadruplicate cultures
with means and standard deviations as shown. Murine anti-human CD20
mAb 1F5 (anti-CD20) was used alone or was cross-linked with
anti-murine .kappa. mAb 187.1 (anti-CD20XL). CD40 activation was
accomplished using soluble human CD154 fused with murine CD8
(CD154) (Hollenbaugh et al., EMBO J. 11: 4212-21 (1992)), and CD40
cross-linking was accomplished using anti-murine CD8 mAb 53-6
(CD154XL). This procedure allowed simultaneous cross-linking of
CD20 and CD40 on the cell surface. The results are presented in
FIG. 5.
[0247] The effect of CD20 and CD40 cross-linking on Ramos cells, a
B lymphoma cell line, was examined. Ramos cells were analyzed for
CD95 (Fas) expression and percent apoptosis eighteen hours after
treatment (no goat anti-mouse IgG (GAM)) and/or cross-linking
(+GAM) using murine mAbs that specifically bind CD20 (1F5) and CD40
(G28-5). Control cells were treated with a non-binding isotype
control (64.1) specific for CD3.
[0248] Treated Ramos cells were harvested, incubated with
FITC-anti-CD95, and analyzed by flow cytometry to determine the
relative expression level of Fas on the cell surface after CD20 or
CD40 cross-linking. Data is plotted as mean fluorescence of cells
after treatment with the stimuli indicated (FIG. 6A).
[0249] Treated Ramos cells from the same experiment were harvested
and binding of annexin V was measured to indicate the percentage
apoptosis in the treated cultures. Apoptosis was measured by
binding of Annexin V 18 hours after cross-linking of CD20 and CD40
using 1F5 and G28-5 followed by cross-linking with GAM. Binding of
Annexin V was measured using a FITC-Annexin V kit (Catalog
#PN-IM2376, Immunotech, Marseille, France). Annexin V binding is
known to be an early event in progression of cells into apoptosis.
Apoptosis, or programmed cell death, is a process characterized by
a cascade of catabolic reactions leading to cell death by suicide.
In the early phase of apoptosis, before cells change morphology and
hydrolyze DNA, the integrity of the cell membrane is maintained but
cells lose the asymmetry of their membrane phospholipids, exposing
negatively charged phospholipids, such as phosphatidylserine, at
the cell surface. Annexin V, a calcium and phopholipid binding
protein, binds preferentially and with high affinity to
phosphatidylserine. Results demonstrating the effect of
cross-linking both CD20 and CD40 on expression of the FAS receptor
(CD95) are presented in FIG. 6B. The effect of cross-linking of
both CD20 and CD40 on Annexin V binding to cells is shown in FIG.
6B.
Example 4
Construction and Characterization of 2H7 scFv-CD154 Fusion
Proteins
[0250] To construct a molecule capable of binding to both CD20 and
CD40, cDNA encoding the 2H7 scFv was fused with cDNA encoding
CD154, the CD40 ligand. The 2H7 scFv cDNA encoded on the
HindIII-Bc1I fragment was removed from the 2H7 scFvIg construct,
and inserted into a pD18 vector along with a BamHI-XbaI cDNA
fragment encoding the extracellular domain of human CD154. The
extracellular domain is encoded at the carboxy terminus of CD154,
similar to other type II membrane proteins.
[0251] The extracellular domain of human CD154 was PCR amplified
using cDNA generated with random primers and RNA from human T
lymphocytes activated with PHA (phytohemagglutinin) The primer sets
included two different 5' or sense primers that created fusion
junctions at two different positions within the extracellular
domain of CD154. Two different fusion junctions were designed that
resulted in a short or truncated form (form S4) including amino
acids 108 (Glu)-261 (Leu)+(Glu), and a long or complete form (form
L2) including amino acids 48 (Arg)-261 (Leu)+(Glu), of the
extracellular domain of CD154, both constructed as BamHI-XbaI
fragments. The sense primer which fuses the two different truncated
extracellular domains to the 2H7scFv includes a BamHI site for
cloning. The sense primer for the S4 form of the CD154 cDNA is
designated CD154BAM108 and encodes a 34 mer with the following
sequence: 5'-gtt gtc gga tcc aga aaa cag ctt tga aat gca a-3' (SEQ
ID NO: 27), while the antisense primer is designated CD154XBA and
encodes a 44 mer with the following sequence: 5'-gtt gtt tct aga
tta tca ctc gag ttt gag taa gcc aaa gga cg-3' (SEQ ID NO: 28).
[0252] The oligonucleotide primers used in amplifying the long form
(L2) of the CD154 extracellular domain encoding amino acids 48
(Arg)-261 (Leu)+(Glu), were as follows: The sense primer designated
CD154 BAM48 encoded a 35-mer with the following sequence: 5'-gtt
gtc gga tcc aag aag gtt gga caa gat aga ag-3' (SEQ ID NO: 29). The
antisense primer designated CD154XBA encoded the 44-mer: 5'-gtt gtt
tct aga tta tca ctc gag ttt gag taa gcc aaa gga cg-3' (SEQ ID NO:
28). Other PCR reaction conditions were identical to those used for
amplifying the 2H7 scFv (see Example 1). PCR fragments were
purified by PCR quick kits (QIAGEN, San Diego, Calif.), eluted in
30 .mu.l ddH.sub.2O, and digested with BamHI and XbaI (Roche)
restriction endonucleases in a 40 .mu.l reaction volume at
37.degree. C. for 3 hours. Fragments were gel purified, purified
using QIAEX kits according to the manufacturer's instructions
(QIAGEN), and ligated along with the 2H7 HindIII-Bc1I fragment into
the pD 18 expression vector digested with HindIII+XbaI. Ligation
reactions were transformed into DH5-alpha chemically competent
bacteria and plated onto LB plates containing 100 .mu.g/ml
ampicillin. Transformants were grown overnight at 37.degree. C.,
and isolated colonies used to inoculate 3 ml liquid cultures in
Luria Broth containing 100 .mu.g/ml ampicillin. Clones were
screened after mini-plasmid preparations (QIAGEN) for insertion of
both the 2H7 scFv and the CD154 extracellular domain fragments.
[0253] The 2H7scFv-CD154 construct cDNAs were subjected to cycle
sequencing on a PE 9700 thermocycler using a 25-cycle program that
included denaturating at 96.degree. C., seconds, annealing at
50.degree. C. for 5 seconds, and extension at 60.degree. C., for 4
minutes. The sequencing primers used were pD18 forward (SEQ ID NO:
30: 5'-gtctatataagcagagctctggc-3') and pD18 reverse (SEQ ID NO: 31:
5'-cgaggctgatcagcgagctctagca-3'') primers. In addition, an internal
primer was used that had homology to the human CD154 sequence (SEQ
ID NO: 32: 5''-ccgcaatttgaggattctgatcacc-3'). Sequencing reactions
included primers at 3.2 pmol, approximately 200 ng DNA template,
and 8 .mu.l sequencing mix. Sequencing reactions were performed
using the Big Dye Terminator Ready Sequencing Mix (PE-Applied
Biosystems, Foster City, Calif.) according to the manufacturer's
instructions. Samples were subsequently purified using Centrisep
columns (Princeton Separations, Adelphia, N.J.). The eluates were
dried in a Savant speed-vacuum dryer, denatured in 20 .mu.l
template Suppression Reagent (ABI) at 95.degree. C. for 2 minutes,
and analyzed on an ABI 310 Genetic Analyzer (PE-Applied
Biosystems). The sequence was edited, translated, and analyzed
using Vector Nti version 6.0 (Informax, North Bethesda, Md.). The
2H7scFv-CD 154 L2 cDNA sequence (SEQ ID NOs: 21 and 149) and
predicted amino acid sequence (SEQ ID NOs: 33 and 150) is presented
in FIG. 7A, and 2H7scFv-CD154 S4 cDNA sequence (SEQ ID NOs: 22 and
151) and predicted amino acid sequence (SEQ ID NOs: 34 and 152) is
presented in FIG. 7B.
[0254] The binding activity of the 2H7 scFv-CD154 fusion proteins
(SEQ ID NOS: 33, 150 and 34, 152) to CD20 and CD40 simultaneously
was determined by flow cytometry. The assay used CHO cell targets
that express CD20. After a 45-minute incubation of CD20 CHO cells
with supernatants from cells transfected with the 2H7 scFv-CD154
expression plasmid, the CD20 CHO cells were washed twice and
incubated with biotin-conjugated CD40-Ig fusion protein in PBS/2%
FBS. After 45 min, cells were washed twice and incubated with
phycoerythrin (PE)-labeled strepavidin at 1:100 in PBS/2% FBS
(Molecular Probes, Eugene Oreg.). After an additional 30 min
incubation, cells were washed 2.times. and were analyzed by flow
cytometry. The results show that the 2H7 scFv-CD154 molecule was
able to bind to CD20 on the cell surface and to capture
biotin-conjugated CD40 from solution (FIG. 8).
[0255] To determine the effect of the 2H7scFv-CD154 on growth and
viability of B lymphoma and lymphoblastoid cell lines, cells were
incubated with 2H7scFv-CD154 L2 (SEQ. ID NO: 33, 150) for 12 hours
and then examined for binding of Annexin V. Binding of Annexin V
was measured using a FITC-Annexin V kit (Immunotech, Marseille,
France, Catalog #PN-1M2376). B cell lines were incubated in 1 ml
cultures with dilutions of concentrated, dialyzed supernatants from
cells expressing secreted forms of the 2H7scFv-CD154 fusion
proteins. The results are presented in FIG. 9.
[0256] The growth rate of the Ramos B lymphoma cell line in the
presence of 2H7scFv-CD 154 was examined by uptake of
.sup.3H-thymidine for the last 6 hours of a 24-hour culture. The
effect of 2H7scFv-CD154 on cell proliferation is shown in FIG.
10.
Example 5
Construction and Characterization of Cytoxb Antibody
Derivatives
[0257] CytoxB antibodies were derived from the 2H7 scFv-IgG
polypeptide. The 2H7 scFv (see Example 1) was linked to the human
IgG1 Fc domain via an altered hinge domain (see FIG. 11). Cysteine
residues in the hinge region were substituted with serine residues
by site-directed mutagenesis and other methods known in the art.
The mutant hinge was fused either to a wild-type Fc domain to
create one construct, designated CytoxB-MHWTG1C, or was fused to a
mutated Fc domain (CytoxB-MHMG1C) that had additional mutations
introduced into the CH2 domain. Amino acid residues in CH2 that are
implicated in effector function are illustrated in FIG. 11.
Mutations of one or more of these residues may reduce FcR binding
and mediation of effector functions. In this example, the leucine
residue 234 known in the art to be important to Fc receptor
binding, was mutated in the 2H7 scFv fusion protein,
CytoxB-[MG1H/MG1C]. In another construct, the human IgG1 hinge
region was substituted with a portion of the human IgA hinge, which
was fused to wild-type human Fc domain (CytoxB-IgAHWTHG1C). (See
FIG. 11.) This mutated hinge region allows expression of a mixture
of monomeric and dimeric molecules that retain functional
properties of the human IgG1 CH2 and CH3 domains. Synthetic,
recombinant cDNA expression cassettes for these molecules were
constructed and polypeptides were expressed in CHODG44 cells
according to methods described in Example 2.
[0258] Purified fusion protein derivatives of CytoxB-scFvlg
molecules were analyzed by SDS-PAGE according to the methods
described in Example 2. Polyacrylamide gels were run under
non-reducing and reducing conditions. Two different molecule weight
marker sets, BioRad prestained markers, (BioRad, Hercules, Calif.)
and Novex Multimark molecular weight markers were loaded onto each
gel. The migration patterns of the different constructs and of
Rituximab.TM. are presented in FIG. 12.
[0259] The ability of the different derivatives of CytoxB-scFvlg
molecules to mediate ADCC was measured using the Bjab B lymphoma
cells as the target and freshly prepared human PBMCs as effector
cells. (See Example 2.) Effector to target ratios were varied as
follows: 70:1, 35:1, and 18:1, with the number of Bjab cells per
well remaining constant but the number of PBMCs were varied. Bjab
cells were labeled for 2 hours with .sup.51Cr and aliquoted at a
cell density of 5.times.10.sup.4 cells/well to each well of
flat-bottom 96 well plates. Purified fusion proteins or rituximab
were added at a concentration of 10 .mu.g/ml to the various
dilutions of PBMCs. Spontaneous release was measured without
addition of PBMC or fusion protein, and maximal release was
measured by the addition of detergent (1% NP-40) to the appropriate
wells. Reactions were incubated for 4 hours, and 100 .mu.l of
culture supernatant was harvested to a Lumaplate (Packard
Instruments) and allowed to dry overnight prior to counting cpm
released. The results are presented in FIG. 13.
[0260] Complement dependent cytotoxicity (CDC) activity of the
CytoxB derivatives was also measured. Reactions were performed
essentially as described in Example 2. The results are presented in
FIG. 14 as percent of dead cells to total cells for each
concentration of fusion protein.
Example 6
In Vivo Studies in Macaques
[0261] Initial in vivo studies with CytoxB derivatives have been
performed in nonhuman primates. FIG. 15 shows data characterizing
the serum half-life of CytoxB in monkeys. Measurements were
performed on serum samples obtained from two different macaques
(J99231 and K99334) after doses of 6 mg/kg were administered to
each monkey on the days indicated by arrows. For each sample, the
level of 2H7scFvIg present was estimated by comparison to a
standard curve generated by binding of purified CytoxB-(MHWTG1C)-Ig
fusion protein to CD20 CHO cells (see Example 2). The data are
tabulated in the bottom panel of the FIG. 15.
[0262] The effect of CytoxB-(MHWTG1C)Ig fusion protein on levels of
circulating CD40+ cells in macaques was investigated. Complete
blood counts were performed at each of the days indicated in FIG.
16. In addition, FACS (fluorescence activated cell sorter) assays
were performed on peripheral blood lymphocytes using a
CD40-specific fluorescein conjugated antibody to detect B cells
among the cell population. The percentage of positive cells was
then used to calculate the number of B cells in the original
samples. The data are graphed as thousands of B cells per
microliter of blood measured at the days indicated after injection
(FIG. 16).
Example 7
Construction and Expression of an Anti-CD19 scFv-Ig Fusion
Protein
[0263] An anti-CD 19 scFv-Ig fusion protein was constructed,
transfected into eukaryotic cells, and expressed according to
methods presented in Examples 1, 2, and 5 and standard in the art.
The variable heavy chain regions and variable light chain regions
were cloned from RNA isolated from hybridoma cells producing
antibody HD37, which specifically binds to CD 19. Expression levels
of a HD37scFv-IgAHWTG1C and a HD37scFv-IgMHWTG1C were measured and
compared to a standard curve generated using purified HD37 scFvlg.
The results are presented in FIG. 17.
Example 8
Construction and Expression of an Anti-L6 scFv-Ig Fusion
Protein
[0264] An scFv-Ig fusion protein was constructed using variable
regions derived from an anti-carcinoma mAb, L6. The fusion protein
was constructed, transfected into eukaryotic cells, and expressed
according to methods presented in Examples 1, 2, and 5 and standard
in the art. Expression levels of L6scFv-IgAHWTG1C and
L6scFv-IgMHWTG1C were measured and compared to a standard curve
generated using purified HD37 scFyIg. The results are presented in
FIG. 18.
Example 9
Characterization of Various scFv-Ig Fusion Proteins
[0265] In addition to the scFv-Ig fusion protein already described,
G28-1 (anti-CD37) scFv-Ig fusion proteins were prepared essentially
as described in Examples 1 and 5. The variable regions of the heavy
and light chains were cloned according to methods known in the art.
ADCC activity of 2H7-MHWTG1C, 2H7-IgAHWTG1C, G28-1-MHWTG1C, G28-1
IgAHWTG1C, HD37-MHWTG1C, and HD37-IgAHWTG1C was determined
according to methods described above (see Example 2). Results are
presented in FIG. 19. ADCC activity of L6scFv-IgAHWTG1C and
L6scFv-IgMHWTG1C was measured using the 2981 human lung carcinoma
cell line. The results are presented in FIG. 20. The murine L6
monoclonal antibody is known not to exhibit ADCC activity.
[0266] The purified proteins were analyzed by SDS-PAGE under
reducing and non-reducing conditions. Samples were prepared and
gels run essentially as described in Examples 2 and 5. The results
for the L6 and 2H7 scFv-Ig fusion proteins are presented in FIG. 21
and the results for the G28-1 and HD37 scFv-Ig fusion proteins are
presented in FIG. 22.
Example 10
Construction and Expression of Anti-CD20 ScFb-Llama Ig Fusion
Proteins
[0267] This Example illustrates the cloning of llama IgG1, IgG2,
and IgG3 constant region domains and the construction of
immunoglobulin fusion proteins with each of the three constant
regions and anti-CD20 scFv.
[0268] The constant regions of llama IgG1, IgG2, and IgG3
immunoglobulins were cloned and inserted into mammalian vector
constructs containing an anti-CD20 single chain Fv, 2H7 scFv. Total
RNA was isolated from peripheral blood mononuclear cells (PBMC)
from llama blood (Triple J Farms, Bellingham, Wash.) by lysing the
lymphocytes in TRIzo.RTM. (Invitrogen Life Technologies, Carlsbad,
Calif.) according to the manufacturer's instructions. One microgram
(1 .mu.g) of total RNA was used as template to prepare cDNA by
reverse transcription. The RNA and 200 ng random primers were
combined and denatured at 72.degree. C. for 10 minutes prior to
addition of enzyme. Superscript II reverse transcriptase
(Invitrogen Life Technologies) was added to the RNA plus primer
mixture in a total volume of 25 .mu.l in the presence of 5.times.
second strand buffer and 0.1 M DTT provided with the enzyme. The
reverse transcription reaction was allowed to proceed at 42.degree.
C. for one hour. The cDNA was amplified by PCR using sequence
specific primers. The 5' primers were designed according to
published sequences for the V.sub.HH and V.sub.H domains of
camelids. The 3' primer, which was used to amplify all three
isotypes, was designed using mammalian CH3 domain sequences as a
guide. The following specific primers were used. The Bcl and XbaI
sites are indicated by underlined italicized sequences.
TABLE-US-00001 5' primer for llama IgG1 constant region LLG1-5'bgl:
(SEQ ID NO: 227) 5'-gtt gtt gat caa gaa cca cat gga gga tgc acg
tg-3' 5' primer for llama IgG2 constant region LLG2-5'bgl: (SEQ ID
NO: 228) 5'-gtt gtt gat caa gaa ccc aag aca cca aaa cc-3' 5' primer
for llama IgG3 constant region LLG3-5'bgl: (SEQ ID NO: 229) 5'-gtt
gtt gat caa gcg cac cac agc gaa gac ccc-3' 3' primer for llama
IgG1, IgG2, and IgG3 constant regions LLG123-3'X: (SEQ ID NO: 238)
5'-gtt gtt tct aga tta cta ttt acc cga aga ctg ggt gat gga-3'
[0269] PCR fragments of the expected size were cloned into
TOPO.RTM. cloning vectors (Invitrogen Life Technologies) and then
were sequenced. The sense sequencing primer, LLseqsense, had the
sequence 5'-ctg aga tcg agt tea get g-3' (SEQ ID NO: 230), and the
antisense primer, LLseqAS, had the sequence 5'-cct cct ttg get ttg
tct c-3' (SEQ ID NO: 231). Sequencing was performed as described in
Example 1. FIG. 23 compares the amino acid sequence of the three
isotype llama constant regions containing the hinge, CH2, and CH3
domains with the amino acid sequence of human IgG1 hinge, CH2, and
CH3 domains.
[0270] After verifying the sequence, the amplified PCR products
were digested with restriction enzymes BclI and XbaI to create
compatible restriction sites. The digested fragments were then
gel-purified, and the DNA was eluted using a QIAquick Gel
Extraction Kit (QIAGEN, Valencia, Calif.). The 2H7scFv-Ig pD18
mammalian expression vector construct (see Example 2) was digested
with BclI and XbaI to remove the human IgG hinge, CH2, and CH3
domains. The pD18 vector is a modified derivative of pcDNA3 that
contains an attenuated DHFR gene, which serves as a selectable
marker for mammalian expression (Hayden et al., Tissue Antigens
48:242-54 (1996)). The purified llama IgG1, IgG2, and IgG3 constant
region PCR products were ligated by T4 DNA ligase (Roche Molecular
Biochemicals, Indianapolis, Ind.) into the double-digested 2H7
scFv-pD18 vector at room temperature overnight according to the
manufacturer's instructions. After ligation, the ligation products
were transformed into E. coli DH5.alpha. bacteria (BD Biosciences,
Palo Alto, Calif.) and plated according to standard molecular
biology procedures and manufacturer's instructions. Isolated
colonies were chosen to screen for transformants containing the
correct inserts.
[0271] For expression of the encoded polypeptides, plasmid DNA from
positive clones was transiently transfected into COS-7 cells using
DEAE-dextran (Hayden et al., Ther Immunol. 1:3-15 (1994)). COS-7
cells were seeded at approximately 3.times.10.sup.6 cells per 150
mm plate and grown overnight until the cells were about 75%
confluent. Cells were then washed once with serum-free DMEM
(Invitrogen Life Technologies, Grand Island, N.Y.). Transfection
supernatant (10 ml) containing 400 .mu.g/ml DEAE-dextran, 0.1 mM
chloroquine, and 5 .mu.g/ml of the DNA constructs were added to the
cells, which were then incubated at 37.degree. C. for 3-4 hrs.
After incubation, cells were pulsed with 10 ml of 10% dimethyl
sulfoxide (DMSO) in 1.times.PBS at room temperature for 2 minutes.
Cells were then placed back into fully supplemented DMEM/10% FBS
(1% L-glutamine, 1% penicillin/streptomycin, 1% sodium pyruvate, 1%
MEM essential amino acids) (Invitrogen Life Technologies). After 24
hours, the media was replaced with serum-free fully supplemented
DMEM (Invitrogen Life Technologies), and the cells were maintained
up to 21 days with media changes every 3-4 days.
[0272] Ig-fusion proteins were purified by passing COS cell culture
supernatants through Protein A Agarose (Repligen, Cambridge, Mass.)
columns. After application of the culture supernatant, the Protein
A columns were then washed with 1.times.PBS (Invitrogen Life
Technologies). Bound Ig-fusion proteins were eluted with 0.1 M
citric acid (pH 2.8), and the collected fractions were immediately
neutralized with Tris base (pH 10.85). The fractions containing
protein were identified by measuring the optical density
(A.sub.280) and then were pooled, dialyzed against 1.times.PBS,
(Invitrogen Life Technologies) and filtered through a 0.2 .mu.m
filter.
[0273] The purified Ig-fusion proteins were analyzed by SDS-PAGE.
Aliquots of 2H7 scFv-llama IgG1, 2H7 scFv-llama IgG2, 2H7
scFv-llama IgG3, and Rituxan.RTM. (Rituximab, anti-CD20 antibody,
Genentech, Inc. and IDEC Pharmaceuticals Corp.) (provided by Dr.
Oliver W. Press, Fred Hutchison Cancer Research Center, Seattle,
Wash.) (5 .mu.g protein) were combined with 25 .mu.l 2.times.
NuPAGE.RTM. SDS Sample Buffer (Invitrogen Life Technologies)
(non-reduced samples). Samples of each protein were also prepared
in reducing sample buffer containing 5% 2-mercaptoethanol
(Sigma-Aldrich, St. Louis, Mo.). Molecular weight markers
(Invitrogen Life Technologies) were applied to the gels in non
reducing buffer only. The proteins were fractionated on NuPAGE.RTM.
10% Bis-Tris gels (Invitrogen Life Technologies). After
electrophoresis (approximately 1 hour), the gels were washed three
times, five minutes each, with Distilled Water (Invitrogen Life
Technologies) and then stained in 50 ml Bio-Safe Coommassie Stain
(BioRad, Hercules, Calif.) overnight at room temperature. After a
wash in Distilled Water, the gels were photographed. The migration
pattern of each Ig-fusion protein is presented in FIG. 24.
[0274] The ability of the 2H7 scFv-llama Ig fusion proteins to bind
to cells expressing CD20 was demonstrated by flow cytometry. Serial
dilutions starting at 25 .mu.g/ml of purified 2H7 scFv-llama IgG1,
2H7 scFv-llama IgG2, and 2H7 scFv-llama IgG3 were prepared and
incubated with CD20-transfected (CD20+) CHO cells (from the
laboratory of Dr. S. Skov, Institute of Medical Microbiology and
Immunology, Copenhagen Denmark in 1% FBS 1.times.PBS media
(Invitrogen Life Technologies) for one hour on ice. After the
incubation, the cells were then centrifuged and washed with 1% FBS
in 1.times.PBS. To detect bound 2H7 scFv-llama Ig, the cells were
incubated for one hour on ice with fluorescein-conjugated goat
anti-camelid IgG (heavy and light chain) (1:100) (Triple J Farms).
The cells were then centrifuged and resuspended in 1%
FBS-1.times.PBS and analyzed using a Coulter Epics XL cell sorter
(Beckman Coulter, Miami, Fla.). The data (percent of maximum
brightness) are presented in FIG. 25.
Example 11
Effector Function of Anti-CD20 scFv-Llama Ig Fusion Proteins
[0275] This Example demonstrates the ability of anti-CD20 llama
IgG1, IgG2, and IgG3 fusion proteins to mediate complement
dependent cytotoxicity (CDC) and antibody dependent cell-mediated
cytotoxicity (ADCC).
[0276] The ability of the 2H7 scFv-llama IgG fusion proteins to
kill CD20 positive cells in the presence of complement was tested
using the BJAB human B cell line. Rabbit complement was obtained
from 3-4 week old rabbits (Pel-Freez, Brown Deer, Wis.). BJAB cells
(2.times.10.sup.6 cells/ml) were combined with rabbit complement
(final dilution 1:10) and purified 2H7 Ig fusion proteins. 2H7
scFv-llama IgG1, 2H7 scFv-llama IgG2, 2H7 scFv-llama IgG3, and 2H7
scFv-human IgG1 wild type hinge-CH2-CH3) (Example 1) were added at
1:3 serial dilutions beginning at a concentration of 30 .mu.g/ml.
After one hour at 37.degree. C., cell viability was determined by
counting live and dead cells by trypan blue exclusion (0.4%)
(Invitrogen Life Technologies) using a hemacytometer (Bright-line,
Horsham, Pa.). The percent killing was calculated by dividing the
number of dead cells by the number of total cells (dead+live
cells). The data presented in FIG. 26 show that all Ig fusion
proteins had CDC activity.
