U.S. patent application number 14/202999 was filed with the patent office on 2014-09-18 for antigen binding constructs to cd8.
This patent application is currently assigned to IMAGINAB, INC.. The applicant listed for this patent is IMAGINAB, INC.. Invention is credited to Giti Agahi, Christian P. Behrenbruch, David T. Ho, Tove Olafsen.
Application Number | 20140271462 14/202999 |
Document ID | / |
Family ID | 51527868 |
Filed Date | 2014-09-18 |
United States Patent
Application |
20140271462 |
Kind Code |
A1 |
Ho; David T. ; et
al. |
September 18, 2014 |
ANTIGEN BINDING CONSTRUCTS TO CD8
Abstract
Antigen binding constructs that bind to CD8, for example
antibodies, including antibody fragments (such as scFv, minibodies,
and cys-diabodies) that bind to CD8, are described herein. Methods
of use are described herein.
Inventors: |
Ho; David T.; (Long Beach,
CA) ; Olafsen; Tove; (Reseda, CA) ; Agahi;
Giti; (Los Angeles, CA) ; Behrenbruch; Christian
P.; (Inglewood, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IMAGINAB, INC. |
Inglewood |
CA |
US |
|
|
Assignee: |
IMAGINAB, INC.
Inglewood
CA
|
Family ID: |
51527868 |
Appl. No.: |
14/202999 |
Filed: |
March 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61780286 |
Mar 13, 2013 |
|
|
|
Current U.S.
Class: |
424/1.49 ;
424/178.1; 435/328; 435/7.92; 530/387.3; 530/391.3; 530/391.7;
536/23.53 |
Current CPC
Class: |
A61K 47/6849 20170801;
C07K 2317/56 20130101; C07K 2317/624 20130101; C07K 2317/626
20130101; G01N 2333/70517 20130101; A61K 2039/505 20130101; G01N
33/566 20130101; C07K 2317/92 20130101; C07K 2317/24 20130101; A61K
51/1027 20130101; C07K 2317/565 20130101; G01N 33/6872 20130101;
C07K 2317/31 20130101; C07K 2317/35 20130101; C07K 16/2815
20130101; C07K 2317/622 20130101 |
Class at
Publication: |
424/1.49 ;
530/391.3; 530/391.7; 530/387.3; 536/23.53; 424/178.1; 435/328;
435/7.92 |
International
Class: |
C07K 16/28 20060101
C07K016/28; G01N 33/53 20060101 G01N033/53; A61K 51/10 20060101
A61K051/10; A61K 47/48 20060101 A61K047/48 |
Claims
1. An antigen binding construct that comprises: a HCDR1 of a HCDR1
of SEQ ID NO: 3 or 6; a HCDR2 of HCDR1 of SEQ ID NO: 3 or 6; a
HCDR3 of HCDR1 of SEQ ID NO: 3 or 6; a LCDR1 of LCDR1 of SEQ ID NO:
9; a LCDR2 of LCDR1 of SEQ ID NO: 9; and a LCDR3 of LCDR1 of SEQ ID
NO: 9.
2. The antigen binding construct of claim 1, wherein the antigen
binding construct binds specifically to CD8.
3. The antigen binding construct of claim 1, further comprising a
detectable marker.
4. The antigen binding construct of claim 1, further comprising a
therapeutic agent.
5. The antigen binding construct of claim 1, wherein the antigen
binding construct is bispecific.
6. The antigen binding construct of claim 1, wherein the antigen
binding construct is a monovalent scFv.
7. A humanized cys-diabody that binds to CD8, the humanized
cys-diabody comprising a polypeptide that comprises: a single-chain
variable fragment (scFv) comprising a variable heavy (V.sub.H)
domain linked to a variable light (V.sub.L) domain; and a
C-terminal cysteine.
8. The humanized cys-diabody of claim 7, wherein the order of the
variable domains, from N terminus to C terminus of the polypeptide
is V.sub.L, V.sub.H.
9. The humanized cys-diabody of claim 7, wherein the order of the
variable domains, from N terminus to C terminus of the polypeptide
is V.sub.H, V.sub.L.
10. The humanized cys-diabody of claim 7, further comprising a
detectable molecule.
11. The humanized cys-diabody of claim 7, wherein the humanized
cys-diabody comprises: a HCDR1 of a HCDR1 of SEQ ID NO: 3 or 6; a
HCDR2 of HCDR1 of SEQ ID NO: 3 or 6; a HCDR3 of HCDR1 of SEQ ID NO:
3 or 6; a LCDR1 of LCDR1 of SEQ ID NO: 9; a LCDR2 of LCDR1 of SEQ
ID NO: 9; and a LCDR3 of LCDR1 of SEQ ID NO: 9.
12. A humanized minibody that binds to CD8, the humanized minibody
comprising a polypeptide that comprises from N-terminus to
C-terminus: a single-chain variable fragment (scFv) that binds to
CD8, the scFv comprising a variable heavy (V.sub.H) domain linked
to a variable light (V.sub.L) domain; a hinge-extension domain
comprising a human IgG1 hinge region; and a human IgG C.sub.H3
sequence.
13. The humanized minibody of claim 12, further comprising a
detectable marker.
14. The humanized minibody of claim 12, wherein the humanized
minibody comprises: a HCDR1 of a HCDR1 of SEQ ID NO: 3 or 6; a
HCDR2 of HCDR1 of SEQ ID NO: 3 or 6; a HCDR3 of HCDR1 of SEQ ID NO:
3 or 6; a LCDR1 of LCDR1 of SEQ ID NO: 9; a LCDR2 of LCDR1 of SEQ
ID NO: 9; and a LCDR3 of LCDR1 of SEQ ID NO: 9.
15. A nucleic acid encoding an antibody of claim 1.
16. A cell line producing an antibody of claim 1.
17. A kit comprising: an antigen binding construct of claim 1; and
a detectable marker.
18. A method of detecting a presence or absence of a CD8, the
method comprising: applying the antigen binding construct of claim
1 to a sample; and detecting a presence or an absence of the
antigen binding construct, thereby detecting a presence of absence
of a CD8.
19. The method of claim 18, wherein the antigen binding construct
is conjugated to a detectable marker.
20. The method of claim 18, wherein applying the antigen binding
construct comprises administering the antigen binding construct to
a subject.
21. The method of claim 18, wherein detecting binding or absence of
binding of the antigen binding construct to CD8 comprises at least
one of positron emission tomography or single-photon emission
computed tomography.
22. The method of claim 18, the method further comprising applying
a secondary antigen binding construct to the sample, wherein the
secondary antigen binding construct binds specifically to the
antigen binding construct.
23. The method of claim 18, wherein the antigen binding construct
is incubated with the sample for no more than 20 hours.
24. The method of claim 18, wherein the antigen binding construct
is incubated with the sample for no more than 6 hours.
25. The method of claim 18, wherein the antigen binding construct
is administered to a host, and wherein a first quantity of antigen
binding construct thereof is unbound to CD8, and a second quantity
of antigen binding construct is bound to CD8, wherein at least
about 80% of the first quantity of antigen binding construct is
eliminated in no more than 12 hours.
26. A method of targeting a therapeutic agent to a CD8, the method
comprising administering to a subject an antigen binding construct
of claim 1, wherein the antigen binding construct is conjugated to
a therapeutic agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Provisional Application No. 61/780,286, filed Mar. 13, 2013, which
is hereby incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM
LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled SeqListIGNAB014A.TXT, created on Feb. 28, 2014, which
is 65,858 bytes in size. The information in the electronic format
of the Sequence Listing is incorporated herein by reference in its
entirety.
FIELD
[0003] Embodiments described herein relate generally to antigen
binding constructs, such as antibodies, including antibody
fragments, that bind to CD8, (such as minibodies, cys-diabodies,
scFv), as well as methods for their use.
BACKGROUND
[0004] CD8 (cluster of differentiation 8) is a transmembrane
glycoprotein which is a specific marker for a subclass of T-cells
(which includes cytotoxic T-cells). CD8 assembles as either a
heterodimer of the CD8 alpha and CD8 beta subunits or a CD8 alpha
homodimer. The assembled dimeric CD8 complex acts as a co-receptor
together with the T-cell receptor (TCR) to recognize antigen
presentation by MHC class I cells. CD8 plays a role in the
development of T-cells and activation of mature T-cells. Changes in
T-cell localization can reflect the progression of an immune
response and can occur over time.
SUMMARY
[0005] Some embodiments provided herein relate to antigen binding
constructs, such as antibodies, including antibody fragments, that
includes a HCDR1 of a HCDR1 sequence in SEQ ID NO: 3 or 6; a HCDR2
of a HCDR2 sequence in SEQ ID NO: 3 or 6; a HCDR3 of a HCDR3
sequence in SEQ ID NO: 3 or 6; a LCDR1 of a LCDR1 sequence in SEQ
ID NO: 9; a LCDR2 of a LCDR2 sequence in SEQ ID NO: 9; and a LCDR3
of a LCDR3 sequence in SEQ ID NO: 9). In some embodiments, the
antigen binding construct binds specifically to CD8. In some
embodiments, the antigen binding construct includes a detectable
marker as described herein. In some embodiments, the antigen
binding construct includes a therapeutic agent as described
herein.
[0006] Some embodiments provided herein relate to a humanized
cys-diabody that binds to CD8. The humanized cys-diabody can
include a polypeptide that comprises from N-terminus to C-terminus:
a single-chain variable fragment (scFv) comprising a variable heavy
(V.sub.H) domain linked to a variable light (V.sub.L) domain; and a
C-terminal cysteine.
[0007] Some embodiments provided herein relate to a humanized
minibody that binds to CD8. The humanized minibody can include a
polypeptide that comprises, from N-terminus to C-terminus: a
single-chain variable fragment (scFv) comprising a variable heavy
(V.sub.H) domain linked to a variable light (V.sub.L) domain; a
hinge-extension domain comprising a human IgG1 hinge region; and a
human IgG C.sub.H3 sequence.
[0008] Some embodiments provided herein relate to a nucleic acid
encoding an antigen binding construct as described herein, for
example a CD8 antibody or antibody fragment.
[0009] Some embodiments provided herein relate to a cell line that
produces an antigen binding construct as described herein, for
example a CD8 antibody or antibody fragment.
[0010] Some embodiments provided herein relate to a kit. The kit
can include an antigen binding construct as described herein, for
example a CD8 antigen binding fragment. In some embodiments, the
kit includes a detectable marker.
[0011] Some embodiments provided herein relate to a method of
detecting the presence or absence of CD8. The method can comprise
applying an antigen binding construct as described herein, for
example a CD8 antigen binding construct to a sample. The method can
include detecting a binding or an absence of binding of the antigen
binding construct to CD8. In some embodiments, the method is
performed in vivo. In some embodiments, the method is performed in
vitro. In some embodiments, part of the method is performed in
vivo, and part of the method is performed in vitro.
[0012] Some embodiments provided herein relate to a method of
targeting a therapeutic agent to a CD8. The method can include
administering to a subject an antigen binding construct as
described herein, for example a CD8 antibody or antibody fragment.
In some embodiments, the antigen binding construct is conjugated to
a therapeutic agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1A illustrates some embodiments of a schematic of a
minibody having bivalent binding to CD8.
[0014] FIG. 1B illustrates some embodiments of a schematic of a
minibody.
[0015] FIG. 1C provides an example of a CD8 alpha.
[0016] FIG. 2A illustrates some embodiments of an alignment of the
murine OKT8 Variable Heavy (V.sub.H) region against a human
antibody and a humanized V.sub.H region (the huOKT8 construct).
[0017] FIG. 2B illustrates some embodiments of an alignment of the
murine OKT8 Variable Light (V.sub.L) region against a humanized
V.sub.L region and the huOKT8 construct.
[0018] FIG. 3A illustrates some embodiments of a schematic of a
cys-diabody showing bivalent binding to an antigen.
[0019] FIG. 3B illustrates a schematic of a cys-diabody showing
bivalent binding to an antigen.
[0020] FIG. 4 illustrates some embodiments of a chimeric OKT8
minibody V.sub.L-V.sub.H sequence.
[0021] FIG. 5 illustrates some embodiments of a chimeric OKT8
minibody V.sub.H-V.sub.L sequence.
[0022] FIG. 6 illustrates some embodiments of a humanized OKT8
minibody V.sub.L-V.sub.H sequence.
[0023] FIG. 7 illustrates some embodiments of a humanized OKT8
minibody V.sub.H-V.sub.L sequence.
[0024] FIG. 8 illustrates some embodiments of a humanized OKT8
cys-diabody V.sub.L-5-V.sub.H sequence.
[0025] FIG. 9 illustrates some embodiments of a humanized OKT8
cys-diabody V.sub.H-5-V.sub.L sequence.
[0026] FIG. 10 illustrates some embodiments of a humanized OKT8
cys-diabody V.sub.L-8-V.sub.H sequence.
[0027] FIG. 11 illustrates some embodiments of a humanized OKT8
cys-diabody V.sub.H-8-V.sub.L sequence.
[0028] FIG. 12A depicts some embodiments of sequences for
cys-diabodies.
[0029] FIG. 12B depicts some embodiments of sequences for
minibodies.
[0030] FIG. 12C depicts some embodiments of sequences for
V.sub.L.
[0031] FIG. 12D depicts some embodiments of sequences for
huV.sub.L.
[0032] FIG. 12E depicts some embodiments of sequences for
V.sub.H.
[0033] FIG. 12F depicts some embodiments of sequences for huV.sub.H
(version "a" from Version 1).
[0034] FIG. 12G depicts some embodiments of sequences for huV.sub.H
(version "b" from Version 1).
[0035] FIG. 12H depicts some embodiments of sequences for huV.sub.H
(version "c" from Version 2).
[0036] FIG. 12I depicts some embodiments of sequences for huV.sub.H
(version "c" from Version 2).
[0037] FIG. 13 illustrates some embodiments of a vector map for
pcDNA.TM. 3.1/myc-His(-) Versions A, B, C.
[0038] FIG. 14 illustrates some embodiments of a method of
detecting a presence or absence of a target.
[0039] FIG. 15 illustrates a western blot analysis of chimeric and
humanized OKT8 minibodies.
[0040] FIG. 16 is a graph displaying binding of the IAb_Mb_CD8
variants to purified rhCD8 by ELISA.
[0041] FIGS. 17A-17D depict the results from the flow cytometry
analysis of the IAb_Mb_CD8 variants.
[0042] FIGS. 18A and 18B are depictions of gels of western blots of
the humanized OKT8 Minibodies.
[0043] FIG. 19 is a graph displaying IAb_Mb_CD8 expression analysis
by ELISA.
[0044] FIG. 20 is a graph depicting the binding of the IAb_Mb_CD8
variants A and B to rhCD8 by ELISA.
[0045] FIG. 21 is a graph depicting the binding of IAb_Mb_CD8
variants C and D to rhCD8 by ELISA.
[0046] FIGS. 22A and 22B are graphs displaying the flow cytometry
analysis of the IAb_Mb_CD8 variants A and B.
[0047] FIGS. 23A and 23B are graphs displaying the flow cytometry
analysis of the IAb_Mb_CD8 variants C and D.
[0048] FIG. 24 depicts an image of a western blot analysis of
IAb_Cys-Dba_CD8 variants.
[0049] FIG. 25 is a graph depicting the binding of IAb_Cys-Dba_CD8
variants to rhCD8 by ELISA.
[0050] FIGS. 26A and 26B display flow cytometry analysis of the
IAb_Cys-Dba_CD8 variants.
[0051] FIG. 27 is a set of graphs depicting the flow cytometry
analysis of the IAb_Cys-Dba_CD8 variants.
DETAILED DESCRIPTION
[0052] Described herein are antigen binding constructs, including
antibodies and fragments thereof, such as cys-diabodies and
minibodies, that bind to a target molecule, CD8. Such antigen
binding constructs can be useful for detecting the presence,
localization, and/or quantities of the target molecule (CD8 and/or
CD8+ cells, for example, certain classes of T-cells). Such antigen
binding constructs can also be useful for targeting therapeutic
agents to cells that express the target molecule. In some
embodiments, methods are provided for detecting the presence or
absence of the target molecule (or "target") using antigen binding
constructs (including antibodies, and constructs such as
cys-diabodies and/or minibodies). In some embodiments, methods are
provided for using the antigen binding constructs for therapeutic
purposes.
DEFINITIONS AND VARIOUS EMBODIMENTS
[0053] "Treating" or "treatment" of a condition may refer to
preventing the condition, slowing the onset and/or rate of
development of the condition, reducing the risk of developing the
condition, preventing and/or delaying the development of symptoms
associated with the condition, reducing or ending symptoms
associated with the condition, generating a complete or partial
regression of the condition, or some combination thereof. The term
"prevent" does not require the absolute prohibition of the disorder
or disease.
[0054] A "therapeutically effective amount" or a "therapeutically
effective dose" is an amount that produces a desired therapeutic
effect in a subject, such as preventing, treating a target
condition, delaying the onset of the disorder and/or symptoms,
and/or alleviating symptoms associated with the condition. This
amount will vary depending upon a variety of factors, including but
not limited to the characteristics of the therapeutic compound
(including activity, pharmacokinetics, pharmacodynamics, and
bioavailability), the physiological condition of the subject
(including age, sex, disease type and stage, general physical
condition, responsiveness to a given dosage, and type of
medication), the nature of the pharmaceutically acceptable carrier
or carriers in the formulation, and/or the route of administration.
One skilled in the clinical and pharmacological arts will be able
to determine a therapeutically effective amount through routine
experimentation, for example by monitoring a subject's response to
administration of a compound and adjusting the dosage accordingly,
given the present disclosure. For additional guidance, see
Remington: The Science and Practice of Pharmacy 21.sup.st Edition,
Univ. of Sciences in Philadelphia (USIP), Lippincott Williams &
Wilkins, Philadelphia, Pa., 2005.
[0055] The term "antigen binding construct" includes all varieties
of antibodies, including binding fragments thereof. Further
included are constructs that include 1, 2, 3, 4, 5, and/or 6 CDRs.
In some embodiments, these CDRs can be distributed between their
appropriate framework regions in a traditional antibody. In some
embodiments, the CDRs can be contained within a heavy and/or light
chain variable region. In some embodiments, the CDRs can be within
a heavy chain and/or a light chain. In some embodiments, the CDRs
can be within a single peptide chain. In some embodiments, the CDRs
can be within two or more peptides that are covalently linked
together. In some embodiments, they can be covalently linked
together by a disulfide bond. In some embodiments, they can be
linked via a linking molecule or moiety. In some embodiments, the
antigen binding proteins are non-covalent, such as a diabody and a
monovalent scFv. Unless otherwise denoted herein, the antigen
binding constructs described herein bind to the noted target
molecule. The term "target" or "target molecule" denotes the CD8
protein. Examples of CD8 proteins are known in the art, and
include, for example the CD8 protein of SEQ ID NO: 24, FIG. 1C.
[0056] The term "antibody" includes, but is not limited to,
genetically engineered or otherwise modified forms of
immunoglobulins, such as intrabodies, chimeric antibodies, fully
human antibodies, humanized antibodies, antibody fragments, and
heteroconjugate antibodies (e.g., bispecific antibodies, diabodies,
triabodies, tetrabodies, etc.). The term "antibody" includes
cys-diabodies and minibodies. Thus, each and every embodiment
provided herein in regard to "antibodies" is also envisioned as
cys-diabody and/or minibody embodiments, unless explicitly denoted
otherwise. The term "antibody" includes a polypeptide of the
immunoglobulin family or a polypeptide comprising fragments of an
immunoglobulin that is capable of noncovalently, reversibly, and in
a specific manner binding a corresponding antigen. An exemplary
antibody structural unit comprises a tetramer. In some embodiments,
a full length antibody can be composed of two identical pairs of
polypeptide chains, each pair having one "light" and one "heavy"
chain (, connected through a disulfide bond. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. For full length chains, the
light chains are classified as either kappa or lambda. For full
length chains, the heavy chains are classified as gamma, mu, alpha,
delta, or epsilon, which in turn define the immunoglobulin classes,
IgG, IgM, IgA, IgD, and IgE, respectively. The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these regions of light and heavy chains respectively. As
used in this application, an "antibody" encompasses all variations
of antibody and fragments thereof. Thus, within the scope of this
concept are full length antibodies, chimeric antibodies, humanized
antibodies, single chain antibodies (scFv), Fab, Fab', and
multimeric versions of these fragments (e.g., F(ab').sub.2) with
the same binding specificity. In some embodiments, the antibody
binds specifically to a desired target.
[0057] "Complementarity-determining domains" or
"complementarity-determining regions ("CDRs") interchangeably refer
to the hypervariable regions of V.sub.L and V.sub.H. The CDRs are
the target protein-binding site of the antibody chains that harbors
specificity for such target protein. In some embodiments, there are
three CDRs (CDR1-3, numbered sequentially from the N-terminus) in
each V.sub.L and/or V.sub.H, constituting about 15-20% of the
variable domains. The CDRs are structurally complementary to the
epitope of the target protein and are thus directly responsible for
the binding specificity. The remaining stretches of the V.sub.L or
V.sub.H, the so-called framework regions (FRs), exhibit less
variation in amino acid sequence (Kuby, Immunology, 4th ed.,
Chapter 4. W.H. Freeman & Co., New York, 2000).
[0058] The positions of the CDRs and framework regions can be
determined using various well known definitions in the art, e.g.,
Kabat (Wu, T. T., E. A. Kabat. 1970. An analysis of the sequences
of the variable regions of Bence Jones proteins and myeloma light
chains and their implications for antibody complementarity. J. Exp.
Med. 132: 211-250; Kabat, E. A., Wu, T. T., Perry, H., Gottesman,
K., and Foeller, C. (1991) Sequences of Proteins of Immunological
Interest, 5th ed., NIH Publication No. 91-3242, Bethesda, Md.),
Chothia Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987);
Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol.
Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol.,
273:927-748 (1997)), ImMunoGeneTics database (IMGT) (on the
worldwide web at imgt.org/) Giudicelli, V., Duroux, P., Ginestoux,
C., Folch, G., Jabado-Michaloud, J., Chaume, D. and Lefranc, M.-P.
IMGT/LIGM-DB, the IMGT.RTM. comprehensive database of
immunoglobulin and T cell receptor nucleotide sequences Nucl. Acids
Res., 34, D781-D784 (2006), PMID: 16381979; Lefranc, M.-P., Pommie,
C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L.,
Thouvenin-Contet, V. and Lefranc, G., IMGT unique numbering for
immunoglobulin and T cell receptor variable domains and Ig
superfamily V-like domains Dev. Comp. Immunol., 27, 55-77 (2003).
PMID: 12477501; Brochet, X., Lefranc, M.-P. and Giudicelli, V.
IMGT/V-QUEST: the highly customized and integrated system for IG
and TR standardized V-J and V-D-J sequence analysis Nucl. Acids
Res, 36, W503-508 (2008); AbM (Martin et al., Proc. Natl. Acad.
Sci. USA, 86:9268-9272 (1989); the contact definition (MacCallum et
al., J. Mol. Biol., 262:732-745 (1996)), and/or the automatic
modeling and analysis tool Honegger A, Phickthun A. (world wide web
at bioc dot uzh dot ch/antibody/Numbering/index dot html).
[0059] The term "binding specificity determinant" or "BSD"
interchangeably refer to the minimum contiguous or non-contiguous
amino acid sequence within a complementarity determining region
necessary for determining the binding specificity of an antibody. A
minimum binding specificity determinant can be within one or more
CDR sequences. In some embodiments, the minimum binding specificity
determinants reside within (i.e., are determined solely by) a
portion or the full-length of the CDR3 sequences of the heavy and
light chains of the antibody. In some embodiments, CDR3 of the
heavy chain variable region is sufficient for the antigen binding
construct specificity.
[0060] An "antibody variable light chain" or an "antibody variable
heavy chain" as used herein refers to a polypeptide comprising the
V.sub.L or V.sub.H, respectively. The endogenous V.sub.L is encoded
by the gene segments V (variable) and J (junctional), and the
endogenous V.sub.H by V, D (diversity), and J. Each of V.sub.L or
V.sub.H includes the CDRs as well as the framework regions. In this
application, antibody variable light chains and/or antibody
variable heavy chains may, from time to time, be collectively
referred to as "antibody chains." These terms encompass antibody
chains containing mutations that do not disrupt the basic structure
of V.sub.L or V.sub.H, as one skilled in the art will readily
recognize. In some embodiments, full length heavy and/or light
chains are contemplated. In some embodiments, only the variable
region of the heavy and/or light chains are contemplated as being
present.
[0061] Antibodies can exist as intact immunoglobulins or as a
number of fragments produced by digestion with various peptidases.
Thus, for example, pepsin digests an antibody below the disulfide
linkages in the hinge region to produce F(ab)'.sub.2, a dimer of
Fab' which itself is a light chain (V.sub.L-C.sub.L) joined to
V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is a Fab with part of the hinge
region. (Paul, Fundamental Immunology 3d ed. (1993). While various
antibody fragments are defined in terms of the digestion of an
intact antibody, one of skill will appreciate that such fragments
may be synthesized de novo either chemically or by using
recombinant DNA methodology. Thus, the term "antibody," as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., Nature 348:552-554 (1990)).
[0062] For preparation of monoclonal or polyclonal antibodies, any
technique known in the art can be used (see, e.g., Kohler &
Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology
Today 4:72 (1983); Cole et al., Monoclonal Antibodies and Cancer
Therapy, pp. 77-96. Alan R. Liss, Inc. 1985; Advances in the
production of human monoclonal antibodies Shixia Wang, Antibody
Technology Journal 2011:1 1-4; J Cell Biochem. 2005 Oct. 1;
96(2):305-13; Recombinant polyclonal antibodies for cancer therapy;
Sharon J, Liebman M A, Williams B R; and Drug Discov Today. 2006
July, 11(13-14):655-60, Recombinant polyclonal antibodies: the next
generation of antibody therapeutics?, Haurum J S). Techniques for
the production of single chain antibodies (U.S. Pat. No. 4,946,778)
can be adapted to produce antibodies to polypeptides of this
invention. Also, transgenic mice, or other organisms such as other
mammals, may be used to express fully human monoclonal antibodies.
Alternatively, phage display technology can be used to identify
high affinity binders to selected antigens (see, e.g., McCafferty
et al., supra; Marks et al., Biotechnology, 10:779-783,
(1992)).
[0063] Methods for humanizing or primatizing non-human antibodies
are well known in the art. Generally, a humanized antibody has one
or more amino acid residues introduced into it from a source which
is non-human. These non-human amino acid residues are often
referred to as import residues, which are typically taken from an
import variable domain. In some embodiments, the terms "donor" and
"acceptor" sequences can be employed. Humanization can be
essentially performed following the method of Winter and co-workers
(see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et
al., Nature 332:323-327 (1988); Verhoeyen et al., Science
239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596
(1992)), by substituting rodent CDRs or CDR sequences for the
corresponding sequences of a human antibody. Accordingly, such
humanized antibodies are chimeric antibodies (U.S. Pat. No.
4,816,567), wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence
from a non-human species. In practice, humanized antibodies are
typically human antibodies in which some complementarity
determining region ("CDR") residues and possibly some framework
("FR") residues are substituted by residues from analogous sites in
rodent antibodies.
[0064] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, and drug; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0065] Antibodies further include one or more immunoglobulin chains
that are chemically conjugated to, or expressed as, fusion proteins
with other proteins. In some embodiments, the antigen binding
constructs can be monovalent scFv constructs. In some embodiments,
the antigen binding constructs can be bispecific constructs. A
bispecific or bifunctional antibody is an artificial hybrid
antibody having two different heavy/light chain pairs and two
different binding sites. Other antigen-binding fragments or
antibody portions of the invention include bivalent scFv (diabody),
bispecific scFv antibodies where the antibody molecule recognizes
two different epitopes, single binding domains (sdAb or
nanobodies), and minibodies.
[0066] The term "antibody fragment" includes, but is not limited to
one or more antigen binding fragments of antibodies alone or in
combination with other molecules, including, but not limited to
Fab', F(ab').sub.2, Fab, Fv, rIgG (reduced IgG), scFv fragments
(monovalent, tri-valent, etc.), single domain fragments
(nanobodies), peptibodies, minibodies, diabodies, and
cys-diabodies. The term "scFv" refers to a single chain Fv
("fragment variable") antibody in which the variable domains of the
heavy chain and of the light chain of a traditional two chain
antibody have been joined to form one chain.
[0067] A pharmaceutically acceptable carrier may be a
pharmaceutically acceptable material, composition, or vehicle that
is involved in carrying or transporting a compound of interest from
one tissue, organ, or portion of the body to another tissue, organ,
or portion of the body. For example, the carrier may be a liquid or
solid filler, diluent, excipient, solvent, or encapsulating
material, or some combination thereof. Each component of the
carrier is "pharmaceutically acceptable" in that it is be
compatible with the other ingredients of the formulation. It also
must be suitable for contact with any tissue, organ, or portion of
the body that it may encounter, meaning that it must not carry a
risk of toxicity, irritation, allergic response, immunogenicity, or
any other complication that excessively outweighs its therapeutic
benefits. The pharmaceutical compositions described herein may be
administered by any suitable route of administration. A route of
administration may refer to any administration pathway known in the
art, including but not limited to aerosol, enteral, nasal,
ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical
cream or ointment, patch), or vaginal. "Transdermal" administration
may be accomplished using a topical cream or ointment or by means
of a transdermal patch. "Parenteral" refers to a route of
administration that is generally associated with injection,
including infraorbital, infusion, intraarterial, intracapsular,
intracardiac, intradermal, intramuscular, intraperitoneal,
intrapulmonary, intraspinal, intrasternal, intrathecal,
intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,
transmucosal, or transtracheal. In some embodiments, the antigen
binding construct can be delivered intraoperatively as a local
administration during an intervention or resection.
[0068] The term "CD8 dependent disorder" includes cancers for which
there is an immunological component (including response to cancer
immunotherapies), autoimmune disorders inflammation disorders,
etc.
[0069] A minibody is an antibody format that has a smaller
molecular weight than the full-length antibody while maintaining
the bivalent binding property against an antigen. Because of its
smaller size, the minibody has a faster clearance from the system
and enhanced penetration when targeting tumor tissue. With the
ability for strong targeting combined with rapid clearance, the
minibody is advantageous for diagnostic imaging and delivery of
cytotoxic/radioactive payloads for which prolonged circulation
times may result in adverse patient dosing or dosimetry.
[0070] The phrase "specifically (or selectively) bind," when used
in the context of describing the interaction between an antigen,
e.g., a protein, to an antibody or antibody-derived binding agent,
refers to a binding reaction that is determinative of the presence
of the antigen in a heterogeneous population of proteins and other
biologics, e.g., in a biological sample, e.g., a blood, serum,
plasma or tissue sample. Thus, under designated immunoassay
conditions, in some embodiments, the antibodies or binding agents
with a particular binding specificity bind to a particular antigen
at least two times the background and do not substantially bind in
a significant amount to other antigens present in the sample.
Specific binding to an antibody or binding agent under such
conditions may require the antibody or agent to have been selected
for its specificity for a particular protein. A variety of
immunoassay formats may be used to select antibodies specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select antibodies
specifically immunoreactive with a protein (see, e.g., Harlow &
Lane, Using Antibodies, A Laboratory Manual (1998), for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity). Typically a specific or
selective binding reaction will produce a signal at least twice
over the background signal and more typically at least than 10 to
100 times over the background.
[0071] The term "equilibrium dissociation constant (K.sub.D, M)"
refers to the dissociation rate constant (k.sub.d, time.sup.-1)
divided by the association rate constant (k.sub.a, time.sup.-1,
M.sup.-1). Equilibrium dissociation constants can be measured using
any known method in the art. The antibodies of the present
invention generally will have an equilibrium dissociation constant
of less than about 10.sup.-7 or 10.sup.-8 M, for example, less than
about 10.sup.-9 M or 10.sup.-10 M, in some embodiments, less than
about 10.sup.-11 M, 10.sup.-12 M, or 10.sup.-13 M.
[0072] The term "isolated," when applied to a nucleic acid or
protein, denotes that the nucleic acid or protein is essentially
free of other cellular components with which it is associated in
the natural state. In some embodiments, it can be in either a dry
or aqueous solution. Purity and homogeneity can be determined using
analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high performance liquid chromatography. A
protein that is the predominant species present in a preparation is
substantially purified. In particular, an isolated gene is
separated from open reading frames that flank the gene and encode a
protein other than the gene of interest. The term "purified"
denotes that a nucleic acid or protein gives rise to essentially
one band in an electrophoretic gel. In some embodiments, this can
denote that the nucleic acid or protein is at least 85% pure, more
preferably at least 95% pure, and most preferably at least 99% pure
of molecules that are present under in vivo conditions.
[0073] The term "nucleic acid" or "polynucleotide" refers to
deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the term encompasses nucleic acids containing
known analogues of natural nucleotides that have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions), alleles, orthologs, SNPs, and
complementary sequences as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); and Rossolini et al., Mol. Cell. Probes 8:91-98
(1994)).
[0074] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0075] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refer to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha.-carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0076] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid that encodes a polypeptide is implicit in each described
sequence.
[0077] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0078] The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0079] "Percentage of sequence identity" can be determined by
comparing two optimally aligned sequences over a comparison window,
wherein the portion of the polynucleotide sequence in the
comparison window may comprise additions or deletions (i.e., gaps)
as compared to the reference sequence (e.g., a polypeptide of the
invention), which does not comprise additions or deletions, for
optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison and multiplying the result by 100 to yield the
percentage of sequence identity.
[0080] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same sequences. Two
sequences are "substantially identical" if two sequences have a
specified percentage of amino acid residues or nucleotides that are
the same (for example, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%
or 99% sequence identity over a specified region, or, when not
specified, over the entire sequence of a reference sequence), when
compared and aligned for maximum correspondence over a comparison
window, or designated region as measured using one of the following
sequence comparison algorithms or by manual alignment and visual
inspection. Some embodiments provided herein provide polypeptides
or polynucleotides that are substantially identical to the
polypeptides or polynucleotides, respectively, exemplified herein
(e.g., the variable regions exemplified in any one FIG. 2A, 2B, or
4-11, 12C-12I; the CDRs exemplified in any one of FIG. 2A, 2B, or
12C to 12I; the FRs exemplified in any one of FIG. 2A, 2B, or
12C-12I; and the nucleic acid sequences exemplified in any one of
FIGS. 12A-12I or 4-11). Optionally, the identity exists over a
region that is at least about 15, 25 or 50 nucleotides in length,
or more preferably over a region that is 100 to 500 or 1000 or more
nucleotides in length, or over the full length of the reference
sequence. With respect to amino acid sequences, identity or
substantial identity can exist over a region that is at least 5,
10, 15 or 20 amino acids in length, optionally at least about 25,
30, 35, 40, 50, 75 or 100 amino acids in length, optionally at
least about 150, 200 or 250 amino acids in length, or over the full
length of the reference sequence. With respect to shorter amino
acid sequences, e.g., amino acid sequences of 20 or fewer amino
acids, in some embodiments, substantial identity exists when one or
two amino acid residues are conservatively substituted, according
to the conservative substitutions defined herein.
[0081] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0082] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity method of Pearson and Lipman (1988) Proc.
Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995 supplement)).
[0083] Two examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1977)
Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad.
Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0084] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0085] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, in some embodiments,
a polypeptide is typically substantially identical to a second
polypeptide, for example, where the two peptides differ only by
conservative substitutions. Another indication that two nucleic
acid sequences are substantially identical is that the two
molecules or their complements hybridize to each other under
stringent conditions, as described below. Yet another indication
that two nucleic acid sequences are substantially identical is that
the same primers can be used to amplify the sequence.
[0086] The terms "subject," "patient," and "individual"
interchangeably refer to an entity that is being examined and/or
treated. This can include, for example, a mammal, for example, a
human or a non-human primate mammal. The mammal can also be a
laboratory mammal, e.g., mouse, rat, rabbit, hamster. In some
embodiments, the mammal can be an agricultural mammal (e.g.,
equine, ovine, bovine, porcine, camelid) or domestic mammal (e.g.,
canine, feline).
[0087] The term "therapeutically acceptable amount" or
"therapeutically effective dose" interchangeably refer to an amount
sufficient to effect the desired result. In some embodiments, a
therapeutically acceptable amount does not induce or cause
undesirable side effects. A therapeutically acceptable amount can
be determined by first administering a low dose, and then
incrementally increasing that dose until the desired effect is
achieved.
[0088] The term "co-administer" refers to the administration of two
active agents in the blood of an individual or in a sample to be
tested. Active agents that are co-administered can be concurrently
or sequentially delivered.
Antigen Binding Constructs (Including Antibodies and Binding
Fragments)
[0089] Antigen binding constructs that bind to the target are
described herein. An antigen binding construct is a molecule that
includes one or more portions of an immunoglobulin or
immunoglobulin-related molecule that specifically binds to, or is
immunologically reactive with the target molecule.
[0090] In some embodiments, the antigen binding constructs allow
for the detection of human CD8 which is a specific biomarker found
on the surface of a subset of T-cells for diagnostic imaging of the
immune system. Imaging of CD8 allows for the in vivo detection of
T-cell localization. Changes in T-cell localization can reflect the
progression of an immune response and can occur over time as a
result various therapeutic treatments or even disease states.
[0091] In some embodiments, this is useful for imaging T-cell
localization for immunotherapy. Adoptive immunotherapy is a form of
therapy where a patient's own T-cells are manipulated in vitro and
re-introduced into the patient. For this form of treatment, imaging
of T-cells is useful for determining the status of the
treatment.
[0092] In addition, CD8 plays a role in activating downstream
signaling pathways that are important for the activation of
cytolytic T cells that function to clear viral pathogens and
provide immunity to tumors. CD8 positive T cells can recognize
short peptides presented within the MHCI protein of antigen
presenting cells. In some embodiments, engineered fragments
directed to CD8 can potentiate signaling through the T cell
receptor and enhance the ability of a subject to clear viral
pathogens and respond to tumor antigens. Thus, in some embodiments,
the antigen binding constructs provided herein can be agonists and
can activate the CD8 target. In some embodiments, an agonist scFv,
minibody, cys-diabody, and/or antibody is provided. In some
embodiments, the agonist antigen binding construct includes one or
more of the CDRs, heavy chain variable regions, or light chain
variable regions provided herein. In some embodiments, the agonist
can activate downstream signaling pathways through CD8 for the
activation of cytolytic T cells that function to clear viral
pathogens and provide immunity to tumors.
[0093] In some situations, using full-length antibodies for imaging
is not optimal since they typically require imaging times to be
scheduled more than 1 week after administration due to the long
serum half-lives of full-length antibodies.
[0094] Another target-based approach for imaging subtypes of immune
cells involves small molecules. For example, one approach for
diagnostic imaging of the endogenous immune system has involved the
use of small molecule tracers which detect changes in the cell's
metabolic pathway such as .sup.18F-fluoroacetate ([.sup.18F]FAC).
Since such tracers detect changes in the metabolic pathway, they
target cell populations with elevated metabolic activities which
primarily include activated T-cells. The limitation of this
approach is that it will only detect the activated subset of
T-cells, whereas imaging with anti-CD8 antibody fragments will
detect the entire population of CD8 expressing T-cells as the
target is expressed on both activated and resting CD8 cells.
[0095] The variable regions of the OKT8 antibody were reformatted
by protein engineering into various alternative antigen binding
constructs. The minibody format is a homodimer with each monomer
having a single-chain variable fragment (scFv) linked to the human
IgG1 C.sub.H3 domain (see FIGS. 1A and 1B). In some embodiments,
the scFv is composed of the variable heavy (V.sub.H) and light
(V.sub.L) domains and is connected by an 18 amino acid GlySer-rich
linker. In some embodiments, the scFv is tethered to the human IgG1
C.sub.H3 domain by the human IgG1 upper and core hinge regions (15
residues) followed by a 10 amino acid GlySer linker. The minibody
(V.sub.H-V.sub.L-C.sub.H3) exists as a stable dimer due to the
association between the C.sub.H3 domains as well as the formation
of disulfide bonds within the hinge regions. To allow for secretion
of the minibody, a signal sequence is fused at the N-terminus of
the variable heavy domain. In some embodiments, the GlySer residues
allow for flexibility. In some embodiments, glutamine and/or lysine
residues can be added to enhance solubility.
[0096] Two variants of the chimeric OKT8 minibody were engineered
that differed in the orientation of the variable regions (V.sub.H
to V.sub.L and V.sub.L to V.sub.H). Every antibody V domain
contains two cysteines that form intra-disulfide bonds. The murine
OKT8 V.sub.H has an extra cysteine in framework 3 (FR3) which may
interfere with the expression of the protein as it may lead to
aggregation and consequently retention in the endoplasmic reticulum
(ER). The chimeric minibodies were made with a serine replacing the
extra cysteine in the framework (C84S of the murine V.sub.H). In
some embodiments, any of the embodiments provided herein can be
adjusted to include the C84S adjustment. Tables 0.1, 0.2, and 0.3
provide a summary of come embodiments of the arrangements of
various antigen binding constructs provided herein.