[0277] The ADCC activity of the 2H7 scFv-llama IgG fusion proteins
was determined using BJAB cells as target cells and human or llama
peripheral blood mononuclear cells (PBMC) as effector cells. BJAB
cells were pre-incubated for approximately 2 hours with .sup.51Cr
(100 .mu.Ci) (Amersham Biosciences, Piscataway, N.J.) in fully
supplemented IMDM (Invitrogen Life Technologies) containing 15%
FBS. The cells were mixed intermittently during the pre-incubation
period. Fresh, resting human PBMC were purified from whole blood
using Lymphocyte Separation Media (LSM) (ICN Pharmaceuticals, New
York, N.Y.). PBMC were combined with labeled BJAB cells
(5.times.10.sup.4 cells per well of 96 well tissue culture plate)
at ratios of 25:1, 50:1, and 100:1. To each combination was added
10 .mu.g/ml of purified 2H7 scFv-llama IgG1, 2H7 scFv-llama IgG2,
2H7 scFv-llama IgG3, Rituximab, or no anti-CD20 antibody. The
mixtures were incubated for 6 hours at 37.degree. C. Supernatant
from each reaction containing .sup.51Cr released from lysed cells
was collected onto a LumaPlate-96 filter plate (Packard, Meriden,
Conn.), which was dried overnight. The amount of .sup.51Cr was
measured by a TopCount NXT plate reader (Packard). FIG. 27 shows
that the 2H7 scFv-llama IgG2 fusion protein was the most effective
llama fusion protein in mediating ADCC. Each data point represents
the average measurement of triplicate wells.
[0278] ADCC activity was affected by the source of effector cells.
Llama PBMC were isolated from llama blood (Triple J Farms) using
LSM. Llama effector cells were added at the same ratios to BJAB
target cells as described for the ADCC assay using human effector
cells. The cells were combined with 10 .mu.g/ml of purified 2H7
scFv-llama IgG1, 2H7 scFv-llama IgG2, 2H7 scFv-llama IgG3,
Rituximab, or no anti-CD20 antibody. The results are presented in
FIG. 28.
Example 12
Construction and Characterization of scFv Ig Fusion Proteins
Expressed on the Cell Surface
[0279] This Example describes a retroviral transfection system for
ectopic surface expression of genetically engineered cell surface
receptors composed of scFvs that bind costimulatory receptors. The
Example also demonstrates the effector function of these various
scFv Ig fusion proteins expressed on the surface of target
cells.
[0280] The heavy and light chain variable regions were cloned from
murine monoclonal antibodies specific for various costimulatory
receptors, and single chain Fv constructs were prepared essentially
as described in Example 1. Antibodies included 2H7, anti-human
CD20; 40.2.220, anti-human CD40; 2E12, anti-human CD28; 10A8,
anti-human CD152 (anti-CTLA-4); and 500A2, anti-murine CD3. The
heavy chain and light chain variable regions of each antibody were
cloned according to standard methods for cloning immunoglobulin
genes and as described in Example 1. Single chain Fv constructs
were prepared as described in Example 1 by inserting a nucleotide
sequence encoding a (gly.sub.4ser).sub.3 peptide linker between the
VL region nucleotide sequence of 40.2.220, 2E12, 10A8, and 500A2,
respectively (SEQ ID NOs: 243 or 462; 249 or 468; 474; 263,
respectively) and the VH region nucleotide sequence of 40.2.220,
2E12, 10A8, and 500A2, respectively (SEQ ID NOs: 245 or 464; 251 or
470; 261 or 476; 257, respectively). The polypeptide sequence for
VL of 40.2.220, 2E12, 10A8, and 500A2 are set forth in SEQ ID NOs:
244 or 463; 250 or 469; 255 or 475; 264, respectively, and the
polypeptide sequence for VH of 40.2.220, 2E12, 10A8, and 500A2 are
set forth in SEQ ID NOs: 246 or 465; 252 or 471; 258 or 477; 262,
respectively. Each scFv polynucleotide (SEQ ID NOs: 247 or 466; 253
or 472; 259 or 478; 399 for 40.2.220, 2E12, 10A8, and 500A2,
respectively) was then fused to human IgG1 mutant hinge
(CCC.fwdarw.SSS) and mutant CH2 (proline to serine mutation at
residue 238 (238 numbering according to EU nomenclature, Ward et
al., 1995 Therap. Immunol. 2:77-94; residue 251 according to Kabat
et al.) and wild type CH3 domains according to the methods
described in Example 5 and 11. Each scFv mutant IgG1 fusion
polynucleotide sequence was then fused in frame to sequences
encoding the transmembrane domain and cytoplasmic tail of human
CD80 (SEQ ID NO: 460), such that when the fusion protein was
expressed in the transfected cell, CD80 provided an anchor for
surface expression of the scFv Ig fusion protein. cDNAs encoding
the scFv-IgG-CD80 fusion proteins (SEQ ID NOs: 268 or 483; 265 or
481; 270 or 485; 272 or 487 for 40.2.220-, 2E12-, 10A8-, and
500A2-scFv-IgG-CD80, respectively) were inserted into the
retroviral vector pLNCX (BD Biosciences Clontech, Palo Alto,
Calif.) according to standard molecular biology procedures and
vendor instructions. The scFv-Ig-CD80 cDNA was inserted between the
5'LTR-neomycin resistance gene-CMV promoter sequences and the 3'LTR
sequence. The retroviral constructs were transfected into Reh, an
acute lymphocytic leukemia cell line (ATCC CRL-8286). Transfected
cells were screened to select clones that were expressing scFv-Ig
fusion proteins on the cell surface.
[0281] CDC and ADCC assays were performed with the transfected Reh
cells to determine if expression of the scFv-Ig polypeptides on the
cell surface augmented effector cell function. Reh cells expressing
anti-human CD152 scFv-mutant IgG-CD80 (SEQ ID NO: 270 or 485); Reh
anti-human CD28 scFv-mutant IgG-CD80 (SEQ ID NOS: 268 or 483); Reh
anti-human CD40 scFv-mutant IgG-CD80 (SEQ ID NOS: 265 or 481); Reh
anti-human CD20 scFv-mutant IgG-CD80 were combined with human PBMC
(see Example 11) and rabbit complement (10 .mu.g/ml) for one hour
at 37.degree. C. Untransfected Reh cells were included as a
control. Viability of the cells was determined by trypan blue
exclusion, and the percent of killed cells was calculated (see
Example 11). FIG. 29 shows the effectiveness of the scFv-IgG-CD80
fusion proteins when expressed on the cell surface of tumor cells
to mediate complement dependent cytotoxicity.
[0282] The same transfected Reh cells tested in the CDC assay plus
Reh cells transfected with the polynucleotide construct that
encodes anti-murine CD3-scFv-Ig-CD80 (SEQ ID NO: 486) were analyzed
for ADCC activity (see Example 11). Untransfected and transfected
Reh cells were pre-labeled with .sup.51 Cr (100 .mu.Ci) (Amersham)
for two hours at 37.degree. C. Human PBMC served as effector cells
and were added to the Reh target cells (5.times.10.sup.4 cells per
well of 96 well plate) at ratios of 5:1, 2.5:1, and 1.25:1. After
five hours at 37.degree. C., culture supernatants were harvested
and analyzed as described in Example 11. Percent specific killing
was calculated according to the following equation: ((experiment
release minus spontaneous release)/(maximum release minus
spontaneous release)).times.100. The data are presented in FIG. 30.
Each data point represents the average of quadruplicate
samples.
[0283] Using the same procedures described above, the same results
with other binding domains were obtained using the following
monoclonal antibodies mAbs as sources of sFv: for CD20, 1F5
(Genbank AY 058907 and AY058906); for CD40, 2.36 and G28.5; for
CD28, 9.3.
[0284] Cell surface expression of antibody binding domains is
accomplished by fusing antibody scFvs to IgA hinge and constant
regions and IgE hinge and constant regions. Polynucleotides
encoding an anti-4-1BB scFv, 5B9 (anti-human 4-1BB) scFv, and 2e12
(anti-human CD40) fused to IgAH IgA T4 (four terminal CH3 residues
deleted) fused to the CD80 transmembrane and cytoplasmic domains
and IgE Fc regions are shown in SEQ ID NOs: 626 and 630. The
encoded polypeptides are shown in SEQ ID NOs: 627 and 631.
Example 13
Construction and Sequence of Human Ig Hinge-Ch2-Ch3 Mutants and 2H7
Variable Region Mutants
[0285] This Example describes construction of scFv fusion proteins
containing mutant human IgG1 and IgA constant regions. This Example
also describes construction of a 2H7 scFv mutant with a single
point mutation in the variable heavy chain region. Mutations were
introduced into variable and constant region domains according to
methods described herein and known in the molecular biology arts.
FIG. 31 presents nomenclature for the Ig constant region
constructs.
[0286] The human IgG1 hinge region of the 2H7 scFv human IgG1
hinge-CH2-CH3 fusion proteins was mutated to substitute cysteine
residues that in a whole immunoglobulin are involved in forming
disulfide bonds between two heavy chain molecules. One mutant, 2H7
scFv fused to a human IgG1 hinge region in which all three cysteine
residues were mutated to serine residues (MTH (SSS)), was prepared
as described in Example 5 (designated in Example 5 as
CytoxB-MHWTG1C (includes wild type IgG1 CH2 and CH3 domains)) (now
referred to as 2H7 scFv MTH (SSS) WTCH2CH3) and comprises the
polynucleotide sequence SEQ ID NO: 4 encoding the polypeptide as
set forth in SEQ ID NO: 17. The polynucleotide sequence encoding
this mutant (SEQ ID NO: 4) was used as a template to create mutant
hinge regions in which the first two cysteine residues were
substituted with serine residues (IgG MTH (SSC)). An
oligonucleotide was designed to substitute the third serine residue
with a cysteine and had the following sequence: 5'-gtt gtt gat cag
gag ccc aaa tct tct gac aaa act cac aca tct cca ccg tgc cca gca cct
g-3' (HuIgGMHncs3, SEQ ID NO: 275). A second mutant was prepared in
which the mutant hinge had serine residues substituting the first
and third cysteine residues (IgG MTH (SCS)). The sequence of the
oligonucleotide to create this mutant was as follows: 5' gtt gtt
gat cag gag ccc aaa tct tct gac aaa act cac aca tgc cca ccg-3'
(HuIgGMHncs2, SEQ ID NO: 276). A third mutant was prepared with
cysteine residues substituted at the second and third positions
(IgG MTH (CSS)), also using the IgG MTH (SSS) mutant as template,
and an oligonucleotide having the sequence, 5'-gtt gtt gat cag gag
ccc aaa tct tgt gac aaa act cac-3' (HuIgGMHncsl, SEQ ID NO:
277).
[0287] The oligonucleotides introducing the mutations into the
hinge region were combined with template and a 3' oligonucleotide
containing an XbaI site (underlined and italicized) (5'-gtt gtt tct
aga tca ttt acc cgg aga cag gga gag get ctt ctg cgt gta g-3' (SEQ
ID NO: 278)) to amplify the mutant hinge-wild type (WT)-CH2-CH3
sequences by PCR. The IgG MTH CSS and IgG MTH SCS mutant sequences
were amplified for 25 cycles with a denaturation profile of
94.degree. C., annealing at 52.degree. C. for 30 seconds, and
extension at 72.degree. C. for 30 seconds. The IgG MTH SSC mutant
sequence was amplified under slightly different conditions:
denaturation profile of 94.degree. C., annealing at 45.degree. C.
for 30 seconds, and extension at 72.degree. C. for 45 seconds. The
amplified polynucleotides were inserted into the TOPO.RTM. cloning
vector (Invitrogen Life Technologies) and then were sequenced as
described in Example 1 to confirm the presence of the mutation.
pD18 vector containing 2H7 say was digested to remove the constant
region sequences essentially as described in Example 10. The mutant
hinge-wild type CH2-CH3 regions were inserted in frame into the
digested vector DNA to obtain vectors comprising 2H7 scFv MTH (CSS)
WTCH2CH3 encoding DNA (SEQ ID NO: 581); 2H7 scFv MTH (SCS) WTCH2CH3
encoding DNA (SEQ ID NO: 583); and 2H7 scFv MTH (SSC) WTCH2CH3
encoding DNA (SEQ ID NO: 585).
[0288] A mutation of leucine to serine at position 11 in the first
framework region of the heavy chain variable region (numbering
according to Kabat et al., Sequences of Proteins of Immunological
Interest, 5.sup.th ed. Bethesda, Md.: Public Health Service,
National Institutes of Health (1991)) was introduced into the 2H7
scFv MTH (SSS) WTCH2CH3 fusion protein (SEQ ID NOS: 4 or 488). The
wild type leucine residue was substituted with serine by
site-directed mutagenesis using the oligonucleotide Vhser11: 5'-gga
ggt ggg agc tct cag gct tat cta cag cag tct ggg gct gag tcg gtg agg
cc-3' (SEQ ID NO: 279). The 3' primer for PCR was huIgG1-3' having
the sequence 5'-gtc tct aga cta tca ttt acc cgg aga cag-3' (SEQ ID
NO: 280) (XbaI site underlined and italicized). After PCR
amplification, the fragments were inserted into the TOPO.RTM.
cloning vector and sequenced to confirm the presence of the VH11
leucine to serine mutation. The 2H7 scFv-IgG MTH (SSS) WTCH2CH3
encoding DNA was shuttled into the PSL1180 cloning vector
(Pharmacia Biotech, Inc., Piscataway, N.J.). The construct
PSL1180-2H7 scFv-IgG MTH (SSS) WTCH2CH3 was digested with Sac and
XbaI to remove the wild type VH domain and the hinge and CH2 and
CH3 domains. The PCR product comprising the VH11 mutant was
digested with Sac and XbaI and then inserted into the digested
PSL1180 construct according to standard molecular biology
procedures. The construct was then digested with Hind III and XbaI,
and inserted into the mammalian expression vector pD18 (see methods
described in Example 1 and Example 10). The mutant is designated
2H7 scFv VH11SER IgG MTH (SSS) WTCH2CH3 (FIG. 31). Four constructs
containing IgA constant region domains were prepared. One construct
contained wild type IgA hinge fused to human IgG1 CH2 and CH3 (IgAH
IgG WTCH2CH3) (FIG. 31). Sequential PCR amplifications were
performed to substitute the human IgG1 hinge of the 2H7 scFv
construct with nucleotide sequences encoding the IgA hinge. The 5'
oligonucleotide primer (huIgA/Gchim5) for the first PCR reaction
had the sequence, 5'-cca tct ccc tca act cca cct acc cca tct ccc
tca tgc gca cct gaa ctc ctg-3' (SEQ ID NO: 281). The primer
(hulgAhg-5') for the second PCR reaction to add more IgA specific
hinge sequence and add a BclI restriction enzyme site (italicized
and underlined) had the sequence, 5'-gtt gtt gat cag cca gtt ccc
tca act cca cct acc cca tct ccc caa ct-3' (SEQ ID NO: 282). The 3'
primer for both amplification steps was huIgG1-3' having the
sequence, 5'-gtc tct aga cta tca ttt acc cgg aga cag-3' (SEQ ID NO:
280). The sequence of the PCR product was confirmed by TOPO.RTM.
cloning as described above. The gel-purified fragment was digested
with BclI and XbaI and then inserted into the 2H7 scFv-pD18 vector
that had been digested BclI and XbaI to remove all the IgG1
constant region domains. Ligation was performed as described in
Example 10 to provide a mammalian expression vector comprising the
nucleotide sequence (SEQ ID NO: 283) encoding a 2H7 scFv IgA
hinge-IgG1 CH2-CH3 polypeptide (SEQ ID NO: 284).
[0289] A second pD 18 mammalian expression vector was constructed
that had a polynucleotide sequence (SEQ ID NO: 1) that encoded a
2H7 scFv fused to wild type IgA hinge, CH2, and CH3 domains (SEQ ID
NO: 285). Human IgA constant regions sequences were obtained by
using random primers to reverse transcribe total RNA isolated from
human tonsil followed by PCR amplification of the cDNA using
sequence specific primers, essentially as described in Example 10.
Human IgA hinge-CH2-CH3 nucleotide sequence (SEQ ID NO: 285)
encoding the IgA-CH2-CH3 polypeptide (IgAH IgACH2CH3, FIG. 31) (SEQ
ID NO: 286) was amplified using the 5' oligonucleotide hulgAhg-5'
(SEQ ID NO: (same as above, SEQ ID NO: 281) and a 3'
oligonucleotide huIgA3' having the sequence, 5'-gtt gtt tct aga tta
tca gta gca ggt gcc gtc cac ctc cgc cat gac aac-3' (SEQ ID NO:
289). Secretion of a 2H7-IgA hinge-IgA CH2-CH3 polypeptide from
transfected mammalian cells required co-expression of human J chain
that covalently binds to two IgA CH3 domains via disulfide bonds.
Total RNA was isolated from tonsil B cells and was reversed
transcribed to generate cDNA as described above. PCR amplification
of the nucleotide sequence encoding the J chain was performed with
J chain specific primers. The 5' PCR primer, HUJCH5n1, had the
sequence, 5'-gtt gtt aga tct caa gaa gat gaa agg att gtt ctt-3'
(SEQ ID NO: 292), and sequence of the 3' primer, HUJCH3, was 5'-gtt
gtt tct aga tta gtc agg ata gca ggc atc tgg-3' (SEQ ID NO: 293).
The cDNA was cloned into TOPO.RTM. for sequencing as described in
Example 10. J chain encoding cDNA (SEQ ID NO: 290) was then
inserted into pD18 and pcDNA3-Hygro (+) (Invitrogen Life
Technology) vectors for co-transfection with 2H7 scFv IgA
hinge-CH2-CH3 constructs. The J chain has the predicted amino acid
sequence set forth in SEQ ID NO: 291.
[0290] Secretion of an scFv IgA constant region construct in the
absence of J chain was accomplished by engineering a truncated CH3
domain with a deletion of the four carboxy terminal amino acids
(GTCY, SEQ ID NO: 294) (IgAH IgA-T4, FIG. 31), which include a
cysteine residue that forms a disulfide bond with the J chain. The
IgA hinge-CH2-CH3 nucleotide sequence containing the deletion in
CH3 (SEQ ID NO: 295) was prepared using a 5' PCR primer
(huIgAhg-5') having the sequence 5'-gtt gtt gat cag cca gtt ccc tca
act cca cct acc cca tct ccc tca act-3' (SEQ ID NO: 310) (BclI site
is underlined and italicized), and a 3' PCR primer (HUIGA3T1)
having the sequence 5'-gtt gtt tct aga tta tca gtc cac ctc cgc cat
gac aac aga cac-3' (SEQ ID NO: 297). This mutated IgA constant
region nucleotide sequence was inserted into a 2H7 scFv pD 18
vector as described for the generation of the previous 2H7 scFv-Ig
constructs (see Example 1 and this example) that comprises the
polynucleotide sequence (SEQ ID NO: 298) encoding a 2H7 IgAH IgA-T4
polypeptide (SEQ ID NO: 299).
[0291] A fourth construct was prepared that encoded a 2H7 scFv-IgA
constant region fusion protein with a deletion of 14 additional
amino acids, most of which are hydrophobic residues, from the
carboxy terminus of IgA CH3. The 2H7 scFv-IgAH IgA-T4 encoding
polynucleotide was used as template to engineer a deletion of the
nucleotide sequence encoding PTHVNVSVVMAEVD (SEQ ID NO: 300). The
5' oligonucleotide primer had the sequence 5'-gtt gtt gat cag cca
gtt ccc tca act cca cct acc cca tct ccc tca act-3' (SEQ ID NO: 310)
(BclI site shown as underlined and italicized). The 3'
oligonucleotide sequence was 5'-gtt gtt tct aga tta tca ttt acc cgc
caa gcg gtc gat ggt ctt-3' (SEQ ID NO: 301). The deleted IgA CH3
region was amplified by using the above oligonucleotides to amplify
the IgA constant region from RNA isolated from human tonsil such
that the cDNA contained the deleted carboxyl terminal encoding
region for the 18 amino acids. The IgAH IgA-T18 constant region was
inserted into a 2H7 scFv pD18 vector that comprises the
polynucleotide sequence (SEQ ID NO: 302) encoding a 2H7 IgAH
IgA-T18 polynucleotide (SEQ ID NO: 303) as described above.
Example 14
Effector Function of CTLA-4 IgG Fusion Proteins
[0292] The Example compares the effector functions of CTLA-4 Ig
fusion proteins in CDC and ADCC assays.
[0293] Two CTLA-4 IgG fusion proteins were constructed. One fusion
protein comprises the extracellular domain of CTLA-4 fused to human
IgG1 wild type hinge, CH2, and CH3 domains and is designated CTLA-4
IgG WTH (CCC) WTCH2CH3 (SEQ ID NO: 307). A pD18 mammalian
expression vector comprising a polynucleotide sequence encoding
CTLA-4 IgG WTH (CCC) WTCH2CH3 (SEQ ID NO: 306) was prepared by
fusing in frame the nucleotide sequence encoding the extracellular
domain of CTLA-4 (SEQ ID NO: 308) (see U.S. Pat. No. 5,844,095) to
the nucleotide sequence encoding IgG WTH (CCC) WTCH2CH3 according
to the methods described in Examples 1 and 10. The extracellular
domain nucleotide sequence also comprises a BclI restriction enzyme
site at the 3' end, and a leader peptide nucleotide sequence (SEQ
ID NO: 313) that encodes an oncoM leader peptide (SEQ ID NO: 314).
A second CTLA-4 IgG fusion protein, designated CTLA-4 IgG MTH (SSS)
MTCH2WTCH3, contained the extracellular domain of CTLA-4 (plus the
oncoM leader peptide sequence) fused to a mutant IgG hinge in which
all three cysteine residues were replaced with serine residues. The
hinge region was fused to a mutant IgG1 CH2 domain that had a
mutation at isotype position 238 (EU numbering, Ward et al., supra,
(position 251 using numbering according to Kabat et al., supra;
position 209 where numbering commences with first residue of IgG1
CH1; i.e., PAPELLGGPS (SEQ ID NO: 537) of wild type IgG1 CH2 is
modified to PAPELLGGSS (SEQ ID NO: 583), which was fused to IgG1
wild type CH3 (U.S. Pat. No. 5,844,095). The CTLA-4 IgG MTH (SSS)
MTCH2WTCH3 polynucleotide comprises the nucleotide sequence in SEQ
ID NO: 315 and the deduced amino acid sequence comprises the
sequence provided in SEQ ID NO: 316. CTLA-4 fusion proteins were
also prepared using CTLA-4 extracellular membrane encoding
sequences without the leader peptide (SEQ ID NO: 313 (DNA) and 314
(AA)).
[0294] To measure CDC activity, purified CTLA-4 IgG WTH (CCC)
WTCH2CH3 (2 .mu.g/ml) or CTLA-4 IgG MTH (SSS) MTCH2WTCH3 (2
.mu.g/ml) was added to Reh cells (see Example 12) and to Reh cells
transfected with the costimulatory molecule CD80 such that CD80 was
expressed on the cell surface (Reh CD80.10, obtained from Dr. E.
Clark, University of Washington, Seattle, Wash.; see Doty et al.,
1998 J. Immunol. 161:2700; Doty et al., 1996 J. Immunol. 157:3270),
in the presence or absence of rabbit complement (10 .mu.g/ml).
Purified CTLA Ig fusion proteins were prepared from culture
supernatants of transiently transfected COS cells according to
methods described in Example 10. The assays were performed
essentially as described in Example 11 and 12. The data presented
in FIG. 32 show that only CD80-transfected Reh cells were killed in
the presence of complement and CTLA-4 IgG WTH (CCC) WTCH2CH3 fusion
protein.
[0295] The purified CTLA-4 Ig fusion proteins were also tested in
ADCC assays. Human PBMC, serving as effector cells, were added to
Reh or Reh CD80.1 target cells at a ratio of 1.25:1, 2.5:1, 5.0:1,
and 10:1. Cells were labeled and the assays performed essentially
as described in Examples 11 and 12. The results are presented in
FIG. 33. Each data point represents the average of four independent
culture wells at each effector:target cell ratio. The data show
that only CTLA-4 IgG WTH (CCC) WTCH2CH3 mediated significant ADCC
of Reh CD80.10 cells.
Example 15
Effector Function of CTLA-4 IgA Fusion Proteins
[0296] CTLA-4 IgA fusion proteins are prepared as described for the
IgG fusion proteins (see Examples 1, 13, and 14). CTLA-4
extracellular domain nucleotide sequence (SEQ ID NO: 313) is fused
in open reading frame to nucleotides encoding IgAH IgACH2CH3 (SEQ
ID NO: 287) to provide the nucleotide sequence (SEQ ID NO: 319)
encoding a CTLA-4 IgAH IgACH2CH3 fusion protein (SEQ ID NO: 320).
The fusion protein is transiently expressed in COS cells (see
Example 10) or stably expressed in CHO cells (see Example 1).
Secretion of the CTLA-4 IgAH IgACH2CH3 fusion protein requires
co-transfection with a construct containing a polynucleotide
sequence (SEQ ID NO: 290) that encodes human J chain (SEQ ID NO:
291). The CTLA-4 IgAH IgACH2CH3 fusion protein is isolated as
described in Examples 10 and 14. To express a CTLA-4 IgA construct
without the presence of J chain, a CTLA-4 IgAH IgA-T4 construct is
prepared and transfected into mammalian cells. In a similar manner
as described for the CTLA-4 extracellular fragment fused to wild
type IgA hinge-CH2CH3, the CTLA-4 extracellular domain nucleotide
sequence (SEQ ID NO: 308) is fused in open reading frame to a
nucleotide sequence (SEQ ID NO: 295) encoding a IgAH IgA-T4
polypeptide (SEQ ID NO: 296) to provide a nucleotide sequence
comprising SEQ ID NO: 319 encoding a CTLA-4 IgAH IgA-T4 polypeptide
(SEQ ID NO: 320). Effector function of each construct is evaluated
by CDC and ADCC as described in Example 14.
Example 16
Binding of Anti-CD20 scFv Human Ig Fusion Proteins to CHO Cells
Expressing CD20
[0297] This Example describes binding of 2H7 scFv Ig fusion
proteins to CHO cells that express CD20. The analysis was performed
by flow cytometry. Culture supernatants were collected from
transiently transfected COS cells expressing 2H7 scFv IgG WTH (CCC)
WTCH2CH3 (SEQ ID NOS: 2, 15; 239, 240; 458, 459); 2H7 scFv IgG MTH
(CSS) WTCH2CH3 (SEQ ID NO: 582); 2H7 scFv IgG MTH (SCS) WTCH2CH3
(SEQ ID NO: 584); and 2H7 scFv VHSER11WTH WTCH2CH3, and two-fold
serial dilutions were prepared. Serial two-fold dilutions of
purified 2H7 scFv IgG MTH (SSC) WTCH2CH3 (SEQ ID NOS: 585, 586)
were prepared starting at a concentration of 5 .mu.g/ml. The
culture supernatants and purified fusion protein samples were
incubated with (CD20+) CHO cells for one hour on ice. The cells
were washed twice and then incubated with 1:100 FITC-conjugated
goat anti-human IgG (CalTag) for 40 minutes. The unbound conjugate
was then removed by washing the cells and flow cytometry analysis
was performed using a Coulter Epics XL cell sorter. Results are
presented in FIG. 34.