TABLE-US-00001 TABLE 0.1 Minibodies 1 2 3 4 5 6 Name Leader Region
1 Linker Region 2 Remainder Chimeric Leader murine V.sub.L 18 aa
linker Murine V.sub.H IgG1 IAb_Mb1_CD8 SEQ ID NO: 34 SEQ ID NO: 40
SEQ ID NO: 36 SEQ ID NO: 44 hinge/linker- SEQ ID NO: C.sub.H3
domain SEQ ID NO: 38 Chimeric Leader murine V.sub.H 18 aa linker
Murine V.sub.L IgG1 IAb_Mb2_CD8 SEQ ID NO: 34 SEQ ID NO: 44 SEQ ID
NO: 36 SEQ ID NO: 40 hinge/linker- C.sub.H3 domain SEQ ID NO: 38
Humanized Leader hu V.sub.L 18 aa linker hu V.sub.H (2.sup.nd) IgG1
IAb_Mb1_CD8 SEQ ID NO: 34 SEQ ID NO: 9 SEQ ID NO: 36 SEQ ID NO: 6
hinge/linker- C.sub.H3 domain SEQ ID NO: 38 Humanized Leader Hu
V.sub.H (2.sup.nd) 18 aa linker hu V.sub.L IgG1 IAb_Mb2_CD8 SEQ ID
NO: 34 SEQ ID NO: 6 SEQ ID NO: SEQ ID NO: 9 hinge/linker- SEQ ID
NO: 36 C.sub.H3 domain SEQ ID NO: 38
TABLE-US-00002 TABLE 0.2 Affinity Matured Minibodies 1 2 3 4 5 6
Name Leader Region 1 Linker Region 2 Remainder IAb_Mb1a_CD8 Leader
huV.sub.L 18aa Linker huV.sub.H IgG1 SEQ ID NO: 34 SEQ ID NO: 42
SEQ ID NO: 36 (version a) hinge/linker- SEQ ID NO: 46 C.sup.H3
domain SEQ ID NO: 38 IAb_Mb2a_CD8 Leader huV.sub.H 18aa Linker
huV.sub.L IgG1 SEQ ID NO: 34 (version a) SEQ ID SEQ ID NO: 42
hinge/linker- SEQ ID NO: 46 NO: 36 C.sup.H3 domain SEQ ID NO: 38
IAb_Mb1b_CD8 Leader huV.sub.L 18aa Linker huV.sub.H IgG1 SEQ ID NO:
34 SEQ ID NO: 42 SEQ ID NO: 36 (version b) hinge/linker- SEQ ID NO:
48 C.sup.H3 domain SEQ ID NO: 38 IAb_Mb2b_CD8 Leader huV.sub.H 18aa
Linker huV.sub.L IgG1 SEQ ID NO: 34 (version b) SEQ ID NO: 36 SEQ
ID NO: 42 hinge/linker- SEQ ID NO: 48 C.sup.H3 domain SEQ ID NO: 38
IAb_Mb1c_CD8 Leader huV.sub.L 18aa Linker huV.sub.H IgG1 SEQ ID NO:
34 SEQ ID NO: 42 SEQ ID NO: 36 (version c) hinge/linker- SEQ ID NO:
50 C.sup.H3 domain SEQ ID NO: 38 IAb_Mb2c_CD8 Leader huV.sub.H 18aa
Linker huV.sub.L IgG1 SEQ ID NO: 34 (version c) SEQ ID NO: 36 SEQ
ID NO: 42 hinge/linker- SEQ ID NO: 50 C.sup.H3 domain SEQ ID NO: 38
IAb_Mb1d_CD8 Leader huV.sub.L 18aa Linker huV.sub.H IgG1 SEQ ID NO:
34 SEQ ID NO: 42 SEQ ID NO: 36 (version d) hinge/linker- SEQ ID NO:
52 C.sub.H3 domain SEQ ID NO: 38 IAb_Mb2d_CD8 Leader huV.sub.H 18aa
Linker huV.sub.L IgG1 SEQ ID NO: 34 (version d) SEQ ID NO: 36 SEQ
ID NO: 42 hinge/linker- SEQ ID NO: 52 C.sub.H3 domain SEQ ID NO:
38
TABLE-US-00003 TABLE 0.3 Cys-Diabodies 1 2 3 4 5 6 Name Leader
Region 1 Linker Region 2 Remainder IAb_Cys- Leader huV.sub.L 5aa
Linker huV.sub.H Cys Tail Db1b_CD8 SEQ ID NO: 26 SEQ ID NO: 42 SEQ
ID NO: 28 (version b) SEQ ID NO: 32 SEQ ID NO: 48 IAb_Cys- Leader
huV.sub.H 5aa Linker huV.sub.L Cys Tail Db2b_CD8 SEQ ID NO: 26
(version b) SEQ ID NO: 28 SEQ ID NO: 42 SEQ ID NO: 32 SEQ ID NO: 48
IAb_Cys- Leader huV.sub.L 8aa Linker huV.sub.H Cys Tail Db3b_CD8
SEQ ID NO: 26 SEQ ID NO: 42 SEQ ID NO: 30 (version a) SEQ ID NO: 32
SEQ ID NO: 48 IAb_Cys- Leader huV.sub.H 8aa Linker huV.sub.L Cys
Tail Db4b_CD8 SEQ ID NO: 26 (version a) SEQ ID NO: 30 SEQ ID NO: 42
SEQ ID NO: 32 SEQ ID NO: 48
[0097] Depicted in Tables 0.1, 0.2, and 03 are arrangements of
sequences for monomers that can be used in minibodies (Table 0.1
and 0.2) and cys-diabodies (Table 0.3). Each row of the table
represents the sequence of a monomer construct, with left-to-right
representing N-terminus to C-terminus. In some embodiments, the
shown sequences of each monomer construct are directly linked to
each other. Thus, in some embodiments, the construct can include
any of the constructs on a single row in Table 0.1, Table 0.2, or
Table 0.3. In some embodiments, the constructs can include any
combination in Table 0.1, Table 0.2, or Table 0.3. In some
embodiments, for example, the first item in the first row, column 2
can be combined with the first row, column 3 to the first row
column 4, to the first row column 5, to the first row, column 6. In
some embodiments, column 3 and column 6 can be swapped with one
another. In some embodiments, the first item in the first row,
column 2 can be combined with the first row, column 3 to the second
row column 4, to the second row column 5, to the second row, column
6. Thus, the tables represent all possible combinations, both
within a single row and across various rows (and with columns
swapped).
[0098] In some embodiments, an antigen binding construct includes a
heavy chain CDR1 (HCDR1) of the HCDR1 in SEQ ID NOs: 3, 6, 44, 46,
48, 50, or 52; a heavy chain CDR2 (HCDR2) of the HCDR2 in SEQ ID
NOs: 3, 6, 44, 46, 48, 50, or 52; a heavy chain CDR3 (HCDR3) of the
HCDR3 in SEQ ID NOs: 3, 6, 44, 46, 48, 50, or 52; a light chain
CDR1 (LCDR1) of the LCDR1 in SEQ ID NOs: 9 or 42; a light chain
CDR2 (LCDR2) of the LCDR2 in SEQ ID NOs: 9 or 42; and/or a light
chain CDR3 (LCDR3) of the LCDR3 in SEQ ID NOs: 9 or 42. In some
embodiments, the antigen binding construct includes 6, 5, 4, 3, 2,
or 1, the above CDRs (some embodiments of the CDRs are indicated in
FIGS. 2A, 2B, 12C-12I). In some embodiments, the antigen binding
construct includes HCDR3. In some embodiments, the antigen binding
construct binds specifically to the target molecule. In some
embodiments, the antigen binding construct competes for binding
with one or more of the antibodies having the herein provided CDRs.
In some embodiments, the antigen binding construct includes at
least the 3 heavy chain CDRs noted herein. In some embodiments, the
antigen binding construct includes heavy chain CDR3. In some
embodiments, the antigen binding construct further includes any one
of the heavy chain CDR2 sequences provided herein.
[0099] In some embodiments, the antigen binding construct is human
or humanized. In some embodiments, the antigen binding construct
includes at least one human framework region, or a framework region
with at least about 80% sequence identity, for example at least
about 80%, 85%, 90%, 93%, 95%, 97%, or 99% identity to a human
framework region. In some embodiments the antigen binding construct
includes a heavy chain FR1 (HFR1) of the HFR1 in SEQ ID NO: 3, 6,
44, 46, 48, 50, or 52; a heavy chain FR2 (HFR2) of the HFR2 in SEQ
ID NO: 3, 6, 44, 46, 48, 50, or 52; a heavy chain FR3 (HFR3) of the
HFR3 in SEQ ID NO: 3, 6, 44, 46, 48, 50, or 52; a heavy chain FR4
(HFR4) of the HFR4 in SEQ ID NO: 3, 6, 44, 46, 48, 50, or 52; a
light chain FR1 (LFR1) of the LFR1 in SEQ ID NO: 9 or 42; a light
chain FR2 (LFR2) of the LFR2 in SEQ ID NO: 9 or 42; a light chain
FR3 (LFR3) of the LFR3 in SEQ ID NO: 9 or 42; and a light chain FR4
(LFR4) of the LFR4 in SEQ ID NO: 9 or 42. In some embodiments, the
antigen binding construct includes 8, 7, 6, 5, 4, 3, 2, or 1 of the
listed FRs.
[0100] In some embodiments, the antigen binding construct includes
a detectable marker. In some embodiments, the antigen binding
construct includes a therapeutic agent.
[0101] In some embodiments, the antigen binding construct is
bivalent. Bivalent antigen binding construct can include at least a
first antigen binding domain, for example a first scFv, and at
least a second antigen binding domain, for example a second scFv.
In some embodiments, a bivalent antigen binding construct is a
multimer that includes at least two monomers, for example at least
2, 3, 4, 5, 6, 7, or 8 monomers, each of which has an antigen
binding domain. In some embodiments, the antigen binding construct
is a minibody. In some embodiments, the antigen binding construct
is a diabody, including, for example, a cys-diabody. The scFv,
and/or minibody and/or the cys-diabody can include any of the CDR
and heavy chain variable region and/or light chain variable region
embodiments provided herein (for example, the CDR sequences
provided in FIGS. 2A, 2B, and 12C-12I). In some embodiments, the
antigen binding construct is a monovalent scFv. In some
embodiments, a monovalent scFv is provided that includes the HCDR1
in the HCDR1 of FIG. 2A, FIG. 12E-12I, or SEQ ID NO: 3, 6, 44, 46,
48, 50, or 52; the HCDR2 in the HCDR2 of FIG. 2A, FIG. 12E-12I, or
SEQ ID NO: 3, 6, 44, 46, 48, 50, or 52; the HCDR3 in the HCDR3 of
FIG. 2A, FIG. 12E-12I, or SEQ ID NO: 3, 6, 44, 46, 48, 50, or 52;
the LCDR1 in the LCDR1 of FIG. 2B, FIG. 12C, FIG. 12D, SEQ ID NO: 9
or 42; the LCDR2 in the LCDR2 of FIG. 2B, FIG. 12C, FIG. 12D, SEQ
ID NO: 9 or 42, and the LCDR3 in the LCDR3 of FIG. 2B, FIG. 12C,
FIG. 12D, SEQ ID NO: 9 or 42. In some embodiments, the monovalent
scFv includes the heavy chain variable region of the heavy chain
variable region in FIG. 2A, FIGS. 4-11, FIGS. 12C-12I, or SEQ ID
NO: 3, 6, 44, 46, 48, 50, or 52. In some embodiments, the
monovalent scFv includes the light chain variable region of the
light chain variable region in FIG. 2B, 4-11, 12C, 12D, SEQ ID NO:
9, 42, or 40. In some embodiments, the monovalent scFv includes the
heavy chain variable region of the heavy chain variable region in
FIG. 2A, FIGS. 4-11, FIGS. 12C-12I, or SEQ ID NO: 3, 6, 44, 46, 48,
50, or 52 and the light chain variable region of the light chain
variable region in in FIG. 2B, 4-11, 12C, 12D, SEQ ID NO: 9, 42, or
40.
[0102] In some embodiments, the antigen binding construct is
bispecific. Bispecific antibodies can include at least a first
binding domain, for example an scFv that binds specifically to a
first epitope, and at least a second binding domain, for example an
scFv that binds specifically to a second epitope. Thus, bispecific
antigen binding constructs can bind to two or more epitopes. In
some embodiments, the first epitope and the second epitope are part
of the same antigen, and the bispecific antigen binding construct
can thus bind to two epitopes of the same antigen. In some
embodiments, the first epitope is part of a first antigen, and the
second epitope is part of a second antigen, and the bispecific
antigen binding construct can thus bind to two different antigens.
In some embodiments, the antigen binding construct binds to two
epitopes simultaneously.
[0103] In some embodiments, the antigen binding construct has a
heavy chain variable region of the heavy chain variable region in
SEQ ID NO: 3, 6, 16, 18, 20, 22, 44, 46, 48, 50, or 52. In some
embodiments, the antigen binding construct has a heavy chain
variable region that includes a sequence with at least about 80%
identity, for example at least about 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ
ID NO: 3. In some embodiments, the antigen binding construct has a
heavy chain variable region that includes a sequence with at least
about 80% identity, for example at least about 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity to SEQ ID NO: 6. In some embodiments, the antigen binding
construct has a heavy chain variable region that includes a
sequence with at least about 80% identity, for example at least
about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identity to SEQ ID NO: 44. In some
embodiments, the antigen binding construct has a heavy chain
variable region that includes a sequence with at least about 80%
identity, for example at least about 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ
ID NO: 46. In some embodiments, the antigen binding construct has a
heavy chain variable region that includes a sequence with at least
about 80% identity, for example at least about 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity to SEQ ID NO: 48. In some embodiments, the antigen binding
construct has a heavy chain variable region that includes a
sequence with at least about 80% identity, for example at least
about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identity to SEQ ID NO: 50. In some
embodiments, the antigen binding construct has a heavy chain
variable region that includes a sequence with at least about 80%
identity, for example at least about 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ
ID NO: 52.
[0104] In some embodiments, the antigen binding construct has a
light chain variable region that includes SEQ ID NO: 9, 16, 18, 20,
22, 40, or 42. In some embodiments, the antigen binding construct
has a light chain variable region that includes a sequence with
least about 80% identity, for example at least about 80%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity to SEQ ID NO: 9. In some embodiments, the antigen binding
construct has a light chain variable region that includes a
sequence with least about 80% identity, for example at least about
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 40. In some embodiments,
the antigen binding construct has a light chain variable region
that includes a sequence with least about 80% identity, for example
at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 42. In some
embodiments, the antigen binding construct is a human antigen
binding construct and has a heavy chain variable region, a light
chain variable region, or a heavy and light chain that is at least
as identical as at least the heavy and/or light chain variable
sequences noted above.
[0105] Some embodiments provided herein include an antigen binding
construct that competes for binding to the target molecule with one
or more antigen binding constructs provided herein. In some
embodiments, the competing antigen binding construct binds to the
same epitope on the target molecule as the reference antigen
binding construct. In some embodiments, the reference antigen
binding construct binds to a first epitope of the target molecule,
and the competing antigen binding construct binds to a second
epitope of the target molecule, but interferes with binding of the
reference antigen binding construct to the target molecule, for
example by sterically blocking binding of the reference antigen
binding construct, or by inducing a conformational change in the
target molecule. In some embodiments, the first epitope overlaps
with the second epitope. In some embodiments, columns 3 and 5 of
Tables 0.1 and/or 0.2 can be swapped. In some embodiments, any of
the heavy chains variable regions provided herein can be combined
with any of the light chain variable regions herein for a scFv,
minibody, and/or diabody. In some embodiments, any of the heavy
and/or light chain variable regions (columns 3 and 5) in tables
0.1, 0.2, and 0.3 can be exchanged with one another or another
light or heavy chain variable region, to produce an antigen binding
construct (such as a scFv, a cys-diabody, a minibody, or an
antibody).
[0106] In some embodiments, the minibody and cys-diabody formats
have advantageous pharmacokinetic characteristics for diagnostic
imaging and certain therapeutic applications while maintaining the
high binding affinity and specificity of a parental antibody.
Compared to imaging with the full-length parental antibody, the
pharmacokinetics are more desirable for these fragments in that
they are able to target the antigen and then rapidly clear the
system for rapid high-contrast imaging. In some embodiments, the
shorter serum half lives for the minibody and the cys-diabody allow
for imaging to occur over a range of times, approximately 8-48
hours post injection for the minibody and 2-24 hours post-injection
for the cys-diabody. The rapid serum clearance together with better
tissue penetration can allow for same day imaging, providing a
significant advantage in the clinic with respect to patient care
management.
[0107] In addition, the cys-diabody antibody format features the
C-terminus cysteine tail. These two sulfhydryl groups (following
mild reduction) provide a strategy for site-specific conjugation of
functional moieties such as radiolabels that need not interfere
with the cys-diabody's binding activity.
Diabodies that Bind to the Target Molecule
[0108] In some embodiments, the antigen binding construct can be a
diabody. The diabody can include a first polypeptide chain which
includes a heavy (V.sub.H) chain variable domain connected to a
light chain variable domain (V.sub.L) on the first polypeptide
chain. In some embodiments, the light and heavy variable chain
domains can be connected by a linker. The linker can be of the
appropriate length to reduce the likelihood of pairing between the
two domains on the first polypeptide chain and a second polypeptide
chain comprising a light chain variable domain (V.sub.L) linked to
a heavy chain variable domain V.sub.H on the second polypeptide
chain connected by a linker that is too short to allow significant
pairing between the two domains on the second polypeptide
chain.
[0109] In some embodiments, the appropriate length of the linker
encourages chain pairing between the complementary domains of the
first and the second polypeptide chains and can promote the
assembly of a dimeric molecule with two functional antigen binding
sites. Thus, in some embodiments, the diabody is bivalent. In some
embodiments, the diabody can be a cysteine linked diabody (a
Cys-Db). A schematic of a Cys-Db binding to two antigen sites is
illustrated in FIGS. 3A and 3B.
[0110] In some embodiments, the linker can be a peptide. In some
embodiments, the linker can be any suitable length that promotes
such assembly, for example, between 1 and 20 amino acids, such as 5
and 10 amino acids in length. As described further herein, some
cys-diabodies can include a peptide linker that is 5 to 8 amino
acids in length. In some embodiments, the linker need not be made
from, or exclusively from amino acids, and can include, for
example, modified amino acids (see, for example, Increased
Resistance of Peptides to Serum Proteases by Modification of their
Amino Groups, Rossella Galati, Alessandra Verdina, Giuliana
Falasca, and Alberto Chersi, (2003) Z. Naturforsch, 58c, 558-561).
In some embodiments, the linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in
length. In some embodiments, the linker can be from 2 to 30
angstroms in length, for example 2.5 to 27 angstroms.
[0111] In some embodiments, the antigen binding construct includes
a humanized cys-diabody. The humanized cys-diabody can include a
single-chain variable fragment (scFv) that includes a variable
heavy (V.sub.H) domain linked to a variable light (V.sub.L) domain,
and a C-terminal cysteine. In some embodiments, the humanized
cys-diabody is a homodimer. In some embodiments, the humanized
diabody is a heterodimer. In some embodiments, individual monomers
are provided that each have a cysteine terminal residue.
[0112] In some embodiments, the scFv of the humanized cys-diabody
has a V.sub.H-V.sub.L orientation or a V.sub.L-V.sub.H orientation.
As used herein, a V.sub.H-V.sub.L (which may also be referred to
herein as "V.sub.HV.sub.L") orientation means that the variable
heavy domain (V.sub.H) of the scFv is upstream from the variable
light domain (V.sub.L) and a V.sub.LV.sub.H orientation means that
the V.sub.L domain of the scFv is upstream from the V.sub.H domain.
As used herein, "upstream" means toward the N-terminus of an amino
acid or toward the 5' end of a nucleotide sequence.
[0113] The antibody variable regions can be linked together by a
linker as described herein. In some embodiments, the linker is a
GlySer linker as described herein.
[0114] In some embodiments, the cys-diabody includes a detectable
marker.
[0115] In some embodiments, the cys-diabody includes a pair of
monomers. Each monomer can include a polypeptide. In some
embodiments, the polypeptides of the monomers are identical (for
example, cys-diabody can be a homodimer). In some embodiments, the
polypeptides of the monomers are different (for example, the
cys-diabody can be a heterodimer).
[0116] In some embodiments, the polypeptide of the monomer includes
SEQ ID NO: 12 (See FIG. 8). In some embodiments, the polypeptide of
the monomer includes a sequence with least about 80% identity, for
example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12
(cys-diabody (V.sub.L-5-V.sub.H)).
[0117] In some embodiments, the polypeptide of the monomer includes
SEQ ID NO: 13 (See FIG. 9). In some embodiments, the polypeptide of
the monomer includes a sequence with least about 80% identity, for
example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93, 94, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13
(cys-diabody (V.sub.H-5-V.sub.L)).
[0118] In some embodiments, the polypeptide of the monomer includes
SEQ ID NO: 14 (V.sub.L-8-V.sub.H)] (See FIG. 10). In some
embodiments, the polypeptide of the monomer includes a sequence
with least about 80% identity, for example at least about 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94, 95%, 96%, 97%, 98%, 99%,
or 100% identity to SEQ ID NO: 14.
[0119] In some embodiments, the polypeptide of the monomer includes
SEQ ID NO: 15 (humanized OKT8 cys-diabody (V.sub.H-8-V.sub.L)) (See
FIG. 11). In some embodiments, the polypeptide of the monomer
includes a sequence with least about 80% identity, for example at
least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94,
95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15.
[0120] In some embodiments, the polypeptide of the monomer includes
any of the combined sections as indicated in Table 0.3, including
polypeptides of the monomer with a sequences of at least about 80%
identity, for example at least about 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93, 94, 95%, 96%, 97%, 98%, 99%, or 100% identity to
the monomers as set forth in Table 0.3.
[0121] In some embodiments, the cysteines are cross-linked with one
another. In some embodiments, the cysteines are reduced, and thus,
these tail forming cysteines do not form a disulfide bond with one
another. In some embodiments, one or more of the "tail forming"
cysteines form a covalent bond with one or more detectable marker,
such as a fluorescent probe.
[0122] As will be appreciated by those of skill in the art, while
the present disclosure generally references "cys-diabodies"
alternative arrangements can be employed to achieve the same or
similar ends. In some embodiments, any covalently modifiable moiety
can be employed in place of one or more of the cysteines. For
example, this can include a GlySer linker, a GlyLeu linker, and/or
an insert cysteine after a short tag. In some embodiments, the
connection can be established via a coiled coil or a leucine
zipper. In some embodiments, the "tail" itself can include
functional groups on its end so that it can selectively bind to a
desired residue and/or location at the ends of each of the
polypetides, in place of the disulfide bond itself. In some
embodiments, rather than the tail providing space between the two
polypeptide chains, the covalently modifiable moieties can be
attached directly to the end of the heavy or light chain
polypeptide, but the two covalently modifiable moieties can be
connected by a linker.
[0123] In some embodiments, a chimeric cys-diabody that binds to
the target molecule is provided. In some embodiments, the chimeric
cys-diabody includes a monomer in the V.sub.L-V.sub.H format, and
includes the sequence of SEQ ID NO: 12 or 14, or a sequence having
at least about 80% identity thereto, for example at least about
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%% identity thereto. In
some embodiments, the chimeric cys-diabody includes a monomer in
the V.sub.H-V.sub.L format, and includes the sequence of SEQ ID NO:
13 or 15, or a sequence having at least about 80% identity thereto,
for example at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99%% identity thereto.
[0124] In some embodiments, any of the constructs provided herein
(including those arrangements noted as cys-diabody embodiments, can
be provided as a scFv embodiment. In such embodiments, the
construct can still include the cysteine on the tail, but simply
not be cross-linked. In other embodiments, the construct need not
have the cysteine in a tail or the tail at all.
Linker and/or Tail Options
[0125] In some embodiments, for individual antibodies, the heavy
and light chain variable domains can associate in different ways.
For this reason, the use of different linker lengths allows for
conformational flexibility and range-of-motion to ensure formation
of the disulfide bonds.
[0126] In some embodiments, the two linker lengths can be somewhere
between (and including) about 1 to 50 amino acids, for example, 2
to 15, 2 to 14, 3 to 13, 4 to 10, or 5 amino acids to 8 amino
acids. In some embodiments, each linker within a pair for a diabody
can be the same length. In some embodiments, each linker within the
pair can be a different length. In some embodiments, any
combination of linker length pairs can be used, as long as they
allow and/or promote the desired combinations. In some embodiments,
a modified amino acid can be used.
[0127] FIGS. 8-11 provide four Cys-Db variants, V.sub.H-5-V.sub.L,
V.sub.H-8-V.sub.L, V.sub.L-5-V.sub.H, and VL8VH (see FIGS. 8-11,
and Table 0.3). Producing and testing the expression and binding of
all four variants allows for identification of a desired format for
protein production for each new Cys-Db. Evaluating the set of
variants can help to make certain that a high-quality, stable
protein is produced where the disulfide bridge is available.
Therefore, engineering a Cys-Db can involve using two distinct
linker lengths, not one--as in the minibody, as well as both
orientations of the variable regions, V.sub.H-V.sub.L and
V.sub.L-V.sub.H.
[0128] In some embodiments, the linker is a GlySer linker. The
GlySer linker can be a polypeptide that is rich in Gly and/or Ser
residues. In some embodiments, at least about 40% of the amino acid
residues of the GlySer linker are Gly, Ser, or a combination of Gly
and Ser, for example at least about 40%, 50%, 60%, 70%, 80%, or
90%. In some embodiments, the GlySer linker is at least about 2
amino acids long, for example at least about 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 amino
acids long. In some embodiments, the linker includes at least one
of SEQ ID NO: 28, 30, and/or 36.
[0129] In some embodiments, a cysteine is added at the C-terminus
of the diabody. This cysteine can allow the diabody complex to form
covalent cysteine bonds and provides the option for available
sulfur residues for site-specific conjugation of functional
moieties such as radiolabels. In some embodiments, a terminal end
of the antibody itself is altered so as to contain a cysteine. In
some embodiments, a tail sequence, for example (Gly-Gly-Cys) is
added at the C-terminus. In some embodiments, the cysteine tail
sequence allows two monomers of a cys-diabody to form disulfide
bonds with each other. In some embodiments, the cysteine tail
sequence allows a cys-diabody to form disulfide linkages with a
detectable moiety such as a detectable marker and/or therapeutic
agent. The sulfhydryl groups of the cysteine tail can undergo mild
reduction prior to site-specific conjugation of a desired
functional moiety, for example a detectable marker and/or
therapeutic agent. In some embodiments, the tail is at least about
1 amino acid long, for example at least about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40
amino acids long. In some embodiments, the tail includes at least
one of SEQ ID NO: 32. In some embodiments, the tail is 3 to 8 amino
acids in length. In some embodiments, the tail can and/or include a
coiled coil and/or a leucine zipper. As noted above, in some
embodiments, the cysteine is located at the c-terminus; however,
this does not require that the cysteine be located as the last
c-terminal amino acid. Instead, this denotes that the cysteine can
be part of any of the residues that are located in the C-terminus
of the protein.
[0130] In some embodiments, the linking option between the two
C-terminuses can be achieved by a cysteine, for direct and/or
indirect, cross-linking.
Minibodies that Bind to the Target Molecule
[0131] A "minibody" as described herein includes a homodimer,
wherein each monomer is a single-chain variable fragment (scFv)
linked to a human IgG1 C.sub.H3 domain by a linker, such as a hinge
sequence. In some embodiments, the hinge sequence is a human IgG1
hinge sequence as shown in FIG. 12B, SEQ ID NOs: 53-60.
[0132] In some embodiments, the hinge sequence is an artificial
hinge sequence. In some embodiments, the hinge sequence can be an
IgG hinge from any one or more of the four classes. The artificial
hinge sequence may include a portion of a human IgG1 hinge and a
GlySer linker sequence.
[0133] In some embodiments, the artificial hinge sequence includes
approximately the first 14 or 15 residues of the human IgG1 hinge
followed by a linker sequence. In some embodiments, the linker can
be any of those provided herein. In some embodiments, the linker
can be a GlySer linker sequence that is 6, 7, 8, 9 or 10 amino
acids in length. In some embodiments, the artificial hinge sequence
includes approximately the first 15 residues of the IgG1 hinge
followed by a GlySer linker sequence that is about 10 amino acids
in length. In some embodiments, association between the C.sub.H3
domains causes the minibody to exist as a stable dimer.
[0134] In some embodiments, the minibody scFv sequence can include
CDR and/or FR, and or variable region sequences that are similar
and/or the same to a diabody sequence described herein (for
Example, as found in FIGS. 2A, 2B, 4, 5, 6, 7, 8, 9, 10, 11, and
12C-12I and tables 0.1 and 0.2). In some embodiments, the minibody
scFv has a sequence (CDR, CDRs, full set of 6 CDRS, heavy chain
variable region, light chain variable region, heavy and light chain
variable regions, etc) that is at identical to a scFv of a
cys-diabody described herein.
[0135] In some embodiments, the minibody has a sequence that is at
least about 80% identical to a sequence in SEQ ID NO: 3, 6, 9, 16,
18, 20, 22, 34, 36, 38, 53-60, 40, 42, 44, 46, 48, 50, and 52,
and/or the sequence for the arrangements in Tables 0.1 and/or 0.2,
for example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93, 94, 95%, 96%, 97%, 98%, or 99% identity.
[0136] In some embodiments, the minibody has a variable chain
region that is at least about 80% identical to a sequence in SEQ ID
NO: 3, 6, 9, 16, 18, 20, 22, 40, 42, 44, 46, 48, 50, and 52, for
example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93, 94, 95%, 96%, 97%, 98%, or 99% identity.
[0137] The scFv can have a V.sub.H-V.sub.L or a V.sub.L-V.sub.H
orientation. In some embodiments, the V.sub.H and V.sub.L are
linked to each other by an amino acid linker sequence. The amino
acid linker can be a linker as described herein. In some
embodiments, the linker is GlySer-rich and approximately 15-20
amino acids in length. In another embodiment, the linker is GlySer
rich and is 18 amino acids in length. In some embodiments, the
linker length varies between (and including) about 1 to 50 amino
acids, for example, 2 to 30, 3 to 20, 4 to 15, or 5 amino acids to
8 amino acids. In some embodiments, the minibody scFv has a
sequence that is at least about 80% identical to a scFv of a
cys-diabody described herein, for example at least about 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94, 95%, 96%, 97%, 98%, or
99% identity. The scFv can have a V.sub.HV.sub.L or a
V.sub.LV.sub.H orientation.
[0138] In some embodiments, each monomer of the minibody includes
the following elements, from N-terminus to C-terminus: (a) an scFv
sequence that includes a V.sub.H domain linked to a V.sub.L domain
and that binds to the target molecule, (b) a hinge-extension domain
comprising a human IgG1 hinge region, and (c) a human IgG C.sub.H3
sequence. In some embodiments, each monomer of the minibody
includes an IgG2, an IgG3, or an IgG4 C.sub.H3. In some
embodiments, the minibody is encoded by a nucleic acid can be
expressed by a cell, a cell line or other suitable expression
system as described herein. Thus, a signal sequence can be fused to
the N-terminus of the scFv to enable secretion of the minibody when
expressed in the cell or cell line.
[0139] In some embodiments, the scFv, minibody, cys-diabody and/or
antibody includes one or more of the residues in the humanized
sequence shown in FIGS. 2A and/or 2B and denoted with an asterisk.
In some embodiments, while one or more of the residues marked with
an asterisk in FIG. 2A or 2B is present; the remaining sequence can
be varied. For example, the sequence can be 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent or
greater identity to the remaining sections of the sequence. In some
embodiments, the human and/or humanized antigen binding construct
will include one or more of the asterisked residues in FIG. 2A, for
example 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, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some
embodiments, the antigen binding construct includes one or more of
the underlined residues in FIG. 2A. In some embodiments, the
antigen binding construct includes one or more of the
non-underlined residues in FIG. 2A. In some embodiments, the
antigen binding construct includes one or more of the
non-underlined residues in FIG. 2A as well as the boxed CDR
sections, whereas other residues are allowed to vary. In some
embodiments, the antigen binding construct
[0140] Alternatively, and/or in addition to, the antigen binding
construct can include one or more of the asterisked residues in
FIG. 2A or 2B, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the antigen
binding construct includes one or more of the non-underlined
residues in FIG. 2A or 2B. In some embodiments, the antigen binding
construct includes one or more of the non-underlined residues in
FIG. 2A or 2B as well as the boxed CDR sections, whereas other
residues are allowed to vary. In some embodiments, the CDR residues
are maintained and the residues with the asterisk are maintained,
but one or more of the other residues are allowed to vary.
[0141] In some embodiments, a chimeric minibody that binds to the
target molecule is provided. In some embodiments, the chimeric
minibody includes a monomer in the V.sub.L-V.sub.H format, and
includes the sequence of SEQ ID NO: 16 or 20, or a sequence having
at least about 80% identity thereto, for example at least about
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%% identity thereto. In
some embodiments, the chimeric minibody includes a monomer in the
V.sub.H-V.sub.L format, and includes the sequence of SEQ ID NO: 18
or 22, or a sequence having at least about 80% identity thereto,
for example at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99%% identity thereto.
[0142] In some embodiments, the polypeptide of the monomer includes
any of the combined sections as indicated in Tables 0.1 and 0.2,
including polypeptides of the monomer with a sequences of at least
about 80% identity, for example at least about 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93, 94, 95%, 96%, 97%, 98%, 99%, or 100%
identity to the monomers as set forth in Tables 0.1 and 0.2.
Nucleic Acids
[0143] In some embodiments, the polypeptides of the antigen binding
constructs can be encoded by nucleic acids and expressed in vivo or
in vitro, or these peptide can be synthesized chemically. Thus, in
some embodiments, a nucleic acid encoding an antigen binding
construct is provided. In some embodiments, the nucleic acid
encodes one part or monomer of a cys-diabody or minibody. In some
embodiments, the nucleic acid encodes two or more monomers, for
example, at least 2 monomers. Nucleic acids encoding multiple
monomers can include nucleic acid cleavage sites between at least
two monomers, can encode transcription or translation start site
between two or more monomers, and/or can encode proteolytic target
sites between two or more monomers.
[0144] In some embodiments, an expression vector contains a nucleic
acid encoding an antigen binding construct as disclosed herein. In
some embodiments, the expression vector includes
pcDNA3.1.TM./myc-His(-) Version A vector for mammalian expression
(Invitrogen, Inc.), or a variant thereof (see FIG. 13). The
pcDNA3.1 expression vector features a CMV promoter for mammalian
expression and both mammalian (Neomycin) and bacterial (Ampicillin)
selection markers (see FIG. 10). In some embodiments, the
expression vector includes a plasmid. In some embodiments, the
vector includes a viral vector, for example a retroviral or
adenoviral vector. In embodiments, the vector includes a cosmid,
YAC, or BAC.
[0145] In some embodiments, the nucleotide sequence encoding at
least one of the minibody monomers comprises at least one of SEQ ID
NOs: 17, 19, 21, 23, 39, 41, 43, 45, 47, 49, 51, or a sequence
having at least about 80% identity, for example about 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94, 95%, 96%, 97%, 98%, 99%,
or greater identity thereto.
[0146] In some embodiments, the nucleotide sequence encoding at
least one of the cys-diabody monomers includes SEQ ID NOs: 77, 78,
10, 11, 39, 41, 43, 45, 47, 49, 51, or a sequence having at least
about 80% identity, for example about 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93, 94, 95%, 96%, 97%, 98%, 99% or greater identity
thereto.
Cell Lines
[0147] In some embodiments, a cell line is provided that expresses
at least one of the antigen binding constructs described herein. In
some embodiments, a mammalian cell line (e.g., CHO-K1 cell line) is
an expression system to produce the minibodies, cys-diabodies or
other antibodies as described herein. In some embodiments, the
minibodies, cys-diabodies and other antibodies or antibody
fragments described herein are non-glycosylated, and a mammalian
expression system is not required, as such post-translational
modifications are not needed. Thus, in some embodiments, one or
more of a wide variety of mammalian or non-mammalian expression
systems are used to produce the antigen binding constructs
disclosed herein (for example, anti-CD8 minibodies and
cys-diabodies) including, but not limited to mammalian expression
systems (e.g., CHO-K1 cells), bacterial expression systems (e.g.,
E. Coli, B. subtilis) yeast expression systems (e.g., Pichia, S.
cerevisiae) or any other known expression system. Other systems can
include insect cells and/or plant cells.
Antigen Binding Construct Modifications
[0148] In some embodiments, the antigen binding construct includes
at least one modification. Exemplary modifications include, but are
not limited to, antigen binding constructs that have been modified
by glycosylation, acetylation, pegylation, phosphorylation,
amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, and linkage to a cellular ligand or other
protein. Any of numerous chemical modifications may be carried out
by known techniques, including, but not limited to, specific
chemical cleavage, acetylation, formylation and metabolic synthesis
of tunicamycin. In some embodiments, the derivative can contain one
or more non-natural amino acids.
[0149] In some embodiments, the antigen binding construct is
conjugated to another substance to form an anti-target conjugate.
The conjugates described herein can be prepared by known methods of
linking antigen binding constructs with lipids, carbohydrates,
protein or other atoms and molecules. In some embodiments, the
conjugate is formed by site-specific conjugation using a suitable
linkage or bond. Site-specific conjugation is more likely to
preserve the binding activity of an antigen binding construct. The
substance may be conjugated or attached at the hinge region of a
reduced antigen binding construct via disulfide bond formation. For
example, introduction of cysteine residues at the C-terminus of a
scFv fragment, such as those that can be introduced in the
cys-diabodies described herein, allows site-specific thiol-reactive
coupling at a site away from the antigen binding site to a wide
variety of agents. Other linkages or bonds used to form the
conjugate can include, but are not limited to, a covalent bond, a
non-covalent bond, a sulfide linkage, a hydrazone linkage, a
hydrazine linkage, an ester linkage, an amido linkage, and amino
linkage, an imino linkage, a thiosemicabazone linkage, a
emicarbazone linkage, an oxime linkage and a carbon-carbon
linkage.
Detectable Markers
[0150] In some embodiments, a modified antigen binding construct is
conjugated to a detectable marker. As used herein, a "detectable
marker" includes an atom, molecule, or compound that is useful in
diagnosing, detecting or visualizing a location and/or quantity of
a target molecule, cell, tissue, organ and the like. Detectable
markers that can be used in accordance with the embodiments herein
include, but are not limited to, radioactive substances (e.g.,
radioisotopes, radionuclides, radiolabels or radiotracers), dyes,
contrast agents, fluorescent compounds or molecules, bioluminescent
compounds or molecules, enzymes and enhancing agents (e.g.,
paramagnetic ions). In addition, some nanoparticles, for example
quantum dots and metal nanoparticles (described below) can be
suitable for use as a detection agent. In some embodiments, the
detectable marker is IndoCyanine Green (ICG).
[0151] Exemplary radioactive substances that can be used as
detectable markers in accordance with the embodiments herein
include, but are not limited to, .sup.18F, .sup.18F-FAC, .sup.32P,
.sup.33P, .sup.45Ti, .sup.47Sc, .sup.52Fe, .sup.59Fe, .sup.62Cu,
.sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.75Sc, .sup.77As,
.sup.86Y, .sup.90Y, .sup.89Sr, .sup.89Zr, .sup.94Tc, .sup.94Tc,
.sup.99 mTc, .sup.99Mo, .sup.105Pd, .sup.105Rh, .sup.111Ag,
.sup.111In, .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.142Pr,
.sup.143Pr, .sup.149Pm, .sup.153Sm, .sup.154-158Gd, .sup.161Tb,
.sup.166Dy, .sup.169Er, .sup.175Lu, .sup.177Lu, .sup.186Re,
.sup.188Re, .sup.189Re, .sup.194Ir, .sup.198Au, .sup.199Au,
.sup.211At, .sup.211Pb, .sup.212Bi, .sup.212Pb, .sup.213Bi,
.sup.223Ra and .sup.225Ac. Exemplary Paramagnetic ions substances
that can be used as detectable markers include, but are not limited
to ions of transition and lanthanide metals (e.g. metals having
atomic numbers of 6 to 9, 21-29, 42, 43, 44, or 57-71). These
metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd,
Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
[0152] When the detectable marker is a radioactive metal or
paramagnetic ion, in some embodiments, the marker can be reacted
with a reagent having a long tail with one or more chelating groups
attached to the long tail for binding these ions. The long tail can
be a polymer such as a polylysine, polysaccharide, or other
derivatized or derivatizable chain having pendant groups to which
may be bound to a chelating group for binding the ions. Examples of
chelating groups that may be used according to the embodiments
herein include, but are not limited to, ethylenediaminetetraacetic
acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA,
NOGADA, NETA, deferoxamine (DfO), porphyrins, polyamines, crown
ethers, bis-thiosemicarbazones, polyoximes, and like groups. The
chelate can be linked to the antigen binding construct by a group
which allows formation of a bond to the molecule with minimal loss
of immunoreactivity and minimal aggregation and/or internal
cross-linking. The same chelates, when complexed with
non-radioactive metals, such as manganese, iron and gadolinium are
useful for MRI, when used along with the antigen binding constructs
and carriers described herein. Macrocyclic chelates such as NOTA,
NOGADA, DOTA, and TETA are of use with a variety of metals and
radiometals including, but not limited to, radionuclides of
gallium, yttrium and copper, respectively. Other ring-type chelates
such as macrocyclic polyethers, which are of interest for stably
binding radionuclides, such as Radium-223 for RAIT may be used. In
certain embodiments, chelating moieties may be used to attach a PET
imaging agent, such as an Aluminum-.sup.18F complex, to a targeting
molecule for use in PET analysis.