Example 17
Immunoblot Analysis of Anti-CD20 scFv Human IgG and IgA Fusion
Proteins
[0298] This Example describes immunoblot analysis of 2H7 scFv IgG
and 2H7 scFv IgA fusion proteins that were immunoprecipitated from
transfected cell culture supernants.
[0299] COS cells were transiently transfected with plasmids
comprising nucleotide sequences for 2H7 scFv IgG WTH (CCC) WTCH2CH3
(SEQ ID NO: 458); 2H7 scFv IgG MTH (CSS) WTCH2CH3 (SEQ ID NO: 581);
2H7 scFv IgG MTH (SCS) WTCH2CH3 (SEQ ID NO: 583); 2H7 scFv IgA H
IgG WTCH2CH3 (SEQ ID NOS: 283, 499); and scFv IgG MTH (SSS)
WTCH2CH3 (SEQ ID NOS: 273, 488) essentially according to the method
described in Example 10. Cells were also transfected with vector
only. After 48-72 hours at 37.degree. C., cell culture supernatants
were harvested and combined with protein A-agarose beads (Repligen)
for one hour at 4.degree. C. The beads were centrifuged and washed
several times in TNEN [20 mM Tris base, 100 mM NaCl, 1 mM EDTA, and
0.05% NP-40, pH 8.0). The immunoprecipitates were combined with 25
.mu.l 2.times. NuPAGE.RTM. SDS Sample Buffer (Invitrogen Life
Technologies) (non-reduced samples). The proteins were fractionated
on NuPAGE.RTM. 10% Bis-Tris gels (Invitrogen Life Technologies).
After electrophoresis (approximately 1 hour), the proteins were
transferred from the gel onto a Immobilon P polyvinylidene fluoride
(PVDF) membrane (Millipore, Bedford, Mass.) using a semi-dry
blotter (Ellard Instrumentation, Monroe, Wash.). The PVDF membrane
was blocked in PBS containing 5% nonfat milk and then probed with
HRP-conjugated goat anti-human IgG (Fc specific) (CalTag). After
washing the immunoblot several times in PBS, the blot was developed
using ECL (Amersham Biosciences). The results are shown in FIG.
35.
Example 18
Binding of Anti-CD20 scFv Human IgA Fusion Proteins to CD20+ CHO
Cells
[0300] This Example describes flow immunocytofluorimetry analysis
of binding of 2H7 scFv IgAH IgACH2CH3 (SEQ ID NOS: 286, 502) and
2H7 scFv IgAFI IgAT4 (SEQ ID NOS: 299, 515) fusion proteins to
(CD20+) CHO cells.
[0301] COS cells were transiently co-transfected as described in
Example 10 with plasmid DNA comprising a polynucleotide sequence
(SEQ ID NO: 285) encoding 2H7 scFv IgAH IgACH2CH3 polypeptide (SEQ
ID NO: 501) and with a separate plasmid comprising a polynucleotide
sequence (SEQ ID NO: 290) encoding a human J chain polypeptide (SEQ
ID NO: 291). COS cells were also transfected with a polynucleotide
sequence (SEQ ID NOS: 298, 514) encoding a 2H7 scFv IgA fusion
protein that had a deletion of four amino acids at the carboxy
terminus of CH3 (2H7 scFv IgAH IgA-T4, SEQ ID NOS: 299, 515). The
transfections were performed as described in Example 10. Culture
supernatants from transfected COS cells were combined with (CD20+)
CHO cells (see Example 1) and incubated for one hour on ice. The
cells were washed twice with PBS-2% FBS and then combined with
FITC-conjugated goat anti-human IgA chain (CalTag) (1:100) for 40
minutes. The cells were again washed and then analyzed by flow
cytometry using a Coulter Epics XL cell sorter. FIG. 36 shows that
co-transfection with J chain was not required for secretion of 2H7
scFv IgAH IgAT4, the 2H7 IgA fusion protein with the truncated CH3
carboxy end (SEQ ID NO: 299, 515).
Example 19
Effector Function of Anti-CD20 scFv Human IgA Fusion Proteins
[0302] This Example illustrates ADCC activity of 2H7 IgG and IgA
fusion proteins against cells that express CD20. BJAB cells were
prelabeled with .sup.51Cr (100 .mu.Ci) (Amersham) for two hours at
37.degree. C. Effector cells were obtained from fresh, resting
human whole blood, which was diluted in an equal volume of
Alsever's solution to prevent coagulation. 2H7 scFv IgG MTH (SSS)
WTCH2CH3 (SEQ ID NO: 489); 2H7 scFv IgG MTH (SCS) WTCH2CH3 (SEQ ID
NO: 586); 2H7 scFv IgG WTH (CCC) WTCH2CH3 (SEQ ID NO: 459); and 2H7
scFv IgAH IgACH2CH3 (SEQ ID NO: 299, 515) fusion proteins were
purified from transiently transfected COS cell supernatants
(100-200 ml) by protein A chromatography as described in Example
10. COS cells transfected with the plasmid encoding 2H7 scFv IgAFI
IgACH2CH3 were co-transfected with a plasmid encoding human J chain
as described in Example 18. Two-fold serial dilutions of the
purified 2H7 Ig fusion proteins starting at 5 .mu.g/ml were added
to the labeled BJAB cells (5.times.10.sup.4 cells per well of 96
well tissue culture plate) in the presence of whole blood (100
.mu.l of whole blood diluted 1:1 in Alsever's solution, final
dilution 1:4) and incubated for five hours at 37.degree. C. Culture
supernatants were harvested and analyzed as described in Example
11. Percent specific killing was calculated according to the
following equation: ((experiment release minus spontaneous
release)/(maximum release minus spontaneous release)).times.100.
The data are presented in FIG. 37. Each data point represents the
average of quadruplicate samples.
[0303] In a second ADCC assay, the number of labeled BJAB target
cells was held constant in each sample, and whole blood was added
at dilutions of 0.25, 0.125, and 0.0625. Purified 2.sub.H7 IgG and
IgA fusion proteins were added at a concentration of 5 .mu.g/ml.
The BJAB cells, whole blood, and fusion proteins were incubated,
the supernatants harvested, and the percent specific killing was
calculated as described above. Percent specific killing for each of
the 2H7 fusion proteins is presented in FIG. 38.
[0304] The ADCC activity of purified 2H7 scFv IgG MTH (SSS)
WTCH2CH3 (5 .mu.g/m-1) and of purified 2H7 scFv IgAH IgACH2CH3 (5
.mu.g/ml) was compared in the presence of different effector cell
populations. PBMC were isolated from whole blood as described in
Examples 11 and 12. PBMC were combined with labeled BJAB target
cells (5.times.10.sup.4 per well of 96 well tissue culture plate)
at ratios of 50:1, 25:1, and 12.5:1. The assay was performed and
the data analyzed as described above. FIG. 39A shows that only the
2H7 scFv IgG MTH (SSS) WTCH2CH3 fusion protein had ADCC activity
when PBMC served as the effector cells. FIG. 39B shows that both
2H7 scFv IgG MTH (SSS) WTCH2CH3 and 2H7 scFv IgAH IgACH2CH3 exhibit
ADCC activity when whole blood was the source of effector cells (as
illustrated in FIG. 38).
Example 20
Expression Level of 2H7 scFv VH11Ser IgG MTH (SSS) WTCH2CH3 Fusion
Protein
[0305] This Example compares the expression level of 2H7 scFv
VH11Ser IgG MTH (SSS) WTCH2CH3 fusion protein (SEQ ID NO: 488) with
other 2H7 scFv IgG constructs that do not contain the mutation in
the variable heavy chain domain. The mammalian expression vector
pD18 comprising nucleotide sequences 2H7 scFv IgG MTH (SSS)
WTCH2CH3 (SEQ ID NO: 488); 2H7 scFv IgG MTH (CSS) WTCH2CH3 (SEQ ID
NO: 581); 2H7 scFv IgG MTH (SCS) WTCH2CH3 (SEQ ID NO: 583); 2117
scFv IgG WTH (CCC) WTCH2CH3 (SEQ ID NO: 458); and 2H7 scFv VHSER11
IgG MTH (SSS) WTCH2CH3 (see Examples 1 and 13) were transiently
transfected into COS cells as described in Example 10. After 72
hours at 37.degree. C., culture supernatants were harvested, and 1
.mu.l of each supernatant was combined with non-reducing sample
buffer (see method described in Example 10). The culture
supernatant samples and aliquots of purified 2H7 scFv IgG MTH (SSS)
WTCH2CH3 (40 ng, 20 ng, 10 ng/5 ng, and 2.5 ng) were fractionated
on 10% Bis-Tris (MOPS) NuPAGE.RTM. gels (Invitrogen Life
Technologies). Multimark.RTM. protein standards (Invitrogen Life
Technologies) were also separated on the gel. The proteins were
transferred to a PDVF membrane and immunoblotted as described in
Example 17. The immunoblot is presented in FIG. 40. The amounts of
the fusion proteins were quantified by densitometry analysis of the
blots using the ScionImage for Windows software and comparison with
the standard curve. The 2H7 scFv IgG WTH (CCC) WTCH2CH3 construct
produced approximately 12 ng/ul or 12 micrograms/ml, the 2H7 scFv
IgG MTH (CSS) WTCH2CH3 produced approximately 10 ng/ul or 10
micrograms/ml, the 2H7 scFv IgG MTH (SCS) WTCH2CH3 construct
produced approximately 1 ng/ul or 1 microgram/ml, and the 2H7 scFv
VHSER11 IgG MTH (SSS) WTCH2CH3 construct produced approximately 30
ng/ml or 30 micrograms/ml.
Example 21
Construction of a 2H7 scFv IgG Fusion Protein with a Mutant CH3
Domain
[0306] Amino acid mutations were introduced into the CH3 domain of
a 2H7 IgG fusion protein. The pD18 vector comprising 2H7 scFv IgG
MTH (SSS) WTCH2CH3 (SEQ ID NO: 488) was digested with BclI and XbaI
to remove the MTH WTCH2CH3 (SEQ ID NO: 6) fragment, which was then
subcloned into pShuttle vector (BD Biosciences Clontech, Palo Alto,
Calif.) that was double-digested with BclI and XbaI. Subcloning was
performed in a kanamycin resistant vector because the ampicillin
resistance gene has an XmnI site, which is required for this
cloning procedure. Five constructs were prepared with the following
substitutions: (1) a phenylalanine residue at position 405
(numbering according to Kabat et al. supra) was substituted with
tyrosine using the oligonucleotide CH3Y405; (2) the phenylalanine
position at 405 was substituted with an alanine residue using the
oligonucleotide CH3A405; (3) the tyrosine residue at position 407
was substituted with an alanine using the oligonucleotide CH3A407;
(4) both wild type amino acids at positions 405 and 407 were
substituted with tyrosine and alanine, respectively using the
oligonucleotide CH3Y405A407; and (5) both wild type amino acids at
positions 405 and 407 were substituted with alanine using the
oligonucleotide CH3A405A407. The oligonucleotides were the 3'
primers for PCR amplification of a portion of the CH3 domain. The
nucleotide sequences for each 3' oligonucleotide were as
follows.
TABLE-US-00002 CH3Y405: (SEQ ID NO: 365) 5'-gtt gtt gaa gac gtt ccc
ctg ctg cca cct gct ctt gtc cac ggt gag ctt gct gta gag gta gaa gga
gcc-3' CH3A405: (SEQ ID NO: 366) 5'-gtt gtt gaa gac gtt ccc ctg ctg
cca cct gct ctt gtc cac ggt gag ctt gct gta gag ggc gaa gga gcc-3'
CH3A407: (SEQ ID NO: 367) 5'-gtt gtt gaa gac gtt ccc ctg ctg cca
cct gct ctt gtc cac ggt gag ctt gct ggc gag gaa gaa gga gcc-3'
CH3Y405A407: (SEQ ID NO: 368) 5'-gtt gtt gaa gac gtt ccc ctg ctg
cca cct gct ctt gtc cac ggt gag ctt gct ggc gag gta gaa gga gcc-3'
CH3A405A407: (SEQ ID NO: 369) 5'-gtt gtt gaa gac gtt ccc ctg ctg
cca cct gct ctt gtc cac ggt gag ctt gct ggc gag ggc gaa gga
gcc-3'
[0307] The template was the mutant hinge MHWTCH2CH3 human IgG1. The
5' PCR oligonucleotide primer was huIgGMHWC, (SEQ ID NO: 332). The
amplified products were TOPO.RTM. cloned and sequenced as described
in Examples 1 and 10. DNA from the clones with the correct sequence
was digested with BclI and XmnI and transferred to pShuttle
containing the MTH WTCH2CH3 sequence, which was also digested with
the same restriction enzymes. The mutated IgG sequences were then
removed by digestion with BclI and XbaI and inserted into a pD18
vector containing 2H7 scFv that was also digested with BclI and
XbaI. The polynucleotide sequences for mutated the CH3 domains,
MTCH3 Y405, MTCH3 A405, MTCH3 A407, MTCH3 Y405A407, and MTCH3
A405A407 are shown in SEQ ID NOs: 370, 371, 372, 373, 374,
respectively, and the polypeptide sequences for each are shown in
SEQ ID NOs: 375, 376, 377, 378, 379 respectively. The
polynucleotide sequences for the 2H7 scFv MTH WTCH2 MTCH3 Y405, 2H7
scFv MTH WTCH2 MTCH3 A405, scFv MTH WTCH2 MTCH3 A407, scFv MTH
WTCH2 MTCH3 Y405A407, and scFv MTH WTCH2 MTCH3 A405A407 are shown
in SEQ ID NOs: 381, 380, 383, 384, 382, respectively, and the
deduced amino acid sequences are shown in SEQ ID NOs: 386, 385,
387, 388 389, respectively.
Example 22
Construction of 2H7 scFv IgG Fusion Proteins with Hinge
Mutations
[0308] A 2H7 scFv IgG fusion protein was constructed with the third
cysteine residue in the IgG1 hinge region substituted with a serine
residue. The template for introduction of the mutations was a
polynucleotide encoding 2H7 scFv WTH WTCH2CH3 (SEQ ID NO: 2, 239).
The oligonucleotide introducing the mutations was a 5' PCR primer
oligonucleotide HIgGMHcys3 having the sequence 5'-gtt gtt gat cag
gag ccc aaa tct tgt gac aaa act cac aca tgt cca ccg tcc cca gca
cct-3' (SEQ ID NO: 589). The oligonucleotide introducing the
mutation into the hinge region was combined with template and a 3'
oligonucleotide containing an XbaI site (underlined and italicized)
(5'-gtt gtt tct aga tca ttt acc cgg aga cag gga gag get ctt ctg cgt
gta g-3' (SEQ ID NO: 278)) to amplify the mutant hinge-wild type
(WT)-CH2-CH3 sequences by PCR. The IgG MTH CCS mutant sequence was
amplified for 30 cycles with a denaturation profile of 94.degree.
C., annealing at 50.degree. C. for 30 seconds, and extension at
72.degree. C. for 30 seconds. The amplified polynucleotides were
inserted into the TOPO.RTM. cloning vector (Invitrogen Life
Technologies) and then were sequenced as described in Example 1 to
confirm the presence of the mutation. pD18 vector containing 2H7
scFv was digested to remove the constant region sequences
essentially as described in Example 10. The mutant hinge-wild type
CH2-CH3 regions were inserted in frame into the digested vector DNA
to obtain vectors comprising 2H7 scFv MTH (CCS) WTCH2CH3 encoding
DNA (SEQ ID NO: 395). The deduced polypeptide sequence is shown in
SEQ ID NO: 398.
Example 23
Construction of Anti-CD20 IgE Fusion Proteins
[0309] A binding domain is fused to IgE constant region sequences
such that the expressed polypeptide is capable of inducing an
allergic response mechanism. The single chain Fv nucleotide
sequence of 40.2.220 (SEQ ID NO: 466), an anti-CD40 antibody, is
fused to IgE CH2-CH3-CH4 according to methods described for other
scFv immunoglobulin constant region constructs (see Examples 1, 5,
10, and 13). To PCR amplify the IgE CH2-CH3-CH4 domains, a 5'
oligonucleotide primer, hIgE5Bc1, having the sequence 5'-gtt gtt
gat cac gtc tgc tcc agg gac ttc acc cc-3', and a 3' oligonucleotide
primer, hIgE3stop, having the sequence 5'-gtt gtt tct aga tta act
ttt acc ggg att tac aga cac cgc tcg ctg g-3' are used.
[0310] The retroviral transfection system for ectopic surface
expression of genetically engineered cell surface receptors
composed of scFvs that bind costimulatory receptors described in
Example 12 is used to construct a 40.2.220 scFv IgE-CD80 fusion
protein. The 40.2.220 scFv IgE fusion polynucleotide sequence is
fused in frame to sequences encoding the transmembrane domain and
cytoplasmic tail of human CD80 (SEQ ID NO: 460), such that when the
fusion protein is expressed in the transfected cell, CD80 provided
an anchor for surface expression of the scFv Ig fusion protein.
cDNA encoding the anti-CD40 scFv-IgE-CD80 fusion proteins is
inserted into the retroviral vector pLNCX (BD Biosciences Clontech)
according to standard molecular biology procedures and vendor
instructions. The 40.2.220 scFv-Ig-CD80 cDNA is inserted between
the 5'LTR-neomycin resistance gene-CMV promoter sequences and the
3'LTR sequence. The retroviral constructs are transfected into a
carcinoma cell line, and transfected cells are screened to select
clones that are expressing the 40.2.220 scFv-Ig-CD80 fusion protein
on the cell surface.
Example 24
Construction of IgA-T4 Mutants that are Expressed on the Cell
Surface
[0311] The retroviral transfection system for ectopic surface
expression of genetically engineered cell surface receptors
composed of scFvs that bind costimulatory receptors described in
Example 12 is used to construct a 2H7 scFv IgA hinge IgA-T4-CD80
fusion protein. The 2H7 scFv IgAH IgA-T4 fusion polynucleotide
sequence (SEQ ID NO: 298) is fused in frame to sequences encoding
the transmembrane domain and cytoplasmic tail of human CD80 (SEQ ID
NO: 460), such that when the fusion protein is expressed in the
transfected cell, CD80 provided an anchor for surface expression of
the scFv Ig fusion protein. cDNA encoding the 2H7 scFv IgAH
IgA-T4-CD80 fusion protein (SEQ ID NO: 299) is inserted into the
retroviral vector pLNCX (BD Biosciences Clontech) according to
standard molecular biology procedures and vendor instructions. The
2H7 scFv IgAH IgA-T4-CD80 cDNA is inserted between the
5'LTR-neomycin resistance gene-CMV promoter sequences and the 3'LTR
sequence. The retroviral construct is transfected into Reh, an
acute lymphocytic leukemia cell line (ATCC CRL-8286). Transfected
cells are screened to select clones that are expressing 2H7 scFv-Ig
fusion proteins on the cell surface.
Example 25
Characterization of an Anti-4-1BB scFv Ig-CD80 Fusion Protein
Expressed on the Cell Surface of Tumor Cells and Growth of the
Tumor Cells In Vivo
[0312] This example describes construction of an anti-murine 4-1BB
(CD137) scFv fusion protein that has an IgG wild type hinge and CH2
and CH3 domains that is fused to the CD80 transmembrane and
cytoplasmic domains. The Example also illustrates the effect of the
cell surface expression of the anti-4-1BB scFv IgG CD80 polypeptide
when the transfected tumor cells are transplanted into mice.
[0313] The heavy and light chain variable regions of a rat
anti-4-1BB (CD137) monoclonal antibody (1D8) were cloned, and a
single chain Fv construct was prepared essentially as described in
Example 1. The heavy chain and light chain variable regions of each
antibody were cloned according to standard methods for cloning
immunoglobulin genes and as described in Example 1. Aingle chain Fv
construct was prepared as described in Example 1 by inserting a
nucleotide sequence encoding a (gly.sub.4ser).sub.3 peptide linker
between the VL region nucleotide sequence of 1D8 (SEQ ID NOS: 336,
549) and the VH region nucleotide sequence of 1D8 (SEQ ID NOS: 333,
547). The polypeptide sequence for 1D8 VL is shown in SEQ ID NOS:
342, 550, and the polypeptide sequence for the VH domain is shown
in SEQ ID NOS: 341, 548. The scFv polynucleotide (SEQ ID NO: 337)
was then fused to human IgG1 wild-type hinge-CH2-CH3 domains
according to the methods described in Example 1. The scFv IgG1
fusion polynucleotide sequence was then fused in frame to sequences
encoding the transmembrane domain and cytoplasmic tail of human
CD80 (SEQ ID NO: 460) essentially as described in Example 12, such
that when the fusion protein was expressed in the transfected cell,
CD80 provided an anchor for surface expression of the scFv Ig
fusion protein. cDNA encoding the scFv-IgG-CD80 fusion protein (SEQ
ID NO: 340) was inserted into the retroviral vector pLNCX (BD
Biosciences Clontech) according to standard molecular biology
procedures and vendor instructions. The scFv-Ig-CD80 cDNA was
inserted between the 51TR-neomycin resistance gene-CMV promoter
sequences and the 3'LTR sequence.
[0314] The retroviral constructs were transfected into the
metastatic M2 clone of K1735, a melanoma cell line, provided by Dr.
I. Hellstrom, PNR1, Seattle, Wash. Transfected cells were screened
to select clones that were expressing scFv-Ig fusion proteins on
the cell surface. To demonstrate that the 1D8 scFv IgG-CD80
construct was expressed on the cell surface of the tumor cells, the
transfected cells were analyzed by flow immunocytofluorimetry.
Transfected cells (K1735-1D8) were incubated for one hour on ice in
phycoerythrin-conjugated F(ab').sub.2 goat anti-human IgG. The
unbound conjugate was then removed by washing the cells and flow
cytometry analysis was performed using a Coulter Epics XL cell
sorter. Results are presented in FIG. 41A.
[0315] The growth of K1735-1D8 transfected cells was examined in
vivo. K1735-WT cells grew progressively when transplanted
subcutaneously (s.c.) in naive C314 mice. Although the same dose of
K1735-1D8 cells initially formed tumors of an approximately 30
mm.sup.2 surface area, the tumors started to regress around day 7
and had disappeared by day 20 as shown in FIG. 41B. Tumor cells
that were transfected with a similarly constructed vector encoding
a non-binding scFv, a human anti-CD28 scFv construct, grew as well
as tumor cells that had not been transfected. The presence of a
foreign protein, that is, human IgG1 constant domains or rat
variable regions, did not make transfected K1735-WT cells
immunogenic; the growth of the K1735-1D8 cells in C3H mice was
identical to that of K1735-WT cells (untransfected).
[0316] To investigate the roles of CD4.sup.+ and CD8.sup.+ T
lymphocytes and NK cells in the regression of K1735-1D8 tumors,
naive mice were injected intraperitoneally (i.p.) with monoclonal
antibodies (mAbs, typically 50 .mu.g in a volume 0.1 ml) to remove
CD8.sup.+, CD4.sup.+ or both CD4.sup.+ and CD8.sup.+ T cells, or
were injected with anti-asialo-GM1 rabbit antibodies to remove NK
cells. Twelve days later, when flow cytometry analysis of spleen
cells from identically treated mice showed that the targeted T cell
populations were depleted, K1735-1D8 cells were transplanted s.c to
each T cell-depleted group. K1735-1D8 had similar growth kinetics
in mice that had been injected with the anti-CD8 MAb or control rat
IgG while removal of CD4.sup.+ T cells resulted in the growth of
K1735-1D8 with the same kinetics as K1735-WT. This failure to
inhibit tumor growth after CD4+ T cell removal was observed
regardless of the presence or absence of CD8+ T cells. K1735-1D8
grew in all NK-depleted mice, although more slowly than in the
CD4-depleted group. The results are presented in FIG. 41C.
Example 26
Therapeutic Effect of Tumor Cells Expressing Anti-4-1BB scFv
IgG-CD80 Fusion Protein
[0317] This Example examines the ability of K1735-1D8 transfected
tumor cells expressing an anti-CD 137 scFv on the cell surface to
generate a sufficient immune response in mice to mediate rejection
of established, untransfected wild type tumors. C3H mice were
transplanted with K1735-WT tumors (2.times.10.sup.6 cells/animal)
and grown for approximately six days. Experiments were performed
using mice with established K1735-WT tumors of 30 mm.sup.2 surface
area. Mice were vaccinated by s.c. injection of K1735-1D8 or
irradiated K1735-WT cells on the contralateral side. Identical
injections were repeated at the time points indicated in FIG. 42.
One group of animals was given four weekly injections of K1735-1D8
cells. According to the same schedule, another group was given
irradiated (12,000 rads) K1735-WT cells, and a third group was
injected with PBS. Thedata are plotted in FIG. 42. The WT tumors
grew progressively in all control mice and in all mice that
received irradiated K1735-WT cells. In contrast, the tumors
regressed in 4 of the 5 mice treated by immunization with
K1735-1D8. The animals remained tumor-free and without signs of
toxicity when the experiment was terminated 3 months later. In the
fifth mouse, the tumor nodule decreased in size as long as the
mouse received K1735-1D8 cells, but the tumor grew back after
therapy was terminated.
[0318] In another experiment with 5 mice/group, mice were injected
intravenously (i.v.) with 3.times.10.sup.5 K1735-WT cells to
initiate lung metastases. Three days later, K1735-1D8 cells were
transplanted s.c. This procedure was repeated once weekly for a
month; control mice were injected with PBS. The experiment was
terminated when one mouse in the control group died, 37 days after
receiving the K1735-WT cells. At that time, lungs of the control
mice each had >500 metastatic foci. In contrast, less than 10
metastatic foci were present in the lungs of the immunized
mice.
[0319] In a third experiment, mixtures of K1735-WT cells and
K1735-1D8 cells were injected into immunocompetent syngeneic C3H
mice. Mice were injected subcutaneously with K7135-WT cells alone
or with a mixture of 2.times.10.sup.6 K1735-WT cells and
2.times.10.sup.5 K1735-1D8 cells. Tumor growth was monitored at
5-day intervals.
Example 27
Expression of Anti-4-1BB scFv IgG-CD80 Fusion Protein on the Cell
Surface of Sarcoma Cells
[0320] This Example demonstrates expression of an anti-CD 137 scFv
on the cell surface of a second type of tumor cell by transfecting
a murine sarcoma cell line with an anti-CD137 scFv IgG-CD80
construct.