[0153] Exemplary contrast agents that can be used as detectable
markers in accordance with the embodiments of the disclosure
include, but are not limited to, barium, diatrizoate, ethiodized
oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide,
iodipamide, iodoxamic acid, iogulamide, iohexyl, iopamidol,
iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid,
iosulamide meglumine, iosemetic acid, iotasul, iotetric acid,
iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,
ipodate, meglumine, metrizamide, metrizoate, propyliodone, thallous
chloride, or combinations thereof.
[0154] Bioluminescent and fluorescent compounds or molecules and
dyes that can be used as detectable markers in accordance with the
embodiments of the disclosure include, but are not limited to,
fluorescein, fluorescein isothiocyanate (FITC), OREGON GREEN.TM.,
rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3,
Cy5, and the like), fluorescent markers (e.g., green fluorescent
protein (GFP), phycoerythrin, and the like), autoquenched
fluorescent compounds that are activated by tumor-associated
proteases, enzymes (e.g., luciferase, horseradish peroxidase,
alkaline phosphatase, and the like), nanoparticles, biotin,
digoxigenin or combination thereof.
[0155] Enzymes that can be used as detectable markers in accordance
with the embodiments of the disclosure include, but are not limited
to, horseradish peroxidase, alkaline phosphatase, acid phosphatase,
glucose oxidase, .beta.-galactosidase, .beta.-glucoronidase or
.beta.-lactamase. Such enzymes may be used in combination with a
chromogen, a fluorogenic compound or a luminogenic compound to
generate a detectable signal.
[0156] In some embodiments, the antigen binding construct is
conjugated to a nanoparticle. The term "nanoparticle" refers to a
microscopic particle whose size is measured in nanometers, e.g., a
particle with at least one dimension less than about 100 nm.
Nanoparticles can be used as detectable substances because they are
small enough to scatter visible light rather than absorb it. For
example, gold nanoparticles possess significant visible light
extinction properties and appear deep red to black in solution. As
a result, compositions comprising antigen binding constructs
conjugated to nanoparticles can be used for the in vivo imaging of
T-cells in a subject. At the small end of the size range,
nanoparticles are often referred to as clusters. Metal, dielectric,
and semiconductor nanoparticles have been formed, as well as hybrid
structures (e.g. core-shell nanoparticles). Nanospheres, nanorods,
and nanocups are just a few of the shapes that have been grown.
Semiconductor quantum dots and nanocrystals are examples of
additional types of nanoparticles. Such nanoscale particles, when
conjugated to an antigen binding construct, can be used as imaging
agents for the in vivo detection of T-cells as described
herein.
Therapeutic Agents
[0157] In some embodiments, an antigen binding construct is
conjugated to a therapeutic agent. A "therapeutic agent" as used
herein is an atom, molecule, or compound that is useful in the
treatment of cancer, inflammation, other disease conditions, or to
otherwise suppress an immune response, for example
immunosuppression in organ transplants. Examples of therapeutic
agents include, but are not limited to, drugs, chemotherapeutic
agents, therapeutic antibodies and antibody fragments, toxins,
radioisotopes, enzymes (e.g., enzymes to cleave prodrugs to a
cytotoxic agent at the site of the antigen binding construct
binding), nucleases, hormones, immunomodulators, antisense
oligonucleotides, chelators, boron compounds, photoactive agents
and dyes, and nanoparticles.
[0158] Chemotherapeutic agents are often cytotoxic or cytostatic in
nature and may include alkylating agents, antimetabolites,
anti-tumor antibiotics, topoisomerase inhibitors, mitotic
inhibitors hormone therapy, targeted therapeutics and
immunotherapeutics. In some embodiments the chemotherapeutic agents
that may be used as detectable markers in accordance with the
embodiments of the disclosure include, but are not limited to,
13-cis-Retinoic Acid, 2-Chlorodeoxyadenosine, 5-Azacitidine,
5-Fluorouracil, 6-Mercaptopurine, 6-Thioguanine, actinomycin-D,
adriamycin, aldesleukin, alemtuzumab, alitretinoin,
all-transretinoic acid, alpha interferon, altretamine,
amethopterin, amifostine, anagrelide, anastrozole,
arabinosylcytosine, arsenic trioxide, amsacrine, aminocamptothecin,
aminoglutethimide, asparaginase, azacytidine, bacillus
calmette-guerin (BCG), bendamustine, bevacizumab, bexarotene,
bicalutamide, bortezomib, bleomycin, busulfan, calcium leucovorin,
citrovorum factor, capecitabine, canertinib, carboplatin,
carmustine, cetuximab, chlorambucil, cisplatin, cladribine,
cortisone, cyclophosphamide, cytarabine, darbepoetin alfa,
dasatinib, daunomycin, decitabine, denileukin diftitox,
dexamethasone, dexasone, dexrazoxane, dactinomycin, daunorubicin,
decarbazine, docetaxel, doxorubicin, doxifluridine, eniluracil,
epirubicin, epoetin alfa, erlotinib, everolimus, exemestane,
estramustine, etoposide, filgrastim, fluoxymesterone, fulvestrant,
flavopiridol, floxuridine, fludarabine, fluorouracil, flutamide,
gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin,
granulocyte--colony stimulating factor, granulocyte
macrophage-colony stimulating factor, hexamethylmelamine,
hydrocortisone hydroxyurea, ibritumomab, interferon alpha,
interleukin-2, interleukin-11, isotretinoin, ixabepilone,
idarubicin, imatinib mesylate, ifosfamide, irinotecan, lapatinib,
lenalidomide, letrozole, leucovorin, leuprolide, liposomal Ara-C,
lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine,
mesna, methotrexate, methylprednisolone, mitomycin C, mitotane,
mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin,
oxaliplatin, paclitaxel, pamidronate, pemetrexed, panitumumab, PEG
Interferon, pegaspargase, pegfilgrastim, PEG-L-asparaginase,
pentostatin, plicamycin, prednisolone, prednisone, procarbazine,
raloxifene, rituximab, romiplostim, ralitrexed, sapacitabine,
sargramostim, satraplatin, sorafenib, sunitinib, semustine,
streptozocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus,
temozolamide, teniposide, thalidomide, thioguanine, thiotepa,
topotecan, toremifene, tositumomab, trastuzumab, tretinoin,
trimitrexate, alrubicin, vincristine, vinblastine, vindestine,
vinorelbine, vorinostat, or zoledronic acid.
[0159] Toxins that may be used as detectable markers in accordance
with the embodiments of the disclosure include, but are not limited
to, ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria
toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
[0160] In some embodiments nanoparticles are used in therapeutic
applications as drug carriers that, when conjugated to an antigen
binding construct, deliver chemotherapeutic agents, hormonal
therapeutic agents, radiotherapeutic agents, toxins, or any other
cytotoxic or anti-cancer agent known in the art to cancerous cells
that overexpress the target on the cell surface.
[0161] Any of the antigen binding constructs described herein may
be further conjugated with one or more additional therapeutic
agents, detectable markers, nanoparticles, carriers or a
combination thereof. For example, an antigen binding construct may
be radiolabeled with Iodine 131 and conjugated to a lipid carrier,
such that the anti-CD8-lipid conjugate forms a micelle. The micelle
can incorporate one or more therapeutic or detectable markers.
Alternatively, in addition, the antigen binding construct may be
radiolabeled with Iodine 131 (for example, at a tyrosine residue)
and conjugated to a drug (for example, at the epsilon amino group
of a lysine residue), and the carrier may incorporate an additional
therapeutic or detectable marker.
Kits
[0162] In some embodiments, kits are provided. In some embodiments,
the kit includes an antigen binding construct as described herein.
In some embodiments, the kit includes a nucleic acid that encodes
an antigen binding construct as described herein. In some
embodiments, the kit includes a cell line that produces an antigen
binding construct as described herein. In some embodiments, the kit
includes a detectable marker as described herein. In some
embodiments, the kit includes a therapeutic agent as described
herein. In some embodiments, the kit includes buffers. In some
embodiments, the kit includes positive controls, for example CD8,
CD8+ cells, or fragments thereof. In some embodiments, the kit
includes negative controls, for example a surface or solution that
is substantially free of CD8. In some embodiments, the kit includes
packaging. In some embodiments, the kit includes instructions.
Methods of Detecting the Presence or Absence of the Target
Molecule
[0163] Antigen binding constructs can be used to detect the
presence or absence of the target molecule in vivo and/or in vitro.
Accordingly, some embodiments include methods of detecting the
presence or absence of the target. The method can include applying
an antigen binding construct to a sample. The method can include
detecting a binding or an absence of binding of the antigen binding
construct to the target molecule, CD8.
[0164] FIG. 14 illustrates some embodiments of methods of detecting
the presence or absence of CD8. It will be appreciated that the
steps shown in FIG. 14 can be performed in any sequence, and/or can
be optionally repeated and/or eliminated, and that additional steps
can optionally be added to the method. An antigen binding construct
as described herein can be applied to a sample 100. An optional
wash 110 can be performed. Optionally, a secondary antigen binding
construct can be applied to the sample 120. An optional wash can be
performed 130. A binding or absence of binding of the antigen
binding construct to the target molecule can be detected 140.
[0165] In some embodiments, an antigen binding construct as
described herein is applied to a sample in vivo. The antigen
binding construct can be administered to a subject. In some
embodiments, the subject is a human. In some embodiments, the
subject is a non-human mammal, for example a rat, mouse, guinea
pig, hamster, rabbit, dog, cat, cow, horse, goat, sheep, donkey,
pig, monkey, or ape. In some embodiments, the antigen binding
construct is infused into the subject. In some embodiments, the
infusion is intravenous. In some embodiments, the infusion is
intraperitoneal. In some embodiments, the antigen binding construct
is applied topically or locally (as in the case of an
interventional or intraoperative application) to the subject. In
some embodiments, a capsule containing the antigen binding
construct is applied to the subject, for example orally or
intraperitoneally. In some embodiments, the antigen binding
construct is selected to reduce the risk of an immunogenic response
by subject. For example, for a human subject, the antigen binding
construct can be humanized as described herein. In some
embodiments, following in vivo application of the antigen binding
construct, the sample, or a portion of the sample is removed from
the host. In some embodiments, the antigen binding construct is
applied in vivo, is incubated in vivo for a period of time as
described herein, and a sample is removed for analysis in vitro,
for example in vitro detection of antigen binding construct bound
to the target molecule or the absence thereof as described
herein.
[0166] In some embodiments, the antigen binding construct is
applied to a sample in vitro. In some embodiments, the sample is
freshly harvested from a subject, for example a biopsy. In some
embodiments, the sample is incubated following harvesting from a
subject. In some embodiments, the sample is fixed. In some
embodiments the sample includes a whole organ and/or tissue. In
some embodiments, the sample includes one or more whole cells. In
some embodiments the sample is from cell extracts, for example
lysates. In some embodiments, antigen binding construct in solution
is added to a solution in the sample. In some embodiments, antigen
binding construct in solution is added to a sample that does not
contain a solution, for example a lyophilized sample, thus
reconstituting the sample. In some embodiments, lyophilized antigen
binding construct is added to a sample that contains solution, thus
reconstituting the antigen binding construct.
[0167] In some embodiments, the antigen binding construct is
optionally incubated with the sample. The antigen binding construct
can be incubated for a period of no more than about 10 days, for
example no more than about 10 days, 9, 8, 7, 6, 5, 4, 3, 2, or 1
day, or no more than about 23 hours, for example no more than about
23 hours, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,
7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25, or 0.1 hour, including ranges
between any two of the listed values. In some embodiments, the
incubation is within a subject to which the antigen binding
construct was administered. In some embodiments, the incubation is
within an incubator. In some embodiments, the incubator is
maintained at a fixed temperature, for example about 21.degree. C.,
room temperature, 25.degree. C., 29.degree. C., 34.degree. C.,
37.degree. C., or 40.degree. C.
[0168] In some embodiments, the antigen binding construct that is
not bound to the target is optionally removed from the sample. In
some embodiments, the sample is washed. Washing a sample can
include removing solution that contains unbound antigen binding
construct, and adding solution that does not contain antigen
binding construct, for example buffer solution. In some
embodiments, an in vitro sample is washed, for example by
aspirating, pipetting, pumping, or draining solution that contains
unbound antigen binding construct, and adding solution that does
not contain antigen binding construct. In some embodiments, an in
vivo sample is washed, for example by administering to the subject
solution that does not contain antigen binding construct, or by
washing a site of topical antigen binding construct administration.
In some embodiments, the wash is performed at least two times, for
example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times. In
some embodiments, following the wash or washes, at least about 50%
of unbound antibody is removed from the sample, for example at
least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%
or greater.
[0169] In some embodiments, unbound antigen binding construct is
eliminated from the sample. Following application of the antigen
binding construct to the sample, antigen binding construct bound to
the target reaches an equilibrium with antigen binding construct
unbound to the target, so that at some time after application of
the antigen binding construct, the amount of antigen binding
construct bound to the target does not substantially increase.
After this time, at least part of the quantity of the antigen
binding construct that is unbound to the target can be eliminated.
In some embodiments, unbound antigen binding construct is
eliminated by metabolic or other bodily processes of the subject to
whom the antibody or fragment was delivered. In some embodiments,
unbound antigen binding construct is eliminated by the addition of
an agent that destroys or destabilized the unbound antigen binding
construct, for example a protease or a neutralizing antibody. In
some embodiments, 1 day after application of the antigen binding
construct, at least about 30% of the antigen binding construct that
was applied has been eliminated, for example at least about 30%,
40%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.9%. In
some embodiments, 2 days after application of the antigen binding
construct, at least about 40% of the antigen binding construct that
was applied has been eliminated, for example at least about 40%,
50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or
99.9%.
[0170] In some embodiments, the presence or absence of the target,
CD8, is detected. The presence or absence of the target can be
detected based on the presence or absence of the antigen binding
construct in the sample. After removal and/or elimination of the
antigen binding construct from the sample, for example by washing
and/or metabolic elimination, remaining antigen binding construct
in the sample can indicate the presence of the target, while an
absence of the antigen binding construct in the sample can indicate
the absence of the target.
[0171] In some embodiments, the antigen binding construct includes
a detectable marker as described herein. Thus, the presence of the
antigen binding construct can be inferred by detecting the
detectable marker.
[0172] In some embodiments, a secondary antigen binding construct
is used to detect the antigen binding construct. The secondary
antigen binding construct can bind specifically to the antigen
binding construct. For example, the secondary antigen binding
construct can include a polyclonal or monoclonal antibody, diabody,
minibody, etc. against the host type of the antibody, or against
the antigen binding construct itself. The secondary antigen binding
construct can be conjugated to a detectable marker as described
herein. The secondary antigen binding construct can be applied to
the sample. In some embodiments, the secondary antigen binding
construct is applied to the sample in substantially the same manner
as the antigen binding construct. For example, if the antigen
binding construct was infused into a subject, the secondary antigen
binding construct can also be infused into the subject.
[0173] In some embodiments, binding or the absence of binding of
the antigen binding construct is detected via at least one of:
positron emission tomography (PET), single-photon emission computed
tomography (SPECT), magnetic resonance imaging (NMR), or detection
of fluorescence emissions. PET can include, but is not limited to
microPET imaging. In some embodiments, binding of the absence of
binding of the antigen binding construct is detected via two or
more forms of imaging. In some embodiments, detection can be via
near-infrared (NIR) and/or Cerenkov.
Methods of Targeting a Therapeutic Agent to a Cell
[0174] Antigen binding constructs can be used to target a
therapeutic molecule, for example a cytotoxin to a target positive
cell, such as a cell expressing CD8. Thus, some embodiments include
methods of targeting a therapeutic agent to a target positive cell.
The method can include administering an antigen binding construct
as described herein to a subject. The subject can be a subject in
need, for example a subject in need of elimination or
neutralization of at least some target positive cells. In some
embodiments, the antigen binding construct includes at least on
therapeutic agent as described herein. In some embodiments, the
therapeutic can be directly conjugated to the antigen binding
construct via a covalent bond, such as a disulfide bond. In some
embodiments, the subject can benefit from the localization of a CD8
positive cell to another cell or agent.
[0175] Optionally, before and/or after administration of the
antigen binding construct that includes at least one therapeutic
agent, the number and/or localization of the target positive cells
of the patient is determined. For example, determining the number
and/or localization of target positive cells prior to
administration can indicate whether the patient is likely to
benefit from neutralization and/or elimination of the target
positive cells. Determining the number and/or localization of the
target positive cells after administration can indicate whether the
target positive cells were eliminated in the patient.
Additional Embodiments
[0176] Some embodiments include detection of human CD8 which is a
specific biomarker found on the surface of a subset of T-cells for
diagnostic imaging of the immune system. Imaging of the target
molecule can allow for the in vivo detection of T-cell
localization. Changes in T-cell localization can reflect the
progression of an immune response and can occur over time as a
result various therapeutic treatments or even disease states. For
example, imaging T-cell localization can be useful in
immunotherapy. Adoptive immunotherapy is a form of therapy where a
patient's own T-cells are manipulated in vitro and re-introduced
into the patient. For this form of treatment, imaging of T-cells
can be useful for monitoring and/or determining the status of the
treatment. Thus, in some embodiments, monitoring the localization
of the target molecule can be a useful for analyzing a mechanism of
action, efficacy, and/or safety in the development of drugs and/or
can aid in the clinical management of disease.
[0177] In some embodiments, the CDRs of an antigen binding
construct that binds specifically to a target, for example for the
antibody OKT8, have been adjusted to minibody and cys-diabody
arrangements. The CDRs of a murine antibody have been grafted onto
a human minibody and cys-diabody framework, thus producing a
chimeric minibody. Antibody V domains typically contain two
cysteines that form intra-disulfide bonds. The OKT8 V.sub.H has an
extra cysteine in framework 3 (FR3) which could interfere with the
expression of the protein as it may lead to aggregation and
consequently retention in the endoplasmic reticulum. Thus, some
embodiments include minibodies made with a serine replacing the
extra cysteine in the framework (see, for example, SEQ ID: 16, 18,
20, and 22).
[0178] In some embodiments, a method of targeting a CD8+ cell to a
first antigen is provided. The method can include applying a
bispecific antigen binding construct to a sample. The bispecific
antigen binding construct can include a CD8 antigen binding
construct as described herein. The bispecific antibody can include
an antigen binding construct that binds to the first antigen, for
example 1, 2, 3, 4, 5, or 6 CDR's, an scFv, or a monomer of a
minibody or cys-diabody. In some embodiments, the bispecific
antibody includes 1, 2, or 3 HCDR's of an antigen binding construct
as described herein, and/or 1, 2, or 3 LCDR's of an antigen binding
construct as described herein. In some embodiments, the bispecific
antigen binding construct includes an scFv of an antigen binding
construct as described herein. In some embodiment, the bispecific
antigen binding construct includes a V.sub.H or V.sub.L sequence as
described herein. In some embodiments, the bispecific an antigen
binding construct includes a minibody or cys-diabody monomer as
described herein. In some embodiments, the bispecific an antigen
binding construct is applied to a sample in vivo, for example an
organ or tissue of a subject. In some embodiments, the bispecific
an antigen binding construct is applied to an in vitro sample.
Without being limited to any one theory, in some embodiments, the
bispecific an antigen binding construct binds to the target on the
target positive cell, and binds to the first antigen (which can be
different from CD8) on the first cell, and thus brings the target
positive cell in proximity to the first cell. For example, a CD8+ T
cell can be brought into proximity of a cancer cell, and can
facilitate an immune response against that cancer cell.
[0179] In some embodiments, the anti-CD8 antigen binding constructs
can be imaging agents that specifically target human CD8+ T-cells.
In some embodiments, the anti-CD8 fragments can directly bind and
detect the localization of the specific subclass of T-cells that
express CD8. In some embodiments, engineered fragments able to
cross link CD8 can potentiate signaling through the T cell receptor
and enhance the ability of a subject to clear viral pathogens and
respond to tumor antigens and vaccines.
[0180] In some embodiments, the minibody and cys-diabody antibody
formats have desired pharmacokinetic characteristics for diagnostic
imaging while maintaining the high binding affinity and specificity
of the parental antibody. Compared to imaging with the full-length
parental antibody, these fragments clear much faster; yet they are
able to target the antigen for rapid high-contrast imaging. The
same favorable pharmacokinetic properties are advantageous for
targeting immune responses allowing for more controlled T cell
stimulation and preventing undesirable effects of overstimulation
(for example, cytokine storms). In preclinical models, the shorter
serum half lives for the minibody and the cys-diabody allow for
optimal imaging at approximately 16-20 hours post injection for the
minibody and 2-6 hours post-injection for the cys-diabody. Same day
imaging can provide a significant advantage in the clinic with
respect to patient care management.
[0181] In addition, the cys-diabody antibody format features the
C-terminus cysteine tail. These two sulfhydryl groups (following
mild reduction) provide a strategy for site-specific conjugation of
functional moieties such as radiolabels that will not interfere
with the cys-diabody's binding activity.
[0182] In some embodiments, these antigen binding constructs can be
diagnostic imaging agents (following labeling with an appropriate
radioisotope such as Iodine-124, Cu-64 or Zr-89 (for PET imaging)
or fluorophore (for fluorescent imaging)). As clinical imaging
agents, these CD8 antigen binding constructs can help to monitor
treatment and be used as a patient selection tool.
[0183] In some embodiments, the antigen binding constructs can be
used for applications where highly specific and high-affinity
binding to CD8 is required. Outside of diagnostic imaging, these
fragments could serve different purposes depending on the
attachment of different functional groups.
[0184] With the attachment of the appropriate infrared or
fluorescent dye, these constructs can be used as the targeting
agent for image-guided intraoperative surgery.
[0185] In some embodiments, in addition to the modifications to the
functional groups attached to the fragments, through the use of
bispecific fragments (where the fragment is able to bind 2
different antigens) it is possible to bring together CD8+ cells to
a second antigen. Bispecific full-length antibodies have been used
in cancer immunotherapy to bring cytotoxic cells of the immune
system to tumor cells. Thus, such embodiments are also contemplated
for the appropriate antigen binding constructs.
[0186] In some embodiments, provided herein are engineered scFv,
minibody, and cys-diabody antibody fragments that are able to bind
and specifically target human CD8 alpha both in vitro and in
vivo.
Example 1
Humanization of CD8 Antibodies and Antibody Fragments
[0187] The murine variable regions of the OKT8 antibody were
humanized by grafting the murine Complimentary Determining Region
(CDR) onto a human framework. The murine V genes were run against
the human V germ-line database. The human V gene with highest
sequence homology was examined for functional residues and
similarity to antigen binding loop (CDRs) structures. The V.sub.L
and V.sub.H CDRs of the murine OKT8 were then incorporated into the
human acceptor variable region framework, replacing the human CDRs.
An alignment of the corresponding murine, human germline, and
humanized sequences is shown for the heavy chain variable regions
(FIG. 2A) and light chain variable regions (FIG. 2B). In these
figures, the CDRs are boxed and the asterisks indicate residues
that differ from each other. Selected mouse residues, known to
function in the loop structure, were kept in the human
framework.
Example 2
Expression of CD8 Minibodies
[0188] The OKT8 minibody constructs (sequence combinations as
outlined in Table 0.1) were transiently transfected into CHO-K1
cells to validate expression. The transfections were performed in a
6-well plate using the Lipofectamine reagent (Invitrogen).
Following a 72-hour incubation at 37.degree. C. in a CO.sub.2
incubator, the supernatants were harvested and filtered to remove
any cells.
[0189] Western blot analysis was performed on supernatant from the
transient transfections to confirm the expression of the antibody
fragments. Supernatant from an empty vector transfection was
included as a negative control, and supernatant from the
transfection of an irrelevant minibody was used as a positive
control. Under non-reducing conditions, the minibodies run at the
expected molecular weight of 80-90 kDa (FIG. 15). A minor band
representing the monomeric form is also detected at approximately
40 kDa. These results confirm the proper expression of the chimeric
and humanized minibodies.
Example 3
Binding
[0190] The chimeric minibody variant 1 demonstrated highest binding
to CD8 by ELISA whereas the humanized variants did not show any
significant binding to CD8 (FIG. 16). Although SPR analysis of the
humanized variants showed binding to soluble CD8, several-fold loss
in affinity was observed compared to chimeric minibody and parental
OKT8 antibody. 96 well plates were coated with the recombinant
human CD8 antigen and incubated with supernatants obtained from the
transient transfection. Binding was detected with horseradish
peroxidase (HRP) conjugated goat anti-human IgG (Fc-specific) and
the chromogenic substrate 3,3',5,5'-Tetramethylbenzidine (TMB)
measuring the absorbance at 405 nm. Dilutions were done in
triplicate. Data is shown as mean of relative absorbance.
[0191] FIGS. 17A-17D display the results of the flow cytometry
analysis of the IAb_Mb_CD8 variants. All histograms show
allophycocyanin (APC) signal vs. counts. Supernatants from
transfection of the variants at different dilutions were incubated
with CD8+ cells. Cells were washed and subsequently stained with a
secondary anti-human IgG (Fc-specific) APC conjugated antibody.
1.times.10.sup.5 cells were stained per point and analysis was
performed with 10,000 events/point.
[0192] The chimeric and humanized minibodies showed
concentration-dependent binding to the CD8+ cells (FIGS. 17A-17D).
Although the humanized minibodies expressed better than the
chimeric, the chimeric minibodies showed a stronger signal by the
flow cytometry suggesting the chimeric minibodies possessed
stronger binding affinity.
Example 4
Maturation of Antigen Binding Constructs
[0193] To improve binding affinity of the humanized antibody
fragments, the two humanized V.sub.H regions were further affinity
matured. For the 1.sup.st version V.sub.H, the affinity maturation
resulted in sub-versions a and b. For the 2.sup.nd version V.sub.H,
the affinity maturation resulted in sub-versions c and d. FIGS.
12F-12I display the resulting antibody variable light (V.sub.L) and
variable heavy (V.sub.H) genes. The DNA with the amino acid
sequences are shown. CDRs are boxed using Chothia definition.
[0194] The affinity matured humanized OKT8 V genes were engineered
into two minibody variants that differed in the orientation of the
V genes in the scFv; the V.sub.L-V.sub.H orientation referred to as
number 1) and V.sub.H-V.sub.L orientation referred to as number 2.
The specific sequence combinations are outlined in Table 0.2.
Example 5
Expression of CD8 Minibodies
[0195] The above IAb_Mb_CD8 expression constructs were transiently
transfected into CHO-K1 cells. Supernatant from the transfections
was analyzed by Western blot to confirm proper expression of the
minibodies. Supernatant from an empty vector transfection was
included as a negative control, and purified protein of an
irrelevant minibody was used as a positive control. All variants
were expressed as evidenced by a band at the expected molecular
weight for the assembled minibody complex (.about.95 kDa) (FIG. 18A
and FIG. 18B). The band present at .about.45 kDa represents the
monomer. Transfection supernatants from transient transfectants
were run on SDS-PAGE and transferred to PVDF membrane. The membrane
was probed with alkaline phosphatase (AP)-conjugated anti-human IgG
(Fc-specific) antibody and developed by incubating with the AP
substrate BCIP/NBT. This is a representative blot of multiple
experiments.
[0196] Quantitative ELISAs were performed to measure IAb_Mb_CD8
variants expression levels from the transient transfection in
CHO-K1 cells. The IAb_Mb_CD8 minibody variants were expressed in a
range between .about.0.5-1.9 .mu.g/ml, with the higher end of the
range being comparable with a previously expressed reference
control minibody (FIG. 19). A goat anti-human IgG (Fc specific) was
used to capture the minibody and an AP-conjugated goat anti-human
IgG (Fc specific) was used for detection. Purified irrelevant
isotype control minibody protein was used as a standard. IAb_Mb_CD8
supernatants were serially diluted to find dilution points which
fit in the linear range of the standard curve.
Example 6
Functional Activity of Minibodies
[0197] To demonstrate the functional activity of the IAb_Mb_CD8
minibody variants, the supernatants from the transient transfection
were tested for binding to purified recombinant human CD8 protein
by ELISA. The concentration of the variants IAb_Mb1b_CD8,
IAb_Mb2b_CD8, and IAb_Mb1a_CD8 were normalized to match the
concentration of IAb_MB2a_CD8 (0.5 .mu.g/ml) based on the
quantitative ELISA presented in FIG. 19. Samples were then serially
diluted to assess binding over a series of concentrations. The
parental OKT8 antibody was also included as a positive control for
the assay (data not shown). All minibody variants showed
concentration dependent binding to soluble recombinant human CD8
(rhCD8).
[0198] FIG. 20 indicates that IAb_Mb1a_CD8 followed by IAb_Mb1b_CD8
has the highest level of binding to the antigen. 96 well plates
were coated with rhCD8 antigen and incubated with supernatants
obtained from the transient transfection. Binding was detected with
HRP-conjugated goat anti-human (Fc-specific) IgG and TMB substrate.
The absorbance measured at 405 nm. Dilutions were done in
triplicate. Data is shown as mean of relative absorbance.
[0199] FIG. 21 indicated that variant 1c has the highest level of
binding followed by variant 1c. 96 well plates were coated with
rhCD8 antigen and incubated with supernatants obtained from the
transient transfection. Binding was detected with HRP-conjugated
goat anti-human (Fc-specific) IgG and TMB substrate. The absorbance
measured at 405 nm. Dilutions were done in triplicate. Data is
shown as mean of relative absorbance.
Example 7
Binding to Cellular Human CD8
[0200] IAb_Mb_CD8 variants were evaluated for binding to cellular
human CD8 using flow cytometry. Supernatants from the transient
transfection were tested for binding to PC3-CD8 cells (PC3 cells
stably transfected with human CD8) (FIGS. 22A and 22B and 23A and
23B). The minibody supernatants were normalized for the flow
cytometry experiment. The parental OKT8 was included as a positive
control for binding (data not shown). PC3 cells was used as
negative control and confirmed that the minibody variants did not
bind (data not shown). IAb_Mb1b_CD8 demonstrated the highest Mean
Fluorescence Intensity (MFI) of the four minibody variants.
[0201] In regard to the results presented in FIGS. 22A and 22B, all
histograms show APC signal vs. counts. Supernatants from
transfection of the variants were incubated with PC3-CD8 cells.
Cells were washed and subsequently stained with APC-conjugated
anti-human IgG (Fc-specific) antibody. 1.times.10.sup.5 cells were
stained per point and analysis was performed with 10,000
events/point.
[0202] In regard to the results presented in FIGS. 23A and 23B, all
histograms show APC signal vs. counts. Supernatants from
transfection of the variants were incubated with PC3-CD8 cells.
Cells were washed and subsequently stained with APC-conjugated
anti-human IgG (Fc-specific) antibody. 1.times.10.sup.5 cells were
stained per point and analysis was performed with 10,000
events/point.
[0203] The results indicated that the constructs still bind to
cellular human CD8.
Example 8
SPR Analysis
[0204] Surface plasma resonance (SPR) was used to determine the
binding affinity for all the IAb_Mb_CD8 variants to recombinant
human CD8 (Table 8.0). The minibody protein in the supernatant was
captured on to the BIAcore chip using an anti-human IgG
(Fc-specific) antibody. The amount of minibody captured on the chip
was normalized to enable a direct comparison of binding affinity
between the variants as a kinetic "scouting" experiment to rank
affinities. The rhCD8 protein was passed over the captured minibody
protein to measure binding. All variants showed strong binding to
CD8 protein that was similar to the parental OKT8 mAb.
TABLE-US-00004 TABLE 8.0 Ligand Analyte ka (1/Ms) kd (1/s) Conc of
analyte KA (1/M) KD (M) OKT8 mAb hCD8 5.6 .times. 10.sup.5 1.0
.times. 10.sup.-3 50 nM 5.5 .times. 10.sup.8 1.8 .times. 10.sup.-9
IAb_Mb1b_CD8 hCD8 6.4 .times. 10.sup.5 1.2 .times. 10.sup.-3 50 nM
5.4 .times. 10.sup.8 1.9 .times. 10.sup.-9 IAb_Mb2b_CD8 hCD8 5.4
.times. 10.sup.5 2.1 .times. 10.sup.-3 50 nM 2.8 .times. 10.sup.8
3.9 .times. 10.sup.-9 IAb_Mb1a_CD8 hCD8 6.5 .times. 10.sup.5 1.2
.times. 10.sup.-3 50 nM 5.3 .times. 10.sup.8 1.9 .times. 10.sup.-9
IAb_Mb2a_CD8 hCD8 4.8 .times. 10.sup.5 2.2 .times. 10.sup.-3 50 nM
2.2 .times. 10.sup.8 4.6 .times. 10.sup.-9 IAb_Mb1d_CD8 hCD8 7.0
.times. 10.sup.5 2.0 .times. 10.sup.-3 50 nM 3.4 .times. 10.sup.8
2.9 .times. 10.sup.-9 IAb_Mb2d_CD8 hCD8 5.4 .times. 10.sup.5 2.9
.times. 10.sup.-3 50 nM 1.8 .times. 10.sup.8 5.5 .times. 10.sup.-9
IAb_Mb1c_CD8 hCD8 7.0 .times. 10.sup.5 1.8 .times. 10.sup.-3 50 nM
4.0 .times. 10.sup.8 2.5 .times. 10.sup.-9 IAb_Mb2c_CD8 hCD8 7.2
.times. 10.sup.5 3.2 .times. 10.sup.-3 50 nM 2.2 .times. 10.sup.8
4.5 .times. 10.sup.-9 Table 8.0 summarizes the measured association
(ka), dissociation (kd), and kD constants for the IAb_Mb_CD8
variants binding to recombinant hCD8. The variants were captured on
a BIAcore chip using anti-human Fc-specific IgG antibody.
[0205] In some embodiments, antigen binding constructs that bind in
the nanomolar range (for example 1 to 2, 2 to 10, 10 to 100, or 100
to 1,000 nM) are provided and include minibody, cys-diabody, and
scFv arrangements (for example).
Example 9
Expression of CD8 Cys-Diabodies
[0206] The cys-diabody constructs as outlined in Table 0.3
(humanized version b) were transiently transfected into CHO-K1
cells to validate expression. The transfections were performed in a
6-well plate using the Lipofectamine reagent. Following a 72-hour
incubation at 37.degree. C. in a CO.sub.2 incubator, the
supernatants were harvested and filtered to remove any cells.
[0207] A western blot analysis was performed using supernatant from
the transient transfections to confirm the expression of the
antibody fragments. Supernatant from an empty vector transfection
was included as a negative control, and supernatant from the
transfection of an irrelevant cys-diabody was used as a positive
control. Under non-reducing conditions, all four variants of the
humanized OKT8 cys-diabody ran at the appropriate molecular weight
of approximately 55 kD (FIG. 24). A minor band representing the
monomeric form is also detected at approximately 25 kD. These
results confirm the proper expression of the humanized OKT8
cys-diabodies. For the western blot, supernatants were collected
following transient transfection into CHO-K1 cells. Transfection
supernatants were electrophoresed by SDS-PAGE under non-reducing
conditions and transferred to a PVDF membrane. The membrane was
probed with an HRP-conjugated anti-His antibody and developed with
the HRP substrate TMB.
Example 10
Binding for CD8 Cys-Diabodies
[0208] The supernatants from the transient transfection of the
IAb_Cys-Dbb_CD8 variants were tested for binding to recombinant
human CD8 (rhCD8) protein by ELISA. Samples were serially diluted
to assess binding over a range of concentrations prior to
incubation with CD8. 96 well plates were coated with rhCD8 antigen.
Coated plates were incubated with supernatants following transient
transfection of the different cys-diabody variants, and then
incubated with HRP-conjugated anti-His antibody. The signal was
detected using TMB and absorbance measured at 405 nm.
[0209] All four variants exhibited concentration-dependent binding
to the rhCD8 protein (FIG. 25). The transfection supernatant from a
negative control cys-diabody that was included in the analysis did
not show any binding to CD8 (FIG. 25).
[0210] All four IAb_Cys-Dbb_CD8 variants were further tested for
binding to cellular human CD8 using flow cytometry. Supernatants
from the transient transfection were tested for binding to a
PC3-CD8 cells (FIGS. 26A and 26B). The parental OKT8 was included
as a positive control for binding (data not shown). The PC3 cells
were included as negative control and confirmed that there was no
non-specific binding of the cys-diabody variants to this cell (data
not shown). IAb_Cys-Db1b_CD8 and IAb_Cys-Db3b_CD8 demonstrated
higher Mean Fluorescence Intensity (MFI) compared to the other two
variants. All histograms in FIGS. 26A and 26B show APC signal vs.
counts. Supernatants from transfection of the variants were
evaluated for binding to PC3-CD8 cells. Cells were subsequently
stained with APC-conjugated anti-His antibody. 1.times.10.sup.5
cells/point and 10,000 events were analyzed for each point.
[0211] The IAb_Cys-Dbb_CD8 variants were evaluated for their
ability to bind endogenous CD8 expressing HPB-ALL cells. All
IAb_Cys-Db 1b and 3b variants bound to the HPB-ALL cells (FIG. 27).
The parental OKT8 was included as a positive control for binding,
and the PC3 cells were included as negative cell line. No binding
was seen by the cys-diabody variants on PC3 cells that did not
express CD8 (data not shown). Supernatants from transfection of the
variants were evaluated for binding to endogenous CD8 expressing
HPB-ALL cells. Cells were subsequently stained with APC-conjugated
anti-His antibody. All histograms show APC signal vs. counts.
1.times.10.sup.5 cells/point and 10,000 events were analyzed for
each point.
Example 11
In Vivo Detection of CD8
[0212] A humanized CD8 cys-diabody of Table 0.3 is conjugated with
a relevant chelator via C-terminal cysteines on the cys-diabody and
subsequently radiolabeled with an isotope of In111, (or in the
alternative, Zr89 or Cu64). Alternatively, the cys-diabody can be
radiolabeled after attaching relevant chelators to Lysine residues
or directly radiolabeled with Iodine.
[0213] The cys-diabody is infused intravenously into a healthy
human subject. The cys-diabody is incubated in the human subject
for 10 minutes post-infusion. Within the same day as the
incubation, the localization of the cys-diabody is detected via a
PET scan or external scintillation system.
[0214] Localization of cys-diabody is used to determine
localization of CD8 in the subject.
Example 12
In Vivo Detection of CD8
[0215] A minibody from Table 0.2 is conjugated with a relevant
chelator via Lysine residues on the minibody and subsequently
radiolabeled with an isotope of In111 (or in the alternative, Zr89
or Cu64). Alternatively, the minibody can be radiolabeled by
directly radiolabeling with Iodine via Tyrosine residues.
[0216] The minibody is infused intravenously into a healthy human
subject. The minibody is incubated in the human subject for 10
minutes post-infusion. On the same day as the incubation, the
localization of the minibody is detected via a PET scan or external
scintillation system.
[0217] Localization of cys-diabody is used to determine
localization of CD8 in the subject.
Example 13
In Vivo Detection of CD8
[0218] A humanized CD8 minibody that is a homodimer of monomers of
SEQ ID NO: 12 is provided. The minibody is infused intravenously
into a healthy human subject. The minibody is incubated in the
human subject for 1 hour post-infusion. A secondary antibody, a
humanized cys-diabody that binds specifically to the CD8 minibody
and is conjugated to 33P is provided. Within the same day as the
incubation, the secondary antibody is infused into to subject. The
secondary antibody is incubated for one hour. The localization of
the minibody is detected via PET imaging, via a marker on the
secondary antibody.
[0219] Localization of minibody is used to determine localization
of CD8 in the subject.
Example 14
Therapeutic Treatment Using a Cys-Diabody
[0220] A CD8 cys-diabody that is a homodimer of monomers of Table
0.3 is provided. The cys-diabody is infused intravenously into a
subject having a CD8 related disorder in an amount adequate to bind
to sufficient levels of CD8 in the subject to provide a lessening
of the symptoms of the CD8 related disorder. The cys-diabody is
conjugated to Yttrium 90.
Example 15
Therapeutic Treatment Using a Minibody
[0221] A CD8 minibody of Table 0.2 is provided. The minibody is
injected into a patient who has been vaccinated with an antigen to
an infectious disease or with a tumor associated antigen. The CD8
directed fragments augment the immune response and enhance the
cytolytic activity of CD8 expressing T cells.