[0321] The 1D8 scFv IgG WTH WTCH2CH3-CD80 polynucleotide (SEQ ID
NO: 340) was transferred from the pLNCX vector into pcDNA3-hygro
vector using restriction enzyme digestion and ligation steps
according to standard molecular biology methods. The construct was
cut with HindIII+ClaI and the sFv fragment was filled in with
Klenow (Roche) and the blunt-ended fragment was ligated into EcoR5
site of pcDNA3. Ag104 murine sarcoma tumor cells were transfected
with the pcDNA3-hygro vector containing the 1D8 scFv IgG CD80
fusion protein. Hygromycin-resistant clones were screened by flow
cytometry using a FITC anti-human IgG antibody to detect expression
of the transgene. Only approximately 15% of the resistant clones
had detectable fusion protein initially. Positive cells identified
by flow cytometry were repeatedly panned on flasks coated with
immobilized anti-human IgG (10 .mu.g/ml) according to standard
methods. Panning was performed by incubating cells on the coated
plates for 30 min at 37 C.; the plates were then washed 2-3.times.
in versene or PBS. After each round, cells were tested for IgG
expression by FACS. The histogram in FIG. 44 shows the staining
pattern after four rounds of panning against anti-human IgG
(black). Untransfected cells were stained and are indicated in
gray. All of the cells in the population were positive.
Example 28
Construction and Characterization of a Bispecific scFv Ig Fusion
Protein and scFv Ig Fusion Proteins with a Mutation in the IgG1 Ch2
Domain
[0322] An anti-CD20 (2H7) scFv IgG fusion protein was constructed
that had a mutant hinge (MT (SSS)) and a mutant CH2 domain in which
the proline at residue (position number 238 according to Ward et
al., supra) was substituted with a serine. The 2H7 scFv IgG MTH
(SSS) MTCH2WTCH3 encoding polynucleotide (SEQ ID NO: 3, 351) was
constructed essentially according to methods described in Examples
1, 5, and 13. The IgG mutant hinge-mutant CH2-wild type CH3 domains
were also fused to an anti-CD20 (2H7)-anti-CD40 (40.2.220)
bispecific scFv. The anti-CD20-anti-CD40 scFv IgGMTH (SSS)
MTCH2WTCH3 encoding polynucleotide sequence is shown in SEQ ID NO:
349 and the encoded polypeptide is shown in SEQ ID NO: 350.
[0323] COS cells were transiently transfected with vectors
comprising the polynucleotide sequences encoding 2H7 seFv IgG MTH
(SSS) MTCH2WTCH3 (SEQ ID NO: 3, 351); anti-CD20-anti-CD40 seFv IgG
MTH (SSS) MTCH2WTCH3 (SEQ ID NO: 349); 2H7 seFv IgG MTH (SSS)
WTCH2CH3 (SEQ ID NO: 4, 225); and 2H7 seFv IgAH IgG WTCH2CH3 (SEQ
ID NO: 5, 283) as described in Example 10. Culture supernatants
were collected and the fusion proteins were purified by protein A
chromatography (see Example 10). The purified polypeptides were
fractionated by SDS-PAGE according to the method described in
Example 10. Rituximab (anti-CD20 monoclonal antibody), and Bio-Rad
prestained molecular weight standards (Bio-Rad, Hercules, Calif.),
and Multimark.RTM. molecular weight standards (Invitrogen Life
Technologies were also applied to the gel. The results are
presented in FIG. 45.
[0324] The 2H7 seFv Ig fusion protein that contains a mutation in
the CH2 domain was compared to fusion proteins that have the wild
type CH2 domain in an ADCC assay. The assays were performed
essentially as described in Examples 11 and 19. Fresh resting PBMC
(effector cells) were added to .sup.51Cr-labeled BJAB cells (target
cells) at the ratios indicated in FIG. 46. Purified 2H7 scFv IgG
MTH (SSS) MTCH2WTCH3, 2H7 seFv IgG MTH (SSS) WTCH2CH3, 2H7 scFv
IgAH IgG WTCH2CH3, and Rituximab, each at 10 .mu.g/ml were added to
the effector/target cell mixtures and incubated for five hours at
37.degree. C. Supernatants were harvested and the amount of
chromium released was determined as described in Examples 11 and
19. Percent specific killing by each fusion protein is presented in
FIG. 46.
Example 29
Tumor Cell Surface Expression of an Anti-Human Cd3 scFv IgG Fusion
Protein
[0325] An anti-human CD3 scFv Ig CD80 fusion protein was prepared
essentially as described in Examples 1 and 12. The fusion protein
comprised an anti-human CD3 scFv fused to wild type IgG1 hinge (SEQ
ID NO: 436, 438) and wild type CH2 (SEQ ID NO: 440) and CH3 (SEQ ID
NO: 442) domains, fused to CD80 transmembrane and cytoplasmic
domains (SEQ ID NO: 461) to enable cell surface expression of the
anti-CD3 scFv. The anti-human CD3 scFv IgG WTH WTCH2CH3-CD80
polynucleotide (SEQ ID NO: 559) encoding the polypeptide (SEQ ID
NO: 560) was transfected in Reh cells and into T51 cells
(lymphoblastoid cell line). Expression of the anti-human CD3 scFv
IgG fusion protein was detected by flow cytometry using FITC
conjugated goat anti-human IgG (see methods in Examples 4, 10, 16,
18). FIG. 47A illustrates expression of the anti-human CD3 fusion
protein on the cell surface of Reh cells, and FIG. 47B shows
expression of the fusion protein on T41 cells.
[0326] ADCC assays were performed with the transfected Reh and T51
cells to determine if expression of the scFv-Ig polypeptides on the
cell surface augmented effector cell function. Untransfected and
transfected Reh cells and untransfected and transfected T51 cells
were pre-labeled with .sup.51Cr (100 .mu.Ci) (Amersham) for two
hours at 37.degree. C. Human PBMC served as effector cells and were
added to the target cells (5.times.10.sup.4 cells per well of 96
well plate) at ratios of 20:1, 10:1, 5:1, and 2.5:1. After four
hours at 37.degree. C., culture supernatants were harvested and
analyzed as described in Examples 11 and 12. Percent specific
killing was calculated as described in Example 12. The results are
presented in FIG. 48.
Example 30
Induction of Cytokine Expression in Tumor Cells Expressing
Anti-CD28 scFv on the Cell Surface
[0327] This Example describes the effect of cell surface expressed
scFv on cytokine mRNA induction in stimulated lymphocytes
co-cultured with tumor cells transfected with an anti-human CD28
scFv IgG-CD80 fusion protein.
[0328] Real time PCR analysis was performed on RNA samples from
human PBMC stimulated with Reh, Reh-anti-CD28 (2e12) (see Example
12 for construction of 2e12 scFv IgG WTH WHTCH3CH2-CD80 and
transfection of Reh cells), and Reh-CD80 (see Example 14) in order
to measure the effects of the surface expressed scFv on cytokine
production by the PBMC effector cells. For the real-time PCR assay,
SYBR Green (QIAGEN) (Morrison et al., Biotechniques 24:954-8, 960,
962 (1998)) was used and measured by an ABI PRISM.RTM. 7000
Sequence Detection System (Applied Biosystems, Foster City, Calif.)
that measures the formation of PCR product after each amplification
cycle. Cells were harvested from cultures and total RNA prepared
using QIAGEN RNA kits, including a QIA shredder column purification
system to homogenize cell lysates, and RNeasy.RTM. mini-columns for
purification of RNA. cDNA was reverse transcribed using equal
amounts of RNA from each cell type and Superscript II Reverse
Transcriptase (LifeTechnologies). SYBR Green real-time PCR analysis
was then performed using the prepared cDNA as template and primer
pairs specific for cytokine gene products. The average length of
the PCR products that were amplified ranged from 150-250 base
pairs. The cDNA levels for many activation response molecules
including IFN.gamma., TNF.alpha., GM-CSF, IL-4, IL-5, IL-6, IL-8,
IL-10, IL-12, IL-15, ICOSL, CD80 and CD86 were assayed. Control
reference cDNAs for constitutively expressed genes, including
GAPDH, .beta.-actin, and CD3s were measured in each assay. The most
significant induction of specific mRNA was observed for
IFN-.gamma., and more modest induction was observed for CTLA-4 and
ICOS.
Example 31
Cloning of an Anti-Human 4-1BB Antibody and Construction of an
Anti-Human 4-1BB scFv Ig Fusion Protein
[0329] A hybridoma cell line expressing a mouse anti-human
monoclonal antibody (designated 5B9) was obtained from Dr. Robert
Mittler, Emory University Vaccine Center, Atlanta, Ga. The variable
heavy and light chain regions were cloned according to known
methods for cloning of immunoglobulin genes and as described
herein. Cells were grown in IMDM/15% FBS (Invitrogen Life
Technologies) media for several days. Cells in logarithmic growth
were harvested from cultures and total RNA prepared using QIAGEN
RNA kits, including a QIA shredder column purification system to
homogenize cell lysates, and RNeasy.RTM. mini-columns for
purification of RNA according to manufacturer's instructions. cDNA
was reverse transcribed using random hexamer primers and
Superscript II Reverse Transcriptase (Invitrogen Life
Technologies).
[0330] cDNA was anchor-tailed using terminal transferase and dGTP.
PCR was then performed using an anchor-tail complementary primer
and a primer that annealed specifically to the antisense strand of
the constant region of either mouse Ck (for amplification of VL) or
the appropriate isotype mouse CH1 (for amplification of VH). The
amplified variable region fragments were TOPO.RTM. cloned
(Invitrogen Life Technologies), and clones with inserts of the
correct size were then sequenced. Consensus sequence for each
variable domain was determined from sequence of at least four
independent clones. The 5B9 VL and VH polynucleotide sequences are
shown in SEQ ID NOs: 355 and 354, respectively, and the deduced
amino acid sequences are shown in SEQ ID NOs: 361 and 360. The scFv
was constructed by a sewing PCR method using overlapping primers
containing a synthetic (gly.sub.4ser).sub.3 linker domain inserted
between the light and heavy chain variable regions (see Example 1).
The 5B9 scFv polypeptide (SEQ ID NO: 356) is encoded by the
polynucleotide sequence comprising SEQ ID NO: 362.
[0331] 5B9 scFv polynucleotide sequence was fused in frame to the
polynucleotide sequence encoding the human IgG1 mutant hinge and
wild type CH2 and CH3 (MTH (SSS) WTCH2CH3, SEQ ID NO: 357)
according to methods described in Examples 5, 10, and 13. COS cells
were transiently transfected with a vector comprising the 5B9 scFv
IgG MTH (SSS) WTCH2CH3 polynucleotide sequence (SEQ ID NO: 357).
Supernatant was collected and binding of the 5B9 scFv IgG MTH (SSS)
WTCH2CH3 polypeptide (SEQ ID NO: 362) was measured by flow
immunocytofluorimetry essentially as described in Examples 4, 10,
16, and 18. Culture supernatant from the 5B9 hybridoma cell line
was also included in the binding assay. Fresh human PBMC were
incubated in the presence of immobilized anti-CD3 for four days
prior to the binding experiment to induce expression of CD l37 on
the surface of activated T cells. Stimulated PBMC were washed and
incubated with COS or hybridoma culture supernatant containing the
5B9 scFv IgG fusion protein or 5B9 murine monoclonal antibody,
respectively, for 1 hour on ice. Binding of 5B9 scFv IgG fusion
protein or 5B9 murine monoclonal antibody was detected with FITC
conjugated anti-human IgG or anti-mouse IgG, respectively. The
results are presented in FIG. 49.
Example 32
Construction of 2H7 scFv IgG Fusion Proteins with Hinge
Mutations
[0332] 2H7 scFv IgG fusion proteins are constructed with the first
cysteine residue and the second cysteine in the IgG1 hinge region
substituted with a serine residue to provide MTH (SCC) and MTH
(CSC). The template for introduction of the mutations is a
polynucleotide encoding 2H7 scFv WTH WTCH2CH3 (SEQ ID NO: 2, 207).
The oligonucleotide introducing the mutations are 5' PCR primer
oligonucleotides HIgGMHcysl (SEQ ID NO: 587) and HIgGMHcys2 (SEQ ID
NO: 391). The encoding polynucleotides of the mutants are presented
in SEQ ID NOs: 393, 394 and the polypeptide sequences are provided
in SEQ ID NO: 396, 397.
[0333] Additional representative sequences of the present invention
are as follows:
TABLE-US-00003 HuIgG1 wild type hinge, CH2, CH3 (SEQ ID NO: 427)
Tctgatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggg
gggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacat-
gcgtggtggtgg
acgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagaca-
aagccgcgg
gaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaa-
ggagtacaagt
gcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaa-
ccacaggtg
tacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttcta-
tcccagcgacat
cgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacg-
gctccttctt
cctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatg-
aggctctgcac aaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga
HuIgG1 wild type hinge, CH2, CH3 (SEQ ID NO: 428)
Sdqepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvd
gvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlpps-
rdeltknqv
sltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhyt-
qkslslspgk Llama IgG1 hinge, CH2, CH3 (SEQ ID NO: 429)
Tgatcaagaaccacatggaggatgcacgtgcccncagtgcccncaatgcccngcnccngaactnccagg
aggcccttctgtctttgtcttccccccgaaacccaaggacgtcctctccatttttggaggccgagtcacgtgcg-
ttgtagtggacgtcg
gaaagaaagaccccgaggtcaatttcaactggtatattgatggcgttgaggtgcgaacggccaatacgaagcca-
aaagaggaaca
gttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccaggactggctgacggggaaggaattca-
agtgcaaggtc
aacaacaaagctctcccggcccccatcgagaggaccatctccaaggccaaagggcagacccgggagccgcaggt-
gtacaccct
ggccccacaccgggaagaactggccaaggacaccgtgagcgtaacatgcctggtcaaaggcttctacccagctg-
acatcaacgtt
gagtggcagaggaacggtcagccggagtcagagggcacctacgccaacacgccgccacagctggacaacgacgg-
gacctact
tcctctacagcaagctctcggtgggaaagaacacgtggcagcggggagaaaccttaacctgtgtggtgatgcat-
gaggccctgca caaccactacacccagaaatccatcacccagtcttcgggtaaatagtaatctaga
Llama IgG1 hinge, CH2, CH3 (In FIG. 23 as Llama IgG1) (SEQ ID NO:
430)
Ephggctcpqcpapelpggpsvfvfppkpkdvlsisgrpevtcvvvdvgkedpevnfnwyidgvevr
tantkpkeeqfnstyrvvsvlpiqhqdwltgkefkckvnnkalpapiertiskakgqtrepqvytlaphreela-
kdtvsvtclvk
gfypadinvewqrngqpesegtyantppqldndgtyflysrlsvgkntwqrgetltgvvmhealhnhytqksit-
qssgk Llama IgG2: (SEQ ID NO: 431)
Tgatcaagaacccaagacaccaaaaccacaaccacaaccacaaccacaacccaatcctacaacagaatcc
aagtgtcccaaatgtccagcccctgagctcctgggagggccctcagtcttcatcttccccccgaaacccaagga-
cgtcctctccattt
ctgggaggcccgaggtcacgtgcgttgtggtagacgtgggccaggaagaccccgaggtcagtttcaactggtac-
attgatggcgc
tgaggtgcgaacggccaacacgaggccaaaagaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgc-
ccatccagc
accaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctctcccggcccccatcgagaag-
accatctcca
aggccaaagggcagacccgggagccgcaggtgtacaccctggccccacaccgggaagagctggccaaggacacc-
gtgagcg
taacatgcctggtcaaaggcttctacccacctgatatcaacgttgagtggcagaggaatgggcagccggagtca-
gagggcacyta
cgccaccacgccaccccagctggacaacgacgggacctacttcctctacagcaagctctcggtgggaaagaaca-
cgtggcagca
gggagaaaccttcacctgtgtggtgatgcacgaggccctgcacaaccactacacccagaaatccatcacccagt-
cttcgggtaaat agtaatctaga Llama IgG2 (SEQ ID NO: 432)
Dqepktpkpqpqpqpqpnptteskcpkcpapellggpsvfifppkpkdvlsisgrpevtcvvvdvgqe
dpevsfnwyidgaevrtantrpkeeqfnstyrvvsvlpiqhqdwltgkefkckvnnkalpapiektiskakgqt-
repqvytlap
hreelakdtvsvtclvkgfyppdinvewqrngqpesegtyattppqldndgtyflysklsvgkntwqqgetftc-
vvmhealhn hytqksitqssgk Llama IgG3 Fc (SEQ ID NO: 433)
Tgatcaagcgcaccacagcgaagaccccagctccaagtgtcccaaatgcccaggccctgaactccttgga
gggcccacggtcttcatcttccccccgaaagccaaggacgtcctctccatcacccgaaaacctgaggtcacgtg-
cttgtggtggac
gtgggtaaagaagaccctgagatcgagttcaagctggtccgtggatgacacagaggtacacacggctgagacaa-
agccaaagga
ggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccaggactggctgacggggaagg-
aattcaagtg
caaggtcaacaacaaagctctcccagcccccatcgagaggaccatctccaaggccaaagggcagacccgggagc-
cgcaggtgt
acaccctggccccacaccgggaagagctggccaaggacaccgtgagcgtaacctgcctggtcaaaggcttcttc-
ccagctgacat
caacgttgagtggcagaggaatgggcagccggagtcagagggcacctacgccaacacgccgccacagctggaca-
acgacggg
acctacttcctctacagcaaactctccgtgggaaagaacacgtggcagcagggagaagtcttcacctgtgtggt-
gatgcacgaggc
tctacacaatcactccacccagaaatccatcacccagtcttcgggtaaatagtaatctagagggccc
Llama IgG3 Fc (SEQ ID NO: 434)
dqahhsedpsskcpkcpgpellggptvfifppkakdvlsitrkpevtclwwtwvkktlrsssswsvddt
evhtaetkpkeeqfnstyrvvsvlpiqhqdwltgkefkckvnnkalpapiertiskakgqtrepqvytlaphre-
elakdtvsvtc
lvkgffpadinvewqrngqpesegtyantppqldndgtyflysklsvgkntwqqgevftcvvmhealhnhstqk-
sitqssgk HuIgG1 wild type hinge (SEQ ID NO: 435)
gatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagca HuIgG1 wild
type hinge (SEQ ID NO: 436) dqepkscdkthtcppcpa HuIgG1 H2, wild type
hinge with leu at second position (results from BglI site) (SEQ ID
NO: 437) gatctggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagca
HuIgG1 H2, wild type hinge with leu at second position. (SEQ ID NO:
438) dlepkscdkthtcppcpa NT HuIgG1 wild type CH2 (SEQ ID NO: 439)
cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccgg
acccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtgga-
cggcgtggag
gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgt-
cctgcaccag
gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccat-
ctccaaagcc aaa HuIgG1 wild type CH2 AA (SEQ ID NO: 440)
pellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwpidgvevhnaktkpreeqynst
yryvsyltylhqdwingkeykckvsnkalpapiektiskak NT HuIgG1 wild type CH3
(SEQ ID NO: 441)
gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggt
cagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccgg-
agaacaacta
caagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagca-
ggtggcagcag
gggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc-
cccgggtaaatga AA HuIgG1 wild type CH3 (SEQ ID NO: 442)
gqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflysk
ltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT HuIgG1 mutated hinge
(C-C-C.fwdarw.S-S-S) (SEQ ID NO: 443)
gatcaggagcccaaatcttctgacaaaactcacacatccccaccgtccccagca AA HuIgG1
mutated hinge (C-C-C.fwdarw.S-S-S) (SEQ ID NO: 444)
dqepkssdkthtsppspa Mutant hinge, but wild type CH2 and CH3-reads
from the hinge + Ig tail, HIgG1MTH WTCH2CH3 (SEQ ID NO: 445):
tgatcaccccaaatcttctgacaaaactcacacatctccaccgtcctcagcacctgaactcctgggtggaccg
tcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt-
ggtggacgtgag
ccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgc-
gggaggagc
agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtac-
aagtgcaaggt
ctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacagg-
tgtacaccc
tgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagc-
gacatcgccgt
ggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctcct-
tcttcctcta
cagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctc-
tgcacaacca ctacacgcagaagagcctctccctgtctccgggtaaatgataatctaga
Protein sequence: Mutant hinge, but wild type CH2 and CH3 (SEQ ID
NO: 446)
Dhpkssdkthtsppssapellggpsyflfppkpkdtlmisrtpeytcyyydvshedpevkfnwyvdgv
evhnaktkpreeqynstyrvvsyltvlhqdwingkeykckvsnkalpapiektiskakgqprepqvytlppsrd-
eltknqvslt
clykgfypsdiayewesngqpennykttppyldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqks-
lslspgk LLG1-5'bgl 35 mer Llama IgG1 5' (SEQ ID NO: 447) 5'-gtt gtt
gat caa gaa cca cat gga gga tgc acg tg-3' LLG2-5'bgl 32 mer, Llama
IgG2-5' (SEQ ID NO: 448) 5'-gtt gtt gat caa gaa ccc aag aca cca aaa
cc-3' LLG3-5'bgl 33 mer, Llama IgG3-5' (SEQ ID NO: 449) 5'-gtt gtt
gat caa gcg cac cac agc gaa gac ccc-3' LLseqsense 19mer, llama
sequencing primer (SEQ ID NO: 450) 5'-ctg aga tcg agt tca get
g-3'
LLseqAS 19 mer (SEQ ID NO: 451) 5'-cct cct ttg get ttg tct c-3' NT
2H7 scFv llama IgG1 (SEQ ID NO: 452)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcaagaaccacatggaggatgcacgtgcccnca-
gtgcccncaat
gcccngcnccngaactnccaggaggcccttctgtctttgtcttccccccgaaacccaaggacgtcctctccatt-
tttggaggccga
gtcacgtgcgttgtagtggacgtcggaaagaaagaccccgaggtcaatttcaactggtatattgatggcgttga-
ggtgcgaacggc
caatacgaagccaaaagaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccagg-
actggctgac
ggggaaggaattcaagtgcaaggtcaacaacaaagctctcccggcccccatcgagaggaccatctccaaggcca-
aagggcaga
cccgggagccgcaggtgtacaccctggccccacaccgggaagaactggccaaggacaccgtgagcgtaacatgc-
ctggtcaa
aggcttctacccagctgacatcaacgttgagtggcagaggaacggtcagccggagtcagagggcacctacgcca-
acacgccgc
cacagctggacaacgacgggacctacttcctctacagcaagctctcggtgggaaagaacacgtggcagcgggga-
gaaaccttaa
cctgtgtggtgatgcatgaggccctgcacaaccactacacccagaaatccatcacccagtcttcgggtaaatag-
taatctaga AA 2H7 scFv llama IgG1 (SEQ ID NO: 453)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssysymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqephggctcpqcpapelpggpsvfvfppkpkdvlsifggrvtc-
vvvdvgk
kdpevnfnwyidgvevrtantkpkeeqfnstyrvvsylpiqhqdwltgkefkckvnnkalpapiertiskakgq-
trepqvytl
aphreelakdtvsvtclvkgfypadinvewqrngqpesegtyantppqldndgtyflysklsvgkntwqrgetl-
tcvvmheal hnhytqksitqssgk NT 2H7 scFv llama IgG2 (SEQ ID NO: 454)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtgt
aagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctgg-
cttctggagtccct
gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgc-
cacttattactgcc
agcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggc-
ggtggtggatc
tggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtga-
agatgtcctgc
aaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatg-
gattggagctattt
atccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctcc-
agcacagcctac
atgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactc-
ttactggtacttcgat
gtctggggcacagggaccacggtcaccgtctcttctgatcaagaacccaagacaccaaaaccacaaccacaacc-
acaaccacaa
cccaatcctacaacagaatccaagtgtcccaaatgtccagcccctgagctcctgggagggccctcagtcttcat-
cttccccccgaaa
cccaaggacgtcctctccatttctgggaggcccgaggtcacgtgcgttgtggtagacgtgggccaggaagaccc-
cgaggtcagttt
caactggtacattgatggcgctgaggtgcgaacggccaacacgaggccaaaagaggaacagttcaacagcacgt-
accgcgtggt
cagcgtcctgcccatccagcaccaggactggctgacggggaaggaattcaagtgcaaggtcaacaacaaagctc-
tcccggcccc
catcgagaagaccatctccaaggccaaagggcagacccgggagccgcaggtgtacaccctggccccacaccggg-
aagagctg
gccaaggacaccgtgagcgtaacatgcctggtcaaaggcttctacccacctgatatcaacgttgagtggcagag-
gaatgggcagc
cggagtcagagggcacytacgccaccacgccaccccagctggacaacgacgggacctacttcctctacagcaag-
ctctcggtgg
gaaagaacacgtggcagcagggagaaaccttcacctgtgtggtgatgcacgaggccctgcacaaccactacacc-
cagaaatcca tcacccagtcttcgggtaaatagtaatctaga AA 2H7 scFv llama IgG2
(SEQ ID NO: 455)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqepktpkpqpqpqpqpnptteskcpkcpapellggpsvflfpp-
kpkdvlsi
sgrpevtcvvvdvgqedpevsfnwyidgaevrtantrpkeeqfnstyrvvsylpiqhqdwltgkefkckvnnka-
lpapiekti
skakgqtrepqvytlaphreelakdtvsvtclvkgfyppdinvewqrngqpesegtyattppqldndgtyflys-
klsvgkntw qqgetftcvvmhealhnhytqksitqssgk NT 2H7 scFv llama IgG3
(SEQ ID NO: 456)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtgt
aagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctgg-
cttctggagtccct
gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgc-
cacttattactgcc
agcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggc-
ggtggtggatc
tggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtga-
agatgtcctgc
aaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatg-
gattggagctattt
atccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctcc-
agcacagcctac
atgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactc-
ttactggtacttcgat
gtctggggcacagggaccacggtcaccgtctcttctgatcaagcgaaccacagcgaagaccccagctccaagtg-
tcccaaatgcc
caggccctgaactccttggagggcccacggtcttcatcttccccccgaaagccaaggacgtcctctccatcacc-
cgaaaacctgag
gtcacgtgcttgtggtggacgtgggtaaagaagaccctgagatcgagttcaagctggtccgtggatgacacaga-
ggtacacacgg
ctgagacaaagccaaaggaggaacagttcaacagcacgtaccgcgtggtcagcgtcctgcccatccagcaccag-
gactggctga
cggggaaggaattcaagtgcaaggtcaacaacaaagctctcccagcccccatcgagaggaccatctccaaggcc-
aaagggcag
acccgggagccgcaggtgtacaccctggccccacaccgggaagagctggccaaggacaccgtgagcgtaacctg-
cctggtcaa
aggcttcttcccagctgacatcaacgttgagtggcagaggaatgggcagccggagtcagagggcacctacgcca-
acacgccgcc
acagctggacaacgacgggacctacttcctctacagcaaactctccgtgggaaagaacacgtggcagcagggag-
aagtcttcacc
tgtgtggtgatgcacgaggctctacacaatcactccacccagaaatccatcacccagtcttcgggtaaatagta-
atctagagggccc AA 2H7 scFv llama IgG3 (SEQ ID NO: 457)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdywgtgttytyssdqahshsedpsskcpkcpgpellggptvflfppkakdvlsitrk-
pevtclwwt
wykktlrsssswsvddteyhtaetkpkeeqfnstyrvvsylpiqhqdwltgkefkckvnnkalpapiertiska-
kgqtrepqv
ytlaphreelakdtvsvtclvkgffpadinvewqrngqpesegtyantppqldndgtyflysklsvgkntwqqg-
evftcvvm healhnhstqksitqssgk 2H7 + Completely WT IgG tail: 2H7 scFv
WTH WTCH2CH3 (SEQ ID NO: 458) Nucleotide sequence:
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtgt
aagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctgg-
cttctggagtccct
gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgc-
cacttattactgcc
agcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggc-
ggtggtggatc
tggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtga-
agatgtcctgc
aaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatg-
gattggagctattt
atccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctcc-
agcacagcctac
atgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactc-
ttactggtacttcgat
gtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatg-
cccaccgtgccc
agcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccc-
ggacccctgagg
tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggag-
gtgcataatgc
caagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg-
actggctgaa
tggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagcca-
aagggcagcc
ccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcc-
tggtcaaagg
cttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctc-
ccgtgctgga
ctccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttct-
catgctccgtgat
gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga
2H7 + Completely WT IgG tail: 2H7 scFv WTH WTCH2CH3 (SEQ ID NO:
459) Protein sequence
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdywgtgttytyssdqepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtp-
evtcyvvd
vshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsnkalpapiektiska-
kgqpre
pqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppyldsdgsfflyskltvdksrwqq-
gnyfscsvm healhnhytqkslslspgk NT CD80 transmembrane domain and
cytoplasmic tail (+restriction sites) (SEQ ID NO: 460)
gcggatccttcgaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctg-
cc
tgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccct-
gtataaatcgat AA CD80 transmembrane domain and cytoplasmic tail (SEQ
ID NO: 461) adpsnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT
40.2.220 VL (anti-human CD40 scFv #1--VL) (SEQ ID NO: 462)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgttctgactcagtctccagccaccctgtctgtgactccaggagatagagtctctctttcctgcagggcc-
agccagagtattagc
gactacttacactggtatcaacaaaaatcacatgagtctccaaggcttctcatcaaatatgcttcccattccat-
ctctgggatcccctcca
ggttcagtggcagtggatcagggtcagatttcactctcagtatcaacagtgtggaacctgaagatgttggaatt-
tattactgtcaacatg
gtcacagctttccgtggacgttcggtggaggcaccaagctggaaatcaaacgg AA 40.2.220
VL (anti-human CD40 scFv #1--VL) (SEQ ID NO: 463)
mdfqvqifsfllisasvimsrgvdivltqspatlsvtpgdrvslscrasqsisdylhwyqqkshesprlliky
ashsisgipsrfsgsgsgsdftlsinsvepedvgiyycqhghsfpwtfgggtkleikr NT
40.2.220 VH (for anti-human CD40 scFv #1--VH) (SEQ ID NO: 464)
cagatccagttggtgcaatctggacctgagctgaagaagcctggagagacagtcaggatctcctgcaaggc
ttctgggtatgccttcacaactactggaatgcagtgggtgcaagagatgccaggaaagggtttgaagtggattg-
gctggataaacac
cccactctggagtgccaaaatatgtagaagacttcaaggacggtttgccttctctttggaaacctctgccaaca-
ctgcatatttacaga
taagcaacctcaaagatgaggacacggctacgtatttctgtgtgagatccgggaatggtaactatgacctggcc-
tactttgcttactg gggccaagggacactggtcactgtctctgatca AA 40.2.220 VH
(for anti-human CD40 scFv #1--VH) (SEQ ID NO: 465)
qiqlvqsgpelkkpgetvrisckasgyaftttgmqwvqempgkglkwigwintplwsakicrrlqgrfa
fsletsantaylqisnlkdedtatyfcvrsgngnydlayfaywgqgtivtvs NT 40.2.220
scFv (anti-human CD40 scFv #1) (SEQ ID NO: 466)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgttctgactcagtctccagccaccctgtctgtgactccaggagatagagtctctctttcctgcagggcc-
agccagagtattagc
gactacttacactggtatcaacaaaaatcacatgagtctccaaggcttctcatcaaatatgcttcccattccat-
ctctgggatcccctcc
aggttcagtggcagtggatcagggtcagatttcactctcagtatcaacagtgtggaacctgaagatgttggaat-
ttattactgtcaaca
tggtcacagctttccgtggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcg-
gaggtgggtc
gggtggcggcggatctcagatccagttggtgcaatctggacctgagctgaagaagcctggagagacagtcagga-
tctcctgcaa
ggcttctgggtatgccttcacaactactggaatgcagtgggtgcaagagatgccaggaaagggtttgaagtgga-
ttggctggataa
acaccccactctggagtgccaaaatatgtagaagacttcaaggacggtttgccttctctttggaaacctctgcc-
aacactgcatattta
cagataagcaacctcaaagatgaggacacggctacgtatttctgtgtgagatccgggaatggtaactatgacct-
ggcctactttgctt actggggccaagggacactggtcactgtctctgatca AA 40.2.220
scFv (anti-human CD40 scFv #1) (SEQ ID NO: 467)
mdfqvqifsfllisasvimsrgvdivltqspatlsvtpgdrvslscrasqsisdylhwyqqkshesprlliky
ashsisgipsrfsgsgsgsdftlsinsvepedvgiyycqhghsfpwtfgggtkleikrggggsggggsggggsq-
iqlvqsgpel
kkpgetvrisckasgyaftttgmqwvqempgkglkwigwintplwsakicrrlqgrfafsletsantaylqisn-
lkdedtatyfc vrsgngnydlayfaywgqgtlytys NT 2e12 VL (with L6 VK leader
peptide) (SEQ ID NO: 468)
atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacat-
tg
tgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaa-
agtgttgaatattat
gtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgcatccaa-
cgtagaatctgg
ggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatg-
atattgcaatgta
tttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacgg
AA 2e12 VL (with L6 VK leader peptide) (SEQ ID NO: 469)
mdfqvqifsfllisasvimsrgvdivhqspaslayslgqratiscrasesveyyvtslmqwyqqkpgqp
pkllisaasnvesgvparfsgsgsgtdfslnihpveeddiamyfcqqsrkvpwtfgggtkleikr
NT 2e12 VH (no leader peptide) (SEQ ID NO: 470)
caggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccg
tctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctg-
ggaatgatatggg
gtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaaggacaactccaagagccaa-
gttttcttaaaaa
tgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttatagtaactttcattactat-
gttatggactactgg ggtcaaggaacctcagtcaccgtctcctca(gatctg) AA 2e12 VH
(SEQ ID NO: 471)
qvqlkesgpglvapsqslsitctvsgfsltgygvnwvrqppgkglewlgmiwgdgstdynsalksrlsit
kdnsksqvflkmnslqtddtaryycardgysnfhyyvmdywgqgtsvtvss NT
2e12scFv(+Restriction sites) (SEQ ID NO: 472)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagcc-
agtgaaagtgttgaa
tattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgc-
atccaacgtagaat
ctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggag-
gatgatattgcaa
tgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggt-
ggcggtggctcg
ggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctc-
acagagcct
gtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaa-
agggtctggagtg
gctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaagg-
acaactccaaga
gccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttat-
agtaactttcattact
atgttatggactactggggtcaaggaacctcagtcaccgtctcctct(gatcag) AA 2e12scFv
(SEQ ID NO: 473)
mdfqvqifsfllisasvimsrgvdivltqspaslayslgqratiscrasesveyyvtslmqwyqqkpgqp
pkllisaasnvesgvparfsgsgsgtdfslnihpveeddiamyfcqqsrkvpwtfgggtkleikrggggsgggg-
sggggsqvq
lkesgpglvapsqslsitctvsgfsltgygvnwvrqppgkglewlgmiwgdgstdynsalksrlsitkdnsksq-
vflkmnslqt ddtaryycardgysnfhyyvmdywgqgtsvtvss 10A8 is anti-CD152
(CTLA-4) 10A8 VL (with L6 VK leader peptide) (SEQ ID NO: 474)
atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacat-
cc
agatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggcaagccaa-
gacattaagaagt
atataggttggtaccaacacaagcctggaaaaggtcccaggctgctcatatattacacatctacattacagcca-
ggcatcccatcaa
ggttcagtggaagtgggtctgggagagattattccctcagcatcagaaacctggagcctgaagatattgcaact-
tattattgtcaaca
gtatgataatcttccattgacgttcggctcggggacaaagttggaaataaaacgg AA 10A8 VL
(SEQ ID NO: 475)
mdfqvqifsfllisasvimsrgvdiqmtqspsslsaslggkvtitckasqdikkyigwyqhkpgkgprlli
yytstlqpgipsrfsgsgsgrdyslsirnlepediatyycqqydnlpltfgsgtkleikr NT
10A8 VH (no leader peptide) (SEQ ID NO: 476)
gatgtacagcttcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtctctcacctgctctgtcac-
t
ggctactccatcaccagtggtttctactggaactggatccgacagtttccgggaaacaaactggaatggatggg-
ccacataagcca
cgacggtaggaataactacaacccatctctcataaatcgaatctccatcactcgtgacacatctaagaaccagt-
ttttcctgaagttga
gttctgtgactactgaggacacagctacatatttctgtgcaagacactacggtagtagcggagctatggactac-
tggggtcaaggaa cctcagtcaccgtctcctctgatca AA 10A8 VH (SEQ ID NO: 477)
dvqlqesgpglvkpsqslsltcsvtgysitsgfywnwirqfpgnklewmghishdgrnnynpslinrisi
trdtsknqfflklssvttedtatyfcarhygssgamdywgqgtsvtvss NT 10A8 scFv (SEQ
ID NO: 478)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggca-
agccaagacatta
agaagtatataggttggtaccaacacaagcctggaaaaggtcccaggctgctcatatattacacatctacatta-
cagccaggcatccc
atcaaggttcagtggaagtgggtctgggagagattattccctcagcatcagaaacctggagcctgaagatattg-
caacttattattgtc
aacagtatgataatcttccattgacgttcggctcggggacaaagttggaaataaaacggggtggcggtggctcg-
ggcggtggtggg
tcgggtggcggcggatctgatgtacagcttcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtc-
tctcacctgctctgt
cactggctactccatcaccagtggtttctactggaactggatccgacagtttccgggaaacaaactggaatgga-
tgggccacataag
ccacgacggtaggaataactacaacccatctctcataaatcgaatctccatcactcgtgacacatctaagaacc-
agtttttcctgaagtt
gagttctgtgactactgaggacacagctacatatttctgtgcaagacactacggtagtagcggagctatggact-
actggggtcaagg aacctcagtcaccgtctcctctgatca AA 10A8 scFv (SEQ ID NO:
479)
mdfqvqifsfllisasvimsrgvdiqmtqspsslsaslggkvtitckasqdikkyigwyqhkpgkgprlli
yytstlqpgipsrfsgsgsgrdyslsimlepediatyycqqydnlpltfgsgtkleikrggggsggggsggggs-
dvqlqesgpgl
vkpsqslsltcsvtgysitsgfywnwirqfpgnklewmghishdgmnynpslinrisitrdtsknqfflklssv-
ttedtatyfcar hygssgamdywgqgtsvtvssd NT 40.2.220-hmtIgG1-hCD80 (SEQ
ID NO: 480)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgttctgactcagtctccagccaccctgtctgtgactccaggagatagagtctctctttcctgcagggcc-
agccagagtattagc
gactacttacactggtatcaacaaaaatcacatgagtctccaaggcttctcatcaaatatgcttcccattccat-
ctctgggatcccctcca
ggttcagtggcagtggatcagggtcagatttcactctcagtatcaacagtgtggaacctgaagatgttggaatt-
tattactgtcaacatg
gtcacagctttccgtggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggctcgggcgga-
ggtgggtcgg
gtggcggcggatctcagatccagttggtgcaatctggacctgagctgaagaagcctggagagacagtcaggatc-
tcctgcaaggc
ttctgggtatgccttcacaactactggaatgcagtgggtgcaagagatgccaggaaagggtttgaagtggattg-
gctggataaacac
cccactctggagtgccaaaatatgtagaagacttcaaggacggtttgccttctctttggaaacctctgccaaca-
ctgcatatttacagat
aagcaacctcaaagatgaggacacggctacgtatttctgtgtgagatccgggaatggtaactatgacctggcct-
actttgcttactgg
ggccaagggacactggtcactgtctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtc-
cccagcacctgaa
ctcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctga-
ggtcacatgcgtg
gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgc-
caagacaaag
ccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaa-
tggcaaggag
tacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagcc-
ccgagaacca
caggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaagg-
cttctatcccag
cgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggact-
ccgacggct
ccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg-
atgcatgaggctc
tgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcc-
tgggccattacctt
aatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaagga-
ggaatgagagattg agaagggaaagtgtacgccctgtataaatcgat AA
40.2.220-hmtIgG1-hCD80 (SEQ ID NO: 481)
mdfqvqifsfllisasvimsrgvdivltqspatlsvtpgdrvslscrasqsisdylhwyqqkshesprlliky
ashsisgipsrfsgsgsgsdftlsinsvepedvgiyycqhghsfpwtfgggtkleikrggggsggggsggggsq-
iqlvqsgpel
kkpgetvrisckasgyaftttgmqwvqempgkglkwigwintplwsakicrrlqgrfafsletsantaylqisn-
lkdedtatyfc
vrsgngnydlayfaywgqgtivtvsdlepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcv-
vvdvshedpe
vkfnwpidgvevhnaktkpreeqynstyrvvsyltvlhqdwingkeykekvsnkalpapiektiskakgqprep-
qvytlpp
srdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsv-
mhealhnhy
tqkslslspgkadpsnllpswaitlisvngifviceltycfaprcreamerlrresvrpv NT
2e12scFv-hmtIgG1-CD80 fusion protein (SEQ ID NO: 482)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagcc-
agtgaaagtgttgaa
tattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgc-
atccaacgtagaat
ctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggag-
gatgatattgcaa
tgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggt-
ggcggtggctcg
ggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctc-
acagagcct
gtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaa-
agggtctggagtg
gctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaagg-
acaactccaaga
gccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggttat-
agtaactttcattact
atgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttctgacaaa-
actcacacatcccc
accgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctca-
tgatctcccggac
ccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg-
gcgtggaggt
gcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcc-
tgcaccagga
ctggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatct-
ccaaagccaaa
gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcct-
gacctgcct
ggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaaga-
ccacgcctcc
cgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggga-
acgtcttctcat
gctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggat-
ccttcgaacctg
ctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgc-
cccaagatgcagagag
agaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat AA
2e12scFv-hmtIgG1-CD80 fusion protein (SEQ ID NO: 483)
mdfqvqifsfllisasvimsrgvdivliqspaslayslgqratiscrasesveyyvtslmqwyqqkpgqp
pkllisaasnvesgvparfsgsgsgtdfslnihpveeddiamyfcqqsrkvpwtfgggtkleikrggggsgggg-
sggggsqvq
lkesgpglvapsqslsitctvsgfsltgygvnwvrqppgkglewlgmiwgdgstdynsalksrlsitkdnsksq-
vflkmnslqt
ddtaryycardgysnfhyyvmdywgqgtsvtvssdlepkssdkthtsppspapellggssvflfppkpkdtlmi-
srtpevtcv
vvdvshedpevkthwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwingkeykekvsnkalpapiekti-
skakgq
prepqvytlppsrdeltknqvsltelvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksr-
wqqgnvfscs
vmhealhnhytqkslslspgkadpsnllpswaitlisvngifviceltycfaprcrerrmerlrresvrpv
NT 10A8 scFv-hmtIgG1-CD80 (SEQ ID NO: 484)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggca-
agccaagacatta
agaagtatataggttggtaccaacacaagcctggaaaaggtcccaggctgctcatatattacacatctacatta-
cagccaggcatccc
atcaaggttcagtggaagtgggtctgggagagattattccctcagcatcagaaacctggagcctgaagatattg-
caacttattattgtc
aacagtatgataatcttccattgacgttcggctcggggacaaagttggaaataaaacggggtggcggtggctcg-
ggcggtggtggg
tcgggtggcggcggatctgatgtacagcttcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtc-
tctcacctgctctgt
cactggctactccatcaccagtggtttctactggaactggatccgacagtttccgggaaacaaactggaatgga-
tgggccacataag
ccacgacggtaggaataactacaacccatctctcataaatcgaatctccatcactcgtgacacatctaagaacc-
agtttttcctgaagtt
gagttctgtgactactgaggacacagctacatatttctgtgcaagacactacggtagtagcggagctatggact-
actggggtcaagg
aacctcagtcaccgtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccag-
cacctgaactcctg
gggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcac-
atgcgtggtggtg
gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagac-
aaagccgcg
ggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggca-
aggagtacaa
gtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag-
aaccacagg
tgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttc-
tatcccagcgac
atcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccga-
cggctccttc
ttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca-
tgaggctctgca
caaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctggg-
ccattaccttaatc
tcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaa-
tgagagattgagaa gggaaagtgtacgccctgtataaatcgat AA 10A8
scFv-hmtIgG1-CD80 (SEQ ID NO: 485)
mdfqvqifsfllisasvimsrgvdiqmtqspsslsaslggkvtitckasqdikkyigwyqhkpgkgprlli
yytstlqpgipsrfsgsgsgrdyslsimlepediatyycqqydnlpltfgsgtkleikrggggsggggsggggs-
dvqlqesgpgl
vkpsqslsltcsvtgysitsgfywnwirqfpgnklewmghishdgmnynpslinrisitrdtsknqfflklssv-
ttedtatyfcar
hygssgamdywgqgtsvtvssdlepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdv-
shedpevk
fnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqv-
ytlppsr
deltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmh-
ealhnhyt
qkslslspgkadpsnllpswaitlisvngifviccltycfaprcrearnerlrresvrpv NT
500A2-hmtIgG1-CD80 (SEQ ID NO: 486)
atgttgtatacatctcagctccttgggcttttactcttctggatttcagcctccagaagtgacatagtgctgac-
tca
gactccagccactctgtctctaattcctggagaaagagtcacaatgacctgtaagaccagtcagaatattggca-
caatcttacactgg
tatcaccaaaaaccaaaggaggctccaagggctctcatcaagtatgcttcgcagtccattcctgggatcccctc-
cagattcagtggc
agtggttcggaaacagatttcactctcagcatcaataacctggagcctgatgatatcggaatttattactgtca-
acaaagtagaagctg
gcctgtcacgttcggtcctggcaccaagctggagataaaacggggtggcggtggctcgggcggaggtgggtcgg-
gtggcggcg
gatctcaggtcaagctgcagcagtccggttctgaactagggaaacctggggcctcagtgaaactgtcctgcaag-
acttcaggctac
atattcacagatcactatatttcttgggtgaaacagaagcctggagaaagcctgcagtggataggaaatgttta-
tggtggaaatggtg
gtacaagctacaatcaaaaattccagggcaaggccacactgactgtagataaaatctctagcacagcctacatg-
gaactcagcagc
ctgacatctgaggattctgccatctattactgtgcaagaaggccggtagcgacgggccatgctatggactactg-
gggtcaggggat
ccaagttaccgtctcctctgatctggagcccaaatcttctgacaaaactcacacatccccaccgtccccagcac-
ctgaactcctggg
gggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacat-
gcgtggtggtgg
acgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagaca-
aagccgcgg
gaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaa-
ggagtacaag
tgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgaga-
accacaggt
gtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttct-
atcccagcgac
atcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccga-
cggctcctt
cttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgc-
atgaggctctgc
acaaccactacacgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgg-
gccattaccttaa
tctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggagg-
aatgagagattgag aagggaaagtgtacgccctgtataaatcgat AA
500A2-hmtIgG1-CD80 (SEQ ID NO: 487)
mlytsqllglllfwisasrsdivltqtpatlslipgervtmtcktsqnigtilhwyhqkpkeapralikyasqs-
i
pgipsrfsgsgsetdftlsinnlepddigiyycqqsrswpvtfgpgtkleikrggggsggggsggggsqvklqq-
sgselgkpga
svklscktsgyiftdhyiswvkqkpgeslqwignvyggnggtsynqkfqgkatltvdkisstaymelssltsed-
saiyycarrp
vatghamdywgqgiqvtvssdlepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvs-
hedpevkf
nwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvy-
tlppsrd
eltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhe-
alhnhytq
kslslspgkadpsnllpswaitlisvngifviccltycfaprcrerrmerlrresvrpv NT 2H7
scFv MTH(SSS)WTCH2CH3 (SEQ ID NO: 488)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtgt
aagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctgg-
cttctggagtccct
gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgc-
cacttattactgcc
agcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggc-
ggtggtggatc
tggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtga-
agatgtcctgc
aaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatg-
gattggagctattt
atccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctcc-
agcacagcctac
atgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactc-
ttactggtacttcgat
gtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatc-
cccaccgtcccc
agcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccc-
ggacccctgagg
tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggag-
gtgcataatgc
caagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg-
actggctgaa
tggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagcca-
aagggcagcc
ccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcc-
tggtcaaagg
cttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctc-
ccgtgctgga
ctccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttct-
catgctccgtgat
gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga
2H7 scFv MTH(SSS)WTCH2CH3 protein sequence (SEQ ID NO: 489):
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdywgtgttytyssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtp-
evtcvvvdv
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsnkalpapiektiskak-
gqprep
qvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppyldsdgsfflyskltvdksrwqqg-
nyfscsvmh ealhnhytqkslslspgk HuIgGMHncs1 (oligo for CSS) (SEQ ID
NO: 490) gtt gtt gat cag gag ccc aaa tct tgt gac aaa act cac
HuIgGMHncs2 (oligo for SCS = ncs2) (SEQ ID NO: 491) gtt gtt gat cag
gag ccc aaa tct tct gac aaa act cac aca tgc cca ccg HuIgGMHncs3
(oligo for SSC = ncs3) (SEQ ID NO: 492) gtt gtt gat cag gag ccc aaa
tct tct gac aaa act cac aca tct cca ccg tgc cca gca cct g
hIgGWT3xba (3' oligo for above mutation introduction) (SEQ ID NO:
493) gtt gtt tct aga tca ttt acc cgg aga cag gga gag gct ctt ctg
cgt gta g Vhser11: (oligo for Leu to Ser at VH11) (SEQ ID NO:
494)
gga ggt ggg agc tct cag gct tat cta cag cag tct ggg gct gag tcg gtg
agg cc huIgG1-3' (3' oligo to amplify IgG1 C regions, 3' end of
CH3) (SEQ ID NO: 495) gtc tct aga cta tca ttt acc cgg aga cag
huIgA/Gchim5 (oligo for pcr#1) (SEQ ID NO: 496) cca tct ccc tca act
cca cct acc cca tct ccc tca tgc gca cct gaa ctc ctg huIgAhg-5'
(oligo for pcr#2) (SEQ ID NO: 497) gtt gtt gat cag cca gtt ccc tca
act cca cct acc cca tct ccc caa ct huIgA3' (SEQ ID NO: 498) gtt gtt
tct aga tta tca gta gca ggt gcc gtc cac ctc cgc cat gac aac 2H7
scFv IgAH IGG WT CH2CH3, 2H7 scFv with IgA hinge and WT CH2 and CH3
(SEQ ID NO: 499)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtgt
aagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctgg-
cttctggagtccct
gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgc-
cacttattactgcc
agcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggc-
ggtggtggatc
tggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtga-
agatgtcctgc
aaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatg-
gattggagctattt
atccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctcc-
agcacagcctac
atgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactc-
ttactggtacttcgat
gtctggggcacagggaccacggtcaccgtctctgatcagccagttccctcaactccacctaccccatctccctc-
aactccacctacc
ccatctccctcatgcgcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacac-
cctcatgatctccc
ggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg-
gacggcgtgg
aggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcacc-
gtcctgcacc
aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaca-
atctccaaag
ccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtc-
agcctgacc
tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta-
caagaccacg
cctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagca-
ggggaacgtctt
ctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaat-
gatctaga 2H7 scFv IgAH IGG WT CH2CH3 protein sequence (SEQ ID NO:
500)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdywgtgttytysdqpvpstpptpspstpptpspscapellggpsvflfppkpkdtlm-
isrtpevtcv
vvdvshedpevkthwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiekti-
skakgq
prepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppyldsdgsfflyskltvdksr-
wqqgnyfscs vmhealhnhytqkslslspgk NT 2H7 scFv IgAH IgACH2CH3 (2H7
scFv IgAhinge and IgA CH2 and CH3) (SEQ ID NO: 501)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcagccagttccctcaactccacctaccccatc-
tccctcaactcca
cctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttagg-
ttcagaagcgat
cctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaaga-
gcgctgttcaa
ggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaa-
ccatgggaaga
ccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacaca-
ttccggcccgag
gtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtgg-
cttcagcccc
aaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccg-
gcaggagccc
agccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacac-
cttctcctg
catggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccaccc-
atgtcaatgt gtctgttgtcatggcggaggtggacggcacctgctactgataatctaga AA 2H7
scFv IgAH IgACH2CH3 (2H7 scFv IgA hinge and IgA CH2 and CH3) (SEQ
ID NO: 502)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdywgtgttytyssdqpvpstpptpspstpptpspscchprlslhrpaledlllgsea-
iltctltglrdas
gytftwtpssgksavqgppdrdlcgcysyssylpgcaepwnhgktftctaaypesktpltatlsksgntfrpev-
hllpppseelal
nelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvg-
healplaftqkti drlagkpthvnvsvvmaevdgtcy IgA hinge-CH2-CH3 (Human IgA
tail, full length) (SEQ ID NO: 503)
tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgcc-
a
cccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacac-
tgaccggcct
gagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgacc-
gtgacctctgt
ggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcac-
tgctgcctaccc
cgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgc-
cgccgccgtc
ggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctgg-
ttcgctggct
gcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcacca-
ccaccttc
gctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggcca-
cgaggccct
gccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtca-
tggcggaggtgg acggcacctgctactgataatctaga IgA hinge-CH2-CH3 Protein
sequence, (Human IgA tail, full length) (SEQ ID NO: 504)
Dqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqg
ppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvt-
ltclargfspkdvl
vrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagk-
pthvnvsvvm aevdgtcy Human J Chain: (SEQ ID NO: 505)
agatctcaagaagatgaaaggattgttcttgttgacaacaaatgtaagtgtgcccggattacttccaggatcat
ccgttcttccgaagatcctaatgaggacattgtggagagaaacatccgaattattgttcctctgaacaacaggg-
agaatatctctgatc
ccacctcaccattgagaaccagatttgtgtaccatttgtctgacctcagctgtaaaaaatgtgatcctacagaa-
gtggagctggataat
cagatagttactgctacccagagcaatatctgtgatgaagacagtgctacagagacctgctacacttatgacag-
aaacaagtgctac
acagctgtggtcccactcgtatatggtggtgagaccaaaatggtggaaacagccttaaccccagatgcctgcta-
tcctgactaatcta ga Human J Chain polypeptide (SEQ ID NO: 506)
rsqederivlvdnkckcaritsriirssedpnediverniriivplnnrenisdptsplrtrfvyhlsdlsckk-
c dpteveldnqivtatqsnicdedsatetcytydrnkcytavvplvyggetkmvetaltpdacyp
HUJCH5nl (J chain 5' primer) (SEQ ID NO: 507) gtt gtt aga tct caa
gaa gat gaa agg att gtt ctt HUJCH3 (J chain 3' primer-antisense)
(SEQ ID NO: 508) gtt gtt tct aga tta gtc agg ata gca ggc atc tgg 4
carboxy terminal amino acids deleted from IgA CH3 (SEQ ID NO: 509)
GTCY IgAH IgAT4 Human IgA tail, truncated (3T1)-(missing last 4
amino acids from carboxy terminus) (SEQ ID NO: 510)
tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgcc
acccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcaca-
ctgaccggcct
gagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgacc-
gtgacctctgt
ggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcac-
tgctgcctaccc
cgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgc-
cgccgccgtc
ggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctgg-
ttcgctggct
gcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcacca-
ccaccttc
gctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggcca-
cgaggccct
gccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtca-
tggcggaggtgg actgataatctaga IgAH IgAT4 Protein sequence: (SEQ ID
NO: 511)
Dqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqg
ppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvt-
ltclargfspkdvl
vrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagk-
pthvnvsvvm aevd HUIGA3T1 (Oligo 3': to delete 4 amino acids at
carboxy end of IgA CH3) (SEQ ID NO: 512) gtt gtt tct aga tta tca
gtc cac ctc cgc cat gac aac aga cac HUIGA3T2: (oligo to delete 14
aa at end of IgA-T4) (SEQ ID NO: 513) gtt gtt tct aga tta tca ttt
acc cgc caa gcg gtc gat ggt ctt NT 2H7 scFv IgAH IgAT4 (SEQ ID NO:
514) (2H7 scFv IgA 3T1 construct)--truncates the CH3 domain at the
3' end
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcagccagttccctcaactccacctaccccatc-
tccctcaactcca
cctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttagg-
ttcagaagcgat
cctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaaga-
gcgctgttcaa
ggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaa-
ccatgggaaga
ccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacaca-
ttccggcccgag
gtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtgg-
cttcagcccc
aaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccg-
gcaggagccc
agccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacac-
cttctcctg
catggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccaccc-
atgtcaatgt gtctgttgtcatggcggaggtggactgataatctaga AA 2H7 scFv
IgAH-T4 (SEQ ID NO: 515)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqpvpstpptpspstpptpspscchprlslhrpaledlllgsea-
iltctltglrdas
gvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpev-
hllpppseelal
nelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvg-
healplaftqkti drlagkpthvnvsvvmaevd 14 amino acids deleted from
IgAH-T4 (so that total of 18 amino acids deleted from wild type IgA
CH3 (SEQ ID NO: 516) PTHVNVSVVMAEVD IgAH IgA-T18 (Human IgA Tail
truncated, 3T2) (SEQ ID NO: 517)
Tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgcc
acccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcaca-
ctgaccggcct
gagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgacc-
gtgacctctgt
ggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcac-
tgctgcctaccc
cgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgc-
cgccgccgtc
ggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctgg-
ttcgctggct
gcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcacca-
ccaccttc
gctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggcca-
cgaggccct gccgctggccttcacacagaagaccatcgaccgcttggcgggtaaa IgAH
IgA-T18 Protein sequence: (SEQ ID NO: 518)
dqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqg
ppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvt-
ltclargfspkdvl
vrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagk
NT 2H7 scFv IgAH IgAT18: (Human IgA Tail truncated, 3T2.) (SEQ ID
NO: 519)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcagccagttccctcaactccacctaccccatc-
tccctcaactcca
cctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttagg-
ttcagaagcgat
cctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaaga-
gcgctgttcaa
ggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaa-
ccatgggaaga
ccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacaca-
ttccggcccgag
gtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtgg-
cttcagcccc
aaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccg-
gcaggagccc
agccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacac-
cttctcctg
catggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaa
AA: 2H7 scFv IgAH IgAT18: (SEQ ID NO: 520)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqpvpstpptpspstpptpspscchprlslhrpaledlllgsea-