Example 16
Therapeutic Treatment Using a Cys-Diabody
[0222] A CD8 cys-diabody that is a homodimer of a monomer of Table
0.3 is provided. The cys-diabody is infused intravenously into a
subject having a CD8 related disorder in an amount adequate to bind
to sufficient levels of CD8 in the subject to provide a lessening
of the symptoms of the CD8 related disorder. The cys-diabody is
conjugated to Lu177Tx. The CD8 cys-diabody binds to a cell
expressing CD8 and the Lu177Tx results in the killing of the
cell.
[0223] In this application, the use of the singular can include the
plural unless specifically stated otherwise or unless, as will be
understood by one of skill in the art in light of the present
disclosure, the singular is the only functional embodiment. Thus,
for example, "a" can mean more than one, and "one embodiment" can
mean that the description applies to multiple embodiments.
INCORPORATION BY REFERENCE
[0224] All references cited herein, including patents, patent
applications, papers, text books, and the like, and the references
cited therein, to the extent that they are not already, are hereby
incorporated by reference in their entirety. In the event that one
or more of the incorporated literature and similar materials
differs from or contradicts this application; including but not
limited to defined terms, term usage, described techniques, or the
like, this application controls.
EQUIVALENTS
[0225] The foregoing description and Examples detail certain
embodiments. It will be appreciated, however, that no matter how
detailed the foregoing may appear in text, the invention may be
practiced in many ways and the invention should be construed in
accordance with the appended claims and any equivalents thereof.
Sequence CWU 1
1
781118PRTMurine 1Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val
Lys Pro Gly Ala1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly
Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Phe Val Arg Gln Arg
Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala
Asn Asp Asn Thr Leu Tyr Ala Ser Lys Phe 50 55 60 Gln Gly Lys Ala
Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80 Met His
Leu Cys Ser Leu Thr Ser Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Gly Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His Trp Gly Gln Gly Thr 100
105 110 Thr Leu Thr Val Ser Ser 115 2120PRTHomo sapiens 2Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20
25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser
Lys Asn Thr Ala Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser 115 120 3118PRTArtificial SequenceAntigen binding
construct or fragment thereof 3Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile
Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser Lys Phe 50 55 60 Gln
Gly Arg Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Gly Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His Trp Gly Gln
Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 4118PRTMurine 4Gln
Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10
15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30 Tyr Ile His Phe Val Arg Gln Arg Pro Glu Gln Gly Leu Glu
Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr
Ala Ser Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr
Ser Ser Asn Thr Ala Tyr65 70 75 80 Met His Leu Cys Ser Leu Thr Ser
Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Tyr Gly Tyr
Tyr Val Phe Asp His Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val
Ser Ser 115 5115PRTHomo sapiens 5Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Thr Val Lys Ile Ser Cys
Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp
Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Leu
Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe 50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Ala Glu Tyr Phe Gln His Trp Gly Gln Gly
Thr Leu Val Thr 100 105 110 Val Ser Ser 115 6118PRTArtificial
SequenceAntigen binding construct or fragment thereof 6Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Thr
Val Lys Ile Ser Cys Lys Val Ser Gly Phe Asn Ile Lys Asp Thr 20 25
30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45 Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser
Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Tyr Tyr Val
Phe Asp His Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser
115 7107PRTMurine 7Asp Val Gln Ile Asn Gln Ser Pro Ser Phe Leu Ala
Ala Ser Pro Gly1 5 10 15 Glu Thr Ile Thr Ile Asn Cys Arg Thr Ser
Arg Ser Ile Ser Gln Tyr 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro
Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu
Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Glu Pro65 70 75 80 Glu Asp
Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Asn Pro Leu 85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 8107PRTHomo
sapiens 8Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Val Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Val Ala
Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 9107PRTArtificial
SequenceAntigen binding construct or fragment thereof 9Asp Val Gln
Ile Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Thr Ser Arg Ser Ile Ser Gln Tyr 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln
His Asn Glu Asn Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 10768DNAArtificial SequenceAntigen binding
construct or fragment thereof 10atggagaccg atacactgct gctgtgggtg
ctgctgctct gggtccctgg cagcacagga 60gacatccaga tgacacagag ccctagctcc
ctgagcgctt ccgtgggaga tagggtgacc 120atcacatgcc ggacctccag
gtccatctcc cagtacctgg cctggtacca gcagaagccc 180ggcaaggtgc
ccaagctgct catctatagc ggcagcaccc tgcagagcgg agtgccttcc
240cggttttccg gatccggctc cggcacagac tttaccctga ccatctccag
cctgcagcct 300gaggatgtcg ccacctacta ctgccaacag cacaacgaga
accccctgac cttcggcggc 360ggaaccaagg tcgagatcaa gggaggaggc
tccggaggag gaggccaagt gcagctggtc 420caatccggcg ccgaagtgaa
aaagcccggc gccaccgtga agatcagctg caaggtgtcc 480ggcttcaaca
tcaaggacac ctatatccac tgggtccagc aagcccccgg aaaaggcctg
540gagtggatgg gacggattga ccccgccaac gacaacacac tctatgcctc
caagttccag 600ggcagggtga caatcaccgc cgacaccagc accgacacag
cttatatgga gctgtcctcc 660ctccggtccg aggataccgc cgtctactac
tgcgccaggg gctacggcta ctacgtgttt 720gaccactggg gccagggcac
cctggtgaca gtgtccagcg gaggctgc 76811768DNAArtificial
SequenceAntigen binding construct or fragment thereof 11atggagaccg
acaccctgct gctctgggtc ctcctgctgt gggtgcctgg cagcacagga 60caggtgcaac
tggtgcagag cggcgccgag gtcaagaaac ctggcgccac cgtgaagatc
120agctgcaagg tgtccggctt caacatcaag gacacctaca tccactgggt
ccaacaagcc 180cccggaaagg gcctggaatg gatgggccgg attgaccccg
ccaacgacaa caccctctat 240gccagcaagt tccagggcag ggtcaccatc
accgccgaca ccagcaccga caccgcctac 300atggagctga gcagcctgcg
gagcgaagac accgccgtgt actactgcgc caggggctac 360ggctactacg
tcttcgacca ttggggacag ggcaccctcg tgacagtgtc cagcggagga
420ggatccggcg gaggaggaga tatccagatg acccagagcc cttccagcct
gtccgcttcc 480gtgggagatc gggtgaccat cacatgcagg acctccaggt
ccatctccca gtacctggcc 540tggtaccaac agaagcccgg caaggtgccc
aagctgctga tctacagcgg cagcacactg 600caatccggcg tcccttcccg
gttttccgga tccggatccg gcaccgactt caccctgacc 660atcagctccc
tgcaacccga ggacgtggcc acctactact gtcagcagca caacgagaac
720cccctcacct ttggcggcgg aaccaaggtc gagatcaagg gcggctgc
76812253PRTArtificial SequenceAntigen binding construct or fragment
thereof 12Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp
Val Pro1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Thr Ser Arg Ser 35 40 45 Ile Ser Gln Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Val Pro 50 55 60 Lys Leu Leu Ile Tyr Ser
Gly Ser Thr Leu Gln Ser Gly Val Pro Ser65 70 75 80 Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu
Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln His Asn 100 105 110
Glu Asn Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser 115
120 125 Gly Gly Gly Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys 130 135 140 Lys Pro Gly Ala Thr Val Lys Ile Ser Cys Lys Val Ser
Gly Phe Asn145 150 155 160 Ile Lys Asp Thr Tyr Ile His Trp Val Gln
Gln Ala Pro Gly Lys Gly 165 170 175 Leu Glu Trp Met Gly Arg Ile Asp
Pro Ala Asn Asp Asn Thr Leu Tyr 180 185 190 Ala Ser Lys Phe Gln Gly
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr 195 200 205 Asp Thr Ala Tyr
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala 210 215 220 Val Tyr
Tyr Cys Ala Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His Trp225 230 235
240 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Cys 245 250
13253PRTArtificial SequenceAntigen binding construct or fragment
thereof 13Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp
Val Pro1 5 10 15 Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys 20 25 30 Lys Pro Gly Ala Thr Val Lys Ile Ser Cys
Lys Val Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile His Trp
Val Gln Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Met Gly Arg
Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr65 70 75 80 Ala Ser Lys Phe
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr 85 90 95 Asp Thr
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala 100 105 110
Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His Trp 115
120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Gly Gly Gly Gly
Asp 130 135 140 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp145 150 155 160 Arg Val Thr Ile Thr Cys Arg Thr Ser Arg
Ser Ile Ser Gln Tyr Leu 165 170 175 Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Val Pro Lys Leu Leu Ile Tyr 180 185 190 Ser Gly Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 195 200 205 Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 210 215 220 Asp Val
Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Asn Pro Leu Thr225 230 235
240 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Cys 245 250
14256PRTArtificial SequenceAntigen binding construct or fragment
thereof 14Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp
Val Pro1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Thr Ser Arg Ser 35 40 45 Ile Ser Gln Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Val Pro 50 55 60 Lys Leu Leu Ile Tyr Ser
Gly Ser Thr Leu Gln Ser Gly Val Pro Ser65 70 75 80 Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu
Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln His Asn 100 105 110
Glu Asn Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly 115
120 125 Gly Gly Ser Gly Gly Gly Gly Gln Val Gln Leu Val Gln Ser Gly
Ala 130 135 140 Glu Val Lys Lys Pro Gly Ala Thr Val Lys Ile Ser Cys
Lys Val Ser145 150 155 160 Gly Phe Asn Ile Lys Asp Thr Tyr Ile His
Trp Val Gln Gln Ala Pro 165 170 175 Gly Lys Gly Leu Glu Trp Met Gly
Arg Ile Asp Pro Ala Asn Asp Asn 180 185 190 Thr Leu Tyr Ala Ser Lys
Phe Gln Gly Arg Val Thr Ile Thr Ala Asp 195 200 205 Thr Ser Thr Asp
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu 210 215 220 Asp Thr
Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Tyr Tyr Val Phe225 230 235
240 Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Cys
245 250 255 15256PRTArtificial SequenceAntigen binding construct or
fragment thereof 15Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu
Leu Trp Val Pro1 5 10 15 Gly Ser Thr Gly Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys 20 25 30 Lys Pro Gly Ala Thr Val Lys Ile
Ser Cys Lys Val Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile
His Trp Val Gln Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Met
Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr65 70 75 80 Ala Ser
Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr 85 90 95
Asp Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala 100
105 110 Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His
Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
Ser Gly Gly 130 135 140 Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser145 150 155 160 Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Thr Ser Arg Ser Ile Ser 165 170 175 Gln Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu 180 185 190 Leu Ile
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe 195 200 205
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 210
215 220 Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu
Asn225 230 235 240 Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Gly Gly Cys 245 250 255 16394PRTArtificial SequenceAntigen
binding construct or fragment thereof 16Met Glu Thr Asp Thr Leu Leu
Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15 Gly Ser Thr Gly Asp
Val Gln Ile Asn Gln Ser Pro Ser Phe Leu Ala 20 25 30 Ala Ser Pro
Gly Glu Thr Ile Thr Ile Asn Cys Arg Thr Ser Arg Ser 35 40 45 Ile
Ser Gln Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn 50 55
60 Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro
Ser65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser 85 90 95 Gly Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr
Cys Gln Gln His Asn 100 105 110 Glu Asn Pro Leu Thr Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys Gly 115 120 125 Ser Thr Ser Gly Gly Gly Ser
Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Ser Glu Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly145 150 155 160 Ala Ser
Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp 165 170 175
Thr Tyr Ile His Phe Val Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp 180
185 190 Ile Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser
Lys 195 200 205 Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser
Asn Thr Ala 210 215 220 Tyr Met His Leu Ser Ser Leu Thr Ser Gly Asp
Thr Ala Val Tyr Tyr225 230 235 240 Cys Gly Arg Gly Tyr Gly Tyr Tyr
Val Phe Asp His Trp Gly Gln Gly 245 250 255 Thr Thr Leu Thr Val Ser
Ser Glu Pro Lys Ser Cys Asp Lys Thr His 260 265 270 Thr Cys Pro Pro
Cys Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly 275 280 285 Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 290 295 300
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr305
310 315 320 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 325 330 335 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 340 345 350 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 355 360 365 Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr 370 375 380 Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys385 390 171185DNAArtificial SequenceAntigen binding
construct or fragment thereof 17atggagacag acaccctgct cctgtgggtg
ctgctcctct gggtccctgg atccaccggc 60gatgtccaga tcaaccaaag ccccagcttt
ctggctgcct cccctggaga gacaatcacc 120atcaattgcc ggaccagccg
gagcatttcc cagtacctcg cctggtacca ggaaaagcct 180ggcaagacca
acaagctgct gatctactcc ggctccacac tccagagcgg cattccctcc
240aggtttagcg gatccggatc cggaaccgac ttcacactca ccatctccgg
cctggagccc 300gaggacttcg ccatgtatta ttgccagcag cacaatgaga
accccctgac cttcggcgct 360ggcaccaagc tggagctgaa aggctccacc
agcggaggcg gatccggagg aggaagcggc 420ggcggaggct cctccgaagt
gcagctgcaa cagagcggcg ccgaactggt gaagcctgga 480gcttccgtga
aactcagctg taccgccagc ggcttcaaca tcaaggatac ctacatccac
540ttcgtgcggc aaaggcctga gcagggcctg gaatggatcg gcaggatcga
ccccgccaac 600gacaacaccc tctacgcctc caagttccaa ggcaaggcca
caatcaccgc tgatacaagc 660tccaacaccg cctacatgca cctcagctcc
ctgaccagcg gagacaccgc cgtgtactac 720tgcggacggg gatacggcta
ctatgtgttc gaccactggg gccaaggcac cacactcacc 780gtgtcctccg
agcccaagtc ctgcgacaag acacacacct gtcccccttg tggaggagga
840tcctccggag gcggctccgg cggacagcct agggagcccc aggtgtacac
actgccccct 900tccagggacg aactcaccaa gaaccaggtg tccctgacct
gcctggtgaa gggattctac 960cccagcgaca tcgccgtgga gtgggagtcc
aacggccaac ccgagaacaa ttacaagacc 1020accccccctg tgctcgattc
cgacggctcc ttcttcctgt actccaagct caccgtggac 1080aagtcccggt
ggcaacaggg caatgtgttc tcctgcagcg tcatgcacga ggccctgcat
1140aaccactaca cccagaaatc cctcagcctc tcccctggaa aatga
118518394PRTArtificial SequenceAntigen binding construct or
fragment thereof 18Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu
Leu Trp Val Pro1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Val 20 25 30 Lys Pro Gly Ala Ser Val Lys Leu
Ser Cys Thr Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile
His Phe Val Arg Gln Arg Pro Glu Gln Gly 50 55 60 Leu Glu Trp Ile
Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr65 70 75 80 Ala Ser
Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser 85 90 95
Asn Thr Ala Tyr Met His Leu Ser Ser Leu Thr Ser Gly Asp Thr Ala 100
105 110 Val Tyr Tyr Cys Gly Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His
Trp 115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Ser Thr
Ser Gly Gly 130 135 140 Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
Ser Asp Val Gln Ile145 150 155 160 Asn Gln Ser Pro Ser Phe Leu Ala
Ala Ser Pro Gly Glu Thr Ile Thr 165 170 175 Ile Asn Cys Arg Thr Ser
Arg Ser Ile Ser Gln Tyr Leu Ala Trp Tyr 180 185 190 Gln Glu Lys Pro
Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser 195 200 205 Thr Leu
Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 210 215 220
Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Glu Pro Glu Asp Phe Ala225
230 235 240 Met Tyr Tyr Cys Gln Gln His Asn Glu Asn Pro Leu Thr Phe
Gly Ala 245 250 255 Gly Thr Lys Leu Glu Leu Lys Glu Pro Lys Ser Cys
Asp Lys Thr His 260 265 270 Thr Cys Pro Pro Cys Gly Gly Gly Ser Ser
Gly Gly Gly Ser Gly Gly 275 280 285 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu 290 295 300 Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr305 310 315 320 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 325 330 335 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 340 345
350 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
355 360 365 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 370 375 380 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys385 390
191185DNAArtificial SequenceAntigen binding construct or fragment
thereof 19atggagaccg acacactcct gctctgggtg ctcctgctgt gggtgcctgg
cagcacagga 60gaagtgcagc tgcagcagtc cggcgccgaa ctcgtcaaac ccggagcctc
cgtcaaactg 120tcctgcacag ccagcggctt caacatcaag gacacctaca
tccatttcgt gcggcaaagg 180cctgaacagg gactcgagtg gatcggcagg
atcgaccccg ccaacgacaa taccctctac 240gcctccaagt tccagggaaa
ggccaccatt accgccgaca catccagcaa caccgcctac 300atgcacctca
gctccctgac atccggcgac accgccgtgt actactgcgg caggggctac
360ggctactacg tgtttgacca ctggggccag ggaacaaccc tgaccgtgtc
cagcggctcc 420acctccggag gcggaagcgg cggaggatcc ggaggaggag
gctcctccga cgtgcaaatc 480aaccagtccc ctagcttcct ggccgctagc
cctggcgaga caatcacaat caattgtcgg 540accagccggt ccatctccca
gtatctggcc tggtaccagg agaagcccgg caagacaaac 600aagctgctca
tctacagcgg cagcaccctc caatccggca tcccttcccg gtttagcggc
660tccggatccg gaaccgactt taccctgacc atcagcggcc tggaacccga
ggatttcgcc 720atgtactact gccagcagca caacgagaat cccctgacct
ttggagccgg cacaaagctc 780gagctgaagg agcccaagag ctgcgacaaa
acccacacct gtcccccttg cggaggagga 840tcctccggcg gcggaagcgg
aggacaaccc agggagcccc aggtctacac cctgcctcct 900agccgggacg
aactgacaaa gaaccaggtg tccctgacct gtctcgtcaa gggcttctac
960ccttccgaca tcgccgtcga gtgggaaagc aacggccagc ccgagaacaa
ttacaagacc 1020acaccccccg tcctggacag cgatggcagc ttcttcctct
actccaagct gaccgtggac 1080aagagccggt ggcaacaagg caacgtgttc
tcctgcagcg tcatgcatga ggccctgcac 1140aatcactaca cccagaagag
cctgagcctc tcccccggca agtga 118520394PRTArtificial SequenceAntigen
binding construct or fragment thereof 20Met Glu Thr Asp Thr Leu Leu
Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15 Gly Ser Thr Gly Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Arg Ser 35 40 45 Ile
Ser Gln Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro 50 55
60 Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro
Ser65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr
Cys Gln Gln His Asn 100 105 110 Glu Asn Pro Leu Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Gly 115 120 125 Ser Thr Ser Gly Gly Gly Ser
Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Ser Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly145 150 155 160 Ala Thr
Val Lys Ile Ser Cys Lys Val Ser Gly Phe Asn Ile Lys Asp 165 170 175
Thr Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp 180
185 190 Met Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser
Lys 195 200 205 Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Asp Thr Ala 210 215 220 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr225 230 235 240 Cys Ala Arg Gly Tyr Gly Tyr Tyr