iltctltglrdas
gvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpev-
hllpppseelal
nelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvg-
healplaftqkti drlagk CTLA-4 IgG WTH WTCH2CH3
(Human-oncoMLP-CTLA4EC-hIgGWT) (SEQ ID NO: 521) Nucleotide
sequence:
gcaacctacatgatggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatc
aagtgaacctcactatccaaggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtac-
ccaccgccata
ctacctgggcataggcaacggaacccagatttatgtaattgatccagaaccgtgcccagattctgatcaaccca-
aatcttgtgacaaa
actcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacc-
caaggacaccct
catgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttca-
actggtacgtg
gacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcag-
cgtcctcac
cgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagccccca-
tcgagaaaac
aatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgacca-
agaaccagg
tcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccg-
gagaacaact
acaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagc-
aggtggcagca
ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgt-
ctccgggtaaatga CTLA-4 IgG WTH WTCH2CH3 Protein sequence: (SEQ ID
NO: 522)
mgvlltqrtllslvlallfpsmasmamhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvt
evcaatymmgneltflddsictgtssgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpep-
cpdsdqpks
cdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpre-
eqynstyr
vvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfyps-
diavewesn
gqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk
Human OncoM leader Peptide + CTLA4 EC (BclI) (SEQ ID NO: 523)
Atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagc
atggcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagta-
tgcatctccagg
caaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacct-
acatgatggg
gaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatcc-
aaggactgagggc
catggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacg-
gaacccagattt atgtaattgatccagaaccgtgcccagattctgatcaa Human OncoM
leader Peptide + CTLA4 EC Peptide sequence: (SEQ ID NO: 524)
mgvlltqrtllslvlallfpsmasmamhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvt
evcaatymmgneltflddsictgtssgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpep-
cpdsdq Human OncoM leader peptide nucleotide (SEQ ID NO: 525)
atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagc
atg Human OncoM leader peptide sequence (SEQ ID NO: 526):
Mgvlltqrtllslvlallfpsm NT Human CTLA4 EC (no LP) (SEQ ID NO: 527)
Gcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagta
tgcatctccaggcaaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtct-
gtgcggcaac
ctacatgacggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtga-
acctcactatcca
aggactgagggccatggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgg-
gcataggcaa cggaacccagatttatgtaattgatccagaaccgtgcccagattct AA Human
CTLA4 EC (no LP) (SEQ ID NO: 528)
Amhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvtevcaatymtgneltflddsictgts
sgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpepcpds NT Human
CTLA4 IgG MTH (SSS) MTCH2CH3 (SEQ ID NO: 529)
Atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagc
atggcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagta-
tgcatctccagg
caaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacct-
acatgatggg
gaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatcc-
aaggactgagggc
catggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacg-
gaacccagattt
atgtaattgatccagaaccgtgcccagattctgatcaacccaaatcttctgacaaaactcacacatccccaccg-
tccccagcacctga
actcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctg-
aggtcacatgcgt
ggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatg-
ccaagacaaa
gccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctga-
atggcaagg
agtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcag-
ccccgagaa
ccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaa-
aggcttctatc
ccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctg-
gactccga
cggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgct-
ccgtgatgcatg
aggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga AA Human
CTLA4 IgG MTH (SSS) MTCH2CH3 (SEQ ID NO: 530)
Mgvlltqrtllslvlallfpsmasmamhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvt
evcaatymmgneltflddsictgtssgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpep-
cpdsdqpks
sdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpre-
eqynstyr
vvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfyps-
diavewesn
gqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk
CTLA-4 IgAH IgACH2CH3 (Human-oncoMLP-CTLA4EC-IgA) (SEQ ID NO: 531)
Nucleotide sequence:
atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagc
atggcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagta-
tgcatctccagg
caaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacct-
acatgatggg
gaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatcc-
aaggactgagggc
catggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacg-
gaacccagattt
atgtaattgatccagaaccgtgcccagattctgatcagccagttccctcaactccacctaccccatctccctca-
actccacctacccca
tctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagc-
gatcctcacgtgc
acactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttca-
aggaccacctg
accgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaag-
accttcacttgc
actgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccga-
ggtccacctgc
tgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagcccc-
aaggatgtgc
tggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagccc-
agccagggc
accaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctg-
catggtgggc
cacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgt-
gtctgttgtcat ggcggaggtggacggcacctgctactgataatctaga CTLA-4 IgAH
IgACH2CH3 Protein sequence (SEQ ID NO: 532):
mgvlltqrtllslvlallfpsmasmamhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvt
evcaatymmgneltflddsictgtssgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpep-
cpdsdqpv
pstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdr-
dlcgcysvssvlpg
caepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwl-
qgsqelprekylt
wasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevdgtc-
y CTLA-4 IgAH IgA-T4 (Human-oncoMLP-CTLA4EC-IgA3T1) (SEQ ID NO:
533) Nucleotide sequence:
atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagc
atggcaatgcacgtggcccagcctgctgtggtactggccagcagccgaggcatcgccagctttgtgtgtgagta-
tgcatctccagg
caaagccactgaggtccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacct-
acatgatggg
gaatgagttgaccttcctagatgattccatctgcacgggcacctccagtggaaatcaagtgaacctcactatcc-
aaggactgagggc
catggacacgggactctacatctgcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacg-
gaacccagattt
atgtaattgatccagaaccgtgcccagattctgatcagccagttccctcaactccacctaccccatctccctca-
actccacctacccca
tctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagc-
gatcctcacgtgc
acactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttca-
aggaccacctg
accgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaag-
accttcacttgc
actgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccga-
ggtccacctgc
tgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagcccc-
aaggatgtgc
tggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagccc-
agccagggc
accaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctg-
catggtgggc
cacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgt-
gtctgttgtcat ggcggaggtggactgataatctaga CTLA-4 IgAH IgA-T4 Protein
sequence (SEQ ID NO: 534):
Mgvlltqrtllslvlallfpsmasmamhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvt
evcaatymmgneltflddsictgtssgnqvnitiqglramdtglyickvelmypppyylgigngtqiyvidpep-
cpdsdqpv
pstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqgppdr-
dlcgcysvssvlpg
caepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwl-
qgsqelprekylt
wasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevd
NT human IgG1 CH2 with 238 mutation pro.fwdarw.ser (SEQ ID NO: 535)
cctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccgga
cccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggac-
ggcgtggagg
tgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtc-
ctgcaccagg
actggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatc-
tccaaagcca aag AA human IgG1 CH2 with 238 mutation pro.fwdarw.ser
(SEQ ID NO: 536)
pellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynst
yrvvsvltvlhqdwlngkeykckvsnkalpapiektiskak Amino acids surrounding
Pro to Ser in CH2 (SEQ ID NO: 537) PAPELLGGPS Amino acids
surrounding Pro to Ser in CH2 (SEQ ID NO: 538) PAPELLGGSS HIgE5Bcl
(SEQ ID NO: 539) gtt gtt gat cac gtc tgc tcc agg gac ttc acc cc
hIgE3stop (SEQ ID NO: 540) gtt gtt tct aga tta act ttt acc ggg att
tac aga cac cgc tcg ctg g hIgE3BB (leaves an open reading frame at
end of gene to read into transmembrane and cytoplasmic tail domain
attached at either the BamHI or SfuI sites) (SEQ ID NO: 541) gtt
gtt ttc gaa gga tcc gct tta ccg gga ttt aca gac acc gct cgc tgg NT
human IgE Fc (CH2-CH3-CH4) ORF (SEQ ID NO: 542):
tgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcg
ggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctgg-
ctggaggacgg
gcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagc-
tcaccctcag
ccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagca-
ccaagaagtgt
gcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtc-
gcccacgatc
acctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcc-
tgtgaacca
ctccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgag-
actggatcg
agggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagacc-
agcggccc
gcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcct-
gcctgatcc
agaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagc-
acgacgcag
ccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaa-
agatgagttc
atctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaa-
agcggatcctt cgaa AA human IgE Fc (CH2-CH3-CH4) ORF (SEQ ID NO:
543):
dhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgtinitwledgqvmdvdlstasttqegelast
qseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdlfirksptitclvvdlapsk-
gtvnltwsrasgk
pvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrsttktsgpraapevyafatpewp-
gsrdkrtlacliq
nfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweqkdeficravheaaspsqtvqrav-
svnpgkadps IFhIgGwtBcl5 (SEQ ID NO: 544) gtt gtt tga tca gga gcc
caa atc ttg tga caa aac tca cac atg ccc acc gtg ccc agc acc (63
mer) hIgGWT3xba (SEQ ID NO: 545) gtt gtt tct aga tca ttt acc cgg
aga cag gga gag gct ctt ctg cgt gta g HuIgGMHWC (sense, 5' primer
for mutating wild type hinge CCC to mutant SSS (SEQ ID NO: 546) gtt
gtt gat cag gag ccc aaa tct tct gac aaa act cac aca tct cca ccg tcc
cca gca cct gaa ctc ctg ggt gga ccg tca gtc ttc c NT 1D8 VH (SEQ ID
NO: 547)
caggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtccctcacatgcactg
tctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatggatg-
ggaataatatattat
gatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagccaagt-
tttcttaaaaatc
aacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaaggagt-
catggtcacagtct cctct AA 1D8 VH (no leader) (SEQ ID NO: 548)
qvqlkeagpglvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisr
dtsksqvflkinslqtddtamyycarihfdywgqgvmvtvss NT 1D8 VL (no leader)
(SEQ ID NO: 549)
gacattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgt
gccagctccagtgtaagttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatga-
cacatccaagctg
gcttctggagttccaaatcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatgga-
gactgaagatgctg
ccacttattactgtcagcagtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacgg
AA 1D8 VL (SEQ ID NO: 550)
divltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiydtsklasgvpnrfsgsgsgtsys
laintmetedaatyycqqwsstpltfgsgtkleikr NT 1D8 scFv (SEQ ID NO: 551)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgcc-
agctccagtgtaa
gttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggct-
tctggagttccaaa
tcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgcca-
cttattactgtcagc
agtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggc-
ggtggtgggt
cgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtcc-
ctcacatgc
actgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatg-
gatgggaataatat
attatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagc-
caagttttcttaaa
aatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaag-
gagtcatggtcaca gtctcctctgatca AA 1D8 scFv (SEQ ID NO: 552)
mdfqvqifsfllisasvimsrgvdivltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiy
dtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikrggggsggggsggggs-
qvqlkeagpg
lvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkins-
lqtddtamyyca rihfdywgqgvmvtvss NT 1D8 scFv IgG WTH WTCH2CH3 (SEQ ID
NO: 553)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgcc-
agctccagtgtaa
gttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggct-
tctggagttccaaa
tcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgcca-
cttattactgtcagc
agtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggc-
ggtggtgggt
cgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtcc-
ctcacatgc
actgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatg-
gatgggaataatat
attatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagc-
caagttttcttaaa
aatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaag-
gagtcatggtcaca
gtctcctctgatcaggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcct-
ggggggaccgtc
agtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtgg-
tggacgtgagcc
acgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgg-
gaggagcag
tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaa-
gtgcaaggtct
ccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtg-
tacaccctg
cccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcga-
catcgccgtgg
agtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttc-
ttcctctacag
caagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgc-
acaaccactac acgcagaagagcctctccctgtctccgggtaaatgatctaga AA 1D8 scFv
IgG WTH WTCH2CH3 (SEQ ID NO: 554)
mdfqvqifsfllisasvimsrgvdivltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiy
dtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikrggggsggggsggggs-
qvqlkeagpg
lvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkins-
lqtddtamyyca
rihfdywgqgymvtvssdqepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshed-
pevkfnw
yvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytl-
ppsrdeltk
nqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhn-
hytqkslsl spgk NT 1D8 scFv IgG MTH MTCH2CH3-CD80 (SEQ ID NO: 555)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgcc-
agctccagtgtaa
gttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggct-
tctggagttccaaa
tcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgcca-
cttattactgtcagc
agtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggc-
ggtggtgggt
cgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtcc-
ctcacatgc
actgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatg-
gatgggaataatat
attatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagc-
caagttttcttaaa
aatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaag-
gagtcatggtcaca
gtctcctctgatctggagcccaaatcttctgacaaaactcacacaagcccaccgagcccagcacctgaactcct-
ggggggatcgtc
agtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtgg-
tggacgtgagcc
acgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgg-
gaggagcag
tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaa-
gtgcaaggtct
ccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtg-
tacaccctg
cccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcga-
catcgccgtgg
agtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttc-
ttcctctacag
caagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgc-
acaaccactac
acgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattacctt-
aatctcagtaaatg
gaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattg-
agaagggaaagt gtacgccctgtataaatcgata AA 1D8 scFv IgG MTH
MTCH2CH3-CD80 (SEQ ID NO: 556)
mdfqvqifsfllisasvimsrgvdivltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiy
dtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikrggggsggggsggggs-
qvqlkeagpg
lvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkins-
lqtddtamyyca
rihfdywgqgvmvtvssdlepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvshed-
pevkfnwy
vdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlp-
psrdeltkn
qvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnh-
ytqkslsls pgkadpsnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT
1D8 scFv IgG WTH WTCH2CH3-CD80 (SEQ ID NO: 557)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgcc-
agctccagtgtaa
gttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggct-
tctggagttccaaa
tcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgcca-
cttattactgtcagc
agtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggc-
ggtggtgggt
cgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtcc-
ctcacatgc
actgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatg-
gatgggaataatat
attatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagc-
caagttttcttaaa
aatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaag-
gagtcatggtcaca
gtctcctctgatctggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcct-
ggggggaccgtc
agtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtgg-
tggacgtgagcc
acgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgg-
gaggagcag
tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaa-
gtgcaaggtct
ccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtg-
tacaccctg
cccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcga-
catcgccgtgg
agtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttc-
ttcctctacag
caagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgc-
acaaccactac
acgcagaagagcctctccctgtctccgggtaaagcggatccttcgaacctgctcccatcctgggccattacctt-
aatctcagtaaatg
gaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattg-
agaagggaaagt gtacgccctgtataaatcgata AA 1D8 scFv IgG WTH
WTCH2CH3-CD80 (SEQ ID NO: 558)
mdfqvqifsfllisasvimsrgvdivltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiy
dtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikrggggsggggsggggs-
qvqlkeagpg
lvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkins-
lqtddtamyyca
rihfdywgqgvmvtvssdlepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshed-
pevkfnw
yvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytl-
ppsrdeltk
nqvsltclvkgfypsdiavewesngqpcnnykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhn-
hytqkslsl spgkadpsnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv
NT Anti human CD3 scFv WTH WTCH2CH3 (SEQ ID NO: 559)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggca-
agtcaggacattc
gcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagatta-
cactcaggagtccca
tcaaggttcagtggcagtgggtctggaacagattattctctcaccattgccaacctgcaaccagaagatattgc-
cacttacttttgcca
acagggtaatacgcttccgtggacgttcggtggaggcaccaaactggtaaccaaacgggagctcggtggcggtg-
gctcgggcg
gtggtgggtcgggtggcggcggatctatcgatgaggtccagctgcaacagtctggacctgaactggtgaagcct-
ggagcttcaat
gtcctgcaaggcctctggttactcattcactggctacatcgtgaactggctgaagcagagccatggaaagaacc-
ttgagtggattgg
acttattaatccatacaaaggtcttactacctacaaccagaaattcaagggcaaggccacattaactgtagaca-
agtcatccagcaca
gcctacatggagctcctcagtctgacatctgaagactctgcagtctattactgtgcaagatctgggtactatgg-
tgactcggactggta
cttcgatgtctggggcgcagggaccacggtcaccgtctcctctgatcaggagcccaaatcttgtgacaaaactc-
acacatgcccac
cgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatg-
atctcccggacc
cctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacgg-
cgtggaggtg
cataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcct-
gcaccagga
ctggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatct-
ccaaagccaa
agggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcc-
tgacctgcc
tggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaag-
accacgcctc
ccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggg-
aacgtcttctca
tgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatc-
taga AA Anti human CD3 scFv WTH WTCH2CH3 (SEQ ID NO: 560)
Mdfqvqifsfllisasvimsrgvdiqmtqttsslsaslgdrvtiscrasqdirnylnwyqqkpdgtvklliy
ytsrlhsgvpsrfsgsgsgtdysltianlqpediatyfcqqgntlpwtfgggtklvtkrelggggsggggsggg-
gsidevqlqqsg
pelvkpgasmsckasgysftgyivnwlkqshgknlewiglinpykglttynqkfkgkatltvdkssstaymell-
sltsedsavy
ycarsgyygdsdwyfdvwgagttvtvssdqepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpev-
tcvvvdvs
hedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakg-
qprepq
vytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgn-
vfscsvmhe alhnhytqkslslspgk NT 2H7-antiCD40 scFv MTH (SSS)
MTCH2WTCH3 (SEQ ID NO: 561) 2h7-40.2.220Ig + restriction sites
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaaggtggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcaatccaactctgaagaagcaaagaaagagga-
ggccaaaaag
gaggaagccaagaaatctaacagcgtcgacattgttctgactcagtctccagccaccctgtctgtgactccagg-
agatagagtctct
ctttcctgcagggccagccagagtattagcgactacttacactggtatcaacaaaaatcacatgagtctccaag-
gcttctcatcaaata
tgcttcccattccatctctgggatcccctccaggttcagtggcagtggatcagggtcagatttcactctcagta-
tcaacagtgtggaac
ctgaagatgttggaatttattactgtcaacatggtcacagctttccgtggacgttcggtggaggcaccaagctg-
gaaatcaaacggg
gtggcggtggctcgggcggaggtgggtcgggtggcggcggatctcagatccagttggtgcaatctggacctgag-
ctgaagaag
cctggagagacagtcaggatctcctgcaaggcttctgggtatgccttcacaactactggaatgcagtgggtgca-
agagatgccagg
aaagggtttgaagtggattggctggataaacaccccactctggagtgccaaaatatgtagaagacttcaaggac-
ggtttgccttctct
ttggaaacctctgccaacactgcatatttacagataagcaacctcaaagatgaggacacggctacgtatttctg-
tgtgagatccggga
atggtaactatgacctggcctactttgcttactggggccaagggacactggtcactgtctctgatcaggagccc-
aaatcttctgacaa
aactcacacatccccaccgtccccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaac-
ccaaggacaccc
tcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc-
aactggtacgtg
gacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcag-
cgtcctcac
cgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagccccca-
tcgagaaaac
aatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgacca-
agaaccagg
tcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccg-
gagaacaact
acaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagc-
aggtggcagca
ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgt-
ctccgggtaaatg atctaga AA 2H7-antiCD40 scFv MTH (SSS) MTCH2WTCH3
(SEQ ID NO: 562) 2H7-40.2.220Ig
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkggggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqsnseeakkeeakkeeakksnsvdivltqspatlsvtpgdrvs-
lscrasqsis
dylhwyqqkshesprllikyashsisgipsrfsgsgsgsdftlsinsvepedvgiyycqhghsfpwtfgggtkl-
eikrggggsg
gggsggggsqiqlvqsgpelkkpgetvrisckasgyaftttgmqwvqempgkglkwigwintplwsakicrrlq-
grfafslet
santaylqisnlkdedtatyfcvrsgngnydlayfaywgqgtlvtvsdqepkssdkthtsppspapellggssv-
flfppkpkdtl
misrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsn-
kalpap
iektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsff-
lyskltvdksr wqqgnvfscsvmhealhnhytqkslslspgk NT 5B9 VH (includes the
VH leader peptide) (SEQ ID NO: 563)
atggctgtcttggggctgctcttctgcctggtgacatttccaagctgtgtcctatcccaggtgcagctgaagca
gtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaacta-
cctatgctgtacact
gggttcgccagtctccaggaaagggtctggagtggctgggagtgatatggagtggtggaatcacagactataat-
gcagctttcatat
ccagactgagcatcaccaaggacgattccaagagccaagttttctttaaaatgaacagtctgcaacctaatgac-
acagccatttatta
ctgtgccagaaatgggggtgataactacccttattactatgctatggactactggggtcaaggaacctcagtca-
ccgtctcctca 5B9 VH missing the leader (SEQ ID NO: 564):
caggtgcagctgaagcagtcaggacctggcctagtgcagtcctcacagagcctgtccatcacctgcacagt
ctctggtttctcattaactacctatgctgtacactgggttcgccagtctccaggaaagggtctggagtggctgg-
gagtgatatggagt
ggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaaggacgattccaagagccaagt-
tttctttaaaatga
acagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggtgataactacccttattactat-
gctatggactactgg ggtcaaggaacctcagtcaccgtctcctca AA 5B9 VH (includes
leader peptide) (SEQ ID NO: 565)
MAVLGLLFCLVTFPSCVLSQVQLKQSGPGLVQSSQSLSITCTVSGFSL
TTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNS
LQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTVSS 5B9 VH no leader peptide
(SEQ ID NO: 566) QVQLKQSGPGLVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLE
WLGVIWSGGITDYNAAFISRLSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDN
YPYYYAMDYWGQGTSVTVSS NT 5B9 VL (SEQ ID NO: 567)
atgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcagatattgtgatgac-
g
caggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcct-
acatagtaatggcatc
acttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagatgtccaaccttgc-
ctcaggagtcccagac
aggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgt-
ttattactgtgct
caaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacgg AA 5B9 VL
(SEQ ID NO: 568) MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKS
LLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEA
EDVGVYYCAQNLELPLTFGAGTKLELKR NT 5B9 scFv (SEQ ID NO: 569)
aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcaga
tattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtcta-
gtaagagtctcctaca
tagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcaga-
tgtccaaccttgcctca
ggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctga-
ggatgtgggtg
tttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggt-
ggcggtggctcgg
gcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcc-
tcacagagc
ctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccagg-
aaagggtctggagtg
gctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaagg-
acgattccaaga
gccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggt-
gataactacccttatt actatgctatggactactggggtcaaggaacctcagtcaccgtctcctct
AA 5B9 scFv (SEQ ID NO: 570)
MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKS
LLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEA
EDVGVYYCAQNLELPLTFGAGTKLELKRGGGGSGGGGSGGGGSSQVQLKQSGPG
LVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISR
LSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTV SS NT 5B9
scFv-hmtIgG1-hCD80 (SEQ ID NO: 571)
aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcaga
tattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtcta-
gtaagagtctcctaca
tagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcaga-
tgtccaaccttgcctca
ggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctga-
ggatgtgggtg
tttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggt-
ggcggtggctcgg
gcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcc-
tcacagagc
ctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccagg-
aaagggtctggagtg
gctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaagg-
acgattccaaga
gccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggt-
gataactacccttatt
actatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatctggagcccaaatcttctgac-
aaaactcacacaag
cccaccgagcccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccc-
tcatgatctccc
ggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg-
gacggcgtgg
aggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcacc-
gtcctgcacc
aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacc-
atctccaaag
ccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtc-
agcctgacc
tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta-
caagaccac
gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagc-
aggggaacgtc
ttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa-
agcggatccttcg
aacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactg-
ctttgccccaagatgca
gagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatactcgag
AA 5B9 scFv-hmtIgG1-hCD80 (SEQ ID NO: 572)
MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKS
LLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEA
EDVGVYYCAQNLELPLTFGAGTKLELKRGGGGSGGGGSGGGGSSQVQLKQSGPG
LVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISR
LSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTV
SSDLEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGKADPSNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLR RESVRPV
NT 2e12 scFv WTH CH2 CH3 (2e12 scFv-WthIgG-CD80) (SEQ ID NO: 573)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagcc-
agtgaaagtgttgaa
tattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgc-
atccaacgtagaat
ctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggag-
gatgatattgcaa
tgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggt-
ggcggtggctcg
ggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctc-
acagagcc
tgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccagga-
aagggtctggagt
ggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaag-
gacaactccaa
gagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggtt-
atagtaactttcatt
actatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatctggagcccaaatcttgtgac-
aaaactcacacatg
cccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccc-
tcatgatctccc
ggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg-
gacggcgtgg
aggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcacc-
gtcctgcacc
aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacc-
atctccaaag
ccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtc-
agcctgacc
tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta-
caagaccac
gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagc-
aggggaacgtc
ttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa-
agcggatccttcg
aacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactg-
ctttgccccaagatgca
gagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgat 2e12
scFv WTH CH2 CH3 2e12 scFv-WthIgG-CD80 (SEQ ID NO: 574)
mdfqvqifsfllisasvimsrgvdivltqspaslavslgqratiscrasesveyyvtslmqwyqqkpgqp
pkllisaasnvesgvparfsgsgsgtdfslnihpveeddiamyfcqqsrkvpwtfgggtkleikrggggsgggg-
sggggsqvq
lkesgpglvapsqslsitctvsgfsltgygvnwvrqppgkglewlgmiwgdgstdynsalksrlsitkdnsksq-
vflkmnslqt
ddtaryycardgysnfhyyvmdywgqgtsvtvssdlepkscdkthtcppcpapellggpsvflfppkpkdtlmi-
srtpevtc
vvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiekt-
iskakg
qprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppyldsdgsfflyskltvdks-
rwqqgnyfsc
svmhealhnhytqkslslspgkadpsnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv
NT 2H7-human IgE Fc (CH2-CH3-CH4) (SEQ ID NO: 575)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaaggtggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctctgatcacgtctgctccagggacttcaccccgcccaccgt-
gaagatcttaca
gtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccag-
ggactatcaac
atcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagct-
ggcctccaca
caaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaagg-
tcacacctttg
aggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttc-
gacctgttca
tccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctgg-
tcccgggcca
gtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccacc-
ctgccggtg
ggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcat-
gcggtcca
cgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgg-
gacaagcg
caccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagc-
tcccggacgcc
cggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccag-
ggccgaatg
ggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcgg-
tgtctgtaaat cccggtaaatgataatctaga AA 2H7 scFv IgE (CH2-CH3-CH4)
(SEQ ID NO: 576)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkggggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvsdhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgfin-
itwledgqvm
dvdlstasttqegelastqseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdlf-
irkspfitclvvdla
pskgtvnltwsrasgkpvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrsttktsg-
praapevyafat
pewpgsrdkrtlacliqnfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweqkdefic-
ravheaasps qtvqravsvnpgk NT 2H7 scFv MH (SSS) MCH2WTCH3 (SEQ ID NO:
577)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacac-
atccccaccgtc
cccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatct-
cccggacccctg
aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg-
gaggtgcata
atgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcac-
caggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaa-
agccaaaggg
cagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgac-
ctgcctggtc
aaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac-
gcctcccgt
gctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacg-
tcttctcatgct
ccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga
AA 2H7 scFv MH (SSS) MCH2WTCH3 (SEQ ID NO: 578)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggssvflfppkpkdtlmisrtp-
evtcvvvdv
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskak-
gqprep
qvytlppsrdeltknqvsltelvkgfypsdiavewesngqpennvkttppyldsdgsfflyskltvdksrwqqg-
nvfscsvmh ealhnhytqkslslspgk NT 5B9 scFv MTHWTCH2CH3 (SEQ ID NO:
579)
aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcaga
tattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtcta-
gtaagagtctcctaca
tagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcaga-
tgtccaaccttgcctca
ggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctga-
ggatgtgggtg
tttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggt-
ggcggtggctcgg
gcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcc-
tcacagagc
ctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccagg-
aaagggtctggagtg
gctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaagg-
acgattccaaga
gccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggt-
gataactacccttatt
actatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcaggagcccaaatcttctgac-
aaaactcacacatc
cccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccc-
tcctgatctccc
ggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg-
gacggcgtgg
aggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcacc-
gtcctgcacc
aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaca-
atctccaaag
ccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtc-
agcctgacc
tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta-
caagaccac
gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagc-
aggggaacgtc
ttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa-
atgatctaga AA 5B9 scFv MTHWTCH2CH3 (SEQ ID NO: 580)
MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKS
LLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEA
EDVGVYYCAQNLELPLTFGAGTKLELKRGGGGSGGGGSGGGGSSQVQLKQSGPG
LVQSSQSLSITCTVSGFSLTTYAVHWVRQSPGKGLEWLGVIWSGGITDYNAAFISR
LSITKDDSKSQVFFKMNSLQPNDTAIYYCARNGGDNYPYYYAMDYWGQGTSVTV
SSDQEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGK Human IgG1 hinge mutations 2H7 scFv-MTH (CSS)
WTCH2CH3 (SEQ ID NO: 581) Nucleotide:
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtgt
aagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctgg-
cttctggagtccct
gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgc-
cacttattactgcc
agcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggc-
ggtggtggatc
tggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtga-
agatgtcctgc
aaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatg-
gattggagctattt
atccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctcc-
agcacagcctac
atgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactc-
ttactggtacttcgat
gtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacacatc-
cccaccgtcccc
agcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccc-
ggacccctgagg
tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggag-
gtgcataatgc
caagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg-
actggctgaa
tggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagcca-
aagggcagcc
ccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcc-
tggtcaaagg
cttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctc-
ccgtgctgga
ctccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttct-
catgctccgtgat
gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga
2H7 scFv-MTH (CSS) WTCH2CH3 protein (SEQ ID NO: 582):
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdywgtgttytyssdqepkscdkthtsppspapellggpsvflfppkpkdtlmisrtp-
evtcyvvdy
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsnkalpapiektiskak-
gqprep
qvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppyldsdgsfflyskltvdksrwqqg-
nyfscsvmh ealhnhytqkslslspgk 2H7 scFv-MTH (SCS) WTCH2CH3 (SEQ ID
NO: 583): Nucleotide:
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtgt
aagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctgg-
cttctggagtccct
gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgc-
cacttattactgcc
agcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggc-
ggtggtggatc
tggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtga-
agatgtcctgc
aaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatg-
gattggagctattt
atccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctcc-
agcacagcctac
atgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactc-
ttactggtacttcgat
gtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatg-
cccaccgtcccc
agcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccc-
ggacccctgagg
tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggag-
gtgcataatgc
caagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg-
actggctgaa
tggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagcca-
aagggcagcc
ccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcc-
tggtcaaagg
cttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctc-
ccgtgctgga
ctccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttct-
catgctccgtgat
gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga
2H7 scFv-MTH (SCS) WTCH2CH3 Protein (SEQ ID NO: 584):
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssysvmhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstavmqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtyssdqepkssdkthtcppspapellggpsvflfppkpkdtlmisrtp-
evtcyvvdv
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskak-
gqprep
qvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqg-
nyfscsvmh ealhnhytqkslslspgk 2H7 scFv-MTH (SSC) WTCH2CH3 (SEQ ID
NO: 585): Nucleotide:
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacac-
atccccaccgtg
cccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatct-
cccggacccctg
aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg-
gaggtgcata
atgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcac-
caggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaa-
agccaaaggg
cagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgac-
ctgcctggtc
aaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac-
gcctcccgt
gctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacg-
tcttctcatgct
ccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga
2H7 scFv-MTH (SSC) WTCH2CH3 Protein (SEQ ID NO: 586):
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppcpapellggpsvflfppkpkdtlmisrtp-
evtcyvvdv
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskak-
gqprep
qvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflvyskltvdksrwqq-
gnyfscsvm h ealhnhytqkslslspgk HIgGMHcys1 (SEQ ID NO: 587) gtt gtt
gat cag gag ccc aaa tct tct gac aaa act cac aca tg HIgGMHcys2 (SEQ
ID NO: 588) gtt gtt gat cag gag ccc aaa tct tgt gac aaa act cac aca
tct cca ccg tgc HIgGMHcys3 (SEQ ID NO: 589) gtt gtt gat cag gag ccc
aaa tct tgt gac aaa act cac aca tgt cca ccg tcc cca gca cct NT
HuIgG1 MTCH3Y405 (SEQ ID NO: 590)
gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggt
cagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccgg-
agaacaacta
caagaccacgcctcccgtgctggactccgacggctccttctacctctatagcaagctcaccgtggacaagagca-
ggtggcagcag
gggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc-
cccgggtaaatga AA HuIgG1 MTCH3Y405 (SEQ ID NO: 591)
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFYLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK NT
HuIgG1 MTCH3A405 (SEQ ID NO: 592)
gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggt
cagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccgg-
agaacaacta
caagaccacgcctcccgtgctggactccgacggctccttcgccctctatagcaagctcaccgtggacaagagca-
ggtggcagca
ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgt-
ccccgggtaaat ga AA HuIgG1 MTCH3A405 (SEQ ID NO: 593)
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFALYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK NT
HuIgG1 MTCH3A407 (SEQ ID NO: 594)
Gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggt
cagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccgg-
agaacaacta
caagaccacgcctcccgtgctggactccgacggctccttcttcctcgccagcaagctcaccgtggacaagagca-
ggtggcagca
ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgt-
ccccgggtaaat ga AA HuIgG1 MTCH3A407 (SEQ ID NO: 595)
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK NT
HuIgG1 MTCH3Y405A407 (SEQ ID NO: 596)
gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggt
cagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccgg-
agaacaacta
caagaccacgcctcccgtgctggactccgacggctccttctacctcgccagcaagctcaccgtggacaagagca-
ggtggcagca
ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgt-
ccccgggtaaat ga AA HuIgG1 MTCH3Y405A407 (SEQ ID NO: 597)
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFYLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK NT
HuIgG1 MTCH3A405A407 (SEQ ID NO: 598)
gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggt
cagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccgg-
agaacaacta
caagaccacgcctcccgtgctggactccgacggctccttcgccctcgccagcaagctcaccgtggacaagagca-
ggtggcagc
aggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctg-
tccccgggtaaa tga AA HuIgG1 MTCH3A405A407 (SEQ ID NO: 599)
gqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfalask
ltvdksrwqqgnvfscsvmhealhnhytqkslslspgk NT 2H7 scFv MTH (SSS)
WTCH2MTCH3Y405 (SEQ ID NO: 600)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacac-
atccccaccgtc
cccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatct-
cccggacccctg
aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg-
gaggtgcata
atgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcac-
caggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaa-
agccaaaggg
cagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgac-
ctgcctggt
caaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca-
cgcctcccg
tgctggactccgacggctccttctacctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaac-
gtcttctcatgc
tccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgatctag-
a AA 2H7 scFv MTH (SSS) WTCH2MTCH3Y405 (SEQ ID NO: 601)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtp-
evtcvvvdv
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsnkalpapiektiskak-
gqprep
qvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfylyskltvdksrwqqg-
nvfscsvm healhnhytqkslslspgk NT 2H7 scFv MTH (SSS) WTCH2MTCH3A405
(SEQ ID NO: 602)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacac-
atccccaccgtc
cccagcacctgaactcctggggggaccgtcagtcttccccttccccccaaaacccaaggacaccctcatgatct-
cccggacccctg
aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg-
gaggtgcata
atgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcac-
caggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaa-
agccaaaggg
cagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgac-
ctgcctggt
caaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca-
cgcctcccg
tgctggactccgacggctccttcgccctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaac-
gtcttctcatg
ctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatga
AA 2H7 scFv MTH (SSS) WTCH2MTCH3A405 (SEQ ID NO: 603)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtp-
evtcvvvdv
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsnkalpapiektiskak-
gqprep
qvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfalyskltvdksrwqqg-
nvfscsvm healhnhytqkslslspgk NT 2H7 scFv MTH (SSS) WTCH2MTCH3A407
(SEQ ID NO: 604)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtgt
aagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctgg-
cttctggagtccct
gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgc-
cacttattactgcc
agcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggc-
ggtggtggatc
tggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtga-
agatgtcctgc
aaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatg-
gattggagctattt
atccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctcc-
agcacagcctac
atgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactc-
ttactggtacttcgat
gtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatc-
cccaccgtcccc
agcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccc-
ggacccctgagg
tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggag-
gtgcataatgc
caagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg-
actggctgaa
tggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagcca-
aagggcagcc
ccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcc-
tggtcaaag
gcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcct-
cccgtgctgg
actccgacggctccttcttcctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttc-
tcatgctccgtg
atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatga AA
2H7 scFv MTH (SSS) WTCH2MTCH3A407 (SEQ ID NO: 605)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdywgtgttytyssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtp-
evtcyvvdy
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykekvsnkalpapiektiskak-
gqprep
qvytlppsreemtknqvsltelvkgfypsdiavewesngqpennykttppyldsdgsfflaskltvdksrwqqg-
nyfscsvm healhnhytqkslslspgk NT 2H7 scFv MTH (SSS)
WTCH2MTCH3Y405A407 (SEQ ID NO: 606)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacac-
atccccaccgtc
cccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatct-
cccggacccctg
aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg-
gaggtgcata
atgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcac-
caggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaa-
agccaaaggg
cagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgac-
ctgcctggt
caaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca-
cgcctcccg
tgctggactccgacggctccttctacctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaac-
gtcttctcatg
ctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatga
AA 2H7 scFv MTH (SSS) WTCH2MTCH3Y405A407 (SEQ ID NO: 607)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtp-
evtcvvvdv
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsnkalpapiektiskak-
gqprep
qvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfylaskltvdksrwqqg-
nvfscsvm healhnhytqkslslspgk NT 2H7 scFv MTH (SSS)
WTCH2MTCH3A405A407 (SEQ ID NO: 608)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtgt
aagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctgg-
cttctggagtccct
gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgc-
cacttattactgcc
agcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggc-
ggtggtggatc
tggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtga-
agatgtcctgc
aaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatg-
gattggagctattt
atccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctcc-
agcacagcctac
atgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactc-
ttactggtacttcgat
gtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatc-
cccaccgtcccc
agcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccc-
ggacccctgagg
tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggag-
gtgcataatgc
caagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg-
actggctgaa
tggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagcca-
aagggcagcc
ccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcc-
tggtcaaag
gcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcct-
cccgtgctgg
actccgacggctccttcgccctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttc-
tcatgctccgt
gatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatga AA
2H7 scFv MTH (SSS) WTCH2MTCH3A405A407 (SEQ ID NO: 609)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtp-
evtcvvvdv
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsnkalpapiektiskak-
gqprep
qvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfalaskltvdksrwqqg-
nvfscsvm healhnhytqkslslspgk NT 2H7 scFv MTH (SCC) WTCH2CH3 (SEQ ID
NO: 610)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtgt
aagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctgg-
cttctggagtccct
gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgc-
cacttattactgcc
agcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggc-
ggtggtggatc
tggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcagtga-
agatgtcctgc
aaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatg-
gattggagctattt
atccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctcc-
agcacagcctac
atgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactc-
ttactggtacttcgat
gtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacacatg-
cccaccgtgccc
agcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccc-
ggacccctgagg
tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggag-
gtgcataatgc
caagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg-
actggctgaa
tggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagcca-
aagggcagcc
ccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcc-
tggtcaaagg
cttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctc-
ccgtgctgga
ctccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttct-
catgctccgtgat
gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga
AA 2H7 scFv MTH (SCC) WTCH2CH3 (SEQ ID NO: 611)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtp-
evtcvvvdv
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskak-
gqprep
qvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqg-
nvfscsvmh ealhnhytqkslslspgk NT 2H7 scFv MTH (CSC) WTCH2CH3 (SEQ ID
NO: 612)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacac-
atctccaccgtg
cccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatct-
cccggacccctg
aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg-
gaggtgcata
atgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcac-
caggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaa-
agccaaaggg
cagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgac-
ctgcctggtc
aaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac-
gcctcccgt
gctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacg-
tcttctcatgct
ccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga
AA 2H7 scFv MTH (CSC) WTCH2CH3 (SEQ ID NO: 613)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqepkscdkthtsppcpapellggpsvflfppkpkdtlmisrtp-
evtcvvvdv
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskak-
gqprep
qvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqg-
nvfscsvmh ealhnhytqkslslspgk NT 2H7 scFv MTH (CCS) WTCH2CH3 (SEQ ID
NO: 614)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttgtgacaaaactcacac-
atgtccaccgtc
cccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatct-
cccggacccctg
aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg-
gaggtgcata
atgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcac-
caggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaa-
agccaaaggg
cagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgac-
ctgcctggtc
aaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac-
gcctcccgt
gctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacg-
tcttctcatgct
ccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga
AA 2H7 scFv MTH (CCS) WTCH2CH3 (SEQ ID NO: 615)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqepkscdkthtcppspapellggpsvflfppkpkdtlmisrtp-
evtcvvvdv
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskak-
gqprep
qvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqg-
nvfscsvmh ealhnhytqkslslspgk NT HuIgAHIgA-T4-ORF (SEQ ID NO: 616)
tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgcc
acccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcaca-
ctgaccggcct
gagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgacc-
gtgacctctgt
ggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcac-
tgctgcctaccc
cgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgc-
cgccgccgtc
ggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctgg-
ttcgctggct
gcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcacca-
ccaccttc
gctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggcca-
cgaggccct
gccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtca-
tggcggaggtgg acgcggatccttcgaac AA HuIgAHIgA-T4-ORF (SEQ ID NO: 617)
Dqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqg
ppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvt-
ltclargfspkdvl
vrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagk-
pthvnvsvvm aevdadpsn NT HuIgAHIgA-T4-ORF (SEQ ID NO: 618)
tgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctcatgctgcc
acccccgactgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcaca-
ctgaccggcct
gagagatgcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgacc-
gtgacctctgt
ggctgctacagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcac-
tgctgcctaccc
cgagtccaagaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgc-
cgccgccgtc
ggaggagctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctgg-
ttcgctggct
gcaggggtcacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcacca-
ccaccttc
gctgtgaccagcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggcca-
cgaggccct
gccgctggccttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtca-
tggcggaggtgg acgcggatccttcgaac AA HuIgAHIgA-T4-ORF (SEQ ID NO: 619)
dqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdasgvtftwtpssgksavqg
ppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvt-
ltclargfspkdvl
vrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagk-
pthvnvsvvm aevdadpsn NT 1D8-IgAH IgA-T4-CD80 (SEQ ID NO: 620)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgcc-
agctccagtgtaa
gttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggct-
tctggagttccaaa
tcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgcca-
cttattactgtcagc
agtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggc-
ggtggtgggt
cgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtcc-
ctcacatgc
actgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatg-
gatgggaataatat
attatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagc-