Val Phe Asp His Trp Gly Gln Gly 245 250 255 Thr Leu Val Thr Val Ser
Ser Glu Pro Lys Ser Cys Asp Lys Thr His 260 265 270 Thr Cys Pro Pro
Cys Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly 275 280 285 Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 290 295 300
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr305
310 315 320 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 325 330 335 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 340 345 350 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 355 360 365 Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr 370 375 380 Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys385 390 211185DNAArtificial SequenceAntigen binding
construct or fragment thereof 21atggagacag acaccctcct gctgtgggtc
ctgctgctgt gggtgcctgg cagcacagga 60gacatccaaa tgacccagtc ccctagcagc
ctcagcgctt ccgtcggaga cagggtcacc 120atcacatgca ggacctccag
gtccatcagc cagtatctgg cctggtatca gcagaaaccc 180ggcaaggtgc
ctaagctgct gatctacagc ggcagcacac tccagagcgg agtgcccagc
240cggttttccg gaagcggatc cggaaccgac ttcaccctga ccatttccag
cctgcaacct 300gaagacgtgg ccacctacta ctgtcagcag cacaacgaga
accccctcac cttcggcgga 360ggcaccaaag tcgagatcaa gggcagcacc
agcggaggag gaagcggcgg aggctccgga 420ggaggaggct cctcccaagt
gcagctcgtc caaagcggcg ctgaggtgaa aaagcccggc 480gccacagtca
aaatctcctg caaggtcagc ggcttcaaca tcaaggatac ctacatccac
540tgggtgcaac aggcccccgg caaaggactc gaatggatgg gccggatcga
ccctgctaac 600gacaacacac tctacgcctc caagttccag ggcagggtga
ccatcaccgc cgatacctcc 660accgacacag cctacatgga gctgagcagc
ctgaggtccg aggacaccgc cgtctattac 720tgcgcccggg gatacggcta
ctacgtgttt gaccattggg gacagggaac actcgtgacc 780gtgagctccg
agcccaagag ctgcgacaag acccacacat gtcctccttg cggaggaggc
840agctccggag gcggatccgg cggacaacct agggagcccc aggtctatac
cctgcccccc 900agcagggacg agctgacaaa gaaccaggtc tccctgacct
gcctggtgaa aggattctac 960cccagcgaca tcgctgtcga atgggagtcc
aacggccagc ccgagaacaa ctacaagaca 1020accccccccg tgctggattc
cgacggcagc ttcttcctct actccaagct gaccgtcgac 1080aagtccaggt
ggcagcaggg caacgtgttt tcctgctccg tgatgcatga ggccctgcac
1140aaccactaca cccagaagtc cctgagcctc agccctggca agtga
118522394PRTArtificial SequenceAntigen binding construct or
fragment thereof 22Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu
Leu Trp Val Pro1 5 10 15 Gly Ser Thr Gly Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys 20 25 30 Lys Pro Gly Ala Thr Val Lys Ile
Ser Cys Lys Val Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile
His Trp Val Gln Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Met
Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr65 70 75 80 Ala Ser
Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr 85 90 95
Asp Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala 100
105 110 Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His
Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Thr
Ser Gly Gly 130 135 140 Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
Ser Asp Ile Gln Met145 150 155 160 Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly Asp Arg Val Thr 165 170 175 Ile Thr Cys Arg Thr Ser
Arg Ser Ile Ser Gln Tyr Leu Ala Trp Tyr 180 185 190 Gln Gln Lys Pro
Gly Lys Val Pro Lys Leu Leu Ile Tyr Ser Gly Ser 195 200 205 Thr Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 210 215 220
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala225
230 235 240 Thr Tyr Tyr Cys Gln Gln His Asn Glu Asn Pro Leu Thr Phe
Gly Gly 245 250 255 Gly Thr Lys Val Glu Ile Lys Glu Pro Lys Ser Cys
Asp Lys Thr His 260 265 270 Thr Cys Pro Pro Cys Gly Gly Gly Ser Ser
Gly Gly Gly Ser Gly Gly 275 280 285 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu 290 295 300 Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr305 310 315 320 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 325 330 335 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 340 345
350 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
355 360 365 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 370 375 380 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys385 390
231185DNAArtificial SequenceAntigen binding construct or fragment
thereof 23atggagaccg atacactgct gctctgggtg ctgctgctgt gggtgcctgg
aagcaccgga 60caggtgcaac tggtccagtc cggcgccgag gtgaaaaagc ctggcgccac
cgtcaagatc 120tcctgtaagg tgagcggctt caacatcaag
gacacctaca tccactgggt gcagcaggct 180cccggaaagg gactggagtg
gatgggcagg atcgaccctg ccaatgacaa caccctctac 240gccagcaagt
tccaaggacg ggtgaccatc acagccgaca catccaccga cacagcctat
300atggagctct ccagcctgag gtccgaggac accgccgtgt actactgtgc
caggggatac 360ggctattacg tgttcgacca ctggggacag ggcaccctgg
tgaccgtgag cagcggaagc 420accagcggcg gaggcagcgg aggcggaagc
ggcggcggcg gatcctccga cattcagatg 480acccaatccc cctccagcct
gtccgctagc gtgggagaca gggtgacaat cacatgtcgg 540acctccaggt
ccatcagcca atatctcgcc tggtatcagc agaagcccgg caaggtgccc
600aagctcctga tctacagcgg ctccaccctc caaagcggag tgccttcccg
gtttagcgga 660agcggcagcg gcacagactt taccctgaca atcagctccc
tgcaacctga ggacgtcgcc 720acatactact gccagcagca caacgagaac
cctctcacct ttggcggcgg caccaaagtg 780gagatcaagg agcccaaatc
ctgcgacaag acacacacct gccccccttg tggaggaggc 840agctccggcg
gcggcagcgg cggacaaccc cgggaacctc aggtgtatac actcccccct
900tccagggatg agctgaccaa gaaccaagtc tccctgacct gtctggtgaa
aggcttctac 960ccctccgaca tcgctgtcga gtgggagagc aacggccagc
ccgaaaacaa ctataagacc 1020accccccccg tgctcgattc cgatggcagc
ttcttcctgt actccaagct cacagtcgac 1080aagagccggt ggcaacaggg
caacgtcttc tcctgtagcg tcatgcacga ggccctccac 1140aaccactaca
cccagaagtc cctctccctg agccccggaa aatga 118524235PRTHomo sapiens
24Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1
5 10 15 His Ala Ala Arg Pro Ser Gln Phe Arg Val Ser Pro Leu Asp Arg
Thr 20 25 30 Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys Gln Val
Leu Leu Ser 35 40 45 Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gln
Pro Arg Gly Ala Ala 50 55 60 Ala Ser Pro Thr Phe Leu Leu Tyr Leu
Ser Gln Asn Lys Pro Lys Ala65 70 75 80 Ala Glu Gly Leu Asp Thr Gln
Arg Phe Ser Gly Lys Arg Leu Gly Asp 85 90 95 Thr Phe Val Leu Thr
Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr 100 105 110 Tyr Phe Cys
Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe 115 120 125 Val
Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg 130 135
140 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
Arg145 150 155 160 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
His Thr Arg Gly 165 170 175 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
Ala Pro Leu Ala Gly Thr 180 185 190 Cys Gly Val Leu Leu Leu Ser Leu
Val Ile Thr Leu Tyr Cys Asn His 195 200 205 Arg Asn Arg Arg Arg Val
Cys Lys Cys Pro Arg Pro Val Val Lys Ser 210 215 220 Gly Asp Lys Pro
Ser Leu Ser Ala Arg Tyr Val225 230 235 2560DNAArtificial
SequenceAntigen binding construct or fragment thereof 25atggaaaccg
acaccctgct gctgtgggtg ctgctgctct gggtcccagg ctccaccggt
602620PRTArtificial SequenceAntigen binding construct or fragment
thereof 26Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp
Val Pro1 5 10 15 Gly Ser Thr Gly 20 2715DNAArtificial
SequenceAntigen binding construct or fragment thereof 27agtggtggag
gaggc 15285PRTArtificial SequenceAntigen binding construct or
fragment thereof 28Ser Gly Gly Gly Gly1 5 2924DNAArtificial
SequenceAntigen binding construct or fragment thereof 29ggcggaggga
gtggcggagg cggc 24308PRTArtificial SequenceAntigen binding
construct or fragment thereof 30Gly Gly Gly Ser Gly Gly Gly Gly1 5
319DNAArtificial SequenceAntigen binding construct or fragment
thereof 31ggcggctgc 9323PRTArtificial SequenceAntigen binding
construct or fragment thereof 32Gly Gly Cys1 3360DNAArtificial
SequenceAntigen binding construct or fragment thereof 33atggaaaccg
acaccctgct gctgtgggtg ctgctgctct gggtcccagg ctccaccggt
603420PRTArtificial SequenceAntigen binding construct or fragment
thereof 34Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp
Val Pro1 5 10 15 Gly Ser Thr Gly 20 3554DNAArtificial
SequenceAntigen binding construct or fragment thereof 35ggctccacat
ccggcggagg ctctggcggt ggatctggcg gaggcggctc atcc
543618PRTArtificial SequenceAntigen binding construct or fragment
thereof 36Gly Ser Thr Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
Gly Gly1 5 10 15 Ser Ser37393DNAArtificial SequenceAntigen binding
construct or fragment thereof 37gagcctaagt cctgcgacaa gacccacacc
tgtccccctt gcggcggagg aagcagcgga 60ggcggatccg gtggccagcc tcgggagcct
caggtgtaca ccctgcctcc ctcccgggac 120gagctgacca agaaccaggt
gtccctgacc tgtctggtca agggcttcta cccttccgat 180atcgccgtgg
agtgggagtc caacggccag cctgagaaca actacaagac cacccctcct
240gtgctggact ccgacggctc cttcttcctg tactccaagc tcacagtgga
taagtcccgg 300tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg
aggccctgca caaccactat 360acccagaagt ccctgtccct gtctcctggc aag
39338131PRTArtificial SequenceAntigen binding construct or fragment
thereof 38Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Gly Gly1 5 10 15 Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln Pro Arg
Glu Pro Gln Val 20 25 30 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser 35 40 45 Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu 50 55 60 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro65 70 75 80 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 85 90 95 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 100 105 110
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 115
120 125 Pro Gly Lys 130 39321DNAMurine 39gatgtccaga taaaccagtc
tccatctttt cttgctgcgt ctcctggaga aaccattact 60ataaattgca ggacaagtag
gagtattagt caatatttag cctggtatca agagaaacct 120gggaaaacta
ataagcttct tatctactct ggatccactc tgcaatctgg aattccatca
180aggttcagtg gcagtggatc tggtacagat ttcactctca ccatcagtgg
cctggagcct 240gaagattttg caatgtatta ctgtcaacag cataatgaaa
acccgctcac gttcggtgct 300gggaccaagc tggagctgaa g 32140107PRTMurine
40Asp Val Gln Ile Asn Gln Ser Pro Ser Phe Leu Ala Ala Ser Pro Gly1
5 10 15 Glu Thr Ile Thr Ile Asn Cys Arg Thr Ser Arg Ser Ile Ser Gln
Tyr 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys
Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Gly Leu Glu Pro65 70 75 80 Glu Asp Phe Ala Met Tyr Tyr
Cys Gln Gln His Asn Glu Asn Pro Leu 85 90 95 Thr Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys 100 105 41321DNAArtificial SequenceAntigen
binding construct or fragment thereof 41gacgtccaga taacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgca ggacaagtag
gagtattagt caatatttag cctggtatca gcagaaacca 120gggaaagttc
ctaagctcct gatctattct ggatccactc tgcaatctgg agtcccatct
180cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 240gaagatgttg caacttatta ctgtcaacag cataatgaaa
acccgctcac gttcggcgga 300gggaccaagg tggagatcaa a
32142107PRTArtificial SequenceAntigen binding construct or fragment
thereof 42Asp Val Gln Ile Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Arg Ser
Ile Ser Gln Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Val Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Val Ala
Thr Tyr Tyr Cys Gln Gln His Asn Glu Asn Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 43354DNAMurine
43gaggtccagc tgcagcagtc tggggcagag cttgtgaagc caggggcctc agtcaagttg
60tcctgcacag cttctggctt caacattaaa gacacctata tacacttcgt gaggcagagg
120cctgaacagg gcctggagtg gattggaagg attgatcctg cgaatgataa
tactttatat 180gcctcaaagt tccagggcaa ggccactata acagcagaca
catcatccaa cacagcctac 240atgcacctct gcagcctgac atctggggac
actgccgtct attactgtgg tagaggttat 300ggttactacg tatttgacca
ctggggccaa ggcaccactc tcacagtctc ctca 35444118PRTMurine 44Glu Val
Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 20
25 30 Tyr Ile His Phe Val Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala
Ser Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser
Ser Asn Thr Ala Tyr65 70 75 80 Met His Leu Cys Ser Leu Thr Ser Gly
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Tyr Gly Tyr Tyr
Val Phe Asp His Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser
Ser 115 45354DNAArtificial SequenceAntigen binding construct or
fragment thereof 45gaagtgcagc tggtggaaag cggcggcggc ctggtgcagc
cgggcggcag cctgcgcctg 60agctgcgcgg cgagcggctt taacattaaa gatacctata
ttcattttgt gcgccaggcg 120ccgggcaaag gcctggaatg gattggccgc
attgatccgg cgaacgataa caccctgtat 180gcgagcaaat ttcagggcaa
agcgaccatt agcgcggata ccagcaaaaa caccgcgtat 240ctgcagatga
acagcctgcg cgcgggagat accgcggtgt attattgcgg ccgcggctat
300ggctattatg tgtttgatca ttggggccag ggcaccctgg tgaccgtgag cagc
35446118PRTArtificial SequenceAntigen binding construct or fragment
thereof 46Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Ile Lys Asp Thr 20 25 30 Tyr Ile His Phe Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Asp
Asn Thr Leu Tyr Ala Ser Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile
Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg
Gly Tyr Gly Tyr Tyr Val Phe Asp His Trp Gly Gln Gly Thr 100 105 110
Leu Val Thr Val Ser Ser 115 47354DNAArtificial SequenceAntigen
binding construct or fragment thereof 47gaagtgcagc tggtggaaag
cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60agctgcgcgg cgagcggctt
taacattaaa gatacctata ttcattttgt gcgccaggcg 120ccgggcaaag
gcctggaatg gattggccgc attgatccgg cgaacgataa caccctgtat
180gcgagcaaat ttcagggcaa agcgaccatt agcgcggata ccagcaaaaa
caccgcgtat 240ctgcagatga acagcctgcg cgcggaagat accgcggtgt
attattgcgg ccgcggctat 300ggctattatg tgtttgatca ttggggccag
ggcaccctgg tgaccgtgag cagc 35448118PRTArtificial SequenceAntigen
binding construct or fragment thereof 48Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His
Phe Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly
Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser Lys Phe 50 55
60 Gln Gly Lys Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Gly Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His
Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
49354DNAArtificial SequenceAntigen binding construct or fragment
thereof 49caggtgcagc tggtgcagag cggcgcggaa gtgaaaaaac cgggcgcgac
cgtgaaaatt 60agctgcaaag tgagcggctt taacattaaa gatacctata ttcattttgt
gcagcaggcg 120ccgggcaaag gcctggaatg gattggccgc attgatccgg
cgaacgataa caccctgtat 180gcgagcaaat ttcagggcaa agcgaccatt
accgcggata ccagcaccga taccgcgtat 240atggaactga gcagcctgcg
cagcggagat accgcggtgt attattgcgg ccgcggctat 300ggctattatg
tgtttgatca ttggggccag ggcaccctgg tgaccgtgag cagc
35450118PRTArtificial SequenceAntigen binding construct or fragment
thereof 50Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Asn
Ile Lys Asp Thr 20 25 30 Tyr Ile His Phe Val Gln Gln Ala Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Asp
Asn Thr Leu Tyr Ala Ser Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile
Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg
Gly Tyr Gly Tyr Tyr Val Phe Asp His Trp Gly Gln Gly Thr 100 105 110
Leu Val Thr Val Ser Ser 115 51354DNAArtificial SequenceAntigen
binding construct or fragment thereof 51caggtgcagc tggtgcagag
cggcgcggaa gtgaaaaaac cgggcgcgac cgtgaaaatt 60agctgcaaag tgagcggctt
taacattaaa gatacctata ttcattttgt gcagcaggcg 120ccgggcaaag
gcctggaatg gattggccgc attgatccgg cgaacgataa caccctgtat
180gcgagcaaat ttcagggcaa agcgaccatt accgcggata ccagcaccga
taccgcgtat 240atggaactga gcagcctgcg cagcgaagat accgcggtgt
attattgcgg ccgcggctat 300ggctattatg tgtttgatca ttggggccag
ggcaccctgg tgaccgtgag cagc 35452117PRTArtificial SequenceAntigen
binding construct or fragment thereof 52Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Thr Val Lys Ile Ser
Cys Lys Val Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His
Phe Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly
Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser Lys Phe 50 55
60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala
Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Gly Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His
Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser 115
5323PRTArtificial SequenceAntigen binding construct or fragment
thereof 53Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala1 5 10 15 Pro Glu Leu Leu Gly Gly Pro 20 5425PRTArtificial
SequenceAntigen binding construct or fragment thereof 54Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Gly1 5 10 15 Gly
Gly Ser Ser Gly Gly Gly Ser Gly 20 25 5519PRTArtificial
SequenceAntigen binding construct or fragment thereof 55Glu Arg Lys
Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val1 5 10 15 Ala
Gly Pro5670PRTArtificial SequenceAntigen binding construct or
fragment thereof 56Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr
Cys Pro Arg Cys1 5 10 15 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro
Pro Cys Pro Arg Cys Pro 20 25 30 Glu Pro Lys Ser Cys Asp Thr Pro
Pro Pro Cys Pro Arg Cys Pro Glu 35 40
45 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro
50 55 60 Glu Leu Leu Gly Gly Pro65 70 5725PRTArtificial
SequenceAntigen binding construct or fragment thereof 57Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys1 5 10 15 Pro
Ala Pro Glu Leu Leu Gly Gly Pro 20 25 5823PRTArtificial
SequenceAntigen binding construct or fragment thereof 58Glu Pro Lys
Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala1 5 10 15 Pro
Glu Leu Leu Gly Gly Pro 20 5920PRTArtificial SequenceAntigen
binding construct or fragment thereof 59Glu Ser Lys Tyr Gly Pro Pro
Cys Pro Ser Cys Pro Ala Pro Glu Phe1 5 10 15 Leu Gly Gly Pro 20
6020PRTArtificial SequenceAntigen binding construct or fragment
thereof 60Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe1 5 10 15 Leu Gly Gly Pro 20 6115DNAArtificial
SequenceRestriction site 61tctagagccg ccacc 156215DNAArtificial
SequenceRestriction site 62tctagagccg ccacc 15636DNAArtificial
SequenceRestriction site 63aagctt 6646DNAArtificial
SequenceRestriction site 64aagctt 66515DNAArtificial
SequenceRestriction site 65tctagagccg ccacc 15666DNAArtificial
SequenceRestriction site 66aagctt 66715DNAArtificial
SequenceRestriction site 67tctagagccg ccacc 15686DNAArtificial
SequenceRestriction site 68aagctt 66915DNAArtificial
SequenceRestriction site 69tctagagccg ccacc 15706DNAArtificial
SequenceRestriction site 70aagctt 67115DNAArtificial
SequenceRestriction site 71tctagagccg ccacc 15726DNAArtificial
SequenceRestriction site 72aagctt 67315DNAArtificial
SequenceRestriction site 73tctagagccg ccacc 15746DNAArtificial
SequenceRestriction site 74aagctt 67515DNAArtificial
SequenceRestriction site 75tctagagccg ccacc 15766DNAArtificial
SequenceRestriction site 76aagctt 677759DNAArtificial
SequenceAntigen binding construct or fragment thereof 77atggagaccg
atacactgct gctgtgggtg ctgctgctct gggtccctgg cagcacagga 60gacatccaga
tgacacagag ccctagctcc ctgagcgctt ccgtgggaga tagggtgacc
120atcacatgcc ggacctccag gtccatctcc cagtacctgg cctggtacca
gcagaagccc 180ggcaaggtgc ccaagctgct catctatagc ggcagcaccc
tgcagagcgg agtgccttcc 240cggttttccg gatccggctc cggcacagac
tttaccctga ccatctccag cctgcagcct 300gaggatgtcg ccacctacta
ctgccaacag cacaacgaga accccctgac cttcggcggc 360ggaaccaagg
tcgagatcaa gtccggagga ggaggccaag tgcagctggt ccaatccggc
420gccgaagtga aaaagcccgg cgccaccgtg aagatcagct gcaaggtgtc
cggcttcaac 480atcaaggaca cctatatcca ctgggtccag caagcccccg
gaaaaggcct ggagtggatg 540ggacggattg accccgccaa cgacaacaca
ctctatgcct ccaagttcca gggcagggtg 600acaatcaccg ccgacaccag
caccgacaca gcttatatgg agctgtcctc cctccggtcc 660gaggataccg
ccgtctacta ctgcgccagg ggctacggct actacgtgtt tgaccactgg
720ggccagggca ccctggtgac agtgtccagc ggaggctgc 75978759DNAArtificial
SequenceAntigen binding construct or fragment thereof 78atggagaccg
acaccctgct gctctgggtc ctcctgctgt gggtgcctgg cagcacagga 60caggtgcaac
tggtgcagag cggcgccgag gtcaagaaac ctggcgccac cgtgaagatc
120agctgcaagg tgtccggctt caacatcaag gacacctaca tccactgggt
ccaacaagcc 180cccggaaagg gcctggaatg gatgggccgg attgaccccg
ccaacgacaa caccctctat 240gccagcaagt tccagggcag ggtcaccatc
accgccgaca ccagcaccga caccgcctac 300atggagctga gcagcctgcg
gagcgaagac accgccgtgt actactgcgc caggggctac 360ggctactacg
tcttcgacca ttggggacag ggcaccctcg tgacagtgtc cagctccggc
420ggaggaggag atatccagat gacccagagc ccttccagcc tgtccgcttc
cgtgggagat 480cgggtgacca tcacatgcag gacctccagg tccatctccc
agtacctggc ctggtaccaa 540cagaagcccg gcaaggtgcc caagctgctg
atctacagcg gcagcacact gcaatccggc 600gtcccttccc ggttttccgg
atccggatcc ggcaccgact tcaccctgac catcagctcc 660ctgcaacccg
aggacgtggc cacctactac tgtcagcagc acaacgagaa ccccctcacc
720tttggcggcg gaaccaaggt cgagatcaag ggcggctgc 759
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