caagttttcttaaa
aatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaag-
gagtcatggtcaca
gtctcctctgatcagccagttccctcaactccacctaccccatctccctcaactccacctaccccatctccctc-
atgctgccacccccg
actgtcactgcaccgaccggccctcgaggacctgctcttaggttcagaagcgatcctcacgtgcacactgaccg-
gcctgagagat
gcctcaggtgtcaccttcacctggacgccctcaagtgggaagagcgctgttcaaggaccacctgaccgtgacct-
ctgtggctgcta
cagcgtgtccagtgtcctgccgggctgtgccgagccatggaaccatgggaagaccttcacttgcactgctgcct-
accccgagtcca
agaccccgctaaccgccaccctctcaaaatccggaaacacattccggcccgaggtccacctgctgccgccgccg-
tcggaggag
ctggccctgaacgagctggtgacgctgacgtgcctggcacgtggcttcagccccaaggatgtgctggttcgctg-
gctgcaggggt
cacaggagctgccccgcgagaagtacctgacttgggcatcccggcaggagcccagccagggcaccaccaccttc-
gctgtgacc
agcatactgcgcgtggcagccgaggactggaagaagggggacaccttctcctgcatggtgggccacgaggccct-
gccgctggc
cttcacacagaagaccatcgaccgcttggcgggtaaacccacccatgtcaatgtgtctgttgtcatggcggagg-
tggacgcggatc
cttcgaacaacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctg-
acctactgctttgcccc
aagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgatac
AA 1D8 scFv IgAH IgA-T4-CD80 (SEQ ID NO: 621)
mdfqvqifsfllisasvimsrgvdivltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiy
dtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikrggggsggggsggggs-
qvqlkeagpg
lvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkins-
lqtddtamyyca
rihfdywgqgvmvtvssdqpvpstpptpspstpptpspscchprlslhrpaledlllgseailtctltglrdas-
gvtftwtpssgks
avqgppdrdlcgcysvssvlpgcacpwnhgktftctaaypcsktpltatlsksgntfrpcvhllpppscclaln-
clvtltclargfs
pkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktid-
rlagkpthvnv
svvmaevdadpsnnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT
human IgE Fc (CH2-CH3-CH4) ORF (SEQ ID NO: 622):
tgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacggcggcg
ggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctgg-
ctggaggacgg
gcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagc-
tcaccctcag
ccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagca-
ccaagaagtgt
gcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtc-
gcccacgatc
acctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcc-
tgtgaacca
ctccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgag-
actggatcg
agggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagacc-
agcggccc
gcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcct-
gcctgatcc
agaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagc-
acgacgcag
ccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaa-
agatgagttc
atctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaa-
agcggatcctt cgaa AA human IgE Fc (CH2-CH3-CH4) ORF (SEQ ID NO:
623):
dhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgfinitwledgqvmdvdlstasttqegelast
qseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdlfirksptitclvvdlapsk-
gtvnltwsrasgk
pvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrsttktsgpraapevyafatpewp-
gsrdkrtlacliq
nfmpedisvqwlhnevqlpdarhsttpqrktkgsgffvfsrlevtraeweqkdeficravheaaspsqtvqrav-
svnpgkadps NT 1D8 scFv-human IgE Fc (CH2-CH3-CH4)-CD80 (SEQ ID NO:
624)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgtgctcactcagtctccaacaaccatagctgcatctccaggggagaaggtcaccatcacctgccgtgcc-
agctccagtgtaa
gttacatgtactggtaccagcagaagtcaggcgcctcccctaaactctggatttatgacacatccaagctggct-
tctggagttccaaa
tcgcttcagtggcagtgggtctgggacctcttattctctcgcaatcaacaccatggagactgaagatgctgcca-
cttattactgtcagc
agtggagtagtactccgctcacgttcgggtctgggaccaagctggagatcaaacggggtggcggtggctcgggc-
ggtggtgggt
cgggtggcggcggatctcaggtgcagctgaaggaggcaggacctggcctggtgcaaccgacacagaccctgtcc-
ctcacatgc
actgtctctgggttctcattaaccagcgatggtgtacactggattcgacagcctccaggaaagggtctggaatg-
gatgggaataatat
attatgatggaggcacagattataattcagcaattaaatccagactgagcatcagcagggacacctccaagagc-
caagttttcttaaa
aatcaacagtctgcaaactgatgacacagccatgtattactgtgccagaatccactttgattactggggccaag-
gagtcatggtcaca
gtctcctctgatcacgtctgctccagggacttcaccccgcccaccgtgaagatcttacagtcgtcctgcgacgg-
cggcgggcacttc
cccccgaccatccagctcctgtgcctcgtctctgggtacaccccagggactatcaacatcacctggctggagga-
cgggcaggtca
tggacgtggacttgtccaccgcctctaccacgcaggagggtgagctggcctccacacaaagcgagctcaccctc-
agccagaagc
actggctgtcagaccgcacctacacctgccaggtcacctatcaaggtcacacctttgaggacagcaccaagaag-
tgtgcagattcc
aacccgagaggggtgagcgcctacctaagccggcccagcccgttcgacctgttcatccgcaagtcgcccacgat-
cacctgtctgg
tggtggacctggcacccagcaaggggaccgtgaacctgacctggtcccgggccagtgggaagcctgtgaaccac-
tccaccaga
aaggaggagaagcagcgcaatggcacgttaaccgtcacgtccaccctgccggtgggcacccgagactggatcga-
gggggaga
cctaccagtgcagggtgacccacccccacctgcccagggccctcatgcggtccacgaccaagaccagcggcccg-
cgtgctgcc
ccggaagtctatgcgtttgcgacgccggagtggccggggagccgggacaagcgcaccctcgcctgcctgatcca-
gaacttcatg
cctgaggacatctcggtgcagtggctgcacaacgaggtgcagctcccggacgcccggcacagcacgacgcagcc-
ccgcaaga
ccaagggctccggcttcttcgtcttcagccgcctggaggtgaccagggccgaatgggagcagaaagatgagttc-
atctgccgtgc
agtccatgaggcagcgagcccctcacagaccgtccagcgagcggtgtctgtaaatcccggtaaagcggatcctt-
cgaagctccca
tcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctactgctttgccccaag-
atgcagagagagaag gaggaatgagagattgagaagggaaagtgtacgccctgtataaatcgata
AA 1D8-scFv-human IgE Fc (CH2-CH3-CH4)-CD80 (SEQ ID NO: 625)
mdfqvqifsfllisasvimsrgvdivltqspttiaaspgekvtitcrasssvsymywyqqksgaspklwiy
dtsklasgvpnrfsgsgsgtsyslaintmetedaatyycqqwsstpltfgsgtkleikrggggsggggsggggs-
qvqlkeagpg
lvqptqtlsltctvsgfsltsdgvhwirqppgkglewmgiiyydggtdynsaiksrlsisrdtsksqvflkins-
lqtddtamyyca
rihfdywgqgvmvtvssdhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgtinitwledgqvmdv-
dlstasttqe
gelastqseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdlfirksptitclvv-
dlapskgtvnltws
rasgkpvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrsttktsgpraapevyafa-
tpewpgsrdkrt
lacliqnfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweqkdeficravheaaspsq-
tvqravsvnp gkadpsklpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT
5B9-IgAH IgA-T4-CD80 (SEQ ID NO: 626)
aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcaga
tattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtcta-
gtaagagtctcctaca
tagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcaga-
tgtccaaccttgcctca
ggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctga-
ggatgtgggtg
tttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggt-
ggcggtggctcgg
gcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcc-
tcacagagc
ctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccagg-
aaagggtctggagtg
gctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaagg-
acgattccaaga
gccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggt-
gataactacccttatt
actatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcagccagttccctcaactcca-
cctaccccatctcc
ctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacc-
tgctcttaggttc
agaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaa-
gtgggaagag
cgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccg-
agccatggaacc
atgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatcc-
ggaaacacattc
cggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcct-
ggcacgtgg
cttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgactt-
gggcatcccgg
caggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaa-
gggggaca
ccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggt-
aaacccaccca
tgtcaatgtgtctgttgtcatggcggaggtggacgcggatccttcgaacaacctgctcccatcctgggccatta-
ccttaatctcagtaa
atggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagaga-
ttgagaagggaa agtgtacgccctgtataaatcgatac AA 5B9-IgAH IgA-T4-CD80
(SEQ ID NO: 627)
mrfsaqllgllvlwipgstadivmtqaafsnpvtlgtsasiscrssksllhsngitylywylqkpgqspqlli
yqmsnlasgvpdrfsssgsgtdftlrisrveaedvgvyycaqnlelpltfgagtklelkrggggsggggsgggg-
ssqvqlkqsg
pglvqssqslsitctvsgfslttyavhwvrqspgkglewlgviwsggitdynaafisrlsitkddsksqvffkm-
nslqpndtaiy
ycarnggdnypyyyamdywgqgtsvtvssdqpvpstpptpspstpptpspscchprlslhrpaledlllgseai-
ltctltglrda
sgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpe-
vhllpppseel
alnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscm-
vghealplaftqk
tidrlagkpthvnvsvvmaevdadpsnnllpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv
NT 5B9-scFv-human IgE Fc (CH2-CH3-CH4)-CD80 (SEQ ID NO: 628)
aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcaga
tattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtcta-
gtaagagtctcctaca
tagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcaga-
tgtccaaccttgcctca
ggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctga-
ggatgtgggtg
tttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggt-
ggcggtggctcgg
gcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcc-
tcacagagc
ctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccagg-
aaagggtctggagtg
gctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaagg-
acgattccaaga
gccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggt-
gataactacccttatt
actatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcacgtctgctccagggacttc-
accccgcccaccg
tgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctct-
gggtacacccca
gggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgca-
ggagggtgag
ctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggt-
cacctatcaag
gtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccgg-
cccagcccgt
tcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtg-
aacctgacctg
gtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccg-
tcacgtcca
ccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgccc-
agggccct
catgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggc-
cggggagcc
gggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaac-
gaggtgcagct
cccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctgg-
aggtgacca
gggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtc-
cagcgagcg
gtgtctgtaaatcccggtaaagcggatccttcgaagctcccatcctgggccattaccttaatctcagtaaatgg-
aatttttgtgatatgct
gcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgc-
cctgtataaat cgata AA 5B9-scFv-human IgE Fc (CH2-CH3-CH4)-CD80 (SEQ
ID NO: 629)
mrfsaqllgllvlwipgstadivmtqaafsnpvtlgtsasiscrssksllhsngitylywylqkpgqspqlli
yqmsnlasgvpdrfsssgsgtdftlrisrveaedvgvyycaqnlelpltfgagtklelkrggggsggggsgggg-
ssqvqlkqsg
pglvqssqslsitctvsgfslttyavhwvrqspgkglewlgviwsggitdynaafisrlsitkddsksqvffkm-
nslqpndtaiy
ycamggdnypyyyamdywgqgtsvtvssdhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgtini-
twledgqv
mdvdlstasttqegelastqseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdl-
firksptitclvvd
lapskgtvnltwsrasgkpvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrsttkt-
sgpraapevyaf
atpewpgsrdkrtlacliqnfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweqkdef-
icravheaasp
sqtvqravsvnpgkadpsklpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv NT
2e12-scFv-IgAH IgA-T4-CD80 (SEQ ID NO: 630)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgtgctcacccaatctccagctcttggctgtgtctctaggtcagagagccaccatctcctgcagagccag-
tgaaagtgttgaa
tattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgc-
atccaacgtagaat
ctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggag-
gatgatattgcaa
tgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggt-
ggcggtggctcg
ggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctc-
acagagcc
tgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccagga-
aagggtctggagt
ggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaag-
gacaactccaa
gagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggtt-
atagtaactttcatt
actatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatcagccagttccctcaactcca-
cctaccccatctcc
ctcaactccacctaccccatctccctcatgctgccacccccgactgtcactgcaccgaccggccctcgaggacc-
tgctcttaggttc
agaagcgatcctcacgtgcacactgaccggcctgagagatgcctcaggtgtcaccttcacctggacgccctcaa-
gtgggaagag
cgctgttcaaggaccacctgaccgtgacctctgtggctgctacagcgtgtccagtgtcctgccgggctgtgccg-
agccatggaacc
atgggaagaccttcacttgcactgctgcctaccccgagtccaagaccccgctaaccgccaccctctcaaaatcc-
ggaaacacattc
cggcccgaggtccacctgctgccgccgccgtcggaggagctggccctgaacgagctggtgacgctgacgtgcct-
ggcacgtgg
cttcagccccaaggatgtgctggttcgctggctgcaggggtcacaggagctgccccgcgagaagtacctgactt-
gggcatcccgg
caggagcccagccagggcaccaccaccttcgctgtgaccagcatactgcgcgtggcagccgaggactggaagaa-
gggggaca
ccttctcctgcatggtgggccacgaggccctgccgctggccttcacacagaagaccatcgaccgcttggcgggt-
aaacccaccca
tgtcaatgtgtctgttgtcatggcggaggtggacgcggatccttcgaacaacctgctcccatcctgggccatta-
ccttaatctcagtaa
atggaatttttgtgatatgctgcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagaga-
ttgagaagggaa agtgtacgccctgtataaatcgatac AA 2e12-scFv-IgAH
IgA-T4-CD80 (SEQ ID NO: 631)
mdfqvqifsfllisasvimsrgvdivltqspaslavslgqratiscrasesveyyvtslmqwyqqkpgqp
pkllisaasnvesgvparfsgsgsgtdfslnihpveeddiamyfcqqsrkvpwtfgggtkleikrggggsgggg-
sggggsqvq
lkesgpglvapsqslsitctvsgfsltgygvnwvrqppgkglewlgmiwgdgstdynsalksrlsitkdnsksq-
vflkmnslqt
ddtaryycardgysnfhyyvmdywgqgtsvtvssdqpvpstpptpspstpptpspscchprlslhrpaledlll-
gseailtctltg
lrdasgvtftwtpssgksavqgppdrdlcgcysvssvlpgcaepwnhgktftctaaypesktpltatlsksgnt-
frpevhllppps
eelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtf-
scmvghealplaf
tqktidrlagkpthvnvsvvmaevdadpsnnllpswaitlisvngifviccltycfaprcrerrrnerlrresv-
rpv NT 2e12-scFv-human IgE Fc (CH2-CH3-CH4)-CD80 (SEQ ID NO: 632)
aagcttatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagt-
cg
acattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagcc-
agtgaaagtgttgaa
tattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgc-
atccaacgtagaat
ctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggag-
gatgatattgcaa
tgtatttctgtcagcaaagtaggaaggttccttggacgttcggtggaggcaccaagctggaaatcaaacggggt-
ggcggtggctcg
ggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctc-
acagagcc
tgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccagga-
aagggtctggagt
ggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactgagcatcaccaag-
gacaactccaa
gagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgccagagatggtt-
atagtaactttcatt
actatgttatggactactggggtcaaggaacctcagtcaccgtctcctcagatcacgtctgctccagggacttc-
accccgcccaccg
tgaagatcttacagtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctct-
gggtacacccca
gggactatcaacatcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgca-
ggagggtgag
ctggcctccacacaaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggt-
cacctatcaag
gtcacacctttgaggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccgg-
cccagcccgt
tcgacctgttcatccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtg-
aacctgacctg
gtcccgggccagtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccg-
tcacgtcca
ccctgccggtgggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgccc-
agggccct
catgcggtccacgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggc-
cggggagcc
gggacaagcgcaccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaac-
gaggtgcagct
cccggacgcccggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctgg-
aggtgacca
gggccgaatgggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtc-
cagcgagcg
gtgtctgtaaatcccggtaaagcggatccttcgaagctcccatcctgggccattaccttaatctcagtaaatgg-
aatttttgtgatatgct
gcctgacctactgctttgccccaagatgcagagagagaaggaggaatgagagattgagaagggaaagtgtacgc-
cctgtataaat cgata AA 2e12-scFv-human IgE Fc (CH2-CH3-CH4)-CD80 (SEQ
ID NO: 633)
mdfqvqifsfllisasvimsrgvdivltqspaslavslgqratiscrasesveyyvtslmqwyqqkpgqp
pkllisaasnvesgvparfsgsgsgtdfslnihpveeddiamyfcqqsrkvpwtfgggtkleikrggggsgggg-
sggggsqvq
lkesgpglvapsqslsitctvsgfsltgygvnwvrqppgkglewlgmiwgdgstdynsalksrlsitkdnsksq-
vflkmnslqt
ddtaryycardgysnfhyyvmdywgqgtsvtvssdhvcsrdftpptvkilqsscdggghfpptiqllclvsgyt-
pgtinitwle
dgqvmdvdlstasttqegelastqseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrps-
pfdlfirksptitc
lvvdlapskgtvnltwsrasgkpvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrvthphlpralmrs-
ttktsgpraape
vyafatpewpgsrdkrtlacliqnfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweq-
kdeficravhe
aaspsqtvqraysvnpgkadpsklpswaitlisvngifviccltycfaprcrerrrnerlrresvrpv
NT 500A2 scFv (SEQ ID NO: 634)
atgttgtatacatctcagctccttgggcttttactcttctggatttcagcctccagaagtgacatagtgctgac-
tca
gactccagccactctgtctctaattcctggagaaagagtcacaatgacctgtaagaccagtcagaatattggca-
caatcttacactgg
tatcaccaaaaaccaaaggaggctccaagggctctcatcaagtatgcttcgcagtccattcctgggatcccctc-
cagattcagtggc
agtggttcggaaacagatttcactctcagcatcaataacctggagcctgatgatatcggaatttattactgtca-
acaaagtagaagctg
gcctgtcacgttcggtcctggcaccaagctggagataaaacggggtggcggtggctcgggcggaggtgggtcgg-
gtggcggcg
gatctcaggtcaagctgcagcagtccggttctgaactagggaaacctggggcctcagtgaaactgtcctgcaag-
acttcaggctac
atattcacagatcactatatttcttgggtgaaacagaagcctggagaaagcctgcagtggataggaaatgttta-
tggtggaaatggtg
gtacaagctacaatcaaaaattccagggcaaggccacactgactgtagataaaatctctagcacagcctacatg-
gaactcagcagc
ctgacatctgaggattctgccatctattactgtgcaagaaggccggtagcgacgggccatgctatggactactg-
gggtcaggggat ccaagttaccgtctcctctgatc AA 500A2 scFv (SEQ ID NO: 635)
mlytsqllglllfwisasrsdivltqtpatlslipgervtmtcktsqnigtilhwyhqkpkeapralikyasqs-
i
pgipsrfsgsgsetdftlsinnlepddigiyycqqsrswpvtfgpgtkleikrggggsggggsggggsqvklqq-
sgselgkpga
svklscktsgyiftdhyiswvkqkpgeslqwignvyggnggtsynqkfqgkatltvdkisstaymelssltsed-
saiyycarrp vatghamdywgqgiqvtvssd 5' oligo: Name: hIgAbcl5 (SEQ ID
NO: 636) Sequence: GTTGTTGATCAGCCAGTTCCCTCAACTCCACCTACC 3' oligo:
Name: IgA3BB (SEQ ID NO: 637)
GTTGTTTTCGAAGGATCCGCGTCCACCTCCGCCATGACAACAGA 5' oligo: Name: IgGWT3
(SEQ ID NO: 638) GTTGTTTTCGAAGGATCCGCTTTACCCGGAGACAGGGAGAGGCT CTT
3' oligo: Name: hIgGWT5 (SEQ ID NO: 639)
GTTGTTAGATCTGGAGCCCAAATCTTGTGACAAAACTCACACATG 5' oligo: Name: FADD5
(SEQ ID NO: 640) Sequence:
GTTGTGGATCCTTCGAACCCGTTCCTGGTGCTGCTGCACTCGGTGTCG 3' oligo: Name:
FADD3 (SEQ ID NO: 641) Sequence:
GTTGTTATCGATCTCGAGTTATCAGGACGCTTCGGAGGTAGATGCGTC FADD-CSSCFV (SEQ
ID NO: 642):
Gtggatccttcgaacccgttcctggtgctgctgcactcggtgtcgtccagcctgtcgagcagcgagctgacc
gagctcaagttcctatgcctcgggcgcgtgggcaagcgcaagctggagcgcgtgcagagcggcctagacctctt-
ctccatgctgc
tggagcagaacgacctggagcccgggcacaccgagctcctgcgcgagctgctcgcctccctgcggcgccacgac-
ctgctgcg
gcgcgtcgacgacttcgaggcgggggcggcggccggggccgcgcctggggaagaagacctgtgtgcagcattta-
acgtcatat
gtgataatgtggggaaagattggagaaggctggctcgtcagctcaaagtctcagacaccaagatcgacagcatc-
gaggacagata
cccccgcaacctgacagagcgtgtgcgggagtcactgagaatctggaagaacacagagaaggagaacgcaacag-
tggcccac
ctggtgggggctctcaggtcctgccagatgaacctggtggctgacctggtacaagaggttcagcaggcccgtga-
cctccagaaca
ggagtggggccatgtccccgatgtcatggaactcagacgcatctacctccgaagcgtcctgataactcgagatc-
gataacaac Peptide sequence (SEQ ID NO: 643):
vdpsnpflvllhsvssslssseltelkflclgrvgkrklervqsgldlfsmlleqndlepghtellrellaslr-
rh
dllrrvddfeagaaagaapgeedlcaafnvicdnvgkdwrrlarqlkvsdtkidsiedryprnltervreslri-
wkntekenatva hlvgalrscqmnlvadlvqevqqardlqnrsgamspmswnsdastseas
Name: HCD28tm5B (SEQ ID NO: 644)
GTTGTGGATCCTCCCTTTTGGGTGCTGGTGGTGGTTGGTGTCCTG GCTTGCTATAGCTTG Name:
HCD28tm3S (SEQ ID NO: 645)
GTTGTTTCGAACCCAGAAAATAATAAAGGCCACTGTTACTAGCA AGCTATAGCAAGCCAG
HCD28tm5' (SEQ ID NO: 646) GTTGTGGATCCTCCCTTTTGGGTGCTGGTGGT
HCD28tm3' (SEQ ID NO: 647) GTTGTTTCGAACCCAGAAAATAATAAAGGCCAC
HCD80tm5' (SEQ ID NO: 648) GTTGTGGATCCTCCTGCTCCCATCCTGG HCD80tm3'
(SEQ ID NO: 649) GTTGTTTCGAACGGCAAAGCAGTAGGTCAGGC Name: MFADD5BB
(SEQ ID NO: 650) Sequence:
GTTGTGGATCCTTCGAACCCATTCCTGGTGCTGCTGCACTCGCTG Name: MFADD3XC (SEQ
ID NO: 651) Sequence: GTTGTTATCGATCTCGAGTCAGGGTGTTTCTGAGGAAGACAC
Murine FADD Nucleotide sequence (full length, but without
flanking-Ig or transmembrane sequences) (SEQ ID NO: 652):
Gtggatccttcgaacatggacccattcctggtgctgctgcactcgctgtccggcagcctgtcgggcaacgat
ctgatggagctcaagttcttgtgccgcgagcgcgtgagcaaacgaaagctggagcgcgtgcagagtggcctgga-
cctgttcacg
gtgctgctggagcagaacgacctggagcgcgggcacaccgggctgctgcgcgagttgctggcctcgctgcgccg-
acacgatct
actgcagcgcctggacgacttcgaggcggggacggcgaccgctgcgcccccgggggaggcagatctgcaggtgg-
catttgac
attgtgtgtgacaatgtggggagagactggaaaagactggcccgcgagctgaaggtgtctgaggccaagatgga-
tgggattgag
gagaagtacccccgaagtctgagtgagcgggtaagggagagtctgaaagtctggaagaatgctgagaagaagaa-
cgcctcggt
ggccggactggtcaaggcgctgcggacctgcaggctgaatctggtggctgacctggtggaagaagcccaggaat-
ctgtgagca
agagtgagaatatgtccccagtactaagggattcaactgtgtcttcctcagaaacaccctgactcgagatcgat
Murine FADD (SEQ ID NO: 653)
vdpsnmdpflvllhslsgslsgndlmelkflcrervskrklervqsgldlftvlleqndlerghtgllrellas-
l
rrhdllqrlddfeagtataappgeadlqvafdivcdnvgrdwkrlarelkvseakmdgieekyprslservres-
lkvwknaekk nasvaglvkalrtcrinlvadlveeaqesysksenmspvlrdstvsssetp
Name: MCASP3-5 (SEQ ID NO: 654) Sequence:
GTTGTGGATCCTTCGAACATGGAGAACAACAAAACCTCAGTGGATTCA Name: MCASP3-3
(SEQ ID NO: 655) Sequence:
GTTGTTATCGATCTCGAGCTAGTGATAAAAGTACAGTTCTTTCGT Name: mcasp8-5 (SEQ
ID NO: 656) Sequence:
GTTGTTTCGAACATGGATTTCCAGAGTTGTCTTTATGCTATTGCTG Name: mcasp8-3 (SEQ
ID NO: 657) Sequence:
GTTGTTATCGATCTCGAGTCATTAGGGAGGGAAGAAGAGCTTCTTCCG Name: hcasp3-5
(SEQ ID NO: 658) Sequence:
GTTGTGGATCCTTCGAACATGGAGAACACTGAAAACTCAGTGGAT Name: hcasp3-3 (SEQ
ID NO: 659) Sequence:
GTTGTTATCGATCTCGAGTTAGTGATAAAAATAGAGTTCTTTTGTGAG Name: hcasp8-5
(SEQ ID NO: 660) Sequence:
GTTGTGGATCCTTCGAACATGGACTTCAGCAGAAATCTTTATGAT Name: hcasp8-3 (SEQ
ID NO: 661)
Sequence: GTTGTTATCGATGCATGCTCAATCAGAAGGGAAGACAAGTTTTTTTCT
HuIgGMHWC (SEQ ID NO: 662) gtt gtt gat cag gag ccc aaa tct tct gac
aaa act cac aca tct cca ccg tcc cca gca cct gaa ctc ctg ggt gga ccg
tca gtc ttc c NT 2H7-human IgE (CH2-CH3-CH4) (SEQ ID NO: 663)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaaggtggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctctgatcacgtctgctccagggacttcaccccgcccaccgt-
gaagatcttaca
gtcgtcctgcgacggcggcgggcacttccccccgaccatccagctcctgtgcctcgtctctgggtacaccccag-
ggactatcaac
atcacctggctggaggacgggcaggtcatggacgtggacttgtccaccgcctctaccacgcaggagggtgagct-
ggcctccaca
caaagcgagctcaccctcagccagaagcactggctgtcagaccgcacctacacctgccaggtcacctatcaagg-
tcacacctttg
aggacagcaccaagaagtgtgcagattccaacccgagaggggtgagcgcctacctaagccggcccagcccgttc-
gacctgttca
tccgcaagtcgcccacgatcacctgtctggtggtggacctggcacccagcaaggggaccgtgaacctgacctgg-
tcccgggcca
gtgggaagcctgtgaaccactccaccagaaaggaggagaagcagcgcaatggcacgttaaccgtcacgtccacc-
ctgccggtg
ggcacccgagactggatcgagggggagacctaccagtgcagggtgacccacccccacctgcccagggccctcat-
gcggtcca
cgaccaagaccagcggcccgcgtgctgccccggaagtctatgcgtttgcgacgccggagtggccggggagccgg-
gacaagcg
caccctcgcctgcctgatccagaacttcatgcctgaggacatctcggtgcagtggctgcacaacgaggtgcagc-
tcccggacgcc
cggcacagcacgacgcagccccgcaagaccaagggctccggcttcttcgtcttcagccgcctggaggtgaccag-
ggccgaatg
ggagcagaaagatgagttcatctgccgtgcagtccatgaggcagcgagcccctcacagaccgtccagcgagcgg-
tgtctgtaaat cccggtaaatgataatctaga AA 2H7 scFv IgE (CH2-CH3-CH4)
(SEQ ID NO: 664)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkggggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdywgtgttvtvsdhvcsrdftpptvkilqsscdggghfpptiqllclvsgytpgtin-
itwledgqvm
dvdlstasttqegelastqseltlsqkhwlsdrtytcqvtyqghtfedstkkcadsnprgvsaylsrpspfdlf-
irksptitclvvdla
pskgtvnltwsrasgkpvnhstrkeekqrngtltvtstlpvgtrdwiegetyqcrythphlpralmrsttktsg-
praapevyafat
pewpgsrdkrtlacliqnfmpedisvqwlhnevqlpdarhsttqprktkgsgffvfsrlevtraeweqkdefic-
ravheaasps qtvqravsvnpgk NT 2H7 scFv MH (SSS) MCH2WTCH3 (SEQ ID NO:
665)
aagcttgccgccatggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataattgccag-
ag
gacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagg-
gccagctcaagtg
taagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctg-
gcttctggagtcc
ctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgct-
gccacttattactg
ccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgg-
gcggtggtgg
atctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagctggtgaggcctggggcctcag-
tgaagatgtcct
gcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaa-
tggattggagct
atttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatc-
ctccagcacagcc
tacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaa-
ctcttactggtacttc
gatgtctggggcacagggaccacggtcaccgtctcttctgatcaggagcccaaatcttctgacaaaactcacac-
atccccaccgtc
cccagcacctgaactcctggggggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatct-
cccggacccctg
aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg-
gaggtgcata
atgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcac-
caggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaa-
agccaaaggg
cagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgac-
ctgcctggtc
aaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac-
gcctcccgt
gctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacg-
tcttctcatgct
ccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga
AA 2H7 scFv MH (SSS) MCH2WTCH3 (SEQ ID NO: 666)
mdfqvqifsfllisasviiargqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiy
apsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggss-
qaylqqsgae
lvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqls-
sltsedsa
vyfcarvvyysnsywyfdvwgtgttvtvssdqepkssdkthtsppspapellggssvflfppkpkdtlmisrtp-
evtcvvvdv
shedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkcykekvsnkalpapiektiskak-
gqprep
qvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqg-
nvfscsvmh ealhnhytqkslslspgk NT 5B9 scFv MTHWTCH2CH3 (SEQ ID NO:
667)
aagcttgccgccatgaggttctctgctcagcttctggggctgcttgtgctctggatccctggatccactgcaga
tattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcaggtcta-
gtaagagtctcctaca
tagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcaga-
tgtccaaccttgcctca
ggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctga-
ggatgtgggtg
tttattactgtgctcaaaatctagaacttccgctcacgttcggtgctgggaccaagctggagctgaaacggggt-
ggcggtggctcgg
gcggtggtgggtcgggtggcggcggatcgtcacaggtgcagctgaagcagtcaggacctggcctagtgcagtcc-
tcacagagc
ctgtccatcacctgcacagtctctggtttctcattaactacctatgctgtacactgggttcgccagtctccagg-
aaagggtctggagtg
gctgggagtgatatggagtggtggaatcacagactataatgcagctttcatatccagactgagcatcaccaagg-
acgattccaaga
gccaagttttctttaaaatgaacagtctgcaacctaatgacacagccatttattactgtgccagaaatgggggt-
gataactacccttatt
actatgctatggactactggggtcaaggaacctcagtcaccgtctcctctgatcaggagcccaaatcttctgac-
aaaactcacacatc
cccaccgtccccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccc-
tcatgatctccc
ggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg-
gacggcgtgg
aggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcacc-
gtcctgcacc
aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaca-
atctccaaag
ccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtc-
agcctgacc
tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta-
caagaccac
gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagc-
aggggaacgtc
ttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa-
atgatctaga AA 5B9 scFv MTHWTCH2CH3 (SEQ ID NO: 668)
mrfsaqllgllvlwipgstadivmtqaafsnpvtlgtsasiscrssksllhsngitylywylqkpgqspqlli
yqmsnlasgvpdrfsssgsgtdftlrisrveaedvgvyycaqnlelpltfgagtklelkrggggsggggsgggg-
ssqvqlkqsg
pglvqssqslsitctvsgfslttyavhwvrqspgkglewlgviwsggitdynaafisrlsitkddsksqvffkm-
nslqpndtaiy
ycarnggdnypyyyamdywgqgtsvtvssdqepkssdkthtsppspapellggpsvflfppkpkdtlmisrtpe-
vtcvvvd
vshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiska-
kgqpre
pqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqq-
gnvfscsvm
healhnhytqkslslspgk
[0334] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for the purpose of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the present invention is not limited except as by the
appended claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140010809A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140010809A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